Adaptive bioreactors and methods of use

Abstract

An adaptive bioreactor that can be an N-1 bioreactor in a seed train system. The adaptive bioreactor can include a housing and a bioprocess container with a flexible sidewall, a top end cap and a bottom end cap, wherein the top end cap and bottom end cap are more rigid than the flexible sidewall and the bioprocess container is attached to the housing by the top end cap.

Description

Docket No. TP389720WO1ADAPTIVE BIOREACTORS AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the U.S. Provisional Patent Application Ser. No.63 / 841,896 filed July 10, 2025, titled “ADAPTIVE BIOREACTORS AND METHODS OF USE,” U.S. Provisional Patent Application Ser. No. 63 / 828,779, filed June 23, 2025. titled “ADAPTIVE BIOREACTORS AND METHODS OF USE,” U.S. Provisional Patent Application Ser. No.63 / 820,301, filed June 09, 2025, titled “ADAPTIVE BIOREACTORS AND METHODS OF USE,” and U.S. Provisional Patent Application Ser. No. 63 / 791,324, filed April 18, 2025, titled “ADAPTIVE BIOREACTORS AND METHODS OF USE,” which are incorporated herein by specific reference.FIELD

[0002] The present disclosure relates to adaptive bioreactors used in cell expansion and seed train operations for the scalable production of biopharmaceuticals.BACKGROUND

[0003] cGMP and GMP production of biopharmaceuticals relies heavily on seed train operations for scalable cell expansion. In seed trains, cells are sequentially expanded by transferring them across multiple lab- and production-scale vessels and equipment with increasing volumes, resulting in higher cell population concentrations. Current seed train operations often start in a small flask. Once the cell density reaches a predetermined value in a first growth vessel, cells and media are sequentially transferred to bench-top and larger production-scale bioreactors, where additional media is added for further cell expansion. Cell expansion is optimized across multiple vessels by controlling numerous process and equipment parameters. Small and large variations exist between equipment parameters between seed train vessels, which complicates optimization of process parameters, scale-up parameters, and predictable yield across bioreactors and other reaction vessels.

[0004] Current seed train operations suffer from numerous shortcomings. For example, transferring cells between different bioreactors is time-consuming and labor-intensive. Equipment and process parameters are not optimized across lab and production scale bioreactors, resulting in reduced and unpredictable yield. Transferring cell suspensions between multiple bioreactors increases the risk of contamination and increases operational and storage costs.Docket No. TP389720WO1

[0005] There is a need to optimize process and equipment parameters across a smaller number of vessels in seed train operations. Improved bioreactors and seed train processes are disclosed herein that address current shortcomings in the industry.SUMMARY OF THE DISCLOSURE

[0006] It is understood that each independent aspect recited herein may include any of the features, options, and possibilities recited in association with the other independent aspects set forth above or as recited elsewhere within this document.

[0007] In various embodiments, a bioreactor is provided that includes a housing and a bioprocess container having a flexible sidewall, a top end cap, and a bottom end cap. The top end cap and the bottom end cap are more rigid than the flexible sidewall, and the top end cap is configured to be secured to the housing. In some embodiments, the bioprocess container is suspended from the housing via the top end cap such that the bottom end cap is not attached to the housing.

[0008] In certain embodiments, the bioreactor further comprises at least one integrated heating system including at least two heating plates located at different heights on a sidewall of the housing. In some embodiments, multiple integrated heating systems are provided, each having heating plates positioned at different heights on different sidewalls of the housing. In particular embodiments, at least one heating plate is positioned approximately 3.0 to 5.0 inches from a bottom end wall of the housing to facilitate operation at low working volumes.

[0009] In various embodiments, the bioprocess container includes an impeller assembly comprising an impeller mounting hub, a drive shaft, and one or more blade assemblies. In some embodiments, the blade assemblies include a first blade assembly and a second blade assembly, wherein the first blade assembly is positioned above the second blade assembly. Each blade assembly may include at least two blades, and the blades may be oriented at an angle relative to a horizontal axis to enhance mixing performance.

[0010] In some embodiments, the bioprocess container further includes one or more sensors positioned on the flexible sidewall. The sensors may be spaced from the bottom end cap or positioned at a height corresponding to one or more blade assemblies to enable monitoring of process parameters at low working volumes.

[0011] In certain embodiments, the housing includes a door configured to facilitate installation and removal of the bioprocess container. The bioprocess container may further include one or moreDocket No. TP389720WO1installation tabs on the flexible sidewall that indicate proper orientation during installation. In some embodiments, the bioprocess container includes a sparger located near the bottom end cap for gas delivery, and the sparger may also include one or more installation tabs to ensure proper orientation.

[0012] In various embodiments, a seed train system is provided that includes an N-l bioreactor and a production scale bioreactor. The N-l bioreactor is associated with a first set of equipment parameters, and the production scale bioreactor is associated with a second set of equipment parameters, wherein at least one equipment parameter is substantially identical between the two sets. The N-l bioreactor may include a housing and a bioprocess container having a flexible sidewall, a top end cap, and a bottom end cap, with the top end cap configured to be secured to the housing. The production scale bioreactor may include a production scale housing and a production scale bioprocess container.

[0013] In some embodiments, the flexible sidewalls of the N-l bioprocess container and the production scale bioprocess container are made from the same material, thereby improving scalability and consistency across the seed train. In certain embodiments, the N- 1 and production scale bioreactors include corresponding impeller shafts and spargers having defined geometries, including diameters and numbers of sparger holes, which may be scaled or matched between systems.

[0014] In various embodiments, the equipment parameters shared between the N-l and production scale bioreactors include one or more parametric ratios. These ratios may include, for example: (i) impeller shaft diameter divided by equivalent bioprocess container diameter; (ii) impeller shaft diameter divided by liquid height; (iii) number of sparger holes divided by sparger surface area; (iv) number of sparger holes divided by sparger diameter; and (v) sparger hole diameter divided by sparger diameter. In some embodiments, the equipment parameters further include a turn-down ratio defined as the ratio of maximum working volume to minimum working volume of the bioreactor, which may be, for example, approximately 5:1.

[0015] In certain embodiments, the seed train system further includes a controller configured to control operation of the N-l bioreactor and the production scale bioreactor based on the first and second sets of equipment parameters.

[0016] In various embodiments, a seed train process is provided that includes growing a first density of cells in an N-l bioreactor having a first set of equipment parameters, transferring the cells to a production scale bioreactor having a second set of equipment parameters, and growing a second density of cells in the production scale bioreactor. At least one equipment parameter is substantially identical between the first and second sets of equipment parameters. In some embodiments, aDocket No. TP389720WO1controller including a processor is used to predict the second density of cells based on the first and second sets of equipment parameters.

[0017] In certain embodiments, the N-l bioreactor and production scale bioreactor used in the process include corresponding bioprocess containers, impeller shafts, equivalent diameters, and spargers with defined geometries and hole configurations. The process may further utilize shared or scaled equipment parameters, including the parametric ratios described above, to enable predictable scale-up and improved process performance.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Various embodiments of the present disclosure are described with reference to the appended drawings. The drawings herein depict only example embodiments of the invention and are not limiting in scope.

[0019] FIG. 1 illustrates a partially exploded view of a bioreactor in accordance with example embodiments.

[0020] FIG. 2 illustrates the bioreactor of FIG. 1 with an installed bioprocess container in accordance with example embodiments.

[0021] FIG. 3 illustrates a bioprocess container in accordance with example embodiments.

[0022] FIG. 4A illustrates a side cross-sectional view of an impeller assembly in accordance with example embodiments.

[0023] FIG. 4B illustrates a top cross-sectional view of an impeller assembly in accordance with example embodiments.

[0024] FIG. 4C illustrates a top view of a top cap of the bioprocess container in accordance with example embodiments.

[0025] FIG. 4D illustrates a top view of a top cap of the bioprocess container in accordance with example embodiments.

[0026] FIG. 4E illustrates a top view of a top cap of the bioprocess container in accordance with example embodiments.

[0027] FIG. 4F illustrates a portion of a front view of the bioprocess container in accordance with example embodiments.

[0028] FIG. 5 illustrates an impeller mounting hub in accordance with example embodiments.Docket No. TP389720WO1

[0029] FTG. 6 illustrates a bottom cap of a bioprocess container in accordance with example embodiments.

[0030] FIG. 6A illustrates a bottom view of a bottom cap of the bioprocess container in accordance with example embodiments.

[0031] FIG. 7 illustrates a bioreactor heating assembly in accordance with example embodiments.

[0032] FIG. 8 illustrates a seed train system, including an N-l bioreactor and production scale bioreactor in accordance with example embodiments.

[0033] FIG. 8A illustrates a sparger system for an N-l bioreactor in accordance with example embodiments.

[0034] FIG. 8B illustrates a sparger system for a production scale bioreactor in accordance with example embodiments.

[0035] FIG. 8C illustrates a series of bioreactors of increasing scale for use in a seed train system.

[0036] FIG. 9 illustrates a block diagram of an example computing device that can automate all operations of an example seed train systems and cell expansion operations, including adaptive N-l bioreactor and production scale bioreactor.

[0037] FIG. 10 illustrates a plot of mean viable cell density versus process time in accordance with example embodiments.

[0038] FIG. 11 illustrates a plot of mean IgG concentration versus process time in accordance with example embodiments.

[0039] FIG. 12 illustrates a plot of Specific productivity versus process time in accordance with example embodiments.

[0040] FIG. 13 illustrates a plot of mean lactate concentration versus process time in accordance with example embodiments.

[0041] FIG. 14 illustrates a plot of online pH versus process time in accordance with example embodiments.

[0042] FIG. 15 illustrates a plot of dissolved oxygen versus process time in accordance with example embodiments.

[0043] FIG. 16 illustrates a plot of lactate dehydrogenase (LDH) versus process time in accordance with example embodiments.Docket No. TP389720WO1DETAILED DESCRIPTION

[0044] The present disclosure relates to adaptive bioreactors used in cell expansion and seed train operations for the scalable production of biopharmaceuticals. This disclosure is not limited to the example apparatus, systems, methods, or process parameters disclosed. Various aspects of the disclosure may vary. Terminology used herein is for the purpose of describing example embodiments of the present disclosure and is not intended to limit the scope of the disclosure.

[0045] The term “comprising” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0046] It will be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a “port” includes one, two, or more ports.

[0047] As used in the specification and appended claims, directional terms, such as “top,” “bottom,” “left,” “right.” “up,” “down,” “upper,” “lower,” “proximal,” “distal” and the like are used herein solely to indicate relative directions and are not otherwise intended to limit the scope of the disclosure or claims.

[0048] Where possible, like numbering of elements have been used in various figures. Furthermore, multiple instances of an element and or sub-elements of a parent element may each include separate letters appended to the element number. For example, two instances of a particular element “10” may be labeled as “10A” and “10B”. In that case, the element label may be used without an appended letter (e.g., “10”) to generally refer to all instances of the element or any one of the elements. Element labels including an appended letter (e.g., “10A”) can be used to refer to a specific instance of the element or to distinguish or draw attention to multiple uses of the element. Furthermore, an element label with an appended letter can be used to designate an alternative design, structure, function, implementation, and / or embodiment of an element. For example, two alternative exemplary embodiments of a particular element may be labeled as “10A” and “10B”. In that case, the element label may be used without an appended letter (e.g., “10”) to generally refer to all instances of the alternative embodiments or any one of the alternative embodiments.

[0049] Various aspects of the present devices and systems may be illustrated by describing components that are coupled, attached, and / or joined together. As used herein, the terms “coupled”, “attached”, and / or “joined” are used to indicate either a direct connection between two componentsDocket No. TP389720WO1or, where appropriate, an indirect connection to one another through intervening or intermediate components. In contrast, when a component is referred to as being “directly coupled”, “directly attached”, and / or “directly joined” to another component, there are no intervening elements present. Furthermore, as used herein, the terms “connection,” “connected,” and the like do not necessarily imply direct contact between the two or more elements.

[0050] The terms “expanded” and “expansion,” as used herein, refer to cellular reproduction. For example, if the number of cells in a cell culture increases from 1,000 cells to 4,000 cells, the cells would be expanded four-fold. Assuming 100% of the cells in the culture medium are both reproducing and reproducing at the same rate, this amount of expansion would occur after two cell divisions. In many instances herein, the terms “cultured” and “expanded” are used interchangeably.

[0051] The term “activation,” as used herein, refers to the state of a cell following sufficient cell surface moiety ligation to induce a measurable morphological, phenotypic, and / or functional change. Within the context of T cells, such activation may be the state of a T cell that has been sufficiently stimulated to induce cellular proliferation. Activation of a T cell may also induce cytokine production and / or secretion, and up- or down-regulation of expression of cell surface molecules, such as receptors or adhesion molecules, or up- or down-regulation of secretion of certain molecules, and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up- or down-regulation of a particular physico-chemical process.

[0052] In some instances, stimulation may comprise a primary response induced by ligation of a cell surface moiety. For example, in the context of receptors, such stimulation may entail the ligation of a receptor and a subsequent signal transduction event. In some instances, expansion of T cells, for example, may comprise stimulating of these T cells. With respect to stimulation of a T cell, such stimulation may refer to the ligation of a T cell surface moiety that in embodiments subsequently induces a signal transduction event, such as binding the TCR / CD3 complex. In some instances, the stimulation event may activate a cell and up- or down-regulate expression of cell surface molecules such as receptors or adhesion molecules, or up- or down-regulate secretion of a molecule, such as down-regulation of Tumor Growth Factor beta (TGF-0) or up-regulation of IL-2, IFN-y etc. In some instances, ligation of cell surface moieties, even in the absence of a direct signal transduction event, may result in the reorganization of cytoskeletal structures, or in the coalescing of cell surface moieties, each of which could serve to enhance, modify, or alter subsequent cell responses.Docket No. TP389720WO1

[0053] The term “stimulatory agent”, as used herein, refers to a molecule that binds to one or more cell type and induces a cellular response. The agent may bind any cell surface moiety, such as a receptor, an antigenic determinant, or other binding site present on the target cell population. The agent may be a protein, peptide, antibody and antibody fragments thereof, fusion proteins, synthetic molecule, an organic molecule (e.g., a small molecule), or the like. In embodiments, in the context of T cell stimulation, antibodies are used as a prototypical example of such an agent.

[0054] Antibodies for use in methods set out herein may be of any species, class or subtype providing that such antibodies can react with the target of interest, e.g., CD3, the TCR, or CD28, as appropriate. Thus “antibodies” for use in methods set out herein include:(a) any of the various classes or sub-classes of immunoglobulin (e.g., IgG, IgA, IgM, IgD or IgE derived from any animal, e.g.. any of the animals conventionally used, e.g., sheep, rabbits, goats, mice, camelids, or egg yolk),(b) monoclonal or polyclonal antibodies,(c) intact antibodies or fragments of antibodies, monoclonal or polyclonal, the fragments being those which contain the binding region of the antibody, e.g., fragments devoid of the Fc portion (e.g.. Fab. Fab‘, F(ab')2, scFv, VHH, or other single domain antibodies), the so called “half molecule” fragments obtained by reductive cleavage of the disulphide bonds connecting the heavy chain components in the intact antibody. Fv may be defined as a fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.(d) antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, fragments of antibodies, “humanized antibodies”, chimeric antibodies, or synthetically made or altered antibody-like structures.

[0055] Also included are functional derivatives or “equivalents” of antibodies e.g., single chain antibodies, CDR-grafted antibodies etc. A single chain antibody (SCA) may be defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a fused single chain molecule.

[0056] ‘ ‘Chimeric antigen receptor” or “CAR” or “CARs” as used herein refers to engineered receptors, which graft an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or any combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. In embodiments, a CAR comprises one or more antigen- specific targeting domains, an extracellular domain, aDocket No. TP389720WO1transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. In embodiments, if the CAR targets two different antigens, the antigen-specific targeting domains may be arranged in tandem. In embodiments, if the CAR targets two different antigens, the antigen-specific targeting domains may be arranged in tandem and separated by linker sequences.

[0057] CARs are engineered receptors, which graft an arbitrary specificity onto an immune cell (e.g., a T cell, such as an activated T cell). These receptors are used to graft the specificity of a monoclonal antibody onto immune cells; with transfer of their coding sequence facilitated by retroviral vectors. The receptors are called chimeric because they are composed of parts from different sources. CARs may be used as a therapy for cancer through adoptive cell transfer. T cells are removed from a patient and modified so they express receptors specific to the patient’s particular cancer. The T cells, which recognize and kill the cancer cells, are reintroduced into the patient. In embodiments, modification of T cells sourced from donors other than the patient may be used to treat the patient.

[0058] Using adoptive transfer of T cells expressing chimeric antigen receptors, CAR-modified T cells can be engineered to target any tumor-associated antigen. Following the collection of a patient’s T cells, the cells are genetically engineered to express CARs specifically directed towards antigens on the patient’s tumor cells before being infused back into the patient.

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, the preferred materials and methods are described herein.

[0060] Various aspects of the present devices, systems, and methods may be illustrated with reference to one or more exemplary embodiments. As used herein, the terms “example,” “embodiment,” and “example embodiment” mean “serving as an example, instance, or illustration,” and should not necessarily be construed as required or as preferred or advantageous over other embodiments disclosed herein.

[0061] The present disclosure relates to adaptive bioreactors used in seed train systems and cell expansion operations. The seed train systems can include one or more bioreactors of various sizes, including an N-l bioreactor that is smaller in size and volume than a production scale bioreactor. The N-l bioreactor is adaptive for several reasons, including its ability to operate at a fraction of the total fluid volume capacity of the bioreactor. The N-l bioreactors heaters, sparger, sensors and impellerDocket No. TP389720WO1assemblies are in numerous locations, including low volume locations, so the adaptive bioreactor can operate at low volume. In example embodiments, the total capacity of the N-l adaptive bioreactor is 5 liters, and the bioreactor can operate and grow cells with 1, 2, 3. 4, and 5 liters of cell culture in the bioreactor, in part, because at least one sensor, sparger, heater, heater shut-off switch, and impeller blade is located at a low volume location. Low volume locations can include a height along the bioreactor that is equivalent to 1-4 liters of cell culture in the bioprocess container or 14 to % the total volume capacity of the bioreactor, including bioprocess container.

[0062] In seed train systems and operations, the N-l adaptive bioreactor has one or more equivalent equipment parameters as the production scale bioreactor in the seed train, allowing greater optimization of equipment and process parameters across the two or more bioreactors in the seed train system. This also leads to an increase in the cell concentration and populations, an increase in scale-up predictability and an increase in the yield of the pharmaceutical end-products. Therefore, the N-l bioreactors and other bioreactors disclosed herein are adaptive in several ways, including adaptive to low volume, high turn-down ratio operations and adaptive to scale-up and process optimization in seed train systems with larger production scale bioreactors. The high turn-down ratio and low volume adaptivity of the N-l bioreactors disclosed herein reduced the number of vessels necessary for seed train operations.

[0063] Equipment parameters can include, but are not limited to volume of the bioreactor or bioprocess container; height of the bioreactor or bioprocess container; diameter of the bioreactor or bioprocess container, equivalent diameter of the bioreactor or bioprocess container, shape of the bioreactor or bioprocess container; bioreactor impeller shape; bioreactor impeller size; bioreactor impeller diameter; bioreactor impeller spacing, bioreactor impeller height relative to working volumes or liquid heights or turndown ratio, bioreactor impeller pitch or angle, bioreactor impeller count, and bioreactor impeller blade count; size, diameter or surface area of the sparger; spacing or distance between of sparger holes / pore; number sparger holes; size or diameter of sparger holes; position of sparger relative to mixing system or impeller; impeller tip speed; minimum and maximum RPM of impeller; mixing dynamics and other equipment parameters. Equipment parameters can also include certain parametric ratios, including but not limited to, impeller diameter divided by equivalent diameter of the bioreactor or bioprocess container (EPl); impeller diameter divided by the liquid height of the bioreactor or bioprocess container (EP2); number of sparger holes divided by the surface area of the sparger (EP3); number of sparger holes divided by the diameter of the sparger (EP4);Docket No. TP389720WO1sparger hole diameter divided by the diameter of the sparger (EP5); liquid column height divided by the width of the bioreactor / bioprocess container (EP6); liquid column height divided by the diameter of the bioreactor / bioprocess container (EP7); liquid column height divided by the equivalent diameter of the bioreactor / bioprocess container (EP8); liquid column height divided by the volume of the gas headspace of the bioreactor / bioprocess container (EP9); height of the bioreactor / bioprocess container divided by the width of the bioreactor / bioprocess container (EP10); height of the bioreactor / bioprocess container divided by the diameter of the bioreactor / bioprocess container (EP11); height of the bioreactor / bioprocess container divided by the equivalent diameter of the bioreactor / bioprocess container (EP 12); height of the bioreactor / bioprocess container divided by the volume of the gas headspace of the bioreactor / bioprocess container (EP 13); turn-down ratio or the ratio of the maximum working volume to the minimum working volume of the bioreactor / bioprocess container (EP 14); Volume of the liquid in the bioreactor / bioprocess container divided by the volume of the gas headspace in the bioreactor / bioprocess container EP15 and others.Impeller diameterEPl =Equivalent diameter of the bioreactor / bioprocess containerImpeller diameterEP2 =Liquid height of the bioreactor / bioprocess containerNumber of sparger holesEP3 =Surface area of the spargerNumber of sparger holesEP4 =Diameter of the spargerSparger hole diameterEP5 =Diameter of the spargerLiquid column HeightEP6 =Width of the bioreactor / bioprocess containerLiquid column HeightEP7 =Diameter of the bioreactor / bioprocess containerDocket No. TP389720WO1Liquid column HeightEP8 =Equivalent Diameter of the bioreactor / bioprocess containerLiquid column HeightVolume of the gas headspace in the bioreactor / bioprocess containerHeight of the bioreactor / bioprocess containerEP10 =Width of the bioreactor / bioprocess containerHeight of the bioreactor / bioprocess containerEP11 =Diameter of the bioreactor / bioprocess containerHeight of the bioreactor / bioprocess containerEP12 =Equivalent Diameter of the bioreactor / bioprocess containerHeight of the bioreactor / bioprocess containerEP13 =Volume of the gas headspace in the bioreactor / bioprocess containerMaximum working volume of the bioreactor / bioprocess containerEP14 =Minimum working volume of the bioreactor / bioprocess containerVolume of the liquid in the bioreactor / bioprocess containerEP15 =Volume of the gas headspace in the bioreactor / bioprocess container

[0064] Process parameters can include but are not limited to cell concentration of cell culture; pH of the cell culture; temperature of the cell culture; pressure of the cell culture; glucose concentration of cell culture; oxygen concentration of the cell culture; carbon dioxide concentration of the cell culture; osmolality of the cell culture; Reynolds number of the cell culture; volumetric mass transfer coefficient (k a) of the cell culture; amino acid concentration of the cell culture; vitamin concentration of the cell culture; growth factor concentration of the cell culture; lactate concentration of the cell culture; ammonia concentration of the cell culture; other waste product concentration of the cell culture; composition of culture medium of the cell culture; mass flow rate of gas into the bioprocess container divided by volume of the bioprocess container, bioreactor impeller power input, power input per volume, impeller rotation number (agitation rate, rpm), pump rate of the impeller, pump rate of the impeller per volume, impeller tip speed, Kolmogorov eddy length, aeration or gassing rate perDocket No. TP389720WO1volume. Cell culture in the context of this disclosure can include cell media, cells and / or cell media and cells combined.

[0065] The N-l bioreactors and production scale bioreactors disclosed herein can be operated by rotating a mixing element, including a motor and impeller assembly, at various rotations per minute (RPM). The RPM can vary with the size of the bioreactor, type / configuration of the mixing element and the type and condition of the cells in the bioreactor. For example, bioreactor impellers can be operated at about 40 to 1,500 RPM (e.g., from about 40 to about 1,000, from about 40 to about 800, from about 40 to about 600, from about 40 to about 400, from about 40 to about 300. from about 40 to about 200, from about 100 to about 1,000, from about 100 to about 800, from about 100 to about 600, from about 100 to about 400, from about 100 to about 300, from about 60 to about 1,000, from about 60 to about 300. etc., RPM). For more sensitive process conditions or when cell types or conditions are fragile, for example after electroporation, the bioreactor impeller and mixing system can be operated at lower RPM of less than 200 RPM, less than 100 RPM, less than 40 RPM, or less than 10 RPM.

[0066] By way of example and not by limitation, the bioreactors and bioprocess containers disclosed herein can be used to contain, process, react and transfer various solutions and / or suspensions including one or more biocomponents. Biocomponents can include fluids, solids, mixtures, solutions, and suspensions including, but not limited to, bacteria, fungi, algae, plant cells, animal cells, white blood cells, T-cells, cell media, protozoans, nematodes, plasmids, viral vectors, blood, plasma, organelles, proteins, nucleic acids, lipids, plasmids, carbohydrates, and / or other biological components. Examples of some common biological components that are grown in example bioreactors include E. coli, yeast, bacillus, T-cells and CHO cells. Solutions and / or suspensions in example bioreactors can also include cell media or cell culture used to feed, grow expand and produce components, such as cells, within the bioreactor. Example cell media components can include water, nutrients, vitamins, salts, glucose, buffering agents, serums, metabolites, vitamins, minerals, amino acids, lipids, fatty acids, growth factors, hormones, cytokines, supplements, metals, antibiotics and / or other cell media components that promote cell replication, cell growth, cell metabolism, cell membrane integrity, protein synthesis, cell division, cell differentiation, cell signaling and communication, environmental stability and other cellular and environmental factors that mimic in vivo conditions. In many cases, the ultimate purpose of expanding and growing cells in example bioreactors and seed train operations disclosed herein is to produce and later recover biologicalDocket No. TP389720WO1products, such as recombinant proteins, viral vectors and antibodies that are generated from the cells in the cell media.

[0067] Bioprocess containers disclosed herein can be used to contain, process, react and transfer various solutions and / or suspensions including one or more biocomponents and cell media for subsequent use (e.g., protein, antibody, and vaccine production from the cells themselves). In many applications, the ultimate purpose of growing cells in bioprocess containers is to produce and later recover biological products, such as recombinant proteins, viral vectors, and antibodies that are generated from the cells in the cell media. Bioprocess containers herein disclosed can be used to grow and expand cells in seed train systems, master batch systems, continuous processes, and batch processes to prepare a specific volume, density, concentration, CFU, and / or aliquot of cells for subsequent use as an inoculant or in a seed train process to scale cell production and recover biological products from the cells.

[0068] Bioprocess containers and bioprocess container sidewalls disclosed herein can be made of a flexible, collapsible material comprised of one or more sheets or layers of a flexible, water impermeable polymeric film such as polyethylene, low-density polyethylene, high-density polyethylene, polypropylene, fluorinated ethylene propylene ethylene- vinyl acetate, an ethylene-vinyl acetate copolymer, polyester, a styrene-butadiene polymer, polyethylene terephthalate, polyurethane, a thermoplastic elastomer, nylon, a cyclic olefin copolymer, an ethylene-vinyl alcohol copolymer, a tie layer, a ply layer and combinations of layers that form a multi-layer polymeric film.

[0069] One or more layers of the polymeric film can have a thickness that is at least or less than 0.02 mm, 0.05 mm, 0.1 mm, 0.2 mm, 0.5 mm, 1 mm, 2 mm, 3 mm or in a range between any two of the foregoing. Other thicknesses can also be used. The film is sufficiently flexible that it can be rolled into a tube without plastic deformation and can be folded over an angle of at least 90°, 180°, 270°, or 360° without plastic deformation.

[0070] The film can be comprised of a single ply material or can comprise two or more layers which are either sealed together or separated to form a double wall container. Where the layers are sealed together, the material can comprise a laminated or extruded material. The laminated material comprises two or more separately formed layers that are subsequently secured together by an adhesive or tie layer. Examples of multi-layer polymeric films that can be used to form the bioprocess containers and corresponding sidewalls disclosed herein, include is the Thermo Scientific CX3-9™ film, CX5-14™ film and Aegis5-14™ film produced by Thermo Fisher Scientific. In an example embodiment,Docket No. TP389720WOImulti-layer polymeric film that forms the sidewall of the bioprocess containers herein comprises a polyester elastomer outer layer, an ultra-low density polyethylene contact layer, and an EVOH barrier layer disposed therebetween.

[0071] Multi-layer polymeric films used in bioprocess containers can be validated for sterility and biocompatibility and approved for use in heavily regulated industries, including the pharmaceutical industry. For example, the example bioprocess containers, cell media and other components of the example bioreactors are regulated by the U.S. Food and Drug Administration and other drug approval agencies. Therefore, multi-layer polymeric films and other components of the example bioreactors disclosed herein are biocompatible and non-toxic to a broad range of biological components, such as cells. Example bioreactors and bioprocess containers can also be sterilizable with ionizing radiation, such as gamma rays and X-rays prior to use in a seed train process or other bioproduction process.

[0072] In example embodiments, the sidewalls of the bioprocess container can be formed and manufactured into a chamber by one or more steps of: cutting one or more sheets of multi-layer polymeric film, arranging one or more sheets on top of each other; and sealing and / or welding the sheets with pressure and heat to seal an outer perimeter edge of the bioprocess container to form a chamber. Holes or ports can be cut into the sidewalls of the bioprocess container to accommodate sensors, fluid conduits and other ports and bioreactor components.

[0073] In example embodiments, bioprocess containers disclosed herein can be formed into a two-dimensional pillow or formed from a continuous tubular extrusion of polymeric material that is cut to length. The ends can be left open, seamed closed, or panels can be sealed over the open ends to form a three-dimensional bioprocess container. Three-dimensional bioprocess containers can have an annular sidewall and a two-dimensional top end wall and a two-dimensional bottom end wall. Three-dimensional bioprocess containers can include a plurality of discrete panels, typically three or more, and more commonly four to six. Each panel is substantially identical and comprises a portion of the sidewall, top end wall, and bottom end wall of the container. Corresponding perimeter edges of each panel are seamed together. The seams are typically formed using methods known in the art such as heat energies, RF energies, sonic welding, or other sealing energies. In alternative embodiments, the panels can be formed in a variety of different patterns, including by methods of manufacturing three-dimensional bags disclosed in United States Patent Publication No. US 2002-0131654 Al, published September 19, 2002, which is incorporated herein by specific reference in its entirety.Docket No. TP389720WO1

[0074] Additional examples of multi-layer polymeric films that can be used to manufacture bioprocess containers are disclosed in United States Patent No. 6,083,587 which issued on July 4, 2000, and United States Patent Publication No. US 2003-0077466 Al, published April 24, 2003, which are hereby incorporated herein by specific reference in their entirety.

[0075] In example embodiments of bioprocess containers, the sidewalls of the bioprocess container are made of one or more sheets of multi-layer polymeric films, and the top cap and bottom caps are made of more rigid material that does not collapse or flex as much as the flexible sidewalls. For example, the top and bottom caps of the bioprocess container can be made from a rigid plastic, such as high-density polyethylene (HDPE), low-density polyethylene (LDPE) or combinations of HDPE and LDPE. In example embodiments, the top and bottom caps each have an outside or top HDPE surface that overlays an over molded LDPE structure or lip. In some embodiments, the one or more sheets of multi-layer polymeric film of the side walls of the bioprocess container can have a yield strength of 9-10 MPa (2% Secant Modulus). In some embodiments, the top and bottom caps have a yield strength of 23-30 MPa and Youngs modulus of 0.4- 1.2 GPa, when made of HDPE and a yield strength of 5-20 MPa and Youngs modulus of 0.1-1.9 GPa, when made of LDPE.

[0076] The bioprocess container can be constructed by attaching the sidewalls to the top cap and bottom cap by sealing the top and bottom ends of the flexible sidewalls with heat to one or more surfaces of the top and bottom cap. In example embodiments, the top and bottom ends of the flexible sidewalls are heat sealed to a perimeter edge of the top and bottom caps of the bioprocess container. For example, the top and bottom ends of the flexible sidewalls are heat sealed to a perimeter edge of the top and bottom caps of the bioprocess container using ultrasonic welding, heat sealing, high frequency welding, laser welding and / or impulse welding and a temperature range of 85 °C to 170 °C. To further secure the caps to the sidewalls of the bioprocess container, the top and bottom ends of the flexible sidewalls can be heat sealed to the perimeter edge of an over molded lip of the top and bottom caps.

[0077] FIG. 1 illustrates a partially exploded view of an example bioreactor 100 with a housing 102 and a bioprocess container 104 in accordance with example embodiments. The bioreactor 100 can be an adaptive bioreactor that operates as an N-l bioreactor in a seed train system with more than one bioreactor. The bioreactor 100 can also have a relatively small footprint. For example, an N-l bioreactor 100 in a seed train can house a bioprocess container with a volume between 1 to 10 liters. In example embodiments, the bioprocess container 104 has a maximum volume of 5 liters. ForDocket No. TP389720WO1example, the bioreactor housing 102 can be cuboidal shaped with a length and width of between 1.0 inch to 2.0 feet. In example embodiments, the bioreactor housing 102 has a total benchtop footprint of 6.0 x 6.0 inches.

[0078] The bioreactor 100 includes a housing 102 that can house the bioprocess container 104. The housing 102 has a top end wall 101, an opposing bottom end wall 103, and sidewalls 105 extending between the top end wall 101 and bottom end wall 103. The housing 102 can include one or more windows or openings 106 in the housing 102 to accommodate bioreactor 100 and bioprocess container 104 components and allow for visual inspection of the bioprocess container 104 and contents of the bioprocess container 104. In example embodiments, the housing 102 and bioprocess container 104 can have a rectangular or square transverse cross section. In example embodiments, the bioreactor 100 housing 102 and bioprocess container 104 are both cuboidal in shape, and the bioreactor is an N-l bioreactor in a seed train system composed of one or more larger volume production scale bioreactors with the same cuboidal shape. Constructing both the N-l bioreactor 100 and production scale bioreactor with the same cuboidal shape provides for (i) natural baffling effects to a fluid in the bioreactors; (ii) avoids vortex formation; formation of dynamic flow patterns; and simplifies the optimization of equipment and process parameters during scale-up and seed train operations. However, in other embodiments, housing 102 and bioprocess container 104 can have other transverse cross section configurations such as circular, elliptical or polygonal.

[0079] The housing 102 can also include a sensor reader 108, such as a sensor reader for a puck sensor, that can interface with and receive, transmit, store and process signals and data generated by one or more sensor(s) 110 (e.g., pH, dissolved oxygen, pressure, and / or temperature sensors) coupled to the bioprocess container 104. The sensor reader 108 can be coupled to or attached to the housing 102 of the bioreactor 100 with a support bracket 136 (shown in FIG. 1). The sensor reader 108 can be coupled to or attached to the housing 102 of the bioreactor 100 with the support bracket 136 (shown in FIG. 1) such that the sensor reader 108 is proximate to the sensor 110 on the bioprocess container 104 when the bioprocess container 104 is installed in the housing 102 (installed bioprocess container 104 shown in FIG. 2).

[0080] A stand 109 is coupled to and extends from the top end wall 101 of the housing 102. The stand can be made of one or more rods that are attached together. Tubing management systems 111 can be attached to the stand 109 through openings or holes 113 in the tubing management systems 111. The tubing management systems 111 include grooves, holes or clips 115 to accommodate tubingDocket No. TP389720WO1and arrange the tubing in an organized manner. Tn example embodiments, the grooves 115 can be U-shaped grooves, circular grooves or grooves with varying clearance or diameter to secure tubing, such as sterile tubing used to introduce or exhaust liquid or gas from the bioprocess container 104.

[0081] The bioreactor impeller assembly includes an impeller mounting hub 132, a drive shaft 128 and a blade assembly 138 (e.g., one or more mixing blades). An impeller motor 114 can drive and rotate the drive shaft 128 of the impeller assembly 130 to mix fluids within the bioprocess container 104. The impeller motor 114 includes a motor mounting sleeve 117 that interfaces with and / or removably couples to the bioreactor impeller assembly 130 and / or bioprocess container as described herein. In example embodiments, one end of the impeller mounting hub 132 is coupled to the drive shaft 128, and the other end of the impeller mounting hub 132 is coupled to a motor shaft (not shown) disposed within the motor mounting sleeve 117 of the impeller motor 114. Rotation of the motor shaft facilitates rotation of the drive shaft 128 and blade assembly 138.

[0082] The impeller motor 114 can be an electric motor. In example embodiments, the impeller motor 114 has an operational voltage range between 10 to 350 volts; a continuous power range between 50 to 1500 watts; a peak power range between 500 to 4000 watts, and a shaft diameter between 0.1 to 6 inches. A motor communication and power cable 112 can be electrically coupled to the impeller motor 114 to drive the motor 114. The motor communication and power cable 112 can be connected to a bioreactor controller 125 or power supply 116 to communicate power and other data signals to and from the impeller motor 114 to control the motor 114 and RPM of the drive shaft 128.

[0083] A power supply 116 can be routed to bioreactor components, through an electrical port in the housing 102 or otherwise. The power supply 116 can be used to power the bioreactor 100 and associated components, including but not limited to sensor(s) 110, sensor reader 108, impeller motor 114, motor communication and power cable 112, heaters 120, controllers and other components. The power supply 116 can transmit power and / or data to and from components of the bioreactor 100, including but not limited to, control signals and data, sensor signals and data and component signals and data. These signals and data can also be transmitted wirelessly between components of the bioreactor 100 and a bioreactor controller 125 to control the operation of the bioreactor 100 and components thereof. The power supply 116 can include one or more AC power supplies, DC power supplies, power distribution boxes, programmable power supplies, uninterruptible power supplies, switched mode power supplies and / or other power supplies.Docket No. TP389720WO1

[0084] The bioreactor 100 housing 102 can be equipped with a door 118 on one of the sidewalls 105. In example embodiment, the door 118 forms one entire sidewall 105 of the housing 102 and includes hinges to swing and actuate the door 118 open and closed to easily accommodate installation of the bioprocess container 104 within the housing 102. The housing 102 can include a door latch 134 (shown in FIG. 2) to secure the door 118 and to open and close the door 118.

[0085] The housing 102 can also include an integrated heating system 120. including one or more heaters 120A, 120B, used to heat the bioprocess container 104 and contents of the bioprocess container 104 to a specific process temperature for processing and / or expanding cells. One or more heater(s) 120 can be coupled to or integrated into one or more sidewall(s) 105 of the housing 102. For example, two heaters 120, including heater housings and heat plates are integrated into the internal surfaces of two sidewalls 105 of the bioreactor 100 housing 102. Each heater 120 can have two heating plates. The split heater configuration heats multiple surfaces and sidewalls 126 of the bioprocess container 104; provides uniform heat distribution across the bioprocess container 104; provides bioreactor operation at low volumes; and provides enhanced temperature control across the bioprocess container 104 and contents therein. In example embodiments, at least one of the heating plates of the heaters 120 is located in a position on the housing 102 so it can heat the contents of the bioprocess container 104 and grow cells at low volumes, such as at a height equivalent to 1 / 5; 1 / 4; 1 / 3; and 1 / 2 of the total volume capacity of cell culture in the bioprocess container 104. In example embodiments, the heating plates of the heaters 120 are spaced from the bottom end wall 103 of the housing 102 by distance of about 3.0 - about 5.0 inches, or more preferably, about 3.5 - about 4.5 inches. However, embodiments are not limited thereto, and the heating plates of the heaters 120 can be spaced from the bottom end wall of the housing 102 by a shorter distance or a longer distance. In example embodiments, the heating plates of the heaters 120 are made of a silicon mesh. In example embodiments, one or more heaters 120 can be used to heat the bioprocess container 104 and contents of the bioprocess container 104 in a temperature range of 17°C to 40°C. The temperature contents of the bioprocess container 104 is measured with a resistive temperature detector (RTD) sensor that is inserted into the multiport of the top cap described later in the disclosure.

[0086] The bioprocess container 104 can be used to contain, process, react and transfer various solutions and / or suspensions including one or more biocomponents and cell media used primarily in seed train systems and cell expansion operations. The bioprocess container 104 can include a top cap or top end cap 122, a bottom cap or bottom end cap 124 and sidewalls 126 extending between the topDocket No. TP389720WO1cap 122 and bottom cap 124. The top cap 122 and bottom cap 124 are preferably made from a rigid material, such as a rigid plastic that is more rigid and less flexible than the sidewalls 126. For example, the top cap 122 and bottom cap 124 can be formed from molded plastic such as HDPE, LDPE and / or combinations of HDPE and LDPE. In some embodiments, the top cap 122 and bottom cap 124 when made of HDPE have a density of 0.94-0.97 g / cm3, tensile strength of 25 to 40 MPa, and impact strength of 100-110 kJ / m2. In other embodiments, the top cap 122 and bottom cap 124 when made of LDPE have a density of 0.91-0.93 g / cm3, tensile strength of 8 to 12MPa, and impact strength of 50 to 150 kJ / m2.

[0087] The sidewalls 126 of the bioprocess container 104 are made of a multi-layer polymeric film as described herein. For example, the bioprocess container sidewalls 126 can be made of a flexible, collapsible material comprised of one or more sheets or layers of a flexible, water impermeable polymeric film such as polyethylene, low-density polyethylene, high-density polyethylene, polypropylene, fluorinated ethylene propylene ethylene-vinyl acetate, an ethylene-vinyl acetate copolymer, polyester, a styrene-butadiene polymer, polyethylene terephthalate, polyurethane, a thermoplastic elastomer, nylon, a cyclic olefin copolymer, an ethylene-vinyl alcohol copolymer, one or more tie layers and combinations of the disclosed polymeric films that form a multi-layer polymeric film. In some embodiments the bioprocess container sidewalls 126 made of a flexible, collapsible material comprised of one or more sheets or layers of above polymers can have tensile strength of 10-20 MPa, Youngs modulus of 1-3 GPa, and impact strength of 15-30kJ7m2.

[0088] The sidewalls 126 of the bioprocess container 104 can include one or more installation tabs 119 that have one or more holes 121 that align with protrusions in the bioreactor 100 housing 102. The installation tabs 119 and holes 121 confine and indicate the installation orientation of the bioprocess container 104 in the bioreactor 100 housing 102. The installation tabs 119 can be perforated for easy removal. Once the bioprocess container 104 is installed in the housing 102 of the bioreactor 100, based on the alignment of installation tabs 119 with protrusions in the housing 102. the installation tabs 119 can be removed along perforations formed in the tabs.

[0089] The sidewalls 126 of the bioprocess container 104 can be formed and manufactured into a chamber by one or more steps of: cutting one or more sheets of multi-layer polymeric film, arranging one or more sheets on top of each other; and sealing and / or welding the sheets with pressure and heat to seal an outer perimeter edge of the bioprocess container to form a chamber or a tube. Holes or ports can be cut into the sidewalls of the bioprocess container to accommodate sensors, fluid conduits andDocket No. TP389720WO1other ports and bioreactor components. The top cap 122 and bottom cap 124 are attached to the top end and bottom end respectively of the bioprocess container 104 sidewall(s) 126 by heat sealing the polymeric film to one or more surfaces of the top cap 122 and the bottom cap 124. For example, the top and bottom ends of the flexible sidewalls 126 are heat sealed to a perimeter edge of the top and bottom caps 122, 124 by ultrasonic welding, heat sealing, high frequency welding, laser welding and / or impulse welding using a temperature range of 85 °C to 170 °C.

[0090] In example embodiments of bioprocess containers 104, the sidewalls 126 of the bioprocess container 104 are made of one or more sheets of flexible, multi-layer polymeric films, and the top cap 122 and bottom cap 124 are made of more rigid material that does not collapse or flex as much as the flexible sidewalls 126. For example, the top and bottom caps 122, 124 of the bioprocess container 104 can be made from a rigid plastic, such as high-density polyethylene (HDPE), low-density polyethylene (LDPE) or combinations of HDPE and LDPE. In example embodiments, the top and bottom caps 122, 124 each have an outside or top HDPE surface that overlays an over molded LDPE structure or lip 127.

[0091] The top cap 122 can include a variety of one or more top ports 140 that can facilitate sterile connection with inlet / outlet conduits, sensors, dip tubes, dip tube and / or sensor combos, tubing, fluid transfer systems, protective sensor sheaths, top spargers or gas overlay, or other component ports 140. The sensor sheaths can be stainless steel rods, plastic, polymer, rigid or flexible and can be used to protect or isolate a sensor, such as a resistive temperature detector (RTD), from the contents of the bioprocess container 104 but also allow the sensor to communicate with the contents of the bioprocess container 104 through an opening.

[0092] In example embodiments, the top port 140 is a multiport 140 that facilitates sterile connection with sensors and inlet / outlet conduits used for liquid transfer and liquid sampling. For example, a foam sensor, an RTD sensor and two tubing inlet / outlet conduits can be fitted, routed and / or positioned through the multiport 140. In example embodiments, a sensor sheath can house the RTD sensor. Other configurations are possible, including those disclosed in WO2024 / 059721 A2 incorporated by reference in its entirety herein. In some embodiments the multiport 140 can include a sample port. The sample port can be configured to allow low void volume samples to be taken for cell viability and density and enables metabolite analysis. This sample port can be a luer lock port that allows for direct sampling or attachment of various sampling manifolds using a standard luer lock connection.Docket No. TP389720WO1

[0093] The top cap 122 of the bioprocess container 104 can also include an impeller mounting hub 132 located at the terminal end of the drive shaft 128 of the bioreactor impeller assembly 130. The impeller mounting hub 132 can be removably coupled to the impeller motor 114 to facilitate rotation of the drive shaft 128. One or more mixing assemblies or blades 138 (e.g., three sets of single blades, dual blades, tri-blades or four blades) are coupled to the drive shaft 128 to produce proper mixing and mixing characteristics of the contents of the bioprocess container 104. In example embodiments, the drive shaft 128 has three sets of dual blades 138. In example embodiments, the blades 138 has a 45-degree pitch or angle from horizontal. At least one of the sets of dual blades 138 is located at a height within the liquid volume so it can mix the contents of the bioprocess container 104 and grow cells at low volumes, such as at a height equivalent to 1 / 5; 1 / 4; 1 / 3; and 1 / 2 of the total volume capacity of cell culture in the bioprocess container 104.

[0094] The blades 138 can have the same or similar dimensions and configurations as the blades described in U.S. Patent Nos. 9,855,537; 10,335,751; 11,654,408; 9,839,886; 10,272,400; 11352598 and U.S. Patent Publication Nos. 2019 / 209.981 and 2021 / 237,009 WO2024 / 059721 incorporated by reference in their entirety herein.

[0095] The bottom cap 124 of the bioprocess container 104 can be a molded component made of material that is more rigid and less flexible than the sidewalls 126, such as rigid HDPE and LDPE. The bottom cap 124 can include one or more bottom ports, including but not limited to tube ports, drain ports, sparger gas ports, sensor ports and / or sample ports for facilitating sterile fluid transfer, sparger coupling and / or sensor coupling (e.g., pressure sensors, temperature sensors, foam sensors, glucose sensors, pH sensors, DO sensors, CO2 sensors, density sensors, cell density sensors, conductivity sensors, spot sensors for measuring fluid and bioreactor process parameters).

[0096] The bottom cap 124 can include a sparger 142 (shown in FIG. 2) configured to facilitate gas mass transfer across the gas-liquid film, and at an interface between sparged gas and liquid cell media, in the bioprocess container 104. The sparger 142 can route or flow various gases, such as air, nitrogen, carbon dioxide, and oxygen into the bioprocess container 104, which dissolves into the liquid cell media to create an environment that optimizes cell growth. The sparger 142 can include a sparger disc with pores or drilled holes of specific sizes or diameters. The holes / pores in the sparger disc can be laser drilled and can have the following hole count to size ratios: 235 x 80 pm holes, 295 x 50 pm holes. 667 x 30 pm holes, 360 x 178pm holes: 570 x 178pm holes; 760 x 233pm holes; 980 x 368pm holes; 1,180 x 445pm holes; 133 x 80 pm holes; 235 x 50 pm holes; 343 x 30 pm holes. In exampleDocket No. TP389720WO1embodiments, the sparger disc has a diameter from 20 mm to 60 mm, the sparger pore sizes ranging from 20 um to 140 um, and the sparger disc has 1-1,000 pores or holes. In other embodiments, combinations of different sized holes are drilled into the sparger disc. The sparger 142 interfaces with or includes a gas line to feed gas through the sparger disc and into the bioprocess container 104. The sparger 142 can be integrated with the bioprocess container 104 to transfer gases into the bioprocess container 104. The sparger 142 can also have a non-circular geometry to better utilize and save space at the base of the bioreactors 100 disclosed herein. The sparger 142 can also have the same features as dome spargers and film spargers disclosed in U.S. Patent Nos. 8,603,805, 9,005,971, 9,259,692, 9,475,012, 9,682,353, 10,328,404 and 9,643,133, 10,350,554, 10,843,141, and U. S. Publication No.2021 / 0069654, which are incorporated herein by specific reference. Other conventional spargers can also be used. The sparger 142 can also include installation tabs 119 with holes that indicate the orientation of installation. The installation tabs 119 indicate and constrain the orientation of the sparger 142 and bioprocess container 104 so that the bioprocess container is installed in the correct orientation within the housing 102. The installation tabs 119 have perforations that allow for easy removal after installation of the sparger 142 into bioprocess container 104.

[0097] In example embodiments, the bottom cap 124 includes a sparger port that interfaces with the sparger 142 and a conduit to introduce gas through the sparger 142. In addition to the drilled hole sparger 142 port, the bottom cap can also have a second gas intake port that allows for the flow of gas through the gas inlet port into the bioprocess container 104. In this way, gas is introduced into the bioprocess container 104 through the sparger 142 port and through a second gas inlet port. A mix of gases can be introduced through both ports, and each port can be dedicated to flow a specific gas or mixture of gases, including but not limited to O2, CO2, 2, and air.

[0098] The bioprocess container 104 includes a sensor 110 coupled to a surface of the bioprocess container 104. In example embodiments, the sensor 110 is coupled to and / or welded to the sidewall 126 of the bioprocess container 104. A variety of sensors 110 can be coupled to the bioprocess container 104, including but not limited to, pressure sensors, temperature sensors, foam sensors, glucose sensors, pH sensors, DO sensors, CO2 sensors, flow sensors, density sensors, cell density sensors, conductivity sensors, spot sensors or the like. In example embodiments, the sensor 110 is a pH sensor, DO sensor, CO2 sensor and a temperature sensor. Sensor measurements, data or signals transmitted from sensors (or associated transmitters) are process, liquid and equipment parameter outputs or inputs, such as RPM of the drive shaft 128, pressure within the bioprocess container 104,Docket No. TP389720WO1temperature within the bioprocess container 104 or cell culture; foam content within the bioprocess container 104; glucose content within the bioprocess container 104 or cell culture; pH within the bioprocess container 104 or cell culture; dissolved oxygen content within the bioprocess container 104 or cell culture; CO2 content within the bioprocess container 104 or cell culture; cell density of the cell culture; and / or conductivity readings associated with the bioprocess container 104 or cell culture.

[0099] The sensor 110 can electronically and / or physically couple to a sensor reader 108 (in wired or wireless fashion) to transmit data and signals indicating bioreactor process, liquid and equipment parameters, such as pressure, temperature, glucose concentration, dissolved oxygen concentration, CO2 concentration, pH, foam content, cell density and conductivity of the contents of the bioprocess container 104. The sensor reader 108 can communicate in a wired or wireless fashion with a controller to transmit signals and data indicating bioreactor process parameters to the controller to facilitate bioreactor process control. In other example embodiments, the sensor 110 can communicate directly with a controller without the sensor reader 108.

[0100] FIG. 2 illustrates the example bioreactor 100 of FIG. 1 with an installed bioprocess container 104 in accordance with example embodiments. As discussed with respect to FIG. 1, the bioreactor 100 includes a housing 102 that can house the bioprocess container 104. The housing 102 has a top end wall 101, an opposing bottom end wall 103, and sidewalls 105 extending between the top end wall 101 and bottom end wall 103. The housing 102 can include one or more windows or openings 106 in the housing 102 to accommodate bioreactor 100 and bioprocess container 104 components and allow for visual inspection of the bioprocess container 104 and contents of the bioprocess container 104.

[0101] A stand 109 is coupled to and extends from the top end wall 101 of the housing 102. The stand 109 can be made of one or more rods that are attached together. Tubing management systems 111 can be attached to the stand 109 through openings or holes 113 in the tubing management systems 111. The tubing management systems 111 include grooves, holes or clips 115 to accommodate tubing and arrange the tubing in an organized manner. In example embodiments, the grooves 115 can be LJ-shaped grooves, circular grooves or grooves with varying clearance or diameter to secure tubing, such as tubing used to introduce, remove or exhaust liquid or gas from the bioprocess container 104.

[0102] The bioreactor impeller assembly includes an impeller mounting hub 132, a drive shaft 128 and a blade assembly 138 (e.g., one or more mixing blades). An impeller motor 114 can drive and rotate the drive shaft 128 of the impeller assembly 130 to mix fluids within the bioprocess containerDocket No. TP389720WO1104. The impeller motor 114 includes a motor mounting sleeve 117 that interfaces with and / or removably couples to the bioreactor impeller assembly 130 and / or bioprocess container as described herein. In example embodiments, one end of the impeller mounting hub 132 is coupled to the drive shaft 128, and the other end of the impeller mounting hub 132 is coupled to a motor shaft (not shown) disposed within the motor mounting sleeve 117 of the impeller motor 114. Rotation of the motor shaft facilitates rotation of the drive shaft 128 and blades 138.

[0103] The impeller motor 114 can be an electric motor. In example embodiments, the impeller motor 114 has an operational voltage range between 10 to 350 volts; a continuous power range between 50 to 1500 watts; a peak power range between 500 to 4000 watts, and a shaft diameter between 0.1 to 6 inches. A motor communication and power cable 112 can be electrically coupled to the impeller motor 114 to drive the motor 114. The motor communication and power cable 112 can be connected to a bioreactor controller 125 or power supply 116 to communicate power and other data signals to and from the impeller motor 114 to control the motor 114 and RPM of the drive shaft 128. In example embodiments, the impeller motor 114 has an operational voltage range between 10 to 350 volts; a continuous power range between 50 to 1500 watts; a peak power range between 500 to 4000 watts, and a shaft diameter between 0.1 to 6 inches.

[0104] The bioprocess container 104 can be installed in the housing 102 by actuating or unlatching the door latch 134; opening the door 118 of the housing 102; and inserting or positioning the bioprocess container 104 in the housing 102. The sidewalls 126 of the bioprocess container 104 can include one or more installation tabs 119 (shown in FIG. 1) with one or more holes 121 that can align with protrusions in the bioreactor 100 housing 102. The installation tabs 119 and holes 121 confine and indicate the installation orientation of the bioprocess container 104 in the bioreactor 100 housing 102. The installation tabs 119 can be perforated for easy removal. Once the bioprocess container 104 is installed in the housing 102 of the bioreactor 100, based on the alignment of installation tabs 119 with protrusions in the housing 102, the installation tabs 119 can be removed along perforations formed in the tabs.

[0105] The bioprocess container 104, with flexible sidewalls 126 formed from a multi-layer flexible film, is supported and secured in the housing 102 from the top end and / or top cap 122. For example, after positioning the bioprocess container 104 in the housing in accordance with installation tabs 119, an over molded lip 127 of the top cap 122 of the bioprocess container 104 can be press fit into the top end wall 101 of the housing 102. The top end and / or top cap 122 of the bioprocess containerDocket No. TP389720WO1104 can also include mounting flanges 123 that sit on top of the housing to hang, suspend, position or secure the bioprocess container 104 in the housing 102 by gravity. Therefore, the bioprocess container 104 hangs and is supported and secured in the housing 102 from the top end and / or top cap 122 by securing or press fitting the top cap 122 to the top end wall 101 of the housing 102. In example embodiments, the bottom end and / or bottom cap 124 of the bioprocess container 104 is not coupled or secured to the housing 102 or the bottom wall 103 of the housing 102. The motor mounting sleeve 117 of the impeller motor 114 can be coupled to the top end and / or top cap 122 or the bioprocess container 104 and coupled to the impeller mounting hub 132.

[0106] In other example embodiments, the bioprocess container 104 can be installed in the housing 102 by actuating or unlatching the door latch 134; opening the door 118 of the housing 102; and inserting or positioning the bioprocess container 104 in the housing 102. The bioprocess container 104, which has flexible sidewalls 126, is supported and secured in the housing 102 from the top end and / or top cap 122. For example, the top end and / or top cap 122 of the bioprocess container 104 can include mounting flanges 123 that sit on top of the housing to position or secure the bioprocess container 104 in the housing 102 by the force of gravity and contact between the flanges 123 and housing 102. In other example embodiments, after positioning the bioprocess container 104 in the housing, an over molded lip 127 (shown in FIG. 1) of the top cap 122 of the bioprocess container 104 can be press fit into the top end wall 101 of the housing 102. The impeller mounting hub 132 (shown in FIG. 1) is removably coupled to the impeller motor 114 to facilitate rotation of the drive shaft 128. In example embodiments, the impeller mounting hub 132 of the bioprocess container 104 is directly or indirectly (via the motor mounting sleeve 117) coupled and secured to the impeller motor 114. In example embodiments, the bottom end and / or bottom cap 124 of the bioprocess container 104 is not coupled or secured to the housing 102 or the bottom wall 103 of the housing 102. In other example embodiment, the bioprocess container 104 and top cap 122 is secured to the housing 102 by gravity after positioning the top cap 122 over the top end wall 101 of the housing 102. In these embodiments, the bioprocess container 104 hangs and is positioned and secured in the housing 102 from the top end only.

[0107] The bioprocess container 104 includes a sensor 110 coupled to a surface of the bioprocess container 104. In example embodiments, the sensor 110 is coupled to, heat sealed and / or welded to the sidewall 126 of the bioprocess container 104. In other example embodiments, the sensor 110 can be adhesively coupled to, sidewall 126 of the bioprocess container 104. In example embodiments, theDocket No. TP389720WO1sensor 110 is spaced from the bottom end cap 124 of the bioreactor 100 by distance of 1.0 - 6.0 inches, with 1.5-3.0 inches being more common. A variety of sensors 110 can be coupled to the bioprocess container 104, including but not limited to, pressure sensors, temperature sensors, foam sensors, glucose sensors, pH sensors, DO sensors, CO2 sensors, flow sensors, density sensors, cell density sensors, conductivity sensors, spot sensors or the like. In example embodiment, the sensor 110 is a pH sensor, DO sensor, CO2 sensor and a temperature sensor. Sensor measurements, data or signals transmitted from sensors (or associated transmitters) are liquid, process and equipment parameter outputs and inputs, such as drive shaft 128 RPM, pressure within the bioprocess container 104, temperature within the bioprocess container 104 or cell culture; foam content within the bioprocess container 104; glucose content within the bioprocess container 104 or cell culture; pH within the bioprocess container 104 or cell culture; dissolved oxygen content within the bioprocess container 104 or cell culture; CO2 content within the bioprocess container 104 or cell culture; cell density of the cell culture; conductivity readings associated with the bioprocess container 104 or cell culture. The sensor 110 is attached to the bioprocess container so it can measure process, liquid and equipment parameters within the cell culture at low volumes, such as at a height equivalent to 1 / 5; 1 / 4; 1 / 3; and 1 / 2 of the total volume capacity of cell culture in the bioprocess container 104. For example, the sensor 110 can be attached 1.0 to 6.0 inches from or above the bottom cap 124 of the bioprocess container to measure process parameters at low volumes and high turn-down ratios.

[0108] The housing 102 can also include a sensor reader 108 (shown in FIG. 1), such as a sensor reader for a puck sensor, that can interface with and receive, transmit, store and process signals and data generated by one or more sensor(s) 110 (e.g., pH, dissolved oxygen pressure, and temperature sensors) coupled to the bioprocess container 104. The sensor reader 108 can be coupled to or attached to the housing 102 of the bioreactor 100 with a support bracket 136 (shown in FIG. 1) attached to the housing 102. The sensor reader 108 can be coupled to or attached to the housing 102 of the bioreactor 100 with the support bracket 136 (shown in FIG. 1) such that the sensor reader 108 (shown in FIG. 1) is proximate to the sensor 110 on the bioprocess container 104 when the bioprocess container 104 is installed in the housing 102 (installed bioprocess container 104 shown in FIG. 2).

[0109] The sensor 110 can electronically and / or physically couple to a sensor reader 108 (shown in FIG. 1) to transmit data and signals indicating bioreactor process parameters, such as pressure, temperature, glucose concentration, dissolved oxygen concentration, CO2 concentration, pH, foam content, cell density and conductivity of the contents of the bioprocess container 104. The sensorDocket No. TP389720WO1reader 108 can communicate in a wired or wireless fashion with a controller to transmit signals and data indicating bioreactor process parameters to the controller to facilitate bioreactor process control. In other example embodiments, the sensor 110 can communicate directly with a controller without the sensor reader 110.

[0110] A power supply 116 can be routed to bioreactor components, through an electrical port in the housing 102 or otherwise. The power supply 116 can be used to power the bioreactor 100 and associated components, including but not limited to sensor(s) 110, sensor reader 108, impeller motor 114, motor communication and power cable 112, heaters 120, controllers and other components. The power supply 116 can transmit power and / or data to and from components of the bioreactor 100, including but not limited to, control signals and data, sensor signals and data and component signals and data. These signals and data can also be transmitted wirelessly between components of the bioreactor 100 and a bioreactor controller 125 to control the operation of the bioreactor 100 and components thereof. The power supply 116 can include one or more AC power supplies. DC power supplies, power distribution boxes, programmable power supplies, uninterruptible power supplies, switched mode power supplies and / or other power supplies.

[0111] As described with respect to FIG. 1, the top cap 122 of the bioprocess container 104, shown also in FIG. 2, can be a molded component made of materials that or more rigid than the sidewalls 126, such as rigid HDPE and LDPE. The top cap 122 can include a variety of top ports 140 that can facilitate sterile connection with sensors, dip tubes, dip tube and / or sensor combos, tubing, fluid transfer systems, protective sensor sheaths, top spargers or gas overlay, or other component ports 140. The sensor sheaths can be stainless steel rods, plastic, polymer, rigid or flexible and can be used to protect or isolate a sensor from the contents of the bioprocess container 104 but also allow the sensor to communicate with the contents of the bioprocess container 104 through an opening.

[0112] The bottom cap 124 can include a sparger 142 (shown in FIG. 2) for facilitating gas mass transfer across the gas-liquid film and interface between the sparged gas and liquid cell media in the bioprocess container 104. The sparger 142 can route or flow various gases, such as air, nitrogen, carbon dioxide and oxygen into the bioprocess container 104, which dissolves into the liquid cell media to create an environment that optimizes cell growth. The sparger 142 can include a sparger disc with pores or drilled holes of specific sizes or diameters. The holes / pores in the sparger disc can be laser drilled and can have the following hole count to size ratios: 360 x 178pm holes; 570 x 178pm holes; 760 x 233pm holes; 980 x 368pm holes; 1,180 x 445pm holes; 133 x 80 pm holes; 235 x 50 pm holes;Docket No. TP389720WO1343 x 30 m holes. Tn example embodiments, the sparger disc has a diameter from 20 mm to 60 mm, the sparger pore sizes ranging from 20 um to 140 um, and the sparger disc has 1-1,000 pores or holes. In other embodiments, combinations of different sized holes are drilled into the sparger disc. The sparger 142 interfaces with or includes a gas line to feed gas through the sparger disc and into the bioprocess container 104. The sparger 142 can be integrated with the bioprocess container 104 to transfer gases into the bioprocess container 104. The sparger 142 can also have a non-circular geometry to better utilize and save space at the base of the bioreactors 100 disclosed herein. The sparger 142 can also have the same features as dome spargers and film spargers disclosed in U.S. Patent Nos. 8,603,805, 9,005,971, 9,259,692, 9,475,012, 9,682,353, 10,328,404 and 9,643,133, 10,350,554, 10,843,141, and U. S. Publication No. 2021 / 0069654, which are incorporated herein by specific reference. Other conventional spargers can also be used. The sparger 142 can also include installation tabs 119 with holes that indicate the orientation of installation. The installation tabs 119 indicate and constrain the orientation of the sparger 142 and bioprocess container 104 so that the bioprocess container is installed in the correct orientation within the housing 102. The installation tabs 119 have perforations that allow for easy removal after installation of the sparger 142 in the bioprocess container 104.

[0113] In example embodiments, the bottom cap 124 includes a sparger port that interfaces with the sparger 142 and a conduit to introduce gas through the sparger 142. In addition to the drilled hole sparger 142 port, the bottom cap can also have a second gas intake port that allows for the flow of gas through the gas inlet port into the bioprocess container 104. In this way, gas is introduced into the bioprocess container 104 through the sparger 142 port and through a second gas inlet port. A mix of gases can be introduced through both ports, and each port can be dedicated to flow a specific gas or mixture of gases, including but not limited to O2, CO2, N2, and air.

[0114] FIG. 3 illustrates a bioprocess container 204 in accordance with example embodiments. The bioprocess container 204 can be used with and installed in the housing 102 illustrated in FIG. 1 and FIG. 2. The bioprocess container 204 can contain, process, react and transfer various solutions and / or suspensions including one or more biocomponents and cell media used primarily in seed train systems and cell expansion operations. The bioprocess container 204 can include a top cap 222, a bottom cap 224 and sidewalls 226 extending between the top cap 222 and bottom cap 224. The top cap 222 and bottom cap 224 are preferably made from a material that is more rigid than the sidewalls 226. such as a rigid HDPE, LDPE or combinations of the same. The top cap 222 and bottom cap 224 can each beDocket No. TP389720WO1made of a molded piece of plastic with flat tops or outside surfaces 229A, 229B overlaying over molded lips 227 A, 227B.

[0115] In example embodiments, the outside surfaces 229 A, 229B are made from HDPE and the over molded lips 227A, 227B are made from LDPE to provide a more flexibility for the top cap installation into the bioreactor housing. In one embodiment, the lip 227A of the top cap 222 can be press fit inside the internal perimeter of the bioreactor housing 102 (shown in FIGS. 1-2) to install and secure the bioprocess container 204 from the top.

[0116] The sidewalls 226 of the bioprocess container 204 are made of a multi-layer polymeric film as described herein. For example, the bioprocess container sidewalls 226 can be made of a flexible, collapsible material comprised of one or more sheets or layers of a flexible, water impermeable polymeric film such as polyethylene, low-density polyethylene, high-density polyethylene, polypropylene, fluorinated ethylene propylene ethylene-vinyl acetate, an ethylene-vinyl acetate copolymer, polyester, a styrene-butadiene polymer, polyethylene terephthalate, polyurethane, a thermoplastic elastomer, nylon, a cyclic olefin copolymer, an ethylene-vinyl alcohol copolymer, one or more tie layers and combinations of the disclosed polymeric films that form a multi-layer polymeric film.

[0117] The sidewalls 226 of the bioprocess container 204 can include one or more installation tabs 219 that have one or more holes 221 that can align with protrusions in the bioreactor housing or just provide an indication of where the holes should be positioned in relation to the housing for proper installation and orientation of the bioprocess container 204. The installation tabs 219 and holes 221 confine and indicate the installation orientation of the bioprocess container 204 in the bioreactor 100 housing 102 (shown in FIGS. 1-2). The installation tabs 219 can be perforated for easy removal. Once the bioprocess container 204 is installed in the housing of the bioreactor, based on the alignment of installation tabs 219, the installation tabs 219 can be removed along perforations formed in the tabs.

[0118] The sidewalls 226 of the bioprocess container 204 can be formed and manufactured into a chamber by one or more steps of: cutting one or more sheets of multi-layer polymeric film, arranging one or more sheets on top of each other; and sealing and / or welding the sheets with pressure and heat to seal an outer perimeter edge of the bioprocess container to form a chamber or a tube. Holes or ports can be cut into the sidewalls of the bioprocess container to accommodate sensors, fluid conduits and other ports and bioreactor components. The top cap 222 and bottom cap 224 are attached to the top end and bottom end respectively of the bioprocess container 204 sidewall(s) 226 by heat sealing theDocket No. TP389720WO1polymeric film to one or more surfaces of the top cap 222 and the bottom cap 224. For example, the top and bottom ends of the flexible sidewalls 226 are heat sealed to a perimeter edge of the cap lips 227 A-B or the top and bottom caps 222, 224 by ultrasonic welding, heat sealing, high frequency welding, laser welding and / or impulse welding the ends to the sidewalls 226 to the cap lips 227 A-B at a temperature range of 85 °C to 170 °C.

[0119] The top cap 222 of the bioprocess container 204 can be a molded component made of material that is more rigid than the sidewalls 226. The top cap 222 can include a variety of top ports 240 that can facilitate sterile connection with sensors, dip tubes, dip tube and / or sensor combos, tubing, fluid transfer systems, protective sensor sheaths, top spargers or gas overlay, or other component ports 240. The sensor sheaths can be stainless steel rods, plastic, polymer, rigid or flexible and can be used to protect or isolate a sensor from the contents of the bioprocess container 204 but also allow the sensor to communicate with the contents of the bioprocess container 204 through an opening.

[0120] In example embodiment, the top port 240 is a multiport 240 that includes three sensor sheaths 241 and two inlet / outlet tubing conduits 243. One sensor sheath 241 can house an RTD sensor or temperature sensor and the other two sensor sheaths 241 can house components of a foam sensor or conductivity sensor that facilitates the measurement of voltage across two leads in the cell culture within the bioprocess container 204. The tubing or conduits can facilitate liquid and gas transfer in and out of the bioprocess container 204.

[0121] The bioprocess container 204, with flexible sidewalls 226 formed from a multi-layer flexible film, is supported and secured in the bioreactor housing (e.g., 102 in FIGS. 1-2) from the top end and / or top cap 222. For example, after positioning the bioprocess container 204 in the housing in accordance with installation tabs 219, an over molded lip 227A of the top cap 222 of the bioprocess container 204 can be press fit into the top end wall of the bioreactor housing (e.g., 101, 102 of FIGS.1-2). The top end and / or top cap 222 of the bioprocess container 204 can also include mounting flanges 223 that sit on top of the bioreactor housing to hang, suspend, position or secure the bioprocess container 204 in the bioreactor housing by gravity. The mounting flanges 223 and bioreactor housing can also include threaded holes 225 to facilitate threaded engagement between the top cap 222 and bioreactor housing. Therefore, the bioprocess container 104 hangs and is supported and secured in the bioreactor housing (e.g., 102 of FIGS. 1-2) from the top end and / or top cap 222 by contacting, securing or press fitting the top cap 222 to the top end wall of the housing (e.g., 101, 102 of FIGS. 1-2). InDocket No. TP389720WO1example embodiments, the bottom end and / or bottom cap 224 of the bioprocess container 204 is not coupled or secured to the bioreactor housing the bottom wall of the housing.

[0122] The bioreactor impeller assembly 230 (also shown and described in FIG. 4A) includes an impeller mounting hub 232, a drive shaft 228 and one or more blade assemblies 238 (example three blade assemblies 238) each consisting of a set of two blades 238 A, 238B. The impeller mounting hub 232 is located at the terminal end of the drive shaft 228 of the bioreactor impeller assembly 230 (also shown and described in FIG. 4A) and can be used to couple to a motor and couple to the top cap 222 of the bioprocess container 204 to mount and position the impeller assembly 230 in the bioprocess container 204. In example embodiments, the impeller mounting hub 232 can be removably coupled to the top cap 222 of the bioprocess container 204 on one end and removably coupled to an impeller motor (for example, impeller motor 114 of FIGS. 1-2) on the other end to facilitate rotation of the drive shaft 228. For example, the impeller mounting hub 232 can include a mounting flange 246 or a bearing housing 246 that interfaces with, attaches to, or rotatably attaches to the top cap 222 of the bioprocess container 204 to mount and position the impeller assembly 230 in the bioprocess container 204. The impeller mounting hub 232 can also include a motor coupler 245 that can removably clip or attach to a shaft of a motor as described previously herein.

[0123] One or more mixing blades 238 (e.g., three sets of single blades, three sets of dual blades, three sets of tri-blades or three sets of four blades) are coupled to the drive shaft 228 having a diameter Do , of the bioreactor impeller assembly 230 to produce proper mixing and mixing characteristics of the contents of the bioprocess container 204. In example embodiments, the drive shaft 228 has three sets of dual blades 238. The blades extending along impeller axis IA can have a pitch or angle Ao of 30-45 degrees from a horizontal or horizontal axis HA. However other blade combinations are possible, including 1-5 sets of one, two, three or four blade combinations can be affixed to the drive shaft 228. The impeller assembly 230 is secured and positioned in the bioprocess container 204 at the top cap 222 (shown in FIG. 3) via the impeller mounting hub 232, mounting flange 246 and / or bearing housing 246 as described herein. At least one of the sets of dual blades 238 is located at a height Ho within the liquid volume so it can mix the contents of the bioprocess container 204 and grow cells at low volumes, such as at a height equivalent to 1 / 5; 1 / 4; 1 / 3; and 1 / 2 of the total volume capacity of cell culture in the bioprocess container 204.

[0124] The bottom cap 224 can include a sparger 242 for facilitating gas mass transfer across the gas-liquid film and interface between the sparged gas and liquid cell media in the bioprocess containerDocket No. TP389720WO1104. The bottom cap 224 can include a sparger port 233 that can be used in conjunction with a conduit and / or pump to pump gas through the sparger 242 and into the bioprocess container 204. The sparger 242 can route or flow various gases, such as air, nitrogen, carbon dioxide and oxygen into the bioprocess container 104, which gas dissolves into the liquid cell media to create an environment that optimizes cell growth. The sparger 242 can include a sparger disc with pores or drilled holes of specific sizes or diameters. The holes / pores in the sparger disc can be laser drilled and can have the following hole to size ratios: 235 x 80 pm holes, 295 x 50 pm holes, 667 x 30 pm holes, 360 x 178pm holes; 570 x 178pm holes; 760 x 233pm holes; 980 x 368pm holes; 1,180 x 445pm holes; 133 x 80 pm holes; 235 x 50 pm holes; 343 x 30 pm holes. In example embodiments, the sparger disc has a diameter from 20 mm to 60 mm, the sparger pore sizes ranging from 20 um to 140 um, and the sparger disc has 1-1,000 pores or holes. In other embodiments, combinations of different- sized holes are drilled into the sparger disc. The sparger 242 interfaces with or includes a gas line to feed gas through the sparger disc and into the bioprocess container 204. The sparger 242 can be integrated with the bioprocess container 104 to transfer gases into the bioprocess container 104. The sparger 242 can also have a noncircular geometry to better utilize and save space at the base of the bioreactors 100 disclosed herein. The sparger 242 can also have the same features as dome spargers and film spargers disclosed in U.S. Patent Nos. 8,603,805, 9,005,971, 9,259,692, 9,475,012, 9,682,353, 10,328,404 and 9,643,133, 10,350,554, 10,843,141, and U. S. Publication No. 2021 / 0069654, which are incorporated herein by specific reference. Other conventional spargers can also be used. The sparger 242 can also include installation tabs 219 with holes that indicate the orientation of installation. The installation tabs 219 indicate and constrain the orientation of the sparger 242 and bioprocess container 204 so that the bioprocess container 204 is installed in the correct orientation within the bioreactor housing (e.g„ 102 of FIGS. 1-2). The installation tabs 219 have perforations that allow for easy removal after installation of the sparger 242 in the bioprocess container 204.

[0125] In example embodiments, the bottom cap 224 includes a sparger port 233 that interfaces with the sparger 242 and a conduit or gas line to introduce gas through the sparger 242. In addition to the drilled hole sparger 242 port, the bottom cap 224 can also have a second gas intake port 231 that allows for the flow of gas through the gas inlet port 231 into the bioprocess container 204. In this way, gas is introduced into the bioprocess container 204 through the sparger port 233, sparger 242 and through a second gas inlet port 231. A mix of gases can be introduced through both ports 231, 233,Docket No. TP389720WO1and each port can be dedicated to flow a specific gas or mixture of gases, including but not limited to O2, CO2, N2 and air.

[0126] The bioprocess container 204 includes a sensor 210 coupled to a surface of the bioprocess container 204. In example embodiments, the sensor 210 is coupled to and / or welded to the sidewall 226 of the bioprocess container 204. A variety of sensors 210 can be coupled to the bioprocess container 204, including but not limited to, pressure sensors, temperature sensors, foam sensors, glucose sensors, pH sensors, DO sensors, CO2 sensors, flow sensors, density sensors, cell density sensors, conductivity sensors, spot sensors or the like. In example embodiment, the sensor 210 is a pH sensor, DO sensor, CO2 sensor and a temperature sensor. Sensor measurements, data or signals transmitted from sensors (or associated transmitters) are liquid, process and equipment parameter outputs and inputs, such as drive shaft 228 RPM, pressure within the bioprocess container 204, temperature within the bioprocess container 104 or cell culture; foam content within the bioprocess container 204; glucose content within the bioprocess container 204 or cell culture; pH within the bioprocess container 204 or cell culture; dissolved oxygen content within the bioprocess container 204 or cell culture; CO2 content within the bioprocess container 204 or cell culture; cell density of the cell culture; conductivity readings associated with the bioprocess container 204 or cell culture. The sensor 210 is located in a position on the bioprocess container so it can measure process, liquid and equipment parameters within the cell culture at low volumes, such as at a height equivalent to 1 / 5; 1 / 4; 1 / 3; and 1 / 2 of the total volume capacity of cell culture in the bioprocess container 204.

[0127] The sensor 210 can electronically and / or physically couple to a sensor reader 108 (shown in FIG. 1) to transmit data and signals indicating bioreactor process parameters, such as pressure, temperature, glucose concentration, dissolved oxygen concentration, CO2 concentration, pH, foam content, cell density and conductivity of the contents of the bioprocess container 204. The sensor reader 208 can communicate in a wired or wireless fashion with a controller to transmit signals and data indicating bioreactor process parameters to the controller to facilitate bioreactor process control. In other example embodiments, the sensor 210 can communicate directly with a controller without the sensor reader 108.

[0128] The bioprocess container 204 can be installed in the bioreactor housing 102 by actuating or unlatching the door latch 134 (shown in FIGS. 1-2); opening the door 118 of the housing 102 (shown in FIGS. 1-2); and inserting or positioning the bioprocess container 204 in the housing 102 (shown in FIGS. 1-2). The sidewalls 226 of the bioprocess container 204 can include one or more installationDocket No. TP389720WO1tabs 219 with one or more holes 221 that can align with protrusions in the bioreactor 100 housing 102 or simply indicate proper orientation of the bioprocess container 204 in the housing. The installation tabs 219 and holes 221 confine and indicate the installation orientation of the bioprocess container 204 in the bioreactor housing 102 (shown in FIGS. 1-2). The installation tabs 219 can be perforated for easy removal. Once the bioprocess container 204 is installed in the housing 102 of the bioreactor 100 (shown in FIGS. 1-2), based on the alignment of installation tabs 219. the installation tabs 219 can be removed along perforations formed in the tabs.

[0129] The bioprocess container 204, with flexible sidewalls 126 formed from a multi-layer flexible film, is supported and secured in the housing 102 (shown in FIGS. 1-2) from the top end and / or top cap 222. For example, after positioning the bioprocess container 204 in the housing in accordance with installation tabs 219, an over molded lip 227A of the top cap 222 of the bioprocess container 204 can be press fit into the top end wall 101 of the housing 102 (shown in FIGS. 1-2). The top end and / or top cap 122 of the bioprocess container 204 can also include mounting flanges 223 that sit on top of the bioreactor housing to hang, suspend, position or secure the bioprocess container 204 in the housing 202 by gravity. Therefore, the bioprocess container 204 hangs and is supported and secured in the housing 102 (shown in FIGS. 1-2) from the top end and / or top cap 222 by securing or press fitting the top cap 122 to the top end wall 101 of the housing 102 (shown in FIGS. 1-2). In example embodiments, the bottom end and / or bottom cap 224 of the bioprocess container 204 is not coupled or secured to the bioreactor housing or the bottom wall of the housing (e.g., 102 of FIGS. 1-2).

[0130] In other example embodiments, the bioprocess container 204 can be installed in the housing 102 (shown in FIGS. 1-2) by actuating or unlatching the door latch 134 (shown in FIGS. 1-2); opening the door 118 of the housing 102 (shown in FIGS. 1-2); and inserting or positioning the bioprocess container 204 in the housing 102 (shown in FIGS. 1-2). The bioprocess container 204. which has flexible sidewalls 226, is supported and secured in the housing 102 from the top end and / or top cap 222. For example, the top end and / or top cap 222 of the bioprocess container 204 can include mounting flanges 223 that sit on top of the housing to position or secure the bioprocess container 204 in the housing (e.g., 102 of FIGS. 1-2) by the force of gravity and contact between the flanges 123 and housing (e.g., 102 of FIGS. 1-2). In other example embodiments, after positioning the bioprocess container 204 in the housing, an over molded lip 227A of the top cap 222 of the bioprocess container 204 can be press fit into the top end wall of the housing (e.g., 101, 102 of FIGS. 1-2). The impellerDocket No. TP389720WO1mounting hub 132 (shown in FIG. 1) is removably coupled to an impeller motor (e.g., 114 of FIGS.1-2) to facilitate rotation of the drive shaft 228. In example embodiments, the impeller mounting hub 232 of the bioprocess container 204 is directly or indirectly (e.g., via the motor mounting sleeve 117 of FIGS. 1-2) coupled and secured to the impeller motor (e.g., 114 of FIGS. 1-2).

[0131] In example embodiments, the bottom end and / or bottom cap 224 of the bioprocess container 204 is not coupled or secured to the bioreactor housing or the bottom wall of the bioreactor housing (e.g., 101, 102 of FIGS. 1-2). In other example embodiment, the bioprocess container 204 and top cap 222 is secured to the bioreactor housing by gravity after positioning the top cap 222 over the top end wall of the housing (e.g., 101, 102 of FIGS. 1-2). In these embodiments, the bioprocess container 204 hangs and is positioned and secured in the bioreactor housing from the top end only.

[0132] FIGs. 4A and 4B illustrate side and top cross-sectional views of an impeller assembly 230 in accordance with example embodiments. The impeller assembly 230 can be used in any of the bioreactors (e.g., bioreactor 100 of FIGS. 1-2) and bioprocess containers (e.g., bioprocess containers 104 and 204 in FIGS. 1-3) disclosed herein. The impeller assembly 230 includes an impeller mounting hub 232 and a drive shaft 228 with one or more sets of mixing blades 238 (e.g., three sets of single blades, three sets of dual blades, three sets of tri-blades or three sets of four blades) coupled to the drive shaft 228. When rotating the drive shaft 228, the mixing blades 238 mix the contents of the bioprocess container 204 using specific mixing characteristics created by shaft location, speed of rotation and mixing blade geometry. In example embodiments, the drive shaft 228 has three sets of dual blades 238. The blades can have a pitch or angle of 30 to 45 degrees from horizontal. However, other blade combinations are possible, including 1-5 sets of one, two, three or four blade combinations can be used on the drive shaft 228. In example embodiments, the drive shaft 238 diameter is between 0.1 to 6 inches.

[0133] In example embodiments, at least one of the sets of dual blades 238 is located on the drive shaft 228 near the bottom end of the shaft so it can mix the contents of the bioprocess container 104 and grow cells at low volumes, such as at a height equivalent to 1 / 5; 1 / 4; 1 / 3; and 1 / 2 of the total volume capacity of cell culture in the bioprocess container 104. For example, at least one of the sets of dual blades 238 can be attached to the drive shaft 228 between 1 to 6 inches from the bottom end of the drive shaft to facilitate mixing at low volumes and high turn-down ratios.

[0134] The impeller mounting hub 232 is located at the terminal end of the drive shaft 228. The impeller mounting hub 232 includes a motor coupler 245, a bearing housing 246, one or more bearingDocket No. TP389720WO1assemblies 248, O-rings 250 and seals 252. The bearing housing 246 houses one or more bearing assemblies 248 and can also function as a mounting flange 246 to mount the impeller assembly 230 to the top cap 222 of the bioprocess container 204. The motor coupler 245 removably couples to a motor shaft (e.g., motor shaft 249 of FIG. 5) by quick snap-fit connection, press fit connection, via flanges or inserts or other coupling or fastening method. The motor coupler 245 can also be coupled in similar fashion to the drive shaft 228, including by a fastener 244, such as a screw or other means. A bearing housing 246 houses a bearing assembly 248 that facilitates rotation of the motor coupler 245 and drive shaft 228 when the impeller mounting hub 232 is coupled to a drive motor or motor shaft (e.g., shaft 249 of FIG. 5). The bearing housing 246 can also function as a mounting flange 246 to mount the impeller assembly 230 to the top cap 222 of the bioprocess container 204. One or more O-rings 250 made from elastomeric material are positioned on one or more sides of the bearing housing 246 to assure that the bearing housing 246 is sealed from liquid encroachment. A primary shaft seal 252 further seals the interface between the bearing housing 246 and drive shaft 228. The primary shaft seal 252 can be made from plastic or another polymer, such as polytetrafluoroethylene. The primary shaft seal 252 can also include an elastomer O-ring, and one or more lips (not shown), such as excluder lips and primary sealing lips that further seal the shaft 228 and bearing housing 246 from the environment.

[0135] The impeller mounting hub 232 can couple to a variety of motors or motor shafts through snap fit connection, press fit connection or through fastening means. In example embodiments, impeller mounting hub 232 couples to a motor through quick snap-fit connection that facilitates easy removal. The impeller mounting hub 232 shown in FIG. 4 can couple to any of the motors (e.g., motor 114 in FIGS. 1-2; motor shaft 249 of FIG. 5) disclosed herein to facilitate rotation of the drive shaft 228. As described herein, the coupling of the impeller mounting hub 232 to the motor also secures, positions and hangs or suspends the bioreactor impeller assembly 230 in the bioprocess container 204. For example, the motor coupler 245 can be removably coupled to the impeller motor 114 (shown in FIGS. 1-2) to secure and fix the top end of the bioreactor impeller assembly 230 in the bioprocess container 204 and to facilitate rotation of the drive shaft 228.

[0136] FIG. 4B illustrates a top view of the impeller assembly 230 with motor coupler 245, bearing housing 246 and a radial axis A across the top end of the impeller assembly 230 and impeller mounting hub 232.

[0137] FIG. 4C illustrates a top view of the top cap 222. The impeller assembly 230 extends along an impeller assembly axis AA, and is parallel to a center axis CA of the bioprocess container 204, butDocket No. TP389720WO1is offset from the center axis CA of the bioprocess container 204 by an offset distance Oo. It may be noted that both impeller assembly axis A A and center axis CA are orthogonal to the plane of the page. In some embodiments, the impeller assembly axis AA can be proximate to the sidewall 126 of the bioprocess container 204. In some embodiments, the impeller assembly axis AA can be proximate to the center axis CA. In some embodiments, the impeller assembly axis AA can be overlapping with the center axis CA. In some embodiments, the offset distance is about 0.4-0.7 inches from the center axis CA of the bioprocess container 204. In some embodiments, the offset distance is about 0.4-0.5 inches from the center axis CA of the bioprocess container 204. In some embodiments, the offset distance is about 0.6-0.7 inches from the center axis CA of the bioprocess container 204. In some embodiments, the offset distance is about 0.5-0.6 inches from the center axis CA of the bioprocess container 204. In some embodiments, the offset distance is less than 0.4 inches from the center axis CA of the bioprocess container 204. In some embodiments, the offset distance is more than 0.7 inches from the center axis CA of the bioprocess container 204.

[0138] FIG. 4D illustrates a top view of the top cap 222 with one or more sensor connections 235, a plurality of ports 237, and the multiport 240. The one or more ports 237 include gas exhaust line port 237A, liquid addition port 237B, glucose, feed, base, or antifoam feed port 237C, and overlay port 237D.

[0139] FIG. 4E illustrates a top view of the top cap 222 with one or more ports 237 being connected to a plurality of line sets 239, including line sets for gas headspace with pressure sensor 239A, gas exhaust line set with filter 239B, line sets for fluid addition 239C, line sets for subsurface fluid addition, foam probe and resistive temperature detector 239D coupled to multiport 240, and line sets for feeding glucose base feed and antifoam 239E.

[0140] FIG. 4F illustrates a top view of the bioprocess container 204 with gas exhaust line set with one or more filters 239B coupling gas exhaust line port 237A, to a gas exhaust vent filter heater system 247 A with a venter heater power cord 247B. The gas exhaust vent filter heater system 247 A provides increased longevity of the exhaust filter on the bioprocess container 204. The heating element is fully insulated with molded silicone and securely surrounds the filter. The exhaust filters are fully encapsulated for consistent temperature regulation.

[0141] FIG. 5 illustrates an impeller mounting hub 232 in accordance with example embodiments. The impeller mounting hub 232 can be used in any of the bioprocess containers and motors (e.g., bioprocess containers 104 and 204, motor 114 and motor mounting sleeve 117 in FIGS. 1-3) disclosedDocket No. TP389720WO1herein. The impeller mounting hub 232 removably couples to a motor 114 and motor drive shaft 249 on one end and an impeller drive shaft 228. The impeller mounting hub 232 transmits rotational force from the motor 114 and to the drive shaft 228 to facilitate mixing of cell culture in the bioprocess container. The motor drive shaft 249 can be located inside a motor mounting sleeve 217 coupled to the motor 114.

[0142] The impeller mounting hub 232 is located at a terminal end of the drive shaft 228 and includes a motor coupler 245, a bearing housing 246, one or more bearing assemblies 248, O-rings 250 and seals 252. The bearing housing 246 houses one or more bearing assemblies 248 and can also function as a mounting flange 246 to mount the impeller assembly 230 to the top cap 222 of the bioprocess container 204 (shown in FIG. 3). In other embodiments, the impeller mounting hub 232 can include an additional mounting bracket 254 that mounts the impeller mounting hub 232 and impeller assembly 230 to the top cap 222 of the bioprocess container. A fastener 244, such as a screw or bolt can be used to fasten or attach the mounting bracket 254 to the top cap 222 of the bioprocess container. The motor mounting sleeve 217 can have protrusions or indentions 258 that correspond to and fit with protrusions or indentions 260 on the impeller mounting hub 232 and / or bearing housing 246. The motor mounting sleeve 217 can be secured over the motor coupler 245 and slide into engagement with the impeller mounting hub 232 via the protrusions or indentions 258, 260.

[0143] The motor coupler 245 removably couples to the motor shaft 249 by quick snap-fit connection, press fit connection, via flanges or inserts or other coupling or fastening method. The motor coupler 245 can also be coupled in similar fashion to the drive shaft 228, including by a fastener 244, such as a screw or other means. A bearing housing 246 houses one or more bearing assemblies 248 that rotate and facilitate rotation of the motor coupler 245 and drive shaft 228 when motor coupler 245 is coupled to the motor shaft 249. The bearing housing 246 can also function as a mounting flange 246 to mount the impeller assembly 230 to the top cap 222 of the bioprocess container 204. One or more O-rings 250 made from elastomeric material are positioned on one or more sides of the bearing housing 246 to assure that the bearing housing 246 is sealed from liquid encroachment. A primary shaft seal 252 further seals the interface between the bearing housing 246 and drive shaft 228. The primary shaft seal 252 can be made from plastic or another polymer, such as polytetrafluoroethylene. The primary shaft seal 252 can also include an elastomer O-ring, and one or more lips (not shown), such as excluder lips and primary sealing lips that further seal the shaft 228 and bearing housing 246 from the environment.Docket No. TP389720WO1

[0144] The impeller mounting hub 232 and motor coupler 245 can couple to a variety of motors or motor shafts through snap fit connection, press fit connection or through fastening means. In example embodiments, the motor coupler 245 couples to a motor shaft 249 through quick snap-fit connection that facilitates easy removal. The impeller mounting hub 232 shown in FIG. 5 can couple to any of the motors (e.g., motor 114 in FIGS. 1-2; motor shaft 249 of FIG. 5) disclosed herein to facilitate rotation of the drive shaft 228. As described herein, the coupling of the impeller mounting hub 232 to the motor also secures, positions and hangs the bioreactor impeller assembly 230 in the bioprocess container.

[0145] FIG. 6 illustrates a bottom cap 224 of a bioprocess container in accordance with example embodiments. The bottom cap 224 can be used in any of the bioreactors (e.g., bioreactor 100 of FIGS.1-2) and sealed to any bioprocess containers (e.g., bioprocess containers 104 and 204 in FIGS. 1-3) disclosed herein. The bottom cap 224 is made from material that is more rigid than the bioprocess container sidewalls. The bottom cap 224 does not bow, flex and / or expand when the bioprocess container is filled with liquid, such a water or media. The bottom cap 222 of a bioprocess container can be made from a rigid plastic, such as high-density polyethylene (HDPE), low-density polyethylene (LDPE) or combinations of HDPE and LDPE. In example embodiments, the bottom cap 224 has an outside or top HDPE surface 224A that overlays an over molded LDPE structure or lip 227.

[0146] The bottom cap 224 is attached to the bottom end of the bioprocess container 204 sidewall(s) 226 by heat sealing the polymeric film to one or more surfaces of the top cap 222 and the bottom cap 224. For example, the bottom end of the flexible sidewalls 226 are heat sealed to a perimeter edge of the cap lip 227 by ultrasonic welding, heat sealing, high frequency welding, laser welding and / or impulse welding the end of the sidewall 226 to the cap lip 227 at a temperature range of 85 °C to 170 °C.The bottom cap lip 227 can have a rounded edge 263 and a sharp or pointed edge 261. The ultimate seal and / or seam between the bioprocess container sidewalls and the lip 227 can be created at the sharp edge 261 of the lip 227 to create a stronger seal. The bioprocess container sidewalls can also be heat sealed to the entire perimeter edge of the bottom cap 224 lip 227. The bioprocess container sidewalls are sealed to the top cap in the same way.

[0147] The bottom cap 224 can includes a sparger 242 for facilitating gas mass transfer across the gas-liquid film and interface between the sparged gas and liquid cell media in the bioprocess container. The bottom cap 224 can include a sparger port (not shown) that can be used in conjunction with a conduit and / or pump to pump gas through the sparger 242 and into the bioprocess container. The sparger 242 can route or flow various gases, such as air, nitrogen, carbon dioxide and oxygen intoDocket No. TP389720WO1the bioprocess container, which gas dissolves into the liquid cell media to create an environment that optimizes cell growth. The sparger 242 can include a sparger disc with pores or drilled holes of specific sizes or diameters. The holes / pores in the sparger disc can be laser drilled and can have the following hole count to size ratios: 235 x 80 pm holes, 295 x 50 pm holes, 667 x 30 pm holes, 360 x 178pm holes; 570 x 178pm holes; 760 x 233pm holes; 980 x 368pm holes; 1,180 x 445pm holes; 133 x 80 pm holes; 235 x 50 pm holes; 343 x 30 pm holes. In example embodiments, the sparger disc has a diameter from 20 mm to 60 mm, the sparger pore sizes ranging from 20 pm to 140 pm, and the sparger disc has 1-1,000 pores or holes. In other embodiments, combinations of different sized holes are drilled into the sparger disc. The sparger 242 interfaces with or includes a gas line to feed gas through the sparger disc and into the bioprocess container 204. The sparger 242 can be integrated with the bioprocess container to transfer gases into the bioprocess container 104. The sparger 242 can also have a non-circular geometry to better utilize and save space at the base of the bioreactors 100 disclosed herein. The sparger 242 can also have the same features as dome spargers and film spargers disclosed in U.S. Patent Nos. 8,603,805, 9,005,971, 9,259,692, 9,475,012, 9,682,353, 10,328,404 and 9,643,133, 10,350,554, 10,843,141, and U. S. Publication No. 2021 / 0069654, which are incorporated herein by specific reference. Other conventional spargers can also be used. The sparger 242 can also include installation tabs 219 with holes 221 that indicate the orientation of installation. The installation tabs 219 indicate and constrain the orientation of the sparger 242 and bioprocess container so that the bioprocess container is installed in the correct orientation within the bioreactor housing (e.g., 102 of FIGS. 1-2). The installation tabs 219 have perforations 265 that allow for easy removal after installation of the sparger 242 and bioprocess container.

[0148] The bottom cap 224 can include one or more bottom ports 231, including but not limited to tube ports, drain ports, sparger gas ports, sensor ports and / or sample ports for facilitating sterile fluid transfer, sparger coupling and / or sensor coupling (e.g., pressure sensors, temperature sensors, foam sensors, glucose sensors, pH sensors, DO sensors, CO2 sensors, density sensors, cell density sensors, conductivity sensors, spot sensors for measuring fluid and bioreactor process parameters). In example embodiments, the bottom cap 224 includes a sparger port (shown as 233 in FIG. 3) that interfaces with the sparger 242 and a conduit or gas line to introduce gas through the sparger 242. In addition to the drilled hole sparger 242 port, the bottom cap 224 can also have other ports, such as a second gas intake port 231 that allows for the flow of gas through the gas inlet port 231 into the bioprocess container 204. In these embodiments, gas can be introduced into the bioprocess container through the spargerDocket No. TP389720WO1port, sparger 242 and through a second gas inlet port 231. A mix of gases can be introduced through both ports, and each port can be dedicated to flow a specific gas or mixture of gases, including but not limited to O2, CO2, N2 and air.

[0149] FIG. 6A illustrates a bottom view of bottom cap 224 with one or more ports 231 and drilled hole sparger port 242. The one or more ports 231 include bottom-mounted sample port 231 A, open pipe sparger 23 IB, perfusion return port 231C, perfusion bleed port 23 ID, and drain or perfusion 231E.

[0150] FIG. 7 illustrates a bioreactor heating assembly 320 in accordance with example embodiments. The heating assembly 320 can be used in any of the bioreactors 100 and housings 102 disclosed herein. For example, the heaters 120 of FIGS. 1-2 can have the same components and configuration as the heating assembly 320 illustrated in FIG. 7. The heating assembly 320 includes heaters 380, electrical wires 382 that provide electricity to the heaters 380, a conductive back plate 384 and an insulative structure 386 that is positioned and attached along the perimeter edge of heating assembly 320. The insulative structure 386 can be an O-ring made of silicone of other insulation material. The heaters 380 can be resistive heaters 380 that heat the conductive back plate 384. The heating assembly 320 is arranged and secured to the housing 102 of the bioreactor 100 so that the conductive back plate 384 contacts the bioprocess container 104 and heats the contents therein. The positioning and staggering of the two heaters 380 in relation to the conductive back plate 384 increases the uniformity of heat across the back plate 384. Each heater 380 also has a thermostat and automatic heater shut off switch. To prevent melting the flexible, film sidewalls 126 of the bioprocess container 104, the automatic heater shut off cuts the power to the heaters when the temperature reaches a certain predetermined limit. In example embodiments, the automatic heater shut off cuts the power to the heaters when the temperature reaches 60, 70 or 80°C. At least one of the heaters 380 is located in a position on the housing 102 so it can heat the contents of the bioprocess container 104 and grow cells at low volumes, such as at a height equivalent to 1 / 5; 1 / 4; 1 / 3; and 1 / 2 of the total volume capacity of cell culture in the bioprocess container 104. For example, one of the heaters 380 can be attached 1 to 6 inches from the bottom wall 103 of the bioreactor housing 102.

[0151] FIG. 8 illustrates a seed train system 400 in accordance with example embodiments. FIG. 8 illustrates a partially exploded view of the adaptive N-l bioreactor 400A and a cross-sectional view of the production scale bioreactor 400B is illustrated in IF. The adaptive N-l bioreactor 400A can be any of the bioreactors described with respect to FIGS 1-5. The adaptive N-l bioreactor 400A is smallerDocket No. TP389720WO1in volume and capacity than the production scale bioreactor 400B. Tn example embodiments, the N-l bioreactor 400A has a 5-to-50-liter capacity with a bioprocess container 404A volume Vi equal to 5 to 50 liters. The production scale bioreactor 400B can have a 50 to 5000 liters capacity with a bioprocess container 404B volume V2 equal to 50 to 5000 liters. Operations of the adaptive N-l bioreactor 400A, the production scale bioreactor 400B, and components thereof can be controlled by a controller 425. Alternatively, adaptive N-l bioreactor 400A can have controller 425 A and the production scale bioreactor can have a controller 425B, and the controllers 425 A, 425B can be in electronic communication with each other to seamlessly control the operations of the adaptive N-l bioreactor 400 A, the production scale bioreactor 400B, and components thereof. In example embodiments, equipment parameters of the adaptive N-l bioreactor and / or production scale bioreactor can be stored in a memory device incorporated in any of the controllers 415, 425 A-B. The equipment parameters can be used to determine, predict and optimize process parameters and ultimate yield of the seed train process and system. In example embodiments, equipment parameters are the matched to be the same for the adaptive N-l bioreactor and production scale bioreactor to optimize the seed train process and better predict yield of the seed train process.

[0152] The adaptive N-l bioreactor 400 A includes several components, including but not limited to a bioreactor housing 402A, a bioprocess container 404A, a sparger 442A, a drive shaft 428A with three sets of dual blades 438A and an impeller mounting hub 432A that couples to a motor 414A that rotates the shaft to mix cell culture in the bioprocess container 404A. The N-l bioreactor 400A and components can have the same functionality as described with respect to the corresponding components described herein and illustrated in FIGS. 1-7. The N-l bioprocess container 404A is cuboidal in shape and has a vertical height of Hi and a width and length of Wi. The drive shaft 428A has a diameter of Di; As shown in FIG. 8 A, the sparger 442A is a disc-shaped drilled holed sparger with a diameter of SDi and SNi sparger holes 444A with each sparger hole 444A having a diameter of NDi; and the blades 438 A have a pitch or angle of Ai relative to the horizontal plane.

[0153] The production scale bioreactor 400B includes several components, including but not limited to a bioreactor housing 402B, bioprocess container 404B, a sparger 442B, drive shaft 428B with three sets of dual blades 438B and impeller mounting hub 432B that couples to a motor 414B that rotates the shaft to mix cell culture in the bioprocess container 404B. The production scale bioprocess container 404B is cuboidal in shape and has a vertical height of H2 and a width and length of W2. The drive shaft 428B has a diameter of D2; As shown in FIG. 8B, the sparger 442B is a disc-Docket No. TP389720WO1shaped drilled holed sparger with a diameter of SD2 and SN2 sparger holes 444B with each sparger hole 444B having a diameter of ND2; and the blades 438B have a pitch or angle of A2 relative to the horizontal plane. The production scale bioreactor has a helical mixing assembly with a flexible driveline 490, several bars or yokes 492 that separate the driveline 490 and form a ladder configuration. The same and similar helical mixing elements are disclosed in U.S. Patent No. 11,584,910 incorporated by reference in its entirety herein.

[0154] One or more equipment parameters between the N-l bioreactor 400 A and production scale bioreactor 400B can be matched to be the same to optimize scale-up and predict yield across both reactors. Equipment parameters can include, but are not limited to volume of the bioreactor or bioprocess container; height of the bioreactor or bioprocess container; diameter of the bioreactor or bioprocess container, equivalent diameter of the bioreactor or bioprocess container, shape of the bioreactor or bioprocess container; bioreactor impeller shape; bioreactor impeller size; bioreactor impeller diameter, bioreactor impeller spacing, bioreactor impeller height relative to working volumes or liquid heights or turndown ratio, bioreactor impeller pitch or angle, bioreactor impeller count, and bioreactor impeller blade count; size, diameter or surface area of the sparger; number sparger holes, and spacing or distance between of sparger holes / pore; size or diameter of sparger holes; position of sparger relative to mixing system or impeller; impeller tip speed; minimum and maximum RPM of impeller; mixing dynamics and other equipment parameters. Equipment parameters can also include certain parametric ratios, including but not limited to, impeller diameter divided by equivalent diameter of the bioreactor or bioprocess container (EPl); impeller diameter divided by the liquid height of the bioreactor or bioprocess container (EP2); number of sparger holes divided by the surface area of the sparger (EP3); number of sparger holes divided by the diameter of the sparger (EP4); sparger hole diameter divided by the diameter of the sparger (EP5); liquid column height divided by the width of the bioreactor / bioprocess container (EP6); liquid column height divided by the diameter of the bioreactor / bioprocess container (EP7); liquid column height divided by the equivalent diameter of the bioreactor / bioprocess container (EP8); liquid column height divided by the volume of the gas headspace of the bioreactor / bioprocess container (EP9); height of the bioreactor / bioprocess container divided by the width of the bioreactor / bioprocess container (EP10); height of the bioreactor / bioprocess container divided by the diameter of the bioreactor / bioprocess container (EP11); height of the bioreactor / bioprocess container divided by the equivalent diameter of the bioreactor / bioprocess container (EP12); height of the bioreactor / bioprocess container divided by the volume of the gasDocket No. TP389720WO1headspace of the bioreactor / bioprocess container (EPl 3); turn-down ratio or the ratio of the maximum working volume to the minimum working volume of the bioreactor / bioprocess container (EP14), Volume of the liquid in the bioreactor / bioprocess container divided by the volume of the gas headspace in the bioreactor / bioprocess container EP 15 and others.Impeller diameterEPl =Equivalent diameter of the bioreactor / bioprocess containerImpeller diameterEP2 =Liquid height of the bioreactor / bioprocess containerNumber of sparger holesEP3 =Surface area of the spargerNumber of sparger holesEP4 =Diameter of the spargerSparger hole diameterEP5 =Diameter of the spargerLiquid column HeightEP6 =Width of the bioreactor / bioprocess containerLiquid column HeightEP7 =Diameter of the bioreactor / bioprocess containerLiquid column HeightEP8 =Equivalent Diameter of the bioreactor / bioprocess containerLiquid column HeightEP9 =Volume ofthe gas headspace in the bioreactor / bioprocess containerHeight of the bioreactor / bioprocess containerEP10 =Width of the bioreactor / bioprocess containerHeight of the bioreactor / bioprocess containerEP11 =Diameter of the bioreactor / bioprocess containerDocket No. TP389720WO1Height of the bioreactor / bioprocess containerEP12 =Equivalent Diameter of the bioreactor / bioprocess containerHeight of the bioreactor / bioprocess containerEP13 =Volume of the gas headspace in the bioreactor / bioprocess containerMaximum working volume of the bioreactor / bioprocess containerEP14 =Minimum working volume of the bioreactor / bioprocess containerVolume of the liquid in the bioreactor / bioprocess containerEP15 =Volume of the gas headspace in the bioreactor / bioprocess container

[0155] In some embodiments, the ratio of impeller diameter to equivalent diameter of bioreactor or bioprocess container is given by the following equations:

[0156] - Impeller diameter -= Q3^%Equivalent diameter of bioreactor or bioprocess container

[0157] Impeller diameter Impeller diameterEquivalent diameter of bioreactor or bioprocess container 1.13 (WD)Hence equivalent diameter of bioreactor or bioprocess container = 1.13 H (WD);wherein WD = Width of bioreactor or bioprocess container * Depth of bioreactor or bioprocess container

[0158] In example embodiments, the values for equipment parameters EPl, EP2, EP3, EP4, and EP5 are the same for the N-l bioreactor 400A and production scale bioreactor 400B. In other example embodiments, one or more of the following equipment parameters are the same for both bioreactors 400A-B in the seed train: drive shaft diameters of Di = D2; sparger diameters of SDi = SD2; number of sparger holes SNi = SN2; sparger hole diameters of NDi = ND2; and impeller blades angle Ai = A2. One or more equipment parameters are matched for both bioreactors 400A-B in the seed train to optimize scale-up and increase predictability of cell growth and yield during scale-up.

[0159] During seed train operations, cell culture, including media and cells are transferred from the N-l bioreactor 400A to production scale bioreactor 400B. Process parameters can also be matched or linearly scaled when transitioning cell expansion to the production scale bioreactor 400B. Process parameters, including volume of cell media, cell density, pH of cell culture, process pressure, process temperature, mixing speed or impeller shaft RPM, glucose levels and other process parameters can beDocket No. TP389720WO1scaled linearly or matched across from the N-l bioreactor 400A and production scale bioreactor 400B in a predictable manner because one or more equipment parameters are the same across both reactors 400A-B in the seed train.

[0160] The flexible bioprocess container 404A-B sidewalls of the N-l bioreactor 400A and production scale bioreactor 400B are also made from the same multilayer polymeric film and have the same cuboidal shape, which also enhances the predictability of yield and scale-up across both reactors 400A-B.

[0161] FIG. 8C illustrates a series of bioreactors of increasing scale that can be used in a seed train system in accordance with example embodiments of the present disclosure. As shown in FIG. 8C, the seed train progression includes a plurality of bioreactors having nominal working volumes of approximately 5 L, 50 L, 500 L, 3,000 L, and 5,000 L, respectively. These bioreactors may correspond to, for example, an adaptive N-l bioreactor and one or more production-scale bioreactors as described with respect to FIG. 8.

[0162] In example embodiments, the 5 L bioreactor represents an adaptive N-l bioreactor configured for low- volume operation, high turn-down ratio, and process development activities, while the 50 L, 500 L, 3,000 L, and 5,000 L bioreactors represent progressively larger production-scale bioreactors configured for scale-up of cell culture processes. The sequence illustrated in FIG. 8C demonstrates a scalable platform in which cell culture can be transferred between vessels of increasing volume while maintaining consistent or substantially similar equipment, process, and operational conditions.

[0163] Each of the bioreactors shown in FIG. 8C may include a housing and a bioprocess container having a flexible sidewall, a top end cap, and a bottom end cap. as described herein. In some embodiments, the bioprocess containers across all scales are formed from the same or substantially similar multilayer polymeric film materials, thereby maintaining consistency in gas permeability, mechanical properties, and biocompatibility across the seed train. This consistency improves the predictability of cell growth, metabolic activity, and product yield during scale-up.

[0164] In example embodiments, the external form factor of the bioreactors in FIG. 8C is substantially similar across scales, with geometric scaling applied to maintain proportional relationships between height, width, and depth. For example, the larger bioreactors (e.g., 3,000 L and 5,000 L) may maintain a similar aspect ratio and cuboidal configuration as the smaller bioreactors (e.g., 5 L and 50 L). This geometric similarity promotes comparable fluid dynamics, including mixingDocket No. TP389720WO1patterns, flow circulation, and gas dispersion characteristics, thereby enabling more predictable scale-up.

[0165] As illustrated in FIG. 8C, each bioreactor includes external components such as tubing assemblies, sensor connections, gas inlet and outlet lines, and control interfaces, which are arranged in a consistent manner across scales. For example, tubing bundles and manifold connections may be positioned along a common side or top of each bioreactor, enabling standardized installation, operation, and process integration. This uniformity reduces operator variability and simplifies process transfer between scales.

[0166] In some embodiments, each bioreactor in FIG. 8C includes a mixing system, such as an impeller assembly or helical mixing assembly, configured to provide adequate mixing and mass transfer for the given working volume. While the physical size and configuration of the mixing elements may differ between smaller and larger bioreactors, the systems can be designed such that one or more key equipment parameters are maintained constant or within a defined range across scales. These parameters can include, but are not limited to: Impeller diameter to equivalent vessel diameter ratio (EPl); Impeller diameter to liquid height ratio (EP2); Sparger hole density and distribution (EP3 and EP4); Sparger hole diameter to sparger diameter ratio (EP5); Power input per unit volume; Tip speed and mixing intensity. Maintaining these parameters within consistent ranges across the bioreactors illustrated in FIG. 8C enables similar hydrodynamic and mass transfer conditions, which in turn supports predictable cell culture performance during scale-up.

[0167] In example embodiments, each bioreactor includes a gas delivery system, such as a sparger located at or near the bottom end cap of the bioprocess container. The sparger configurations across scales may be designed to maintain similar gas bubble size distributions, gas holdup, and volumetric mass transfer coefficients (kLa). For example, the number of sparger holes, hole diameter, and sparger surface area may be scaled proportionally to maintain equivalent gas transfer characteristics across the 5 L to 5,000 L range.

[0168] Additionally, each bioreactor shown in FIG. 8C may include one or more sensors configured to monitor process parameters such as pH, dissolved oxygen (DO), temperature, pressure, and cell density. In example embodiments, the placement of these sensors is scaled relative to liquid height and working volume to ensure that measurements are representative of the bulk fluid conditions. For example, sensors may be positioned at similar relative heights (e.g., corresponding to 14 to % of working volume) across all bioreactor sizes.Docket No. TP389720WO1

[0169] In some embodiments, the control systems associated with the bioreactors in FIG. 8C are configured to operate using common or harmonized control strategies. For example, controllers may regulate parameters such as temperature, pH, and dissolved oxygen using similar feedback control loops, gas flow strategies, and setpoints across all scales. As described with respect to FIG. 8, a centralized controller or networked controllers may store equipment parameters and process recipes and apply them consistently across the seed train system.

[0170] The progression illustrated in FIG. 8C enables a seed train process in which cells are initially cultured in the smallest bioreactor (e.g., 5 L), expanded to a target density, and then transferred sequentially to larger bioreactors (e.g., 50 L, 500 L, 3,000 L, and 5,000 L). At each stage, the process conditions can be maintained or scaled in a predictable manner due to the alignment of equipment parameters and system design across the different bioreactors.

[0171] In example embodiments, the adaptive N-l bioreactor (e.g., 5 L) is capable of operating at multiple working volumes (e.g., 1-5 L), thereby reducing the need for intermediate vessels in the seed train. This high turn-down capability allows a single vessel to replace multiple traditional seed train steps, simplifying operations and reducing contamination risk. The scalability illustrated in FIG. 8C further enables direct or semi-direct transfer from the N-l bioreactor to larger production- sc ale vessels.

[0172] In example embodiments, each of the bioreactors illustrated in FIG. 8C includes a housing and a bioprocess container, as described herein. The bioprocess containers across all scales are formed from the same or substantially similar multilayer polymeric film, thereby maintaining consistent material properties, including gas permeability, flexibility, and biocompatibility, across the entire scale-up progression.

[0173] In addition, each of the bioreactors illustrated in FIG. 8C has a substantially similar cuboidal shape, including a generally rectangular or square transverse cross-section. The cuboidal geometry is maintained across the 5 L, 50 L, 500 L, 3,000 L, and 5,000 L bioreactors through geometric scaling of height, width, and depth. This consistent shape across scales provides comparable fluid dynamic behavior, including natural baffling effects, reduced vortex formation, and similar mixing patterns, thereby improving predictability of scale-up and process performance.

[0174] The combination of (i) the same multilayer polymeric film for the flexible sidewalls and (ii) the same cuboidal geometry across all bioreactor sizes enhances the ability to match or proportionally scale equipment parameters and process conditions between the N-l bioreactor and production scale bioreactors. As a result, the system enables more reliable prediction of cell growth, metabolic activity,Docket No. TP389720WO1and product yield when transitioning from smaller-scale to larger-scale bioreactors within the seed train.

[0175] Furthermore, the consistent design architecture across the bioreactors is shown in FIG. 8C provides several operational advantages, including reduced process development time through simplified scale-up models; improved reproducibility of cell growth and product yield; reduced risk of contamination from fewer transfers and standardized components; and lower operational costs from reduced equipment variability and simplified workflows. In some embodiments, the systems illustrated in FIG. 8C may be used for a variety of cell culture applications, including, but not limited to, the expansion of mammalian cells (e.g., CHO cells), T cells, stem cells, and microbial cultures. The scalability demonstrated across the 5 L to 5,000 L range enables seamless transition from research and development to commercial manufacturing.

[0176] FIG. 9 depicts a block diagram of an example computing device 900 that can perform or control some or all operations of seed train systems and cell expansion operations, including bioreactor 100, adaptive N-l bioreactor 400A, production scale bioreactor 400B, user computing device(s), processing unit(s) and / or controller(s) 125, 425 in accordance with the example embodiments. The example of seed train systems and cell expansion operations, including a bioreactor 100, or adaptive N-l bioreactor 400A, production scale bioreactor 400B, and system controller, including controllers 125, 425, modules, libraries, and data repositories, disclosed herein can include or be implemented by one or more computing devices 900. In some embodiments, the example user computing device 900, and system controller 125, 425 include a single computing device 900 or multiple computing devices 900. Further, as discussed below, a computing device 900 (or multiple computing devices 900) that implements the operations of seed train systems and cell expansion operations, can include bioreactor 100, adaptive N-l bioreactor 400A, production scale bioreactor 400B, a user’s local computing device, a service provider’s local computing device, or a remote computing device. Controllers 125, 425, can also be contained in a unitary computing system or server with a user interface or distributed over servers and systems.

[0177] The computing device 900 of FIG. 9 is illustrated as having a number of components, but any one or more of these components may be omitted or duplicated, as suitable for the application and setting. In some embodiments, some or all of the components included in the computing device 900 can be attached to one or more motherboards and enclosed in a housing (e.g., including plastic, metal, and / or other materials). In some embodiments, some of these components may be fabricated onto aDocket No. TP389720WO1single system-on-a-chip (SoC) (e.g., an SoC may include one or more processing devices 902 and one or more storage devices 904). Additionally, in various embodiments, the computing device 900 may not include one or more of the components illustrated in FIG. 9, but may include interface circuitry (not shown) for coupling to one or more components using any suitable interface (e.g., a Universal Serial Bus (USB) interface, a High-Definition Multimedia Interface (HDMI) interface, a Controller Area Network (CAN) interface, a Serial Peripheral Interface (SPI) interface, an Ethernet interface, a wireless interface, or any other appropriate interface). For example, the computing device 900 may not include a display device 910, but may include display device interface circuitry (e.g., a connector and driver circuitry) to which a display device 910 may be coupled.

[0178] The computing device 900 can include a processing medium or device 902 (e.g., one or more processing devices). As used herein, the term "processing device" refer to any device or portion of a device that processes electronic data from registers and / or memory to transform that electronic data into other electronic data that may be stored in registers and / or memory. The processing device 902 can include one or more digital signal processors (DSPs), application-specific integrated circuits (ASICs), central processing units (CPUs), graphics processing units (GPUs), crypto processors (specialized processors that execute cryptographic algorithms within hardware), server processors, or any other suitable processing devices.

[0179] The computing device 900 can also include a storage device 904 (e.g., one or more storage devices). The storage device 904 can include one or more memory devices such as random-access memory (RAM) (e.g., static RAM (SRAM) devices, magnetic RAM (MRAM) devices, dynamic RAM (DRAM) devices, resistive RAM (RRAM) devices, or conductive-bridging RAM (CBRAM) devices), hard drive-based memory devices, solid-state memory devices, networked drives, cloud drives, or any combination of memory devices. In some embodiments, the storage device 904 can include memory that shares a die with a processing device 902. In such an embodiment, the memory can be used as cache memory and can include embedded dynamic random-access memory (eDRAM) or spin transfer torque magnetic random-access memory (STT-MRAM), for example. In some embodiments, the storage device 904 can include non-transitory computer-readable media having instructions thereon that, when executed by one or more processing devices (e.g., the processing device 902), cause the computing device 900 to perform any appropriate ones of or portions of the methods and operations disclosed herein. Equipment and process parameters can be stored in one or more memory devices ofDocket No. TP389720WO1the storage device 904. The parameters are called / recalled from memory and processed by the processer 402 to control and optimize seed train operations.

[0180] The computing device 900 can include an interface device 906 (e.g., one or more interface devices 906). The interface device 906 can include one or more communication chips, connectors, and / or other hardware and software to govern communications between the computing device 900 and other computing devices. For example, the interface device 906 can include circuitry for managing wireless communications for the transfer of data to and from the computing device 900. The term "wireless" and its derivatives are used to describe circuits, devices, systems, methods, techniques, communications channels, etc., that can communicate data through the use of modulated electromagnetic radiation through a nonsolid medium. The term does not imply that the associated devices do not contain any wires, although in some embodiments they might not. Circuitry included in the interface device 906 for managing wireless communications can implement any of a number of wireless standards or protocols, including but not limited to Institute for Electrical and Electronic Engineers (IEEE) standards including Wi-Fi (IEEE 802.11 family), IEEE 802.16 standards (e.g.. IEEE 802.16-2005 Amendment), Long-Term Evolution (LTE) project along with any amendments, updates, and / or revisions (e.g., advanced LTE project, ultra-mobile broadband (UMB) project (also referred to as "3GPP2"), etc.). In some embodiments, circuitry included in the interface device 906 for managing wireless communications may operate in accordance with a Global System for Mobile Communication (GSM), General Packet Radio Service (GPRS), Universal Mobile Telecommunications System (UMTS), High Speed Packet Access (HSPA), Evolved HSPA (E-HSPA), or LTE network. In some embodiments, circuitry included in the interface device 906 for managing wireless communications can operate in accordance with Enhanced Data for GSM Evolution (EDGE), GSM EDGE Radio Access Network (GERAN), Universal Terrestrial Radio Access Network (UTRAN), or Evolved UTRAN (E-UTRAN). In some embodiments, circuitry included in the interface device 906 for managing wireless communications can operate in accordance with Code Division Multiple Access (CDMA), Time Division Multiple Access (TDMA), Digital Enhanced Cordless Telecommunications (DECT), Evolution-Data Optimized (EV-DO), and derivatives thereof, as well as any other wireless protocols that are designated as 3G, 4G, 5G, and beyond. In some embodiments, the interface device 906 can include one or more antennas (e.g., one or more antenna arrays) to receipt and / or transmission of wireless communications.Docket No. TP389720WO1

[0181] In some embodiments, the interface device 906 can include circuitry for managing wired communications, such as electrical, optical, or any other suitable communication protocols. For example, the interface device 906 can include circuitry to support communications in accordance with Ethernet technologies. In some embodiments, the interface device 906 can support both wireless and wired communication, and / or may support multiple wired communication protocols and / or multiple wireless communication protocols. For example, a first set of circuitries of the interface device 906 can be dedicated to shorter-range wireless communications such as Wi-Fi or Bluetooth, and a second set of circuitries of the interface device 906 can be dedicated to longer-range wireless communications such as global positioning system (GPS), EDGE, GPRS, CDMA, WiMAX, LTE, EV-DO, or others. In some embodiments, a first set of circuitries of the interface device 906 can be dedicated to wireless communications, and a second set of circuitries of the interface device 906 can be dedicated to wired communications.

[0182] The computing device 900 can include battery / power circuitry 908. The battery / power circuitry 908 can include one or more energy storage devices (e.g.. batteries or capacitors) and / or circuitry for coupling components of the computing device 900 to an energy source separate from the computing device 900 (e.g., AC line power).

[0183] The computing device 900 can include a display device 910 (e.g., multiple display devices). The display device 910 can include any visual indicators, such as a heads-up display, a computer monitor, a projector, a touchscreen display, a liquid crystal display (LCD), a light-emitting diode display, or a flat panel display.

[0184] The computing device 900 can include other input / output (I / O) devices 912. The other I / O devices 912 can include one or more audio output devices (e.g.. speakers, headsets, earbuds, alarms, etc.), one or more audio input devices (e.g., microphones or microphone arrays), location devices (e.g., GPS devices in communication with a satellite-based system to receive a location of the computing device 900. as known in the art), audio codecs, video codecs, printers, sensors (e.g., thermocouples or other temperature sensors, humidity sensors, pressure sensors, vibration sensors, accelerometers, gyroscopes, etc.), image capture devices such as cameras, keyboards, cursor control devices such as a mouse, a stylus, a trackball, or a touchpad, bar code readers, Quick Response (QR) code readers, or radio frequency identification (RFID) readers, for example.

[0185] The computing device 900 can have any suitable form factor for its application and setting, such as a handheld or mobile computing device (e.g., a cell phone, a smartphone, a mobile internetDocket No. TP389720WO1device, a tablet computer, a laptop computer, a netbook computer, an Ultrabook computer, a personal digital assistant (PDA), an ultra-mobile personal computer, etc.), a desktop computing device, or a server computing device or other networked computing components.

[0186] The bioreactors disclosed herein have a number of unique benefits. The present disclosure relates to adaptive bioreactors used in seed train systems and cell expansion operations. The seed train systems can include one or more bioreactors of various sizes, including an N-l bioreactor that is smaller in size and volume than a production scale bioreactor. The N-l bioreactor is adaptive for several reasons, including its ability to operate at a fraction of the total fluid volume capacity of the bioreactor. The N-l bioreactors heaters, sparger, sensors and impeller assemblies are in numerous locations, including low volume locations, so the adaptive bioreactor can operate at low volume. In example embodiments, the total capacity of the N-l adaptive bioreactor is 5 liters, and the bioreactor can operate and grow cells with 1, 2, 3, 4, and 5 liters of cell culture in the bioreactor, in part, because at least one sensor, sparger, heater, heater shot-off switch, and impeller blade is located at a low volume location. Low volume locations can include a height along the bioreactor that is equivalent to 1-4 liters of cell culture in the bioprocess container or *4 to % the total volume capacity of the bioreactor, including bioprocess container.

[0187] In seed train systems and operations, the N- 1 adaptive bioreactor has one or more equivalent equipment parameters as the production scale bioreactor in the seed train, allowing greater optimization of equipment and process parameters across the two or more bioreactors in the seed train system. This also leads to an increase in the cell concentration and populations, an increase in scale-up predictability and an increase in the yield of the pharmaceutical end-products. Therefore, the N-l bioreactors and other bioreactors disclosed herein are adaptive in several ways, including adaptive to low volume, high turn-down ratio operations and adaptive to scale-up and process optimization in seed train systems with larger production scale bioreactors. The high turn-down ratio and low volume adaptivity of the N-l bioreactors disclosed herein reduced the number of vessels necessary for seed train operations.

[0188] Using the systems and methods disclosed here, equipment, process and operational parameters can be matched or maintained consistently across the adaptive N-l bioreactor and the production scale bioreactor. When scaling from adaptive N-l bioreactor to production scale bioreactor, the most crucial factor is the ability to accurately predict growth conditions in a bioreactor. The scalable system described herein enables users to achieve this by adjusting or matching parameters ofDocket No. TP389720WO1various components such as the sparger design, impeller design, and drive system. Although the components may differ between adaptive N-l bioreactor and the production scale bioreactor, they can be configured to produce the same or similar results. It is important to note that these systems are interdependent, and adjustments to one component will require consideration of the others to obtain same or similar operational parameters in the production bioreactor.

[0189] Any number of different biocomponents, cell media components, cells and cell culture media can be used in conjunction with bioreactors disclosed herein, biocomponents, cell media components, cells and cell culture media can be varied and optimized based on the ultimate purpose of the cell expansion and seed train operations (e.g., use of expanded cells, protein production, antibody production, vaccine production, therapeutic production).

[0190] Cells (e.g., animal cells, such as mammalian cells) that can be expanded using the devices (e.g., bioreactors) and methods set out herein include immortalized cells (e.g., hybridoma cells) and primary cells (e.g., T cells, B cells, hepatocytes, etc.). Stem cells (e.g., induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, etc.) and other cell types can be expanded using devices and methods set out herein, including expansion of immune system cells such as T cells (e.g., CD4+ T cells, CD8+ T cells, regulatory T cells, Thl7 T cells, gamma delta T cells, memory T cells (e.g., central memory T cells), natural killer T cells, mucosal associated invariant T cells, etc.), natural killer (NK) cells, B cells, dendritic cells, antigen presenting cells, etc.

[0191] Some specific examples of cell types that may be expanded using devices, methods and seed train operations set out herein include African green monkey cells (e.g., BSC cells), HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells. RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK- 21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells and chicken embryo cells. In some instances, a CHO cell line or one or more of several specific CHO cell variants optimized for large-scale protein production (e.g., CHO-Kl) is expanded.

[0192] T cells, for example, can be expanded in a number of different culture media, including X-VIVO 15™ (Lonza, cat. no. BE02-060Q) and OPTMIZER™ CTS™ SFM, AIM-V, and RPMI 1640 (Thermo Fisher Scientific, cat. nos. A1048501, 0870112DK, 11875119). Further, T cells may be expanded with serum or without serum. Additional, T cells can be expanded with a serum replacement,Docket No. TP389720WO1such as CTS™ Immune Cell Serum Replacement (ICSR) (Thermo Fisher Scientific, catalog number A2596101).

[0193] T cells can be activated before, during, and / or after expansion. For example, T cells can be activated in bioreactors during expansion. By way of further example, T cells can be activated through contact with anti-CD3 and anti-CD28 antibodies. Such antibodies can be bound to one of more solid support (e.g., beads). Further, T cells can also be contacted with one or more cytokine (e.g., interleukin- 2, etc.) before, during, and / or after expansion. Thus, provided herein are methods for the adaptive bioreactors and seed train operations disclosed herein can be used specifically T-cell expansion.

[0194] Example cell media formulations that can be used in adaptive bioreactors and seed train operations disclosed herein include Eagle’s MEM (minimal essential media), Ham’s Fl 2, F-12 K, Dulbecco’s, Dulbecco’s Modified Eagle Medium, DMEM / Ham’s F12 1:1, Trowell’s T8, A2, Waymouth, Williams E, MCDB 104 / 110, RPMI-1640 Medium, RPMI-1641 Medium, Iscove’s Modified Dulbecco’s Medium, McCoy’s 5 A, Leibovitz’s L-15, EX- CELL™ 300 Series (JRH Biosciences, Lenexa, KS), protamine-zinc-insulin media. Media may contain serum or be serum free.

[0195] Cells can be expanded in “fed-batch cell culture” processes. “Fed-batch culture” refers to a batch culture wherein the animal cells and culture medium are supplied to the adaptive bioreactor and additional nutrients are fed continuously or in discrete increments, to the cell culture during culturing, with or without periodic cell and / or product harvest before termination of culture. Fed-batch culture includes “semi-continuous fed-batch culture” wherein periodically whole culture (which may include cells and media) is removed and replaced by fresh medium.

[0196] Fed-batch culture is distinguished from simple “batch culture” whereas all components for cell culturing (including the animal cells and all culture nutrients) are supplied to the culturing vessel at the start of the culturing process in batch culture.

[0197] Cells can also be expanded in perfusion processes. In perfusion culturing, the cells are restrained in the culture by (e.g., filtration) and the culture medium is continuously or intermittently introduced and removed from the culturing vessel.

[0198] Typically, cells are expanded in example bioreactors with an O2 concentration between 15% and 50% within the bioprocess container. In some embodiments, cells are expanded in example bioreactors with an O2 concentration between 20% and 50% within the bioprocess container. CO2 concentration can be controlled between 2% and 7%. Spargers and gas ports disclosed herein pumpDocket No. TP389720WO1the necessary gases in the bioprocess container to provide an optimal growth environment for cells during cell expansion.

[0199] Bioreactors set out herein can be used to maintain the viability of cells by operating in at various mixing RPMs (e.g., below 40 RPMs or below 10 RPMs), after these cells have been exposed to electric fields (e.g., electroporation). For example, bioreactor impellers can also be operated at about 40 to 1.500 RPM (e.g., from about 40 to about 1,000, from about 40 to about 800, from about 40 to about 600, from about 40 to about 400, from about 40 to about 300, from about 40 to about 200, from about 100 to about 1,000, from about 100 to about 800, from about 100 to about 600, from about 100 to about 400, from about 100 to about 300, from about 60 to about 1,000, from about 60 to about 300, etc., RPM). Exemplary methods of maintaining cell viability include exposing cells to an electric field followed by incubation of these cells while operating the bioreactors set out herein in static mode. This incubation can be in culture media or a medium designed to allow for the cells to remain in low metabolic state during the incubation period (e.g., an osmotically stabilizing solution containing minimally sufficient nutrients to prevent significant decrease in cell viability).

[0200] In example embodiments, the N-l bioreactor set out herein can have the equipment parameter EP 14 or turndown ratio of 5: 1. which allows scaling of experiments from 1 L to 5 L without replacing the vessel. The N-l bioreactor can also enable an easy transition from benchtop to production-scale operation because its design is similar to that of 500 L and 5,000 L bioreactors, which have turndown ratios of 10:1 and higher. This versatility and scalability can help process development laboratories optimize operations and prepare for large-scale production, ultimately improving overall scalability. Hence, above N-l bioreactor provide for scalable design to transition from development to production.

[0201] In example embodiments, bioreactors set out herein can have high operational efficiency by including a low risk of contamination, up to at least 20% decrease of workflow duration, and at least 27% increase in productivity. While, pre- irradiated components significantly decrease the contamination risk, avoiding cleaning or autoclaving reduce setup and turnaround times. Additionally, avoiding cleaning or autoclaving results in (i) reduced environmental impact by decreasing water consumption and carbon footprint; and (ii) lower capital expenditure, and reduced recurring maintenance and cleaning needs. For instance, autoclaving four or more traditional bioreactors requires 250-300 L of water per sterilization cycle, and a laboratory glassware washer consumes 55-65 L per wash cycle. In other words, 61,000 to 73,000 L of water would be required annually toDocket No. TP389720WO1sterilize and clean traditional bioreactors in a laboratory that operates an autoclave and washer 200 times per year. In contrast, bioreactors disclosed in this disclosure does not require cleaning or sterilization, thereby reducing the significant environmental impact of process development.Example 5 L - 5000 L Scalability experiment using 5L Single-Use Bioreactor

[0202] An example of scaling up a CHO-based fed-batch bioprocess from the 5 L S.U.B. (Single Use Bioreactor) to the 5000 L S.U.B. is outlined in the following, showing the potential for accelerating scale-up and scale-down via straightforward and predictable control parameters. In some embodiments, the 5 L S.U.B. can be the adaptive N-l bioreactor 100, and the 50 L / 500 L / 5000 L S.U.B. can be the production scale bioreactor described above. This example can provide a starting point for basic recommendations on scaling processes using the 5L S.U.B. N-l bioreactor. In some embodiments, a fed-batch process utilizing IgG-producing CHO-K1 cells was performed in four different bioreactors while scaling the process up by a factor of lOOOx.

[0203] IgG producing CHO-K1 cells is thawed, passaged, and expanded into shake flasks containing Gibco™ Efficient-Pro™ AGT™ medium following standard procedures. Cell expansion proceeds as necessary to provide sufficient material to inoculate 5 L, 50 L, 500 L, and 5000 L BioProcess Containers (BPCs) loaded in the respective 5 L, 50 L, 500 L, and 5000 L S.U.B. s at the target initial volumes listed in Error! Reference source not found.. Thermo Scientific HyPerforma™ G3Lab™ and G3Pro™ Universal Bioreactor Controllers are used to control the 5 L, 50 L, 500 L, and 5000 L S.U.B.s, respectively. For each of the reactors, the mediums used are listed in Table- 1 below.Docket No. TP389720WO1Media Use Targetr EssikDnc,o-,™ rro Basal medium N / AMediumGibco ™ Efficient Pro Feed Supplemental feed 2.25% current vessel.j volume, adjusted daily,starting on day 32M Glucose Supplement glucose as 3 g / 'L throughout culture neededGibco™ FoamAway™ Antifoaming agent As neededIrradiated AOFTable- 1

[0204] Example control parameters for the four reactors tested are provided in Table-2 below. Similar parameters are selected for the four reactors to showcase ease of scalability and process transfer between the 5 L S.U.B. and the 5000 L S.U.B. Table-2 also outlines the control strategies for pH and DO (Dissolved Oxygen) control. The controller output for each gas used to control DO and pH was the same across reactors, but the related output scaling for corresponding mass flow controllers (MFCs) was scaled down by a factor of 10 for the 5 L S.U.B. This approach is taken to demonstrate the potential for simplicity in scaling gas strategies used for these systems. Single-use probes and adapters pre-integrated into the BPCs eliminated the need to perform any invasive probe installation for either DO or pH probes.Pitranwicr 5I. S,('.K : S.r.K 5MH. S.I'.B. 1. S.I JR.Docket No. TP389720WO1

[0205] After pertinent calibrations is completed, sterile medium is conditioned, and control loops were enabled in the S.U.B.s. By this point, sufficient seed train material is ready for inoculation, and the reactors are seeded at a target density of 0.3 x 106cells / mL. On day three of the process, efficient-Pro AGT Feed 1 begins with a target addition rate of 2.25% of the current vessel volume, with the feed rate updated daily. A 36% (w / v) glucose solution was supplemented as needed throughout the process, targeting a glucose concentration of 3 g / L in all reactors. Gibco™ FoamAway™ Irradiated AOF Antifoaming Agent was used to control excess foam during operation through periodic dosing,Docket No. TP389720WO1or as triggered via the use of the integrated foam probe. Daily samples from both reactors are taken to analyze cell density, cell viability, cell size, metabolites, and IgG production.

[0206] The results show that a cell culture process can be reliably and efficiently transferred from the research and process development scale to the production scale successfully. The analyzed data included viable cell densities (VCD) and viability (FIG. 10), IgG titers (FIG. 11), specific productivity (FIG. 12), lactate concentrations (FIG. 13), online pH values (FIG. 14), dissolved oxygen (DO) (FIG.15), and lactate dehydrogenase (LDH) (FIG. 16). Samples were taken daily around the same time feed rates were adjusted. The values depicted were recorded using the offline daily sample, except for DO and pH, which were recorded or monitored online continuously using sensors attached to the BPC.

[0207] FIG. 10 illustrates a plot of mean viable cell density (VCD) and cell viability as a function of process time (in days) for the 5 L, 50 L, 500 L, and 5,000 L bioreactors. As shown, viable cell density increases over time for all bioreactor scales, reaching peak values at approximately similar time points, followed by a gradual decline as the culture progresses toward termination. The VCD profiles across all scales are closely aligned, indicating that cell growth kinetics are consistent and reproducible during scale-up. Throughout the fed-batch culture, viable cell densities trended similarly between the reactors, with an average peak VCD of over 55 x 106cells / mL achieved in the 5 L S.U.B (FIG. 10). Notably, the replicates for each S.U.B. with the highest peak VCD performed remarkably similarly, with a peak VCD of 62.2 x 106cells / mL for the 5 L reactor. Cell viability also trended comparably throughout the run. In addition to VCD, FIG. 10 also depicts cell viability as a percentage over the course of the process. Cell viability remains high (e.g., above approximately 80-90%) during the exponential growth phase across all reactor scales, with a gradual decline observed during later stages of the culture. The similarity in viability profiles across scales indicates that shear conditions, nutrient availability, and metabolic stress are comparable between the different bioreactor sizes.

[0208] FIG. 11 illustrates a plot of mean immunoglobulin G (IgG) concentration (g / L) versus process time for the same set of bioreactors. The IgG titer increases over time in each bioreactor, reflecting protein production by the cultured cells. As shown, the IgG concentration profiles for the 5 L, 50 L, 500 L, and 5,000 L bioreactors are substantially similar, with only minor variations between scales. The close alignment of the titer curves indicates that productivity and expression performance are maintained during scale-up. This consistency demonstrates that the matched equipment parameters and process conditions enable reliable translation of product yield from small-scale to productionscale systems. Protein titers were nearly identical between reactors, with mean IgG concentration atDocket No. TP389720WO1culture termination at 3.66 g / L for the 5 L cultures. Productivity was maintained throughout the duration of the culture in both systems. The tight confidence intervals around mean IgG trends are indicative of the high degree of reproducibility between replicates and across scales.

[0209] FIG. 12 illustrates a plot of specific productivity (Qp), expressed as picograms of IgG produced per cell per day, versus process time. The specific productivity profiles across all bioreactor scales show similar trends, with Qp increasing during early and mid-phase culture and fluctuating within a comparable range across scales. The data indicate that cellular productivity is not adversely affected by scale, and that the cells maintain similar metabolic and biosynthetic behavior in each bioreactor. Variations observed in Qp are within expected biological variability and do not indicate any systematic deviation between scales. Specific productivity as a 3-point moving average refers to the grams of product produced per viable cell per day (FIG. 12). Overall, the reactors trended similarly, with the titer spread being only 9% on the last day. This shows that productivity between all scales was comparable.

[0210] FIG. 13 illustrates a plot of mean lactate concentration (g / L) versus process time. Lactate is a key metabolic byproduct and an indicator of cellular metabolic state. As shown, lactate concentration initially increases during early culture stages, reflecting glycolytic metabolism, followed by a decrease or stabilization as cells transition to lactate consumption or more efficient metabolic pathways. The lactate profiles across all bioreactor scales exhibit similar trends, including comparable peak concentrations and timing of metabolic shifts. This alignment indicates that metabolic behavior is consistent across scales and that nutrient and oxygen transfer conditions are effectively maintained during scale-up. Metabolic profiles appeared to align well among the 5 L, 50 L, 500 L, and 5000 L cultures as well. For example, lactate build-up and the lactate metabolism switch trended similarly by the culture replicates in all scales, though the preliminary lactate accumulation was marginally higher in the 5 L compared to the others.

[0211] FIG. 14 illustrates a plot of online pH versus process time for each bioreactor scale. The pH profiles remain tightly controlled around a setpoint (e.g., approximately 7.0-7.2) throughout the culture duration, with only minor fluctuations observed. The similarity in pH control across the 5 L, 50 L, 500 L, and 5,000 L bioreactors demonstrates effective and consistent control strategies, including gas flow (e.g., CO2 addition) and buffering capacity. The ability to maintain stable pH across scales is critical for ensuring consistent cell growth and productivity.Docket No. TP389720WO1

[0212] FIG. 15 illustrates a plot of dissolved oxygen (DO) concentration (%) versus process time. The DO profiles show that oxygen levels are maintained near a target setpoint (e.g., approximately 40%) throughout the culture, with transient deviations corresponding to changes in cellular oxygen demand or control responses. The DO trends are highly similar across all bioreactor scales, indicating that oxygen transfer and gas control strategies (e.g., air, O2, and N2 flow rates) are effectively scaled. This consistency confirms that the sparger design, mixing system, and control parameters provide comparable oxygen availability across different reactor volumes.

[0213] FIG. 16 illustrates a plot of lactate dehydrogenase (LDH) concentration (U / L) versus process time. LDH is commonly used as a marker of cell stress, damage, or lysis. As shown, LDH levels increase over time in all bioreactors, particularly during later stages of the culture when cell viability begins to decline. The LDH profiles across all scales are similar in both magnitude and trend, indicating comparable levels of cellular stress and mechanical forces (e.g., shear stress from mixing and aeration). The absence of significant divergence in LDH levels suggests that the hydrodynamic environment is well-controlled and consistent across scales.

[0214] Furthermore, LDH concentration, which is often used as an indicator of cell stress or damage, was highly parallel between the 5 L, 50 L, 500 L, and the 5000 L S.U.B.s. This may suggest very similar degrees of shear stress being applied to the systems by the agitation and aeration mechanisms and controls, a key consideration for scale-up and scale-down models and benefit of utilizing the 5 L S.U.B.

[0215] The 5 L S.U.B. simplifies and streamlines common scalability challenges that process development labs encounter, including aeration, mixing and agitation, and heat and mass transfer. Strategic design of the 5 L S.U.B. allowed for highly scalable growth kinetics and protein production with simple and user-friendly process development approaches. The 5 L S.U.B. shows scalability of cell culture performance from 5 L benchtop to 5000 L production volumes.

[0216] Various alterations and / or modifications of the inventive features illustrated herein, and additional applications of the principles illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, can be made to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the claims, and are to be considered within the scope of this disclosure. Thus, while various aspects and embodiments have been disclosed herein, other aspects and embodiments are contemplated. While a number of methodsDocket No. TP389720WO1and components similar or equivalent to those described herein can be used to practice embodiments of the present disclosure, only certain components and methods are described herein.

[0217] It will also be appreciated that systems, processes, and / or products according to certain embodiments of the present disclosure may include, incorporate, or otherwise comprise properties features (e.g.. components, members, elements, parts, and / or portions) described in other embodiments disclosed and / or described herein. Accordingly, the various features of certain embodiments can be compatible with, combined with, included in, and / or incorporated into other embodiments of the present disclosure. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include said features without necessarily departing from the scope of the present disclosure.

[0001] The present disclosure can be embodied in other specific forms without departing from its spirit or essential characteristics of the scope of the invention. The described embodiments are illustrative and not restrictive. The scope of the invention is indicated by the appended claims in light of the detailed description, figures and claims disclosed herein. While certain embodiments and details have been included herein and in the attached disclosure for purposes of illustrating embodiments of the present disclosure, it will be apparent to those skilled in the art that various changes in the methods, products, devices, and apparatus disclosed herein may be made without departing from the scope of the disclosure or of the invention, which is defined in the appended claims. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

Docket No. TP389720WO1What is claimed is:

1. A bioreactor, comprising:a housing; anda bioprocess container having a flexible sidewall, a top end cap and a bottom end cap, wherein the top end cap and the bottom end cap are more rigid than the flexible sidewall, and the top end cap of the bioprocess container is configured to be secured to the housing.

2. The bioreactor recited in claim 1, wherein the bioprocess container is suspended from the housing via the top end cap, and the bottom end cap is not attached to the housing.

3. The bioreactor recited in claim 1 or 2, further comprising at least one integrated heating system comprising at least two heating plates located at different heights on a sidewall of the housing.

4. The bioreactor recited in claim 1 or 2, further comprising at least two integrated heating systems, each comprising at least two heating plates located at different heights on two sidewalls of the housing.

5. The bioreactor recited in claim 3 or 4, wherein a first heating plate of the at least two heating plates is located about 3.0 to about 5.0 inches from a bottom end wall of the housing.

6. The bioreactor recited in any preceding claim, wherein the bioprocess container includes an impeller assembly comprising an impeller mounting hub. a drive shaft, a blade assembly having a first blade assembly and a second blade assembly on the drive shaft, wherein the first blade assembly is located above the second blade assembly.

7. The bioreactor recited in claim 6, wherein the first and second blade assemblies each comprise at least two blades.

8. The bioreactor recited in claim 7. wherein at least two blades are each at an angle from a horizontal axis.Docket No. TP389720WO19. The bioreactor recited in claim 1, wherein the bioprocess container includes a sensor on the flexible sidewall of the bioprocess container spaced from the bottom end cap.

10. The bioreactor recited in any of claims 5-7, wherein the bioprocess container further includes a sensor on the flexible sidewall at a same height as the second blade assembly.

11. The bioreactor recited in claim 1, wherein the housing further comprises a door on a sidewall, wherein the door is configured to accommodate installation of the bioprocess container or removal of the bioprocess container.

12. The bioreactor recited in claim 1, wherein the bioprocess container includes at least one installation tab on the flexible sidewall, the installation tab indicating proper orientation or placement of the bioprocess container within the housing upon installation.

13. The bioreactor recited in any preceding claim, wherein the bioprocess container further includes a sparger near the bottom end cap.

14. The bioreactor recited in claim 13, further comprising an installation tab on the sparger, the installation tab indicating proper orientation or placement of the bioprocess container within the housing upon installation.

15. A seed train system, comprising:an N-l bioreactor having or associated with a first set of equipment parameters; and a production scale bioreactor having or associated with a second set of equipment parameters, the production scale bioreactor being operable along with the N-l bioreactor;wherein the first and second sets of equipment parameters include at least one substantially identical equipment parameter.

16. The seed train system recited in claim 15, wherein the N-l bioreactor further comprises: a N-l housing; andan N-l bioprocess container with a flexible sidewall, a top end cap and a bottom end cap, wherein the top end cap and the bottom end cap are more rigid than the flexible sidewall, and the N-l bioprocess container is configured to be secured to the housing via the top end cap.Docket No. TP389720WO117. The seed train system recited in claim 16, wherein the production scale bioreactor includes:a production scale housing; anda production scale bioprocess container.

18. The seed train system recited in claim 17, wherein the production scale bioprocess container has a flexible sidewall made from a same material as the flexible sidewall of the N-l bioprocess container.

19. The seed train system recited in claim 17,wherein the N-l bioreactor comprises an N-l impeller shaft and an N-l sparger having N-l sparger holes, the N-l bioprocess container having a first diameter and the N-l sparger having a second diameter; andwherein the production scale bioreactor comprises a production scale impeller shaft and a production scale sparger having production scale sparger holes, the production scale bioprocess container having a third diameter, and the production scale sparger having a fourth diameter.

20. The seed train system recited in claim 17, whereinthe first and second set of equipment parameters each comprise respective equipment parameter 1, equipment parameter 2, equipment parameter 3, equipment parameter 4 or equipment parameter 5, the equipment parameter 1 is a diameter of the N-l or production scale impeller shaft divided by an equivalent diameter of the N-l or production scale bioprocess container,the equipment parameter 2 is the diameter of the N-l or production scale impeller shaft divided by a height of liquid in the N-l or production scale bioprocess container;the equipment parameter 3 is a number of sparger holes of the N-l or production scale sparger divided by a surface area of the N-l or production scale sparger;the equipment parameter 4 is the number of sparger holes of the N-l or production scale sparger divided by a diameter of the of the N-l or production scale sparger; andthe equipment parameter 5 is a sparger hole diameter of the N-l or production scale sparger divided by the diameter of the of the N-l or production scale sparger.Docket No. TP389720WO121. The seed train system recited in claim 15, further comprising a controller that controls the N-1 and production scale bioreactors based on the first and second set of equipment parameters.

22. A seed train process, comprising:growing a first density of cells in an N-l bioreactor having or associated with a first set of equipment parameters;transferring the first density of cells into a production scale bioreactor having or associated with a second set of equipment parameters; andgrowing a second density of cells in the production scale bioreactor,wherein the first and second sets of equipment parameters include at least one substantially identical equipment parameter.

23. The seed train process recited in claim 22, further comprising a controller with a processor that can predict the second density of cells based on the first and second sets of equipment parameters.

24. The seed train process recited in claim 22, wherein the N-l bioreactor further comprises:an N-l bioprocess container, an N-l impeller shaft, an N-l equivalent diameter of the N-l bioprocess container, and an N-l sparger comprising N-l sparger holes and sparger diameter; andwherein the production scale bioreactor further comprises:a production scale bioprocess container, a production scale impeller shaft, a production scale equivalent diameter of the production scale bioprocess container, and a production scale sparger comprising production scale sparger holes and sparger diameter.

25. The seed train process recited in claim 24, further wherein the first and second sets of equipment parameters comprise an equipment parameter 1, an equipment parameter 2, an equipment parameter 3, an equipment parameter 4 or an equipment parameter 5.

26. The seed train process recited in claim 25, wherein the equipment parameter 1 is a diameter of the N-l or production scale impeller shaft divided by an equivalent diameter of the N-l or production scale bioprocess container, the equipment parameter 2 is the diameter of the N-l or production scaleDocket No. TP389720WO1impeller shaft divided by a height of liquid in the N-l or production scale bioprocess container; the equipment parameter 3 is a number of sparger holes of the N-l or production scale sparger divided by a surface area of the N-l or production scale sparger; the equipment parameter 4 is the number of sparger holes of the N-l or production scale sparger divided by a diameter of the of the N-l or production scale sparger; and the equipment parameter 5 is a sparger hole diameter of the N-l or production scale sparger divided by a diameter of the of the N-l or production scale sparger.

27. The seed train system recited in claim 19. wherein the equipment parameter 1 is a turn-down ratio, or a ratio of maximum working volume to minimum working volume of the N-l or the production scale bioreactor / bioprocess container.

28. The seed train system recited in claim 27, wherein the turn-down ratio is 5:1.

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