Bioreactor Systems

Abstract

The present disclosure relates to a large-scale bioreactor system comprising a vessel with an internal reaction chamber having a volume and cell-supporting capacity that significantly exceeds the volume and cell-supporting capacity of currently available bioreactor systems.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 339,124, filed May 6, 2022, and U.S. Patent Application No. 63 / 342,379, filed May 16, 2022, each of which is incorporated by reference in its entirety into this disclosure for any purpose.

[0002] The present disclosure relates to a large-scale bioreactor system comprising a vessel with an internal reaction chamber having a volume and cell-supporting capacity that significantly exceeds the volume and cell-supporting capacity of currently available bioreactor systems. [Background technology]

[0003] Currently available bioreactor systems are limited in both size (e.g., volume) and cell maintenance capacity. For example, those skilled in the art recognize that currently available bioreactors cannot support high volume, high density non-bacterial cell cultures. The present disclosure provides solutions to these problems. For example, in some embodiments, the present disclosure provides bioreactor systems that include vessels with internal reaction chambers of at least about 125,000 L to 315,000 L and viable cell densities (e.g., greater than 50 million cells per milliliter). Thus, the present disclosure provides solutions to these and other art-recognized and unrecognized problems. Summary of the Invention

[0004] The present disclosure relates to a large-scale bioreactor system comprising a vessel (e.g., a bioreactor vessel) with an internal reaction chamber having a volume and cell-sustaining capacity significantly exceeding the volume and cell-sustaining capacity of currently available bioreactors. In preferred embodiments, the present disclosure provides aspects 1-15 described below. In preferred embodiments, the bioreactor vessel comprises an internal reaction chamber configured to accommodate at least about 125,000 liters (L), at least about 250,000 L, or at least about 315,000 L of a reaction mixture including cells, liquid, and gas. In preferred embodiments, the bioreactor vessel has a liquid depth (LD) to bioreactor vessel diameter (D) ratio of 1.5 to 3, optionally about 2.4, and in an optionally preferred embodiment about 2.36. Other embodiments are disclosed herein as will be appreciated by those of skill in the art. [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 illustrates a first exemplary bioreactor train. [Diagram 2] FIG. 1 shows a second exemplary bioreactor train. [Diagram 3] FIG. 2 illustrates a first exemplary impeller. [Figure 4] FIG. 1 illustrates a second exemplary impeller. [Diagram 5] FIG. 13 illustrates a third exemplary impeller. [Figure 6] FIG. 13 illustrates a fourth exemplary impeller. [Figure 7A] FIG. 1 illustrates an exemplary sparger design. [Figure 7B] FIG. 1 illustrates an exemplary sparger design showing the openings through which gas traverses from the supply to the interior chamber of the bioreactor vessel. [Figure 8] FIG. 1 illustrates an exemplary bioreactor vessel and associated components (bioreactor system). [Figure 9] FIG. 1 illustrates an exemplary manufacturing process. [Figure 10]FIG. 1 illustrates an exemplary bioreactor system with automation. [Figure 11] FIG. 1 illustrates an exemplary bioreactor system with automation. [Figure 12] FIG. 1 illustrates an exemplary bioreactor system with automation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The present disclosure relates to a bioreactor system comprising at least one vessel (e.g., bioreactor vessel) comprising an internal reaction chamber having a reaction mixture volume or volume of at least about 30,000 liters (L), at least about 50,000 L, at least about 75,000 L, at least about 100,000 L, at least about 125,000 L, at least about 250,000 L, or at least about 315,000 L, preferably at least about 125,000 L to 315,000 L, and even more preferably at least about 250,000 L.

[0007] In some embodiments, the vessel may have a configuration and / or be used in a system disclosed in, for example, but not limited to, US Pat. No. 8,658,419 (ABEC Corp.), US Pat. No. 9,228,165 (B2) (ABEC Corp.), US Pat. No. 1,051,9415 (B2), and / or WO 2019 / 070648 (A2). In some embodiments, the disposable feed vessel may be a conical or tulip bottom vessel and / or preferably has a volume of at least 20 L. Other types of suitable feed vessels that may be used as disclosed herein are also known in the art, as will be understood by those skilled in the art. The materials used to manufacture the apparatus described herein may be of the same composition or different compositions. The reaction vessels and / or heat exchange components described herein are typically, but not necessarily, made from corrosion-resistant alloys (e.g., metals). For example, suitable materials may include, but are not limited to, dimple jacket materials and / or sheet / plate materials. Suitable materials include, for example, carbon steel, stainless steel (e.g., 304, 304L, 316, 316L, 317, 317L, AL6XN), aluminum, Inconel® (e.g., Inconel 625, Chronin 625, Altemp 625, Haynes 625, Nickelvac 625, and Nicrofer 6020), Incoloy®, Hastelloy (e.g., A, B, B2, B3, B142T, Hybrid-BC1, C, C4, C22, C22HS, C2000, C263, C276, D, G, G2, G3, G30, G50, H9M, N, R235, S, W, X), and Monel®, titanium, Carpenter 20®, and the like. However, it will be understood that other materials other than or in addition to corrosion-resistant alloys may be suitable, such as, but not limited to, plastics, rubbers, and mixtures of such materials.The "mixture" of materials may refer to either the actual mixture itself to form a composite material, or the use of various materials within the system (e.g., alloy reactor shell and rubber baffle components). With regard to the channel-forming materials mentioned above, any of the suitable materials listed above may be prepared to form channels through which the heat transfer medium may be distributed.

[0008] The reaction vessel comprises an internal chamber and, in preferred embodiments, is associated with and / or at least includes a heat transfer system comprising a heat transfer device for controlling the temperature of a chemical, pharmaceutical, or biological process taking place within the internal reaction chamber of the vessel. In some embodiments, the heat transfer system provides for the distribution of a heat transfer medium such that heat resulting from or required by the process is transferred from or to the reaction mixture. In some embodiments, the reaction vessel comprises a jacket and / or a jacketed tank head (e.g., a dimpled jacket) forming a fluid channel through which a heat transfer fluid can be circulated. In some embodiments, the reaction vessel may be at least partially surrounded by a fluid channel. The jacketed tank head may also function as a lid for the reaction vessel. The jacketed tank head may further serve to support and / or relieve pressure on the DC (e.g., the top of the DC) contained within the reactor vessel.

[0009] In some embodiments, the one or more heat exchange systems may include a jacket through which a heat transfer fluid is circulated. The jacket may include, for example, channels through which the heat transfer fluid is circulated. In some embodiments, the jacket may be a "dimpled" material. A dimpled jacket may typically be installed around a reaction vessel, such as a fermentation tank, and used as part of a heat transfer system. The dimpled jacket material may be used in the apparatus described herein in a typical manner (e.g., wrapped around a reaction vessel). In certain embodiments described herein, the dimpled jacket material may additionally or alternatively be used within a baffle structure. Dimpled jacket materials are commercially available, and any such material may be suitable for use as disclosed herein. Typically, the dimpled jacket material has a substantially uniform pattern of dimples (e.g., recesses, depressions) pressed or molded into a base material (e.g., a metal sheet). The dimpled jacket material may be made, for example, mechanically ("mechanical dimpled jacket") or by expansion (e.g., expansion-resistance spot welding (RSW)). To prepare a mechanical dimple material, a metal sheet having a stamped substantially uniform array of dimples (each dimple typically includes a central hole) is welded to a base metal through the central hole. Expanded RSW dimple material (e.g., expanded HTS or HTS) is typically made by resistance spot welding an array of spots on a thin sheet of metal to a more robust (e.g., thicker) substrate (e.g., metal). The edges of the composite material are sealed by welding, and the interior is expanded under high pressure until the thin material forms a pattern of dimples. Mechanical dimple materials, when used as jackets, typically have high pressure ratings and low to moderate pressure loss, while RWS dimple jackets typically exhibit moderate pressure ratings and high to moderate pressure loss. A heat transfer fluid typically flows between the sheets of dimple material. Other suitable dimple materials are available to those skilled in the art and are suitable for use as described herein.

[0010] In a preferred embodiment, the reaction vessel disclosed herein includes one or more heat transfer systems that efficiently transfer heat, withstand the liquid pressure encountered in the reaction vessel, and can be easily and efficiently sanitized. The suitable heat transfer baffles described herein can be incorporated into the heat transfer system to solve these problems. A preferred exemplary heat transfer baffle is disclosed and / or claimed in U.S. Pat. No. 8,658,419 (B2), the contents of which are incorporated herein in their entirety, as is the disclosure shown in FIGS. 16-18. In certain embodiments, the baffle has at least one internal channel (e.g., 9 in FIGS. 16-18) and at least two external channels (e.g., 10 in FIGS. 16-18). Typically, the heat transfer medium is circulated through one or more distribution channels (e.g., 9 in FIGS. 16-18), but not through one or more relief channels (e.g., 10 in FIGS. 16-18), which may also function as vent(s) for the distribution channels. The distribution channel 9 is typically formed between the support material 11 and the dimple jacket material 12 of each subassembly. The relief channel(s) 10 are typically formed by joining two subassemblies, each of which comprises a support material 11 fixably attached to the dimple jacket material 12. In such an embodiment, the dimple jacket material and the support material of each subassembly are typically joined to each other by welding or other processes, such that the materials are fixably attached to each other. The subassemblies are typically joined to each other using a closure bar 13. The closure bar is typically joined to the support material by welding or other processes that result in a substantially seamless joint. The width of the closure bar can be adjusted to set the width of the relief channel as desired (e.g., to set the juxtaposed dimple jacket materials closer together or further apart). One or more relief holes can be made in the closure bar to allow the relief channel(s) to communicate with the outside of the reaction vessel.The incorporation of distribution and relief channels into the baffles provides excellent heat transfer capabilities and the structural integrity required to withstand the liquid forces encountered in the reaction vessel. The baffles may protrude from the inner wall of the reaction vessel at regular or irregular intervals. The baffles may also be installed at any suitable angle to the inner wall of the reaction vessel (e.g., 60° to the inner wall, 30° to the radius of the reaction vessel). A suitable angle may be an angle understood by those skilled in the art to be suitable or sufficient to attenuate forces encountered by the baffles (e.g., liquid forces) resulting from motion (e.g., rotational and / or vortex motion) of the vessel contents resulting from agitation (e.g., mechanical or other) of the vessel contents. A suitable angle is an angle that prevents damage to the baffles from forces resulting from such motion. Suitable angles include, for example, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, or 90° relative to either the inner wall of the vessel or the radius of the vessel. If the reaction vessel contains a mechanism (e.g., mechanical or other mechanism) for stirring or mixing the reaction, such as a set of rotating blades (e.g., axial or radial flow impellers), the baffle is fixed to or protrudes from the inner wall such that the mechanism and the baffle do not contact each other. For example, if one or more devices for mixing the reaction components are located at the bottom center of the vessel, the baffle can be installed above the top of the means. If multiple mechanical mechanisms are utilized, the baffle is typically configured to avoid those mechanisms. For example, where the mechanism includes one or more sets of rotating blades, the baffle(s) may be positioned above the blades, below the blades, between the blades, or beside the blades. The baffle design ensures sufficient clearance from the mechanical mechanism. The baffle assembly is fixably attached to the vessel via one or more attachment arms 7, typically by welding or other process that results in a substantially seamless joint.As discussed above, the use of the attachment arm advantageously allows for efficient cleaning and / or sanitization of the baffle in that little or no residue remains at the joint between the inner surface of the reaction vessel and the baffle after the attachment process (e.g., welding). Similarly, the baffle may be assembled, attached, or secured to the reaction vessel by any suitable method that creates a substantially seamless attachment point (e.g., a seamless joint or boundary between materials) to form a surface that can be easily and efficiently sanitized. A "substantially seamless attachment point," "seamless joint," or "tight joint" typically indicates that the boundary between the baffle and the reaction vessel is substantially undetectable by either visual means and / or other means (e.g., a microscope). It may also indicate that no residue from a previous reaction remains at the boundary after standard cleaning procedures typically used by those skilled in the art to "sanitize" such equipment. Thus, the system is suitable for sanitization using industry accepted "clean-in-place" and "sterilize-in-place" systems using any suitable cleaning agent, including but not limited to detergents, brushes, and / or steam. Such a boundary allows for simple and efficient disinfection. In some preferred embodiments, the container of the present disclosure may include any suitable number of baffles, preferably 1 to 10, more preferably 4 to 8, and most preferably 8 baffles. In some preferred embodiments, the container does not include a baffle.

[0011] In preferred embodiments, the optimal heat transfer surface area utilized in a particular system may be determined based on an estimate of the cell culture metabolic load based on oxygen uptake rate (OUR) and any mechanical contribution provided by the agitator (e.g., at a volumetric power output of 2 HP / kGal). In preferred embodiments, the heat transfer surface area is formed on the sidewalls of the bioreactor vessel at least up to the maximum working volume and working head. In preferred embodiments, the heat transfer fluid travels through parallel flow paths along the sidewalls and through different heat transfer zones. In some embodiments, an additional heat exchange system may be applied to the harvest line, for example, if low temperatures are required during harvesting of cells from the reaction mixture.

[0012] The heat transfer systems described herein may be made of any material through which a heat transfer fluid (e.g., gas and / or liquid, such as preferably cold water (e.g., 10°C-12°C depending on the application) can be transported (e.g., from the heat transfer system into the internal reaction chamber) such that heat can be conducted to and / or absorbed from another portion of the system by radiation, convection, conduction, or direct contact. Suitable heat transfer media include, but are not limited to, fluids and gases. Suitable fluids and gases include, but are not limited to, steam (top to bottom), hot and cold water, glycol, heat transfer oil, refrigerant, or other pumping fluids having the desired operating temperature range. It is also possible to use multiple types of heat transfer media, for example, such that one type of media is directed to one area of ​​the reaction vessel and another type of media is directed to a different area of ​​the reaction vessel (e.g., as in the zone system described above). Mixtures of heat transfer media (e.g., 30% glycol) may also be desirable.

[0013] In preferred embodiments, the reaction mixture comprises cells, preferably non-bacterial cells, more preferably mammalian, fish, avian, and / or insect cells, at a cell density of about 20 million to about 100 million cells per milliliter, preferably at least about 35 million to 50 million cells per milliliter. For example, in some embodiments, the bioreactor vessel of the present disclosure is configured to support the parameters set forth in Table 1A, Table 1B (preferred embodiments) and / or Table 2A and Table 2B (preferred embodiments). [Table 1] [Table 2] [Table 3] [Table 4]

[0014] A typical bioreactor has an aspect ratio, defined herein as the liquid depth (LD) to bioreactor vessel diameter (D) ratio of about 1.0 to 1.5, preferably 1.25 to 1.5, or about 2 for larger scale bioreactors. Bioreactors disclosed herein have aspect ratios of about 1.5 to about 3.0, with a preferred aspect ratio of about 2.4 (in some preferred embodiments, about 2.36). These aspect ratios, particularly aspect ratios of about 2.36 or greater, support processes exhibiting the parameters set forth in Tables 1 and 2 (e.g., about 35 million cells / mL, reactor volume of about 2,250,000 L). The systems disclosed herein are further configured to be manufactured in one location (e.g., shipped as needed), adjustable depending on the particular cells being grown, maintain sterility, work with commercially available filters, and the like. In some preferred embodiments, bioreactors of the present disclosure may include the dimensions and parameters set forth in Table 3. [Table 5]

[0015] In some embodiments, a "seed train" technique is used to feed an initial volume of cells into a bioreactor. This "seed train" is typically composed of several smaller bioreactors that allow the cell volume to fully expand to the desired density as the bioreactor contents are transferred to subsequent bioreactors. All or a portion of the bioreactor contents may be transferred to the subsequent bioreactors. For example, exemplary bioreactor trains are shown in Figures 1 and 2, respectively.

[0016] The bioreactor vessels disclosed herein are typically, though not necessarily, constructed of metal and usually, though not necessarily, made of a corrosion resistant alloy. For example, suitable materials can include, but are not limited to, sheet / plate materials (and / or dimpled jacket materials, e.g., for heat transfer systems). Suitable exemplary materials include, for example, carbon steel, stainless steel (e.g., 304, 304L, 316, 316L, 317, 317L, AL6XN), aluminum, Inconel® (e.g., Inconel 625, Chronin 625, Altemp 625, Haynes 625, Nickelvac 625, and Nicrofer 6020), Incoloy®, Hastelloy (e.g., A, B, B2, B3, B142T, Hybrid-BC1, C, C4, C22, C22HS, C2000, C263, C276, D, G, G2, G3, G30, G50, H9M, N, R235, S, W, X), and Monel®, titanium, Carpenter 20®, and the like. However, it is understood that other materials other than or in addition to the corrosion-resistant alloys may be suitable, such as, but not limited to, plastics, rubbers, and mixtures of such materials. A "mixture" of materials may refer to either the actual mixture itself or the use of various materials within a system (e.g., alloy reactor shell and rubber baffle components) to form a composite material.

[0017] The reaction mixture typically includes a liquid cell culture medium suitable for maintaining the viability and growth of the cells of interest. As mentioned above, the bioreactor vessels disclosed herein may be adapted for the growth of various types of cells, including, but not limited to, mammalian, fish, avian, and / or insect cells. Exemplary cell culture media are any of those typically used to culture such cells, modified as necessary (e.g., to densities as disclosed herein) to allow viability and growth within the bioreactor vessel. Typically, the cell culture medium and any other liquids introduced into the bioreactor vessel during the cell growth / expansion process are sterile. The cell growth process can take different forms, such as batch (i.e., the entire volume of medium is introduced all at once), fed-batch (media and nutrients are added throughout the growth time) or process intensification forms (i.e., media / nutrients are exchanged using external devices such as filters to allow higher cell densities). As will be appreciated by those skilled in the art, other processes may also be appropriate. After reaction (e.g., growth to 50 million cells / mL), harvesting of cells is typically done directly from the bioreactor vessel to minimize risk of contamination. In some embodiments, after reaction (e.g., growth to 50 million cells / mL), harvesting of cells may be done directly from the bioreactor vessel to a sterile transfer line(s) to minimize risk of contamination. In some embodiments, the entire contents of the bioreactor vessel can be harvested at the same time, or in some embodiments, only a portion can be harvested and then additional medium can be introduced into the system to continue cell growth in a method known as draw and fill.

[0018] In some embodiments, the system may include plumbing (e.g., tubing) that can be cleaned and sterilized independently from other sections of the system so that the bioreactor can aseptically accept liquid additions to the product stream. Exemplary liquid additives may be, for example, cell culture medium or individual components thereof, cell culture fluid for inoculation, basic or acidic solutions to control pH, glucose or another sugar for cell growth, antifoam agents, etc. In some embodiments, each of these liquid additives may be supplied from a previous bioreactor, such as a series of bioreactors, other reservoirs of appropriate size for the reaction, and / or a header system that can feed multiple of these bioreactors.

[0019] Cleaning of the internal reaction chamber (i.e., production reactor) may be accomplished through a cleaning-in-place (CIP) skid that performs associated acid and caustic cleaning and clean water rinses through spray balls and spray tubes within the vessel. Table 4 shows the total flow rates based on three different types of spray balls: static spray ball, single-shaft dynamic spray ball, and multi-shaft dynamic spray ball. In a preferred embodiment, the flow rates are based on ASME BPE flow rate guidelines. "Empty" sterilization utilizing clean steam (i.e., steam prepared from a purified water source) or cooking grade steam may also be used to clean the internal reaction chamber (i.e., production reactor), as known in the art. Time and temperature may be adjusted to meet the desired sterilization requirements. An exemplary preferred sterilization may include heating an empty vessel and its sterilization boundary to 125°C and holding this temperature for 30 minutes. The steam is replaced with clean air and the vessel is cooled to allow for the addition of media. Time and temperature may be adjusted to meet the user's desired sterilization requirements. [Table 6]

[0020] Adequate mixing and gas dispersion is important to maintain optimal cell viability and growth in the bioreactor vessel (i.e., within the internal reaction chamber). Such mixing and dispersion is typically accomplished using an agitation system comprising an agitator comprising one or more impellers, preferably a low shear impeller, in some preferred embodiments a hydrofoil impeller, or in some preferred embodiments a Rushton impeller. Table 5 shows exemplary agitator sizes for the bioreactor vessels disclosed herein. While specific impellers are listed in Table 10, it should be understood that any suitable impeller(s) may be used. In a preferred embodiment, the agitator comprises two to about six impellers, preferably four impellers, to provide adequate mixing throughout the reaction mixture. Additionally, the number of heat transfer baffles may range from zero to eight (or more, as appropriate), with four baffles being a preferred embodiment. [Table 7]

[0021] From these exemplary designs and preferred ranges of OTR of about 5 to about 40 mmol / L / hr (about 20 mmol / L / hr in some preferred embodiments (see Tables 6-7)) and baseline (see Tables 2A and 2B (20 mmol / L / hr and 1 sHP / kGal)) of about 0.5 to about 3.0 sHP / kGal (about 1 sHP / kGal in some preferred embodiments (see Tables 6-7)). Gas generation rates using both air and supplemental oxygen air may be utilized. Tables 6 and 7 show the output of these exemplary designs. In embodiments where the gas flow includes supplemental oxygen (Table 6), the total flow rate is on the order of 0.1 vvm. It is known that the air only flow (Table 7) will correspondingly increase based on the mole fraction of oxygen delivered. Considering the higher aspect ratio, in some embodiments, an agitator designed around a hydrofoil impeller is preferred. The low shear impeller in the bioreactor vessel agitator used to obtain the data in Table 7 was replaced with a hydrofoil type impeller and mass transfer correlations were derived therefrom as shown in Table 8 (supplemental oxygen) and Table 9 (air only). [Table 8] [Table 9] [Table 10] [Table 11]

[0022] Suitable impellers can be any available to one of skill in the art. Exemplary hydrofoil impeller designs that may be used with the bioreactor vessels disclosed herein are shown in Figures 3-6. The properties of the impellers shown in Figures 3-4 are listed in Table 10, and the properties of the impellers shown in Figures 5-6 are listed in Table 11. [Table 12] [Table 13]

[0023] Manufacturability is another important determinant of the feasibility of the agitator at these scales. Given the size of the exemplary impellers shown in Figures 3-6, a one-piece construction as desired in a sterile environment may pose manufacturing challenges (e.g., in-tank couplings for shaft handling and manufacturing would be required). In some embodiments, the individual impeller blades may be bolted to a welded hub for handling. All connections are preferably, but not necessarily, of sanitary design as outlined in ASME BPE, Appendix 10.3. Fixed bearings may also be used to handle shaft flexure, and may be used at the bottom of the bioreactor vessel (e.g., in embodiments where the agitator gearbox is mounted to the top of the bioreactor vessel). In some embodiments, the agitator may be sealed to the bioreactor vessel by a cartridge-type mechanical seal. In some embodiments, a pressurized lubricated seal utilizing clean steam condensate as a lubricant may be utilized. In some embodiments, a single dry running seal with a sanitary gland may be utilized.

[0024] In some embodiments, the reactor system of the present disclosure may include at least two spargers, each of which includes a fluid channel and at least one section with a plurality of through-holes through which at least one component is introduced into the reaction mixture through the bottom section of the internal reaction chamber, and optionally the sections with the plurality of through-holes together form a circular, elliptical, hexagonal, square, rectangular or other shaped structure (see preferred embodiments shown in Figures 7A and 7B, which respectively show the orientation of the sparger positioned at the bottom of the bioreactor vessel and the spacing of the holes represented as points through which the gas passes). In some preferred embodiments, a single sparger may be included (e.g., having a circular, elliptical, hexagonal, square, rectangular or other shape). In preferred embodiments, the spacing of the through-holes in the sparger(s) decreases (e.g., there are more through-holes and they are closer together) as the sparger structure extends away from the end of the source of gas entering the sparger.

[0025] In some embodiments, the fluid channels used as supply lines to the bioreactor vessel (e.g., its internal reaction chamber) may conform to typical carbon steel or stainless steel piping specifications and are preferably sanitary (especially those with direct product contact, connection of other vessels supplying sterile fluids to the bioreactor vessel, gas supply, gas exhaust, CIP distribution and clean steam distribution). Other utilities such as plant steam and chilled water are not considered to be of sanitary design. In preferred embodiments, the line sizing for the sanitary lines allows for the use of ASME BPE tubing, which is the standard for sanitary applications in the biopharmaceutical industry. Diaphragm valves may be used within the sterile boundary of the bioreactor vessel. Other valve types such as sanitary butterfly valves, ball valves and anti-mixing valves may be used in structures within the sterile boundary to guide other fluids if sanitary structures are desired. In preferred embodiments, the material of these lines is 316L stainless steel, in accordance with typical biopharmaceutical industry applications. The surface finish of the bioreactor vessel, especially the internal reaction chamber, preferably meets a minimum mechanical polish of 30μ inch Ra. Suitable surfaces may have a lower Ra (i.e., <30μ inches) when combined with techniques such as electropolishing. Any elastomers in the sanitary lines are preferably USP / FDA compliant (e.g., EPDM and / or platinum cured silicone). In a preferred embodiment, the bioreactor vessel may have a shell of 316L stainless steel with mechanical polishing that meets a minimum of 30μ inches Ra (e.g., unless combined with electropolishing), and the jacket material may be 304L stainless steel and may be insulated using an insulating exterior material. In some embodiments, alternative duplex stainless steels (e.g., UNS S32205) may be used to fabricate the bioreactor vessel. Depending on the final pressure and load rating of the vessel, such alternatives may reduce costs by reducing thickness. In a preferred embodiment, ports for analytical probes such as dissolved oxygen, pH, and pCO2 may be included along the sidewall of the bioreactor vessel.

[0026] In preferred embodiments, the systems described herein may further include one or more manual and / or automatic control systems (i.e., not requiring continuous or constant direct human intervention), including, but not limited to, one or more remotely controlled control systems. For example, the control system may continuously monitor one or more conditions occurring within any of the components of the system, and preferably between at least any two components of the system. Such control systems typically comprise one or more general-purpose computers that include software for processing such information and manually or automatically adjusting desired parameters of the reactions required for a particular process. Thus, in some preferred embodiments, the control system is automated (e.g., using software). In some preferred embodiments, the systems described herein may include one or more automation systems for controlling and monitoring process conditions and process sequencing. In some preferred embodiments, the automation system includes hardware (automation system hardware), including, but not limited to, commercially available programmable logic controllers (PLCs), distributed control systems (DCSs), and / or one or more human machine interfaces (HMIs). In preferred embodiments, the automation system hardware is programmed to control and monitor process conditions and process sequencing. Process control monitoring parameters that may be controlled by such manual and / or preferably automated systems include, but are not limited to, dissolved oxygen, pCO2, temperature, liquid level, foam detection / control, gas generation / mass flow, headspace pressure, pH, agitator speed, viable cell density, exhaust gas analysis, and spectroscopic methods including ultraviolet (UV) and Raman, which may incorporate specific control algorithms such as exponential feeding. Large-scale bioreactor process sequences that may be controlled may include clean-in-place (CIP), sterilize-in-place (SIP), pressure hold testing, vessel filling, cell growth, reagent addition and / or cell harvest processes.Process control monitoring may further include integration / interfacing with external process systems that feed or service the large-scale bioreactor, including reagent addition tanks, CIP systems, SIP systems, liquid sterilization systems, and recovery systems. Process control, monitoring, and sequencing data may be collected and stored as batch records. An exemplary automated control system is shown in Figures 10-12. Such a system preferably includes redundant Ethernet-based plant control network connections to two redundant network switches (not shown) in each remote I / O panel, and redundant Ethernet connections from the switches to the HMI and to the Ethernet I / O (not shown) in the remote I / O panels (Figures 10-12, A, B (Ethernet)).

[0027] An exemplary bioreactor system including an automation system is shown in Figures 10-12. As shown in Figure 10, certain preferred embodiments of a bioreactor system and / or subsystems of a bioreactor system, with its various subsystems, may include a medium preparation subsystem ("Medium Prep") connected to a nutrient preparation subsystem ("Nutrient Prep"), and a large scale medium preparation subsystem ("Large Scale Media Prep") that may include vessels of the same or different sizes, one or more clean-in-place skids, connected by a redundant plant control network. As shown in Figure 11, certain preferred embodiments of a bioreactor system and / or subsystems of a bioreactor system, with its various subsystems, may include a large scale medium preparation subsystem ("Large Scale Media Prep") that may include vessels of the same or different sizes, one or more clean-in-place skids, connected by a redundant plant control network. As shown in Figure 12, certain preferred embodiments of a bioreactor system and / or subsystems of a bioreactor system may include one or more cell culture trains and / or nutrient vessels (e.g., glucose storage), and / or a large bioreactor cell CIP system (Figure 12) that may include vessels of the same or different sizes, one or more clean-in-place skids, with their various subsystems connected by a redundant plant control network. Preferred embodiments of a bioreactor system include feed lines, return lines, etc. In some embodiments, certain preferred bioreactor systems may include multiple bioreactor vessels fluidly connected in series, e.g., 25,000 L, 32,000 L, 40,000 L, 50,000 L, 125,000 L, and / or 250,000 L.In certain preferred embodiments, the various vessels, subsystems, and / or bioreactor systems include at least two trains (e.g., two, three, four, or five) of vessels fluidly connected in series, with vessels being 500 L, 2,000 L, 8,000 L, 32,000 L, 125,000 L, and 250,000 L (in preferred embodiments, each train includes at least one 125,000 L vessel feeding two 250,000 L vessels), and may also be fluidly connected to a glucose storage vessel (e.g., 30,000 L). As will be appreciated by those skilled in the art, other embodiments of the control system may also be used.

[0028] Thus, the present disclosure provides the following preferred aspects and embodiments. (1) A bioreactor system, (a) A container having an internal chamber, a. configured to contain at least about 30,000 liters (L), at least about 50,000 L, at least about 75,000 L, at least about 100,000 L, at least about 125,000 L, at least about 250,000 L, or at least about 315,000 L of a reaction mixture comprising cells, liquid, and / or gas; b. a liquid depth (LD) to bioreactor vessel diameter (D) ratio of 1.5 to 3, and optionally about 2.4 (e.g., about 2.36 in some preferred embodiments); c. a vessel comprising an interior chamber having a top section and a bottom section, at least the bottom section being in contact with the reaction mixture; (b) at least one heat transfer system at least partially surrounding at least one region of the inner reaction chamber and configured to maintain a reaction mixture within said region at a preselected temperature; (c) at least one fluid channel (sparger) supplying at least one component of the reaction mixture, the at least one component being selected from the group consisting of air, oxygen, carbon dioxide (CO2), and / or nitrogen through a bottom section; (d) at least one fluid channel supplying air to an upper section of the inner reaction chamber; (e) at least one agitator for mixing the reaction mixture, the at least one agitator comprising a plurality of low shear impellers, optionally hydrofoil impellers or Rushton impellers; (f) at least one fluid channel for removing exhaust from an upper section of the inner reaction chamber; (g) at least one cleaning and / or sterilization system for cleaning and / or sterilizing the inner reaction chamber, the at least one cleaning and / or sterilization system being fluidly connected to the upper section of the inner reaction chamber; A bioreactor system comprising: (2) A bioreactor system of aspect 1 comprising at least two spargers, each of which comprises a fluid channel and at least one section comprising a plurality of through-holes through which at least one component is introduced into the reaction mixture through a bottom section of the internal reaction chamber, optionally the sections comprising the plurality of through-holes together forming a circular, elliptical, hexagonal, square, rectangular or other shaped structure (see preferred embodiment depicted in FIG. 7A and FIG. 7B, which respectively show the orientation of the sparger positioned at the bottom of the bioreactor vessel and the spacing of the holes represented as points through which gas passes). In some preferred embodiments, a single sparger may be included (e.g., having a circular, elliptical, hexagonal, square, rectangular or other shape). In preferred embodiments, the spacing of the through-holes in the sparger(s) decreases (e.g., there are more through-holes and they are closer together) as the sparger structure extends away from the end of the source of gas entering the sparger. (3) The bioreactor system of any of the above aspects, comprising a single agitator with multiple impellers, optionally four impellers, further optionally, the impellers are hydrofoil impellers or Rushton impellers. (4) The bioreactor system of any of the preceding aspects, wherein the inner reaction chamber is configured to accommodate at least about 125,000 liters (L), at least about 250,000 L, or at least about 315,000 L of a reaction mixture comprising cells, liquid, and gas. (5) The bioreactor system of any of the above aspects, wherein the reaction mixture comprises cells at a density of about 20 million to about 100 million cells per milliliter, optionally about 50 million cells per milliliter. (6) The bioreactor system of any of the above aspects, wherein at least one fluid channel (sparger) supplies oxygen into the reaction mixture at a transfer rate of at least about 20 mmol / L / hr. (7) The bioreactor system of any of the preceding aspects, wherein the heat transfer system includes a heat transfer fluid having a temperature of at least about 10° C. to 12° C. (e.g., higher during a control phase), and optionally, the heat transfer fluid is water. (8) The bioreactor system of any of the above aspects, wherein the heat transfer system comprises a dimpled jacket. (9) The bioreactor system of any of the preceding aspects, wherein the at least one cleaning and / or sterilization system adds a cleaning fluid (optionally, an acid) to the interior of the inner reaction chamber, and further optionally, the cleaning system comprises at least one spray ball and / or spray tube. (10) The bioreactor system of any of the above aspects, wherein the reaction mixture is generated from a series of seed trains in which the volume of the reaction mixture is incrementally increased, optionally starting from a volume of at least about 250 L. However, in some embodiments, the series of seed trains can begin at a much lower level, such as at vial level (e.g., 25 mL). (11) The bioreactor system of any of the preceding aspects, wherein the reaction mixture is maintained by perfusion. (12) The bioreactor system of any of the preceding aspects, wherein foaming of the reaction mixture, if present, is controlled using a chemical antifoam agent and / or a mechanical antifoam system. (13) The bioreactor system of any of the preceding aspects, wherein the liquid in the reaction mixture comprises a cell culture medium. (14) An exemplary bioreactor system comprising the components depicted in Figure 8. This exemplary bioreactor system comprises an internal reaction chamber configured to contain at least about 30,000 liters (L), at least about 50,000 L, at least about 75,000 L, at least about 100,000 L, at least about 125,000 L, at least about 250,000 L, or at least about 315,000 L of a reaction mixture including cells, liquid, and / or gases, a vessel comprising a plurality of impellers connected to a shaft connected to an agitator motor, fluid channels connected to a sterile filter and at least one sparger fluidly connected to a gas flow controller and fluid channels through which air, oxygen, carbon dioxide, and / or nitrogen flow into the sparger (along with a source / vessel supplying them), and a heat transfer fluid to cool the reaction mixture ("liquid volume"). at least one heat transfer system comprising a jacket through which the reaction mixture flows, at least one pump, at least one source of heat transfer fluid and / or a heat ("steam") or cooling source through which the heat transfer fluid flows; a section (e.g., headspace) of the vessel above the reaction mixture (liquid volume) which may comprise at least one air source and fluid channels through which air is introduced into the headspace; at least one spray ball(s) or other structure capable of directing a cleaning fluid, steam, or other gas (and a source thereof) into the empty vessel to clean the vessel after the reaction has ended and the vessel has been emptied; and an exhaust system comprising at least one sterilizing filter, at least one pressure control valve, and which exhausts the gas (e.g., wet gas) to the environment. (15) A method for producing a bioreactor system according to any one of the above aspects, comprising the steps of: (a) modifying a structural shell comprising at least one section of a vessel with a heat transfer system, optionally a dimple jacket, stiffening rings, and / or fixtures, to produce a modified structural shell; (b) seam welding a plurality of modified structural shells to connect them to one another, thereby forming seams at interfaces between the modified structural shells, and grinding the seams; (c) insulating, coating, painting, and / or installing the exterior cladding to the modified structural shell connected in step (b); (d) transporting the products of steps (a), (b) and / or (c) using at least one crane and / or truck or rail (see, e.g., FIG. 9 ); A manufacturing method comprising: (16) The bioreactor system and / or method of any of the preceding aspects, wherein the bioreactor system comprises and / or is operably connected to an automatic control system.

[0029] As will be appreciated by one of ordinary skill in the art, other embodiments, aspects, and advantages of the system and methods of using same are provided herein.

[0030] The term "about, approximately, etc.," when preceding a list of numerical values ​​or ranges, applies independently to each individual value in the list or range as if each individual value in the list or range were immediately following the term. These terms mean that the value they refer to is exactly, close to, or similar to that value. "Optionally or optionally" means that the event or circumstance described thereafter may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur. Ranges may be expressed herein as from about one particular value and / or to about another particular value. When such ranges are expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations by using the antecedent "about" or "approximately," it will be understood that the particular value forms another embodiment. It will further be understood that each endpoint of a range is significant both in relation to the other endpoint and independently of the other endpoint. Ranges (eg, 90-100%) are meant to include the range itself, as well as each individual value within the range, as if each value were individually recited.

[0031] All references cited in this disclosure are incorporated herein by reference in their entirety.Specific embodiments are further described in the following examples.These embodiments are presented merely as examples and are not intended to limit the scope of claims in any way.Although specific embodiments are described in terms of preferred embodiments, it is understood that variations and modifications may occur to those skilled in the art.Therefore, it is intended that the appended claims encompass all such equivalent variations that fall within the scope of the following claims.

Claims

1. A bioreactor system, (a) a vessel having an internal reaction chamber, configured to contain at least about 30,000 liters (L), at least about 50,000 L, at least about 75,000 L, at least about 100,000 L, at least about 125,000 L, at least about 250,000 L, or at least about 315,000 L of a reaction mixture comprising cells, liquid, and / or gas; a liquid depth (LD) to bioreactor vessel diameter (D) ratio of 1.5 to 3, optionally about 2.4, optionally about 2.36; a vessel comprising an internal chamber having a top section and a bottom section, at least the bottom section being in contact with the reaction mixture; (b) at least one heat transfer system at least partially surrounding at least one region of the inner reaction chamber and configured to maintain the reaction mixture within said region at a preselected temperature; (c) at least one fluid channel (sparger) for supplying at least one component of the reaction mixture, said at least one component being air, oxygen, carbon dioxide (CO) through said bottom section; ２ ), and / or nitrogen; (d) at least one fluid channel supplying air to an upper section of the inner reaction chamber; (e) at least one agitator for mixing the reaction mixture, the at least one agitator comprising a plurality of low shear impellers, optionally hydrofoil impellers or Rushton impellers; (f) at least one fluid channel for removing exhaust from the upper section of the inner reaction chamber; (g) at least one cleaning and / or sterilization system for cleaning and / or sterilizing the internal reaction chamber, the at least one cleaning and / or sterilization system being fluidly connected to the upper section of the internal reaction chamber; A bioreactor system comprising:

2. 2. The bioreactor system of claim 1, comprising at least two spargers, each comprising a fluid channel and at least one section comprising a plurality of through-holes through which the at least one component is introduced into the reaction mixture through the bottom section of the inner reaction chamber, optionally wherein the sections comprising a plurality of through-holes together form a hexagonal structure.

3. 3. A bioreactor system according to claim 1, comprising a single agitator with multiple impellers, optionally four impellers, further optionally the impellers being hydrofoil impellers or Rushton impellers.

4. 4. The bioreactor system of claim 1, wherein the internal reaction chamber is configured to accommodate at least about 125,000 liters (L), at least about 250,000 L, or at least about 315,000 L of a reaction mixture comprising cells, liquid, and gas.

5. 5. The bioreactor system of any of claims 1 to 4, wherein the reaction mixture comprises cells at a density of about 20 million to about 100 million cells per milliliter, optionally about 50 million cells per milliliter.

6. The bioreactor system of any one of claims 1 to 5, wherein the at least one fluid channel (sparger) supplies oxygen to the reaction mixture at a transfer rate of at least about 20 mmol / L / hr.

7. 7. The bioreactor system of any of claims 1 to 6, wherein the heat transfer system comprises a heat transfer fluid having a temperature of at least about 10°C to 12°C, optionally wherein the heat transfer fluid is water.

8. The bioreactor system of any one of claims 1 to 7, wherein the heat transfer system comprises a dimpled jacket.

9. 9. The bioreactor system according to any of claims 1 to 8, wherein the at least one cleaning and / or sterilization system applies a cleaning liquid (optionally an acid) to the inside of the inner reaction chamber, further optionally the cleaning system comprises at least one spray ball and / or spray tube.

10. 10. The bioreactor system of any of claims 1 to 9, wherein the reaction mixture is produced from a series of seed trains in which the volume of the reaction mixture is incrementally increased, optionally starting at a volume of about 25 mL, to a volume of at least about 250 L.

11. The bioreactor system of any one of claims 1 to 10, wherein the reaction mixture is maintained by perfusion.

12. 12. The bioreactor system of any of claims 1 to 11, wherein foaming of the reaction mixture, if present, is controlled using a chemical antifoam agent and / or a mechanical antifoam system.

13. The bioreactor system of any one of claims 1 to 12, wherein the liquid in the reaction mixture comprises a cell culture medium.

14. A method for producing a bioreactor system according to any one of claims 1 to 13, comprising: (a) modifying a structural shell comprising at least one section of said bioreactor vessel with a heat transfer system, optionally a dimple jacket, stiffening rings, and / or fittings, to create a modified structural shell; (b) seam welding a plurality of modified structural shells to connect them to one another, thereby forming seams at interfaces between the modified structural shells, and grinding the seams; (c) insulating, coating, painting, and / or installing an exterior cladding to the modified structural shell connected in step (b); (d) transporting the products of steps (a), (b) and / or (c) using at least one crane and / or truck or rail; A manufacturing method comprising:

15. A bioreactor system and / or method according to any of claims 1 to 14, wherein the bioreactor system comprises and / or is operatively connected to an automatic control system.

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