Bioreactor system

Abstract

The present disclosure relates to a large scale bioreactor system comprising a vessel comprising an internal reaction chamber having a volume and cell maintenance capacity significantly higher than currently available bioreactor systems.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Serial No. 63 / 339,124, filed on May 6, 2022, and U.S. Serial No. 63 / 342,379, filed on May 16, 2022, each of which is incorporated in its entirety into the present disclosure for any purpose. Technical Field

[0003] The present disclosure relates to large-scale bioreactor systems that include a vessel that includes an internal reaction chamber having a volume and cell-maintaining capacity significantly greater than currently available bioreactors. Background Art

[0004] The currently available bioreactor systems are limited in size (e.g., volume) and cell maintenance capacity. For example, those of ordinary skill in the art recognize that the currently available bioreactors cannot support large-volume high-density non-bacterial cell cultures. The present disclosure provides solutions to these problems. For example, in some embodiments, the present disclosure provides a bioreactor system comprising a container having an internal reaction chamber of at least about 125,000L to 315,000L, and a viable cell density of, for example, greater than 50 million cells per milliliter. The present disclosure thus provides solutions to these and other problems known and unknown in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 . A first exemplary bioreactor train.

[0006] Figure 2 . Second exemplary bioreactor train.

[0007] Figure 3 .First exemplary impeller.

[0008] Figure 4 .Second exemplary impeller.

[0009] Figure 5 .Third exemplary impeller.

[0010] Figure 6 .Fourth exemplary impeller.

[0011] Fig. 7A .Exemplary sprinkler designs.

[0012] Figure 7B .Exemplary sparger design showing the openings through which gas traverses from a supply source to the interior chamber of a bioreactor vessel.

[0013] Figure 8 .Exemplary bioreactor vessels and related components (bioreactor systems).

[0014] Fig. 9 .Exemplary manufacturing process.

[0015] Fig.10 .Exemplary bioreactor system with automation.

[0016] Fig.11 .Exemplary bioreactor system with automation.

[0017] Fig.12 .Exemplary bioreactor system with automation. Summary of the invention

[0018] The present disclosure relates to a large-scale bioreactor system, which includes a container (e.g., a bioreactor container) comprising an internal reaction chamber, the volume and cell maintenance capacity of which are significantly higher than currently available bioreactors. In preferred embodiments, the present disclosure provides aspects 1-15 described below. In preferred embodiments, the bioreactor container includes an internal reaction chamber, which is configured to accommodate at least about 125,000 liters (L), at least about 250,000L or at least about 315,000L of a reaction mixture comprising cells, liquids and gases. In preferred embodiments, the ratio of the liquid depth (LD) of the bioreactor container to the diameter (D) of the bioreactor container is 1.5-3, the ratio being optionally about 2.4, optionally, about 2.36 in preferred embodiments. Other embodiments as will be understood by one of ordinary skill in the art are also disclosed herein. DETAILED DESCRIPTION

[0019] The present disclosure relates to bioreactor systems comprising at least one container (e.g., a bioreactor container) comprising an internal reaction chamber having a reaction mixture capacity 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.

[0020] In some embodiments, the container may have a form disclosed in, for example, but not limited to, U.S. Patent No. 8,658,419 (ABEC, Inc.), U.S. Patent No. 9,228,165B2 (ABEC, Inc.), US10,519,415B2, and / or WO 2019 / 070648 A2 and / or used in the system disclosed therein. In some embodiments, the disposable feed container may be a cone-bottom or tulip-bottom container and / or preferably has a capacity of at least 20L. Other types of suitable feed containers that can be used as disclosed herein are also known in the art, as understood by those of ordinary skill in the art. The components of the materials used to produce the apparatus described herein may be the same or different. The reactor vessel and / or heat exchange component described herein are typically but not necessarily constructed of a corrosion-resistant alloy (e.g., metal). For example, suitable materials may include, but are not limited to, honeycomb jacket materials and / or sheet / plate raw materials. Suitable materials include, for example, carbon steel, stainless steel (e.g., 304, 304L, 316, 316L, 317, 317L, AL6XN), aluminum, (e.g., Inconel 625, Chronin 625, Altemp 625, Haynes 625, Nickelvac 625, and Nicrofer 6020), Hastelloy alloys (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 Titanium, Carpenter etc. However, it should be understood that other materials besides corrosion resistant alloys may also be suitable, such as, but not limited to, plastics, rubbers, and mixtures of such materials. A "mixture" of materials may refer to the actual mixture itself that forms the combined material or the use of various materials within the system (e.g., an alloy reactor shell and rubber baffle components). With respect to the materials for constructing the channels mentioned above, any of the above-mentioned suitable materials may be prepared so as to form a channel through which a heat transfer medium can be distributed.

[0021] The reaction vessel includes an internal chamber, and in a preferred embodiment, is at least associated with a heat transfer system and / or includes a heat transfer system, which includes a heat transfer device for controlling the temperature of a chemical, pharmaceutical or biological process carried out in the internal reaction chamber of the container. In some embodiments, the heat transfer system provides the distribution of a heat transfer medium so that the heat caused by the process or required for the process is transferred from the reaction mixture or transferred to the reaction mixture. In some embodiments, the reaction vessel includes a jacket and / or a jacketed tank head, which provides a fluid channel through which a heat transfer fluid can circulate (e.g., a honeycomb jacket). In some embodiments, the reaction vessel can be at least partially surrounded by a fluid channel. The jacketed tank head can also serve as the lid of the reaction vessel. The jacketed tank head can also be used to support and / or release the pressure on the DC (e.g., on the top of the DC) contained in the reactor vessel.

[0022] In some embodiments, one or more heat exchange systems may include a jacket through which a heat transfer fluid circulates. For example, the jacket may include a channel through which a heat transfer fluid circulates. In some embodiments, the jacket may be a "honeycomb" material. Honeycomb jackets are typically installed around reaction vessels, such as fermenters, and can be used as part of a heat transfer system. Honeycomb jacket materials can be used in the devices described herein in a typical manner, such as wrapping a reaction vessel. In certain embodiments described herein, honeycomb jacket materials may also or alternatively be used in baffle structures. Honeycomb jacket materials are commercially available, and any such material may be suitable for use as disclosed herein. Typically, honeycomb jacket materials have a substantially uniform honeycomb pattern (e.g., depressions, indentations) pressed or formed in a parent material (e.g., a metal sheet). Honeycomb jacket materials can be made, for example, mechanically ("mechanical honeycomb jacket") or by inflation (e.g., inflation resistance spot welding (RSW)). To prepare a mechanical honeycomb material, a metal sheet in which a substantially uniform array of honeycombs (where each honeycomb typically contains a central hole) is pressed is welded to a parent metal through the central hole. Gas-filled RSW honeycomb materials (e.g., gas-filled HTS or HTS) are typically made by resistance spot welding an array of points on a thin metal sheet to a more solid (e.g., thicker) base material (e.g., metal). The edges of the combined material are sealed by welding, and the interior is inflated at high pressure until the thin material forms a honeycomb pattern. When used as a jacket, mechanical honeycomb materials typically have a high pressure level and a low to medium pressure drop, while RSW honeycomb jackets typically exhibit a medium pressure level and a high to medium pressure drop. Heat transfer fluids typically flow between sheets of material with honeycombs. Other suitable honeycomb materials are available to those skilled in the art and should be suitable for use as described herein.

[0023] In a preferred embodiment, the reactor vessel disclosed herein includes one or more heat transfer systems that effectively transfer heat, withstand the hydraulic pressure encountered in the reactor vessel, and can be simply and effectively sterilized. Suitable heat transfer baffles described herein can be incorporated into the heat transfer system to solve these problems. Preferred exemplary heat transfer baffles are disclosed and / or claimed in U.S. Patent No. 8,658,419B2, which is incorporated herein in its entirety, such as those disclosed herein are shown in Figures 16-18. In certain embodiments, the baffle has at least one internal channel (e.g., 9 in Figures 16-18) and at least two external channels (e.g., 10 in Figures 16-18). Typically, the heat transfer medium circulates through one or more distribution channels (e.g., 9 in Figures 16-18) rather than one or more release channels (e.g., 10 in Figures 16-18), and the release channels can also be used as vents for the distribution channels. The distribution channels 9 are typically formed between the support material 11 and the honeycomb jacket material 12 of each subassembly. The release channel 10 is usually formed by adjoining two subassemblies, each of which includes a support material 11 fixedly attached to a honeycomb jacket material 12. In such embodiments, the honeycomb jacket material and the support material of each subassembly are usually adjacent to each other by welding or other processes that make the materials fixedly attached to each other. The subassemblies are usually adjacent to each other using a closing rod 13. The closing rod is usually adjacent to the support material by welding or other processes that produce a substantially seamless joint. The width of the closing rod can be adjusted to set the width of the release channel as required (for example, the juxtaposed honeycomb jacket materials are set closer together or farther away). One or more release holes can be made in the closing rod so that the release channel can be communicated with the outside of the reaction vessel. The distribution channel and the release channel are combined into the baffle to provide excellent heat transfer capabilities and the structural integrity required to withstand the liquid pressure encountered in the reaction vessel. The baffle can protrude from the inner wall of the reaction vessel at regular or irregular intervals. The baffle can also be installed at any suitable angle relative to the inner wall of the reactor vessel (for example, 60° relative to the inner wall of the reactor vessel and 30° relative to the radius of the reactor vessel). Suitable angles can be angles that are understood by the skilled person to be suitable or sufficient to attenuate forces (e.g., hydraulic forces) encountered by the baffle, which are caused by movement (e.g., rotational movement and / or vortex movement) caused by agitation (e.g., mechanical or otherwise) of the container contents. Suitable angles are angles that will prevent damage to the baffle due to forces caused by such movement. 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 the inner wall of the container or the radius of the container.When the reaction vessel includes a mechanism (e.g., mechanical mechanism or other mechanism) for stirring or mixing the reaction, such as a rotating blade set, etc. (e.g., an axial flow or radial flow impeller), the baffle is attached to the inner wall or protrudes from the inner wall so that the mechanism and the baffle do not contact each other. For example, in the case where one or more devices for mixing reaction components are located at the bottom center of the container, the baffle can be installed above the highest point of the member. In the case of using multiple mechanical mechanisms, the baffle is usually configured to avoid these mechanisms. For example, in the case where the mechanism includes one or more rotating blade sets, the baffle can be located above, below, between or beside the blades. The baffle design will ensure that there is enough clearance with the mechanical mechanism. The baffle assembly is usually fixedly attached to the container by one or more attachment arms 7 by welding or other processes that produce a substantially seamless joint. As described above, the use of attachment arms advantageously provides effective cleaning and / or disinfection of the baffle, because after the attachment process (e.g., welding), very little residue or no residue remains at the joint between the inner surface of the reaction vessel and the baffle. Similarly, the baffles may be bonded to, attached to, or fixed to the reaction vessel by any suitable method, so long as the method provides a substantially seamless attachment point (e.g., a seamless joint or boundary between materials) to provide a surface that can be simply and effectively disinfected. "Substantially seamless attachment point", "seamless joint" or "joint without cracks" generally indicates that the boundary between the baffle and the reaction vessel is substantially undetectable by visual and / or other means (e.g., microscope). It may also be indicated that after the standard cleaning procedures that technicians typically use to "disinfect" such equipment, the boundary does not retain any residues from previous reactions. Therefore, the system is suitable for disinfection 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 enables simple and effective disinfection by itself. In some preferred embodiments, the container of the present disclosure may include any suitable number of baffles, preferably one to ten, more preferably four to eight, and most preferably eight. In some preferred embodiments, the container does not include any baffles.

[0024] In preferred embodiments, the optimal heat transfer surface area utilized by a particular system can be determined based on an estimate of the metabolic load of the cell culture based on the oxygen uptake rate (OUR) and any mechanical contribution provided by the agitator (e.g., 2HP / kGal per unit volume). In preferred embodiments, the heat transfer surface area is provided on the sidewalls of the bioreactor vessel, at least to the maximum working volume and to the working head. In preferred embodiments, the heat transfer fluid moves along the sidewalls through parallel flow paths and through different heat transfer areas. In some embodiments, a supplemental heat exchange system can be applied to the harvest line if, for example, lower temperatures are required during harvesting of cells from the reaction mixture.

[0025] The heat transfer systems described herein can be constructed of any material through which a heat transfer fluid (e.g., a gas and / or preferably a liquid, such as cold water (e.g., at a temperature of 10-12° C., depending on the application)) can be transported so that heat can be conducted to and / or absorbed from another part of the system by radiation, convection, conduction, or direct contact (e.g., from the heat transfer system into the internal reaction chamber). Suitable heat transfer media include, but are not limited to, fluids and gases. Suitable fluids and gases include, but are not limited to, steam (from top to bottom), hot and cold water, ethylene glycol, heat transfer oils, refrigerants, or other pumpable fluids having a desired operating temperature range. Multiple types of heat transfer media can also be used, such that, for example, one type of medium is directed to one area of ​​the reaction vessel, while another type of medium is directed to a different area of ​​the reaction vessel (e.g., as in the zoned system described above). Mixtures of heat transfer media (e.g., 30% ethylene glycol) can also be desirable.

[0026] In preferred embodiments, the reaction mixture comprises cells, preferably non-bacterial cells, more preferably mammalian, fish, avian and / or insect cells, at a 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 vessels of the present disclosure are configured to support the parameters shown in Table 1A, Table 1B (preferred embodiments), and / or Table 2A and Table 2B (preferred embodiments):

[0027] Table 1A

[0028] parameter value unit Target cell density 20-100 <![CDATA[10 per milliliter 3 -10 6 cells]]> pH 6-9 unit temperature 20-60 ℃ Dissolved oxygen 20-60 Saturated %

[0029] Table 1B (Preferred Embodiment)

[0030] parameter value unit Target cell density 35 <![CDATA[10 cells per milliliter 6 > pH 6.8-7.8 unit temperature 33-40 ℃ Dissolved oxygen 30-50 Saturated %

[0031] Table 2A

[0032]

[0033]

[0034] Table 2B (preferred embodiment)

[0035]

[0036] Typical bioreactor aspect ratios (defined herein as the ratio of liquid depth (LD) to bioreactor vessel diameter (D)) are about 1.0-1.5, preferably 1.25-1.5, or for larger bioreactors, up to about 2. The bioreactors disclosed herein have aspect ratios of about 1.5 to about 3.0, preferably an aspect ratio of about 2.4 (in some preferred embodiments, about 2.36). These aspect ratios, particularly aspect ratios of about 2.36 and higher, support processes that exhibit the parameters described in Tables 1 and 2 (e.g., about 35 million cells per milliliter, about 2,250,000 L reactor capacity). The systems disclosed herein are also configured to be manufactured (e.g., and shipped if necessary) at one site, can be adjusted for the specific cells being grown, maintained sterile, and operated with commercially available filters, etc. In some preferred embodiments, the bioreactors disclosed herein may include the dimensions and parameters shown in Table 3:

[0037] Table 3

[0038]

[0039]

[0040] In some embodiments, a "seed train" technique is used to provide an initial volume of cells to a bioreactor. Such a "seed train" is typically composed of many smaller bioreactors that allow the volume of cells to be fully expanded to a desired density when the contents of the bioreactor are transferred to a subsequent bioreactor. All or part of the contents of a bioreactor may be transferred to a subsequent bioreactor. For example, an exemplary bioreactor train is shown in Figure 1 and Figure 2 middle.

[0041] The bioreactor vessels disclosed herein are typically, but not necessarily, constructed of metal, and are typically, but not necessarily, constructed of corrosion-resistant alloys. For example, suitable materials may include, but are not limited to, sheet / plate stock (and / or honeycomb jacket material for, e.g., heat transfer systems). Suitable exemplary materials include, for example, carbon steel, stainless steel (e.g., 304, 304L, 316, 316L, 317, 317L, AL6XN), aluminum, (e.g., Inconel 625, Chronin 625, Altemp 625, Haynes 625, Nickelvac 625, and Nicrofer 6020), Hastelloy alloys (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 Titanium, Carpenter etc. However, it should be understood that other materials besides corrosion resistant alloys may also be suitable, such as, but not limited to, plastics, rubbers, and mixtures of such materials. A "mixture" of materials may refer to the actual mixture itself that forms the combined material or to the use of various materials within the system (e.g., an alloy reactor housing and rubber baffle components).

[0042] The reaction mixture generally includes a liquid cell culture medium suitable for maintaining the viability and growth of the cells of interest. As described above, the bioreactor container disclosed herein can adapt to the growth of various types of cells, including but not limited to mammals, fish, birds and / or insect cells. Exemplary cell culture medium can be any cell culture medium that is commonly used to cultivate such cells and is modified as needed to allow the viability and growth (e.g., to reach the density disclosed herein) in the bioreactor container. Typically, the cell culture medium and any other liquid introduced into the bioreactor container during cell growth / amplification are sterile. The cell expansion process can be in different forms, such as batches (i.e., a disposable introduction of the culture medium of the entire volume), batch feed (adding culture medium and nutrients during the entire growth time) or process intensification forms (i.e., using an external device (such as a filter) to exchange culture medium / nutrients to allow higher cell density). As those of ordinary skill in the art will appreciate, other processes may also be suitable. After the reaction (e.g., growing to 50 million cells per ml), cells are usually harvested directly from the bioreactor container to minimize the risk of contamination. In some embodiments, after the reaction (e.g., growth to 5000 cells per ml), cells can be harvested directly from the bioreactor vessel into a sterile transfer line to minimize the 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 culture medium can be introduced into the system to continue cell expansion in a manner known as draw and fill.

[0043] In some embodiments, the system can include conduits (e.g., tubing) that can be cleaned and sterilized independently of other sections of the system to allow the bioreactor to aseptically receive liquid additives added to the product stream. Exemplary liquid additives can be, for example, cell culture media or various components thereof, cell culture fluids for inoculation, alkaline or acidic solutions to control pH, glucose or other sugars for cell growth, defoamers, and / or the like. In some embodiments, each of these liquid additives can be fed from a previous bioreactor (e.g., in a series of bioreactors), other holding vessels of appropriate size for the reaction, and / or a mother tube system that can supply multiple of these bioreactors.

[0044] The cleaning of the internal reaction chamber (i.e., production reactor) can be achieved by a clean-in-place (CIP) skid, which provides relevant acid and caustic washes and clean water flushing in the container through a spray ball and a spray rod. Table 4 shows the total flow rate based on three different types of spray balls (static, uniaxial dynamic and multiaxial dynamic). In a preferred embodiment, the flow rate is based on the ASME BPE flow guide. The internal reaction chamber (i.e., production reactor) can also be cleaned using "empty" sterilization, which utilizes clean steam (i.e., steam prepared from a purified water source) or cooking-grade steam as known in the art. Time and temperature can be adjusted to adapt to the desired sterilization requirements. Exemplary suitable sterilization can include heating the empty container and its sterile boundaries to 125°C and keeping this temperature for 30 minutes. The steam will be replaced by clean air, and the container will cool to allow the addition of media. Time and temperature can be adjusted to adapt to the sterilization requirements required by the user.

[0045] Table 4

[0046]

[0047]

[0048] Adequate mixing and gas dispersion are key to maintaining optimal cell viability and growth within a bioreactor vessel (i.e., within the internal reaction chamber). Such mixing and dispersion are typically achieved using a stirring system that includes an agitator that includes one or more impellers, preferably a low shear impeller, a hydrofoil impeller in some preferred embodiments, or a Rushton impeller in some preferred embodiments. Table 5 provides exemplary agitator sizes for bioreactor vessels disclosed herein. Although Table 10 lists specific impellers, it should be understood that any suitable impeller may be used. In a preferred embodiment, the agitator may include two (2) to about six (6) impellers, preferably four (4) impellers, to provide adequate mixing throughout the reaction mixture. In addition, the number of heat transfer baffles may range from zero to eight (or more if appropriate), with four baffles being a preferred embodiment.

[0049] Table 5

[0050]

[0051] Based on these exemplary designs and preferred ranges of about 5 to about 40 mmol / L / hr (in some preferred embodiments, about 20 mmol / l / h (see Tables 6-7)) OTR and about 0.5 to about 3.0 sHP / kGal (in some preferred embodiments, about 1 sHP / kGal (see Tables 6-7)) baselines (see Tables 2A and 2B (20 mmol / l / h and 1 sHP / kGal)), the gas flow rate can be estimated. The gassing rate using air or air supplemented with oxygen can be used. Tables 6 and 7 show the output of these exemplary designs. For 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 flow rate alone (Table 7) rises accordingly based on the mole fraction of oxygen delivered. Given the high aspect ratio, in some embodiments, an agitator designed around a hydrofoil impeller is preferred. The low shear impeller included in the bioreactor vessel agitator used to generate the data in Table 7 was replaced with a hydrofoil impeller, and mass transfer correlations were derived therefrom as shown in Table 8 (supplemented oxygen) and Table 9 (air only).

[0052] Table 6

[0053]

[0054] Table 7

[0055]

[0056] Table 8

[0057]

[0058] Table 9

[0059]

[0060] Suitable impellers may be any impeller available to one of ordinary skill in the art. An exemplary hydrofoil impeller design that may be used with the bioreactor vessels disclosed herein is shown in Figure 3-6 middle. Figure 3-4 The characteristics of the impeller shown are shown in Table 10, and Figure 5-6 The characteristics of the impeller shown are shown in Table 11.

[0061] Table 15

[0062] <![CDATA[Working volume of production reactor, m 3 > 125 250 315 Container ID, in 162 204 220 Upper impeller type Wide blade hydrofoil Wide blade hydrofoil Wide blade hydrofoil Middle impeller volume 3 3 3 Middle impeller type Wide blade hydrofoil Wide blade hydrofoil Wide blade hydrofoil Lower impeller type Rushton Rushton Rushton Number of baffles 4 4 4 Pumping direction Down Down Down Total number of impellers 5 5 5

[0063] Table 16

[0064]

[0065]

[0066] Manufacturability is another key determinant of the feasibility of agitators at these scales. Figure 3-6 The size of the exemplary impeller shown in the , as may be required in a sterile environment, the monolithic construction may bring manufacturing challenges (e.g., the need for in-tank connection for shaft processing and manufacturing). In some embodiments, the individual impeller blades can be bolted to the welded hub for processing purposes. It is preferred, but not necessarily necessary, that all connections will be sanitary designs as outlined in ASMEBPE Appendix 10.3. Support bearings can also be used to handle shaft deflection, and they can be used for the bottom of the bioreactor container (e.g., in an embodiment where the agitator gearbox is mounted on the top of the bioreactor container). In some embodiments, the agitator can be sealed to the bioreactor container by a container-type mechanical seal. In some embodiments, a pressurized lubricated seal using clean steam condensate as a lubricant can be used. In some embodiments, a single dry-running seal with a sanitary gland can be used.

[0067] In some embodiments, the reactor system of the present disclosure may include at least two spargers, each spargers including a fluid channel and at least one segment including a plurality of perforations through which at least one component is introduced into the reaction mixture through the bottom segment of the internal reaction chamber, optionally wherein the segments including a plurality of perforations together provide a circular, oval, hexagonal, square, rectangular or other shaped structure (see Fig. 7A and Figure 7B, which show the orientation of the sparger at the bottom of the bioreactor vessel and the spacing of the holes for gas to traverse, represented as dots, respectively. In some preferred embodiments, a single sparger (e.g., having a circular, oval, hexagonal, square, rectangular, or other shape) may be included. In preferred embodiments, the spacing of the perforations in the sparger becomes smaller (e.g., the perforations are more numerous and closer together) as the sparger structure extends away from the end of the gas source that enters the sparger.

[0068] In some embodiments, fluid passages used as supply lines to a bioreactor vessel (e.g., its internal reaction chamber) may conform to typical carbon or stainless steel piping specifications and are preferably sanitary (particularly those with direct product contact, connections to other containers that provide sterile liquids to the bioreactor vessel, gas supply, gas exhaust, CIP distribution, and clean steam distribution). Applications such as plant steam and cooling water are not considered sanitary designs. In a preferred embodiment, the line size of the sanitary lines allows the use of ASME BPE tubing, which is the standard for sanitary applications in biopharmaceuticals. Diaphragm valves can be used within the sterile boundaries of the bioreactor vessel. Other types of valves, such as sanitary butterfly valves, ball valves, and mixproof valves, can also be used in the construction within the sterile boundaries and guide other fluids where sanitary construction is required. In a preferred embodiment, the material of these lines will follow typical biopharmaceutical applications and be 316L stainless steel. The surface finish of the bioreactor vessel, and particularly the surface finish of the internal reaction chamber, preferably meets a minimum Ra mechanical polish of 30 microinches. If combined with a technique such as electrolytic polishing, a suitable surface can have a lower Ra (i.e., <30 microinches). Any elastomer in the sanitary piping is preferably compliant with USP / FDA (e.g., EPDM and / or Platinum Cured Silicone). In a preferred embodiment, the bioreactor vessel can have a shell of 316L stainless steel, which can be mechanically polished to a minimum of 30 microinch Ra (unless combined with, for example, electropolishing), and the jacket material can be 304L stainless steel and insulated with an insulating jacket material. In some embodiments, an alternative duplex steel (e.g., UNSS32205) can be used to construct the bioreactor vessel. Depending on the ultimate pressure and load rating of the vessel, this alternative can be cost-effective due to the reduced thickness. In a preferred embodiment, ports for analytical probes (such as dissolved oxygen, pH, and pCO2) along the sidewall of the bioreactor vessel can be included.

[0069] In preferred embodiments, the systems described herein may also include one or more manual and / or automated control systems (i.e., without continuous direct human intervention, 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 situations occurring within any component of the system, preferably between at least any two components of the system. Such control systems typically include one or more general-purpose computers, which include software for processing such information and manually or automatically adjusting the desired parameters of the reaction as required for a particular process. Therefore, 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 automated systems for controlling and detecting process conditions and process sequencing. In some preferred embodiments, the automated system includes hardware (automated 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 automated system hardware is programmed to control and monitor process conditions and process sequencing. Process control and monitoring parameters that can 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 evolution / mass flow, headspace pressure, pH, agitator speed, viable cell density, exhaust gas analysis and spectroscopy, including ultraviolet (UV) and Raman; and can be combined with specific control algorithms, such as exponential feeding. Large bioreactor process sequences that can be controlled may include cleaning in place (CIP), sterilization in place (SIP), pressure hold testing, container filling, cell growth, reagent addition and / or cell harvesting processes. Process control and monitoring may also include integration / interaction with external process systems that supply or service large bioreactors, including reagent addition tanks, CIP systems, SIP systems, liquid sterilization systems, and harvesting systems. Process control, monitoring and sequencing data can be collected and stored as batch records. An exemplary automated control system is shown in Figure 10-12 Such a system preferably includes redundant Ethernet-based device control network connections of two redundant network switches (not shown) in each remote panel and redundant Ethernet connections of the switches to the HMI and to Ethernet I / O (shown) within the remote I / O panel ( Figure 10-12 , A, B (Ethernet)).

[0070] An exemplary bioreactor system including an automated system is shown in Figure 10-12 .like Fig.10As shown, certain preferred embodiments of the bioreactor system and / or subsystems of the bioreactor system may include: a media preparation ("Media Prep") subsystem connected to a nutrient preparation ("Nutrient Prep") subsystem; and a large media preparation subsystem ("Large Media Prep"), which may include containers of the same or different sizes, clean-in-place skids (connected via a redundant equipment control network); and various subsystems thereof. Fig.11 As shown, certain preferred embodiments of the bioreactor system and / or subsystems of the bioreactor system may include: a large media preparation subsystem ("Large Media Prep"), which may include containers of the same or different sizes, one or more clean-in-place skids (connected via a redundant equipment control network); and various subsystems thereof. Fig.12 As shown, certain preferred embodiments of the bioreactor system and / or subsystems of the bioreactor system may include: one or more cell culture trains and / or nutrient containers (e.g., glucose hold) and / or a large bioreactor cell CIP system ( Fig.12 ), which may include containers of equal or different sizes, one or more clean-in-place skids (connected by a redundant equipment control network); and various subsystems thereof. Preferred embodiments of the bioreactor system include supply lines, return lines, etc. In some embodiments, certain preferred bioreactor systems may include a plurality of, for example, 25,000, 32,000, 40,000, 50,000, 125,000 and / or 250,000 L fluidly connected in series bioreactor vessels. In certain preferred embodiments, the various containers, subsystems, and / or bioreactor systems include at least two (e.g., two, three, four, or five) fluidly connected container trains in series, wherein the containers are 500L, 2,000L, 8,000L, 32,000L, 125,000L, and 250,000L (in a preferred embodiment, each train includes at least one 125,000L container that feeds two 250,000L containers), which may also be fluidly connected to a glucose holding container (e.g., 30,000L). As will be appreciated by one of ordinary skill in the art, other embodiments of the control system may also be used.

[0071] Therefore, the present disclosure provides the following preferred aspects and preferred embodiments:

[0072] 1. A bioreactor system comprising:

[0073] a) A container including an internal reaction chamber:

[0074] a. The internal reaction chamber is configured to accommodate at least about 30,000 liters (L), at least about 50,000

[0075] 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

[0076] L or at least about 315,000 L of a reaction mixture comprising cells, liquids, and / or gases;

[0077] b. a ratio of liquid depth (LD) to bioreactor vessel diameter (D) of 1.5-3, optionally about 2.4 (e.g., about 2.36 in some preferred embodiments); and

[0078] c. a top section and a bottom section, wherein at least the bottom section is in contact with the reaction mixture;

[0079] b) at least one heat transfer system at least partially surrounding at least one region of the internal reaction chamber and configured to maintain the reaction mixture in the region at a preselected temperature;

[0080] c) at least one fluid channel (sparger) which provides at least one component of the reaction mixture through the bottom section, the at least one component being selected from the group consisting of air, oxygen, carbon dioxide (CO2) and / or nitrogen;

[0081] d) at least one fluid channel that provides air to the top section of the internal reaction chamber;

[0082] e) at least one agitator for mixing the reaction mixture, the agitator comprising a plurality of low shear impellers, optionally hydrofoil impellers or Rushton impellers;

[0083] f) at least one fluid channel for removing exhaust gases from the top section of the internal reaction chamber; and,

[0084] 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 top section of the internal reaction chamber.

[0085] 2. A bioreactor system according to aspect 1, comprising at least two spargers, each of the spargers comprising a fluid channel and at least one segment comprising a plurality of perforations, through which the at least one component is introduced into the reaction mixture through the bottom segment of the internal reaction chamber, optionally wherein the segments comprising a plurality of perforations together provide a circular, oval, hexagonal, square, rectangular or other shaped structure (see Fig. 7A and 7B , which show the orientation of the sparger at the bottom of the bioreactor vessel and the spacing of the holes for gas to traverse, represented as dots, respectively. In some preferred embodiments, a single sparger may be included (e.g., having a circular, oval, hexagonal, square, rectangular, or other shape). In preferred embodiments, the spacing of the perforations in the sparger becomes smaller (e.g., the perforations are more numerous and closer together) as the sparger structure extends away from the end of the gas source that enters the sparger.

[0086] 3. The bioreactor system of any one of the preceding aspects, comprising a single agitator, the agitator comprising a plurality of impellers, optionally four impellers, further optionally wherein the impellers are hydrofoil impellers or Rushton impellers.

[0087] 4. The bioreactor system of any of the preceding aspects, wherein the internal reaction chamber is configured to hold 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.

[0088] 5. The bioreactor system of any of the preceding 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.

[0089] 6. The bioreactor system of any of the preceding aspects, wherein the at least one fluid channel (sparger) provides oxygen to the reaction mixture at a delivery rate of at least about 20 mmol / L / hour.

[0090] 7. A bioreactor system as described in any of the preceding aspects, wherein the heat transfer system comprises a heat transfer fluid having a temperature of at least about 10°C-12°C (higher during, for example, a control period), optionally wherein the heat transfer fluid is water.

[0091] 8. The bioreactor system of any of the preceding aspects, wherein the heat transfer system comprises a jacket with honeycombs.

[0092] 9. A bioreactor system as described in any of the preceding aspects, wherein the at least one cleaning and / or sterilization system applies a cleaning solution, optionally an acid, to the interior of the internal reaction chamber, further optionally wherein the cleaning system comprises at least one spray ball and / or spray bar.

[0093] 10. The bioreactor system of any of the preceding aspects, wherein the reaction mixture is produced from a series of seed trains through which the volume of the reaction mixture is gradually increased, optionally starting from about at least 250 L. However, in some embodiments, a series of seed trains may start at a much lower level, such as a vial level (e.g., 25 ml).

[0094] 11. The bioreactor system of any one of the preceding aspects, wherein the reaction mixture is maintained by perfusion.

[0095] 12. The bioreactor system of any one of the preceding aspects, wherein foaming of the reaction mixture, if present, is controlled using a chemical defoamer and / or a mechanical defoaming system.

[0096] 13. The bioreactor system of any one of the preceding aspects, wherein the liquid in the reaction mixture comprises a cell culture medium.

[0097] 14. An exemplary bioreactor system comprising Figure 8 Components shown. This exemplary bioreactor system includes: a vessel including an internal reaction chamber configured to hold 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, liquids, and / or gases; a plurality of impellers connected to a shaft that is in turn connected to an agitator motor; at least one sparger fluidly connected to a fluid channel connected to a sterile filter and a fluid channel for air, oxygen, carbon dioxide, and / or nitrogen to pass through to flow into the sparger (and a source / container providing them) and a gas flow controller; at least one heat transfer system comprising: a jacket; , a heat transfer fluid flows through the jacket to cool the reaction mixture ("liquid volume"); at least one pump; at least one heat transfer fluid source and / or heat source (e.g., "steam") or cooling source, which is connected to the fluid channel through which the heat transfer fluid flows; a section of the container above the reaction mixture (liquid volume) (e.g., head space), which may include at least one air source and fluid channel, and air is introduced into the head space through the fluid channel; at least one spray ball or other structure, through which a cleaning fluid, steam or other gas (and its source) can flow into the empty container to clean the container after the reaction is completed and the container is emptied; and an exhaust system, which includes at least one sterile filter, at least one pressure control valve, wherein the exhaust system exhausts gas (e.g., humid gas) to the environment.

[0098] 15. A method for manufacturing a bioreactor system according to any one of the preceding aspects, the method comprising:

[0099] a. Changing the structural shell to produce a changed structural shell, the structural shell comprising: at least one section of the container having a heat transfer system, the heat transfer system is optionally a honeycomb jacket; reinforcement rings; and / or accessories;

[0100] b. seam welding a plurality of changed structural shells to connect them to each other, thereby producing seams at the interfaces between the changed structural shells, and polishing the seams;

[0101] c. insulating, coating, painting and / or installing the outer sheath to the connected altered structural housing connected in step b); and

[0102] d. using at least one crane and / or track or rail to transport the product of steps a), b) and / or c) (see, for example, Fig. 9 ).

[0103] 16. The bioreactor system and / or method according to any one of the preceding claims, wherein the bioreactor system comprises and / or is operably connected to an automated control system.

[0104] As will be appreciated by one of ordinary skill in the art, other embodiments, aspects, advantages of the system, and methods of using the system are also provided herein.

[0105] When before a numerical list or range, the terms "about", "approximately", etc. independently refer to each individual value in the list or range, as if the term is immediately before each individual value in the list or range. The terms mean that the values ​​they refer to are exactly the same, close or similar to them. Optional or optional means that the event or situation described later may occur, or may not occur, and the description includes the situation where the event or situation occurs and the situation where it does not occur. The range can be expressed as from about one specific value and / or to about another specific value in this article. When such a range is expressed, another aspect includes from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation by using the leading word about or approximately, it should be understood that the specific value forms another aspect. It should be further understood that the end value of each range is meaningful with respect to another end value and independently of another end value. A range (e.g., 90-100%) is meant to include the range itself and each independent value in the range, as if each value is listed separately.

[0106] All references cited in this disclosure are incorporated herein by reference in their entirety. Certain embodiments will be further described in the following examples. These embodiments are provided only as examples and are not intended to limit the scope of the claims in any way. Although certain embodiments have been described according to preferred embodiments, it should be understood that various variations and modifications may be conceived by those skilled in the art. Therefore, the appended claims are intended to cover all such equivalent variations within the scope of the following claims.

Claims

1. A bioreactor system comprising: a) Vessel including an internal reaction chamber: The internal reaction chamber is configured to hold 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 ratio of liquid depth (LD) to bioreactor vessel diameter (D) of 1.5-3, optionally about 2.4, optionally about 2.36; and a top section and a bottom section, wherein at least the bottom section is in contact with the reaction mixture; b) at least one heat transfer system at least partially surrounding at least one region of the internal reaction chamber and configured to maintain the reaction mixture in the region at a preselected temperature; c) at least one fluid channel (sparger) which provides at least one component of the reaction mixture through the bottom section, the at least one component being selected from the group consisting of air, oxygen, carbon dioxide (CO2) and / or nitrogen; d) at least one fluid channel that provides air to the top section of the internal reaction chamber; e) at least one agitator for mixing the reaction mixture, the agitator comprising a plurality of low shear impellers, optionally hydrofoil impellers or Rushton impellers; f) at least one fluid channel for removing exhaust gases from said top section of said internal reaction chamber; as well as 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 top section of the internal reaction chamber.

2. A bioreactor system as described in claim 1, comprising at least two spargers, each of the spargers comprising a fluid channel and at least one section comprising a plurality of perforations, wherein the at least one component is introduced into the reaction mixture through the bottom section of the internal reaction chamber through the perforations, optionally wherein the sections comprising a plurality of perforations together provide a substantially hexagonal structure.

3. A bioreactor system as claimed in any one of the preceding claims, comprising a single agitator comprising a plurality of impellers, optionally four impellers, further optionally wherein the impellers are hydrofoil impellers or Rushton impellers.

4. The bioreactor system of any of the preceding claims, wherein the internal reaction chamber is configured to hold 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 one of the preceding claims, 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. A bioreactor system as claimed in any one of the preceding claims, wherein the at least one fluid channel (sparger) provides oxygen to the reaction mixture at a delivery rate of at least about 20 mmol / L / hour.

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

8. A bioreactor system as claimed in any preceding claim, wherein the heat transfer system comprises a honeycombed jacket.

9. A bioreactor system as described in any of the preceding claims, wherein the at least one cleaning and / or sterilization system applies a cleaning solution, optionally an acid, to the interior of the internal reaction chamber, further optionally wherein the cleaning system comprises at least one spray ball and / or spray bar.

10. A bioreactor system as described in any of the preceding claims, wherein the reaction mixture is produced by a series of seed trains, the volume of the reaction mixture being gradually increased through the seed trains, optionally starting from a volume of about 25 ml to at least 250 L.

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

12. The bioreactor system of any one of the preceding claims, wherein foaming of the reaction mixture, if present, is controlled using a chemical defoamer and / or a mechanical defoaming system.

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

14. A method for manufacturing a bioreactor system as claimed in any one of the preceding claims, the method comprising: a. modifying the structural shell to produce a modified structural shell, the structural shell comprising: at least one section of the bioreactor vessel having a heat transfer system, the heat transfer system optionally being a honeycomb jacket; reinforcement rings; and / or fittings; b. seam welding a plurality of changed structural shells to connect them to each other, thereby producing seams at the interfaces between the changed structural shells, and polishing the seams; c. insulating, coating, painting and / or installing the outer sheath to the connected altered structural housing connected in step b); and d. Transporting the products of steps a), b) and / or c) using at least one crane and / or tracks or rails.

15. The bioreactor system and / or method according to any one of the preceding claims, wherein the bioreactor system comprises and / or is operably connected to an automated control system.

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