Modular bioreactors, bioreactor systems, and related methods

Abstract

The bioreactor includes a housing with multiple removable parts for easy assembly or disassembly in a modular structure. The removable parts include a fixed bed and a support for the fixed bed. Multiple supports may be provided. The multiple supports may interface with each other or with the lid of the bioreactor. The support interfaces with the housing to prevent relative rotation. The multiple supports include a support frame that supports the fixed bed and allows fluid to flow through the support frame. The fixed bed forms a peripheral chamber between the support and the housing to hold the fixed bed. One or more probes and tubes may be inserted into the bioreactor to test internal parameters or to add or remove liquids therefrom.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 328,461, filed April 7, 2022, U.S. Provisional Patent Application No. 63 / 330,967, filed April 14, 2022, U.S. Provisional Patent Application No. 63 / 410,252, filed September 27, 2022, and U.S. Provisional Patent Application No. 63 / 412,709, filed October 3, 2022, the disclosures of which are incorporated herein by reference. This application is also related to U.S. Provisional Patent Application No. 62 / 758,152, U.S. Provisional Patent Application No. 62 / 733,375, and U.S. Provisional Patent Application No. 62 / 608,261, each of which is incorporated herein by reference. The disclosures of U.S. Patent Application Publication No. 2018 / 0282678, International Patent Application No. PCT / EP2018 / 076354, U.S. Provisional Patent Application No. 62 / 711,070, and U.S. Provisional Patent Application No. 62 / 725,545 are also incorporated by reference herein.

[0002] Technical Field The present disclosure relates generally to cell culture technology, and more specifically to modular bioreactors, bioreactor systems, and related methods. [Background technology]

[0003] Bioreactors are commonly used for cell culture. They often contain complex housing configurations made of multiple parts that require excessive welding or gluing to assemble. This can increase manufacturing complexity, risk of failure, and ultimately cost to the end user.

[0004] An additional problem concerns the ability to maximize cell density in the cell growth region of the bioreactor. Many past proposals for fixed-bed bioreactors have used packed beds. While such packed beds can be effective in promoting cell growth and can provide certain advantages, the volume of space within the bioreactor required to create such beds is large. Bioreactors with unstructured packed or fluidized beds are also difficult to easily scale while achieving desirable cell growth, and there is currently a demand for bioreactors that can be utilized in a variety of operating conditions in the field (including, for example, in a sterile hood where clearance may be limited).

[0005] Cell culture can be costly in terms of both time and resources for a given process. If such efforts fail or in suboptimal conditions for the given process, it can result in a waste of time and resources, or at least a suboptimal use of available resources (including researcher time, physical components, various materials involved in the given process, etc.). For this reason, small-scale runs can be useful for purposes such as process development, experimental design, and evaluation of the viability of a given process or optimization of its parameters. If such evaluations were to be performed on a larger scale, more resources would be used each time a given process is run for experimental purposes.

[0006] Furthermore, evaluating process parameters in a single bioreactor system may require extra time to run experiments serially or additional physical resources to control multiple separate single bioreactor systems. For example, evaluating the effect of a change in a given parameter (temperature, pH, DO, oxygenation / kLa, etc.) or a given set of parameters on a given process may require significant time for serial runs to vary that parameter or significant resources and space to run the process simultaneously with different parameters to optimize the process.

[0007] Thus, there is a recognized need for improved bioreactors that are easy and inexpensive to manufacture and can be easily scaled to accommodate a variety of operating conditions. Bioreactors can be assembled in a highly reproducible manner. The associated bioreactor systems can also facilitate scale-up once optimal conditions and / or parameters are determined. Summary of the Invention

[0008] In a first embodiment, an apparatus for culturing cells is disclosed, the apparatus comprising a bioreactor including a housing having walls forming an internal compartment, a fixed bed for culturing cells, a fixed bed support for the fixed bed adapted to be removably positioned within the internal compartment of the housing, and one or more positioners for uniformly spacing the fixed bed support from the walls of the housing.

[0009] In this or other embodiments, the fixed bed support includes a central portion and a peripheral portion extending radially outward from the central portion and including one or more positioners, the peripheral portion having an outer diameter corresponding to an inner diameter of the housing. The peripheral portion may include one or more protrusions. The peripheral portion may include an outer ring connected to the central portion via one or more protrusions.

[0010] In this or other embodiments, the periphery comprises an annular, disk-shaped surface having a plurality of openings. The periphery may comprise a mesh or screen. The periphery may be adapted to support a fixed bed from below. The periphery may be adapted to allow a fluid to pass therethrough.

[0011] In another aspect of this or other embodiments, the central portion can include a separator forming a plurality of spaces within the central portion for accommodating the conduits within the housing. The plurality of spaces can vary in size. At least one of the conduits can be adapted to direct a fluid flow to or from the interior of the central portion. The separator can be configured to position at least one of the conduits adapted to direct a fluid flow away from an interior wall of the central portion to prevent contact between the conduit and a falling film of liquid flowing down the interior wall.

[0012] In another aspect of this or other embodiments, the central portion of the support forms a vessel for housing an agitator. The agitator may include an impeller adapted to rotate around an agitator support adapted to receive and hold the fixed bed support. The agitator support may include a tubular post that connects to a flexible drain connected to the lid of the bioreactor.

[0013] In another aspect of this or other embodiments, the peripheral portion of the fixed bed support can include one or more protrusions in the form of a plurality of radially extending arms. The plurality of radially extending arms can be connected to a rim having an outer diameter corresponding to an inner diameter of the housing. The plurality of radially extending arms can engage and support the fixed bed.

[0014] In a further aspect of this or other embodiments, the housing includes a receptacle for receiving at least one of the one or more protrusions. The central portion and the peripheral portion of the fixed bed support can comprise a single, integral structure.

[0015] In another aspect of this or other embodiments, the fixed bed includes one or more layers of woven or nonwoven material wrapped around a central portion of the fixed bed support.

[0016] In another aspect of this or other embodiments, the apparatus may further include a seal for sealing between the inner wall of the housing and the fixed bed support.

[0017] In another aspect of this or other embodiments, the fixed bed comprises a plurality of fixed bed sections, and the fixed bed support further comprises a plurality of interlocking support sections for supporting each of the plurality of fixed bed sections. Each interlocking support section may be adapted to interlock with an adjacent support. The apparatus may further comprise a first seal for sealing each of the adjacent interlocking support sections to one another. The apparatus may further comprise a second seal for sealing each of the plurality of sections with an inner wall of the housing.

[0018] In another aspect of this or other embodiments, the apparatus further comprises a top frame for placement above the fixed bed. The top frame may be of sufficient height to form at least one pocket that allows fluid from a top end of the fixed bed to accumulate therein, and a sensor to sense a property of the fluid in the at least one pocket. The top frame may be adapted to engage a lid of the bioreactor. The top frame may form a plurality of pockets having different volumes. The top frame may be adapted to receive or align one or more samplers for sampling the fixed bed.

[0019] In a further aspect of this or other embodiments, the apparatus further includes a lid removably connected to the housing. The lid may be adapted to hold the fixed bed vertically within the housing. The lid may include a depending portion for engaging the fixed bed or a fixed bed support. The lid may be adapted for threaded engagement with the housing. The apparatus may further include a gasket between the lid and the housing.

[0020] In another aspect of this or other embodiments, the device can include a port in the wall of the housing above the upper end of the fixed bed when the device is placed within the fixed bed.

[0021] In another aspect of this or other embodiments, the housing may comprise a unitary rigid structure that defines an interior compartment.

[0022] In another embodiment of the present disclosure, an apparatus for culturing cells is disclosed, the apparatus comprising a housing and a lid that together define a container having an internal compartment, and an assembly for placement within the internal compartment, the assembly including a fixed bed adapted for culturing cells, the assembly adapted to interface with the container to maintain the position of the fixed bed within the internal compartment.

[0023] In one aspect of this or other embodiments, the assembly includes a top adapted to interface with the lid. The top may include a top frame for placement above the fixed bed. The top frame may be of sufficient height to form at least one pocket for accumulating fluid from a top end of the fixed bed and a sensor for sensing a property of the fluid in the at least one pocket. The top frame may include a recess for engaging a protrusion extending from the lid of the bioreactor. The top frame may form a plurality of pockets, each of the plurality of pockets having a different volume.

[0024] In another aspect of this or other embodiments, the fixed bed comprises a plurality of fixed bed sections, each fixed bed section associated with one of the plurality of supports adapted to couple with an adjacent support. The upper frame may be configured to couple with at least one of the plurality of supports. Each of the plurality of supports comprises a central portion and a peripheral portion adapted to allow fluid to reach the fixed bed, the peripheral portion having an outer diameter corresponding to an inner diameter of the housing.

[0025] In another aspect of this or other embodiments, the device may further include at least one O-ring between at least two of the plurality of supports or between the lid and at least one of the plurality of supports. When the lid is attached to the housing, the lid may be adapted to apply a downward pressure to the assembly. The downward pressure may be sufficient to maintain the at least one O-ring in place without the use of an adhesive.

[0026] In a further aspect of this or other embodiments, the assembly may include a lower portion for housing the agitator, the lower portion adapted to couple with at least one support for supporting the fixed bed.

[0027] In a third embodiment of the present disclosure, an apparatus for culturing cells is described, the apparatus comprising a bioreactor including a housing having walls forming an internal compartment, a fixed bed for culturing cells, and a support for the fixed bed adapted to be removably positioned within the internal compartment, the support including one or more central projections at least partially forming a chamber including an agitator and extending towards the wall of the housing.

[0028] In one aspect of this or other embodiments, the projection engages the fixed bed.

[0029] In another aspect of this or other embodiments, the housing includes one or more receivers for receiving the one or more protrusions. Engagement between the one or more protrusions and the one or more receivers may be adapted to prevent rotation of the support within the housing.

[0030] In a fourth embodiment, an apparatus for culturing cells is disclosed that includes a housing having walls forming an internal compartment, a fixed bed for culturing cells in the internal compartment, one or more probes extending into the internal compartment adjacent to the fixed bed or into the fixed bed, and an upper frame overlying the fixed bed for holding the fixed bed and organizing the one or more probes.

[0031] In one aspect of this or other embodiments, the upper frame includes one or more indicia to indicate the position or orientation of the one or more probes.

[0032] In another aspect of this or other embodiments, one or more probes are attached to an upper frame.

[0033] In a fifth embodiment, an apparatus for culturing cells is disclosed that includes a one-piece housing, at least one fixed bed for culturing cells, and a plurality of fixed bed supports adapted to be coupled for placement within the one-piece housing.

[0034] In one aspect of this or other embodiments, each of the plurality of fixed bed supports comprises an annular portion and a support frame extending radially outward from the annular portion. The support frame may have an outer diameter corresponding to an inner diameter of the housing. The support frame may comprise a generally planar extension. The support frame may be adapted to allow fluid flow therethrough. The support frame may extend from a bottom of the annular portion. The support frame may comprise a plurality of radially extending arms connected to a peripheral ring. The support frame includes a mesh or screen. The support frame may be adapted to support at least one fixed bed from below. The support frame may be adapted to serve as a base for placing the fixed bed on the fixed bed support.

[0035] In another aspect of this or other embodiments, each fixed bed support includes a projection or receiver for interlocking with a corresponding projection or receiver on an adjacent fixed bed support.

[0036] In a sixth embodiment, an apparatus for culturing cells is disclosed, the apparatus comprising: a housing having walls forming an internal compartment; a fixed bed for culturing cells; an annular fixed bed support supporting the fixed bed, the fixed bed support adapted to be removably positioned within the internal compartment of the housing and defining a peripheral chamber between the annular fixed bed support and the housing; an impeller for circulating a fluid through the fixed bed in the peripheral chamber; and an impeller support extending at least partially through the impeller to center the impeller within the housing, the impeller being attached to the impeller support by a snap-fit ​​connection.

[0037] In a seventh embodiment, a method of manufacturing a fixed bed bioreactor is disclosed, the method comprising connecting one or more fixed bed supports into a single housing.

[0038] In one aspect of this or other embodiments, the linking step includes interconnecting a first fixed bed support to a second fixed bed support.

[0039] In another aspect of this or other embodiments, the method further includes disposing an impeller within a portion of the first fixed bed support.

[0040] In another aspect of this or other embodiments, the coupling step includes coupling the second fixed bed support with a lid covering the housing.

[0041] In a further aspect of this or other embodiments, the method further includes forming a first seal between the first fixed bed support and the second fixed bed support.

[0042] In another aspect of this or other embodiments, the method further includes forming a second seal between the second fixed bed support and the housing.

[0043] In another aspect of this or other embodiments, the method further includes wrapping a fixed bed around each fixed bed support.

[0044] In an eighth embodiment, a bioreactor for culturing cells is disclosed. The bioreactor includes a housing having a wall defining an internal compartment, a plurality of fixed beds for culturing cells, and a plurality of annular fixed bed supports. Each of the plurality of fixed bed supports is adapted to support at least one of the plurality of fixed beds respectively. Each of the plurality of fixed bed supports includes an annular section and a support frame extending radially from the annular section. The support frame has an outer diameter corresponding in size to an inner diameter of the wall of the housing, and the support frame is adapted to support at least one of the plurality of fixed beds from below such that a fluid flows through the support frame. The plurality of fixed bed supports are adapted to be connected to each other to form peripheral chambers between the plurality of annular fixed bed supports and the wall of the housing, and to form a central chamber within the annular section.

[0045] The bioreactor further includes a lid connected to the housing for enclosing the plurality of fixed beds and the plurality of fixed bed supports in the internal compartment, a plurality of probes extending into the internal compartment adjacent to at least one of the fixed beds or into the fixed beds, and an upper frame overlying the plurality of fixed bed supports and forming a plurality of pockets within which fluid exiting upper ends of the plurality of fixed beds can accumulate, wherein at least one of the plurality of probes is adapted to sense a property of a fluid within a respective one of the plurality of pockets.

[0046] The bioreactor further includes an impeller for circulating fluid within the bioreactor and a vessel for housing the impeller. The vessel includes a plurality of openings adapted for fluid flow from within the vessel to the peripheral chamber and a plurality of positioners in the form of arms extending radially therefrom adapted to position the vessel within the housing and space the vessel from a wall of the housing. The upper frame is adapted to couple to at least one of the plurality of annular fixed bed supports and to couple to the lid to prevent relative rotation therebetween.

[0047] In a ninth embodiment, a bioreactor for culturing cells is disclosed. The bioreactor includes a housing having a wall defining an internal compartment, a removable fixed bed for culturing cells, and a removable fixed bed support adapted to support the fixed bed. The fixed bed support is annular in shape and includes a plurality of arms extending radially outward, the radially extending arms defining an outer diameter sized to correspond to an inner diameter of the wall of the housing, and for positioning and centering the fixed bed support within the housing. The plurality of arms are configured to support the fixed bed from below. The fixed bed support forms a peripheral chamber between the outer wall of the fixed bed support and the housing, and also forms a central chamber within the fixed bed support. The fixed bed is adapted to be positioned within the peripheral chamber. The housing includes one or more receivers in a wall of the housing for receiving at least one of the plurality of arms, the one or more receivers adapted to support the fixed bed support within the internal compartment and prevent relative rotation of the fixed bed support within the housing.

[0048] The bioreactor further includes a lid connected to the housing for sealing the fixed bed and fixed bed support within the internal compartment, and at least one probe extending into the internal compartment at a position above the fixed bed in the peripheral chamber.

[0049] The bioreactor further includes an impeller configured to rotate on the impeller support, the impeller circulating a fluid within the bioreactor. The impeller is disposed within a chamber formed between a lower portion of the fixed bed support and the floor of the housing. The impeller is adapted to circulate a fluid from a central chamber of the fixed bed support outwardly to a peripheral chamber and upwardly through the fixed bed therein.

[0050] The bioreactor further includes a discharge line connected to the impeller support for discharging liquid from the bioreactor. [Brief description of the drawings]

[0051] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0052] [Figure 1] FIG. 1 is a perspective view of a first embodiment of a bioreactor according to one aspect of the present disclosure.

[0053] [Figure 1A] FIG. 1A is a cross-sectional view of the bioreactor of FIG.

[0054] [Diagram 2] FIG. 2 is an exploded view of the bioreactor of FIG.

[0055] [Diagram 3] FIG. 3 shows a spiral fixed bed that can be used in conjunction with a bioreactor.

[0056] [Figure 3A] FIG. 3A shows certain details of the helical fixed bed. [Figure 3B] FIG. 3B shows certain details of the helical fixed bed. [Figure 3C] FIG. 3C shows certain details of the helical fixed bed.

[0057] [Figure 3D] FIG. 3D shows an alternative arrangement for forming a structured fixed bed. [Figure 3E] FIG. 3E shows an alternative arrangement for forming a structured fixed bed. [Figure 3F] FIG. 3F shows an alternative arrangement for forming a structured fixed bed. [Figure 3G] FIG. 3G shows an alternative arrangement for forming a structured fixed bed.

[0058] [Figure 4] FIG. 4 shows a vessel housing the agitator within the bioreactor of FIG. [Figure 4A] FIG. 4A shows a vessel housing the agitator within the bioreactor of FIG.

[0059] [Figure 4B] FIG. 4B shows an impeller assembly for use in a bioreactor according to one embodiment of the present disclosure. [Figure 4C] FIG. 4C shows an impeller assembly for use in a bioreactor according to one embodiment of the present disclosure.

[0060] [Diagram 5] FIG. 5 is an exploded perspective view of a fixed bed and a support for the fixed bed according to one embodiment of the present disclosure.

[0061] [Figure 6] FIG. 6 is a perspective view of the support with fixed bed of FIG.

[0062] [Figure 6A] FIG. 6A is a bottom view of the support of FIG.

[0063] [Figure 7] FIG. 7 is a side view of the bioreactor of FIG.

[0064] [Figure 7A] FIG. 7A is a cross-sectional view taken along line 7A-7A of FIG.

[0065] [Figure 8] FIG. 8 is a perspective view of a frame for providing upper support for a fixed bed according to one embodiment of the present disclosure.

[0066] [Figure 9] FIG. 9 is a perspective cross-sectional view of the bioreactor of FIG.

[0067] [Figure 9A] FIG. 9A is an enlarged view of a portion of the bioreactor of FIG.

[0068] [Figure 10] FIG. 10 is a partial cross-sectional view of a further embodiment of a bioreactor according to another aspect of the present disclosure.

[0069] [Figure 10A] FIG. 10A is an exploded perspective view of the bioreactor of FIG.

[0070] [Figure 11] FIG. 11 is an exploded perspective view of a further embodiment of a bioreactor according to another aspect of the present disclosure.

[0071] [Figure 11A] FIG. 11A is a top view of the bioreactor of FIG.

[0072] [Figure 11B] FIG. 11B is a cross-sectional side view of the bioreactor taken along line 11B-11B of FIG. 11A.

[0073] [Figure 12] FIG. 12 is another side view of the bioreactor of FIG.

[0074] [Figure 12A]FIG. 12A is a cross-sectional view taken along line 12A-12A of FIG.

[0075] [Figure 13] FIG. 13 is a cross-sectional view of the lower portion of the bioreactor of FIG.

[0076] [Figure 14] FIG. 14 is a cross-sectional view of the bioreactor of FIG.

[0077] [Figure 15] FIG. 15 is a top view of the bioreactor of FIG.

[0078] [Figure 15A] FIG. 15A is a cross-sectional view taken along line 15A-15A of FIG.

[0079] [Figure 15B] FIG. 15B is an enlarged partial view of FIG. 15A.

[0080] [Figure 16] FIG. 16 is a flow chart showing the steps for assembling a bioreactor according to the present disclosure.

[0081] [Figure 17] FIG. 17 is a schematic diagram of the bioreactor system.

[0082] [Figure 18] FIG. 18 is a schematic diagram of the cell density sampling locations in the bioreactor.

[0083] [Figure 19] FIG. 19 shows the time course of pH and DO parameters in the bioreactor.

[0084] [Figure 20] FIG. 20 shows the time course of metabolite levels in bioreactors of different sizes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] Reference is now made to Figures 1, 1A, and 2, which together show one embodiment of a bioreactor 100 for culturing cells according to one aspect of the present disclosure. The bioreactor 100 includes an exterior case or housing 112, which is shown as transparent in Figure 1 to allow the internal structure to be appreciated. The housing 112 forms an interior compartment in which cell culture can be completed using various components or techniques, as further outlined in the following description.

[0086] According to one aspect of the present disclosure, the housing 112 may, in some embodiments, form a vessel that includes a unitary or monolithic structure, such as a pot or bucket having an open top. Providing such a vessel may eliminate the cost and complexity of fastening multiple pieces together, such as by welding or using adhesives, to form the housing 112. Additionally, such a structure may eliminate the need for airtight seals associated with the body of the housing 112, eliminating the potential for leakage and / or contamination and improving the integrity of the bioreactor.

[0087] This one-piece housing 112 may be manufactured using injection molding techniques, 3D printing, or other methods that result in no seams to minimize exposure to contamination. In some applications, the housing 112 may be translucent or transparent. In other applications, the housing 112 may be opaque and made of any material, although plastic is preferred to allow for disposable placement if desired.

[0088] A cover or lid 114 can be placed over the open top of the housing 112 to cover or seal its interior compartment. In one embodiment, the lid 114 is designed for easy removal, such as by being secured in place by interlocking engagement with the housing 112 (including possible friction or bayonet fit), although removable fasteners such as interlocking tabs and / or clips, clamps, and / or screws can also be used. This allows for easier opening of the bioreactor 100 and may eliminate the need for a sampler (which tends to increase costs and may be difficult to implement, especially in smaller vessels, given size constraints). The combination of the housing 112 and lid 114 forms a vessel that houses the remaining elements of the bioreactor.

[0089] The lid 114 includes various openings or ports P with removable closures or caps C to allow selective introduction or removal of materials, fluids, gases, probes, sensors, samplers, etc., providing flexibility in design. In particular, the lid 114 may include a holder 114b for receiving a suitable sensor (e.g., temperature, capacitance, dielectric constant, biomass, metabolites such as glucose or lactate, pressure, flow measurement, liquid level, pH or DO probes, etc.). As best shown in FIG. 1A, an internal connector 114c for a conduit or tube forms part of the lid 114. The lid 114 may further be provided with a corresponding connector 114d for a media extraction tube T. A removable cap 114e with a suitable seal, such as an O-ring, allows auxiliary access if required, as shown in FIGS. 1 and 9A. A sampling port for receiving a sampler, such as in the form of a probe, may also be optionally provided in the lid 114.

[0090] Within the internal compartment formed by the housing 112, several compartments or chambers receive and deliver the flow of fluids, gases, or both throughout the bioreactor 100. As shown in FIG. 1A, the chambers may include a first chamber 116 at or near the base of the bioreactor 100. In some embodiments, this first chamber 116 may include an agitator for inducing fluid flow within the bioreactor 100. In some embodiments, the agitator may take the form of a "drop-in" rotatable, non-contact magnetic impeller 118, thereby forming a centrifugal pump within the bioreactor 100. Instead of such an impeller 118, the agitator may be in the form of a stir bar, an external pump forming part of a fluid circulation system, or any other device for inducing fluid circulation within the bioreactor.

[0091] Agitation causes the fluid to flow upward (as shown by arrow V in FIG. 1A) into a second chamber, which may be a peripheral chamber 120 formed on and extending along the exterior or periphery of the bioreactor 100. Alternatively, the bioreactor 100 may be adapted to allow fluid to flow in the opposite direction.

[0092] In some embodiments, the bioreactor 100 is adapted to accommodate a cell culture bed 122 in any form, including a packed bed, a fixed bed, a structured fixed bed, a fluidized bed, etc. For example, FIG. 3 shows a fixed bed 122 in the form of a structured spiral bed, which, in use, can accommodate and hold growing cells. In some embodiments, the spiral bed can be in the form of a cartridge that can be dropped or placed into the peripheral chamber 120, and can be part of a support that forms such a cartridge (and can be adapted to interface with additional modular structures, as further outlined in the following description). The bed 122 is either pre-attached to the housing 112 during manufacturing at the facility before being shipped to the end user, or is attached at the point of use by the end user.

[0093] Fluid leaving the second peripheral chamber 120 is directed to a headspace formed by an upper chamber 121 on one (upper) side of the bed 122, where it is exposed to a gas (such as oxygen). The fluid then flows radially inward to a third central chamber 126 and back to the lower part of the bed 122. In some embodiments, this central chamber 126 may be essentially cylindrical and may be formed by one or more non-perforated conduits or tubes 128 (which may include annular portions of multiple fixed bed supports, each containing a portion of a fixed bed, as described in further detail below), and the flow may be such that a waterfall-like arrangement is formed. The central chamber 126 returns fluid that falls or otherwise enters it to the first base chamber 116 (arrow R indicates the return path) and recirculates it through the bioreactor 100, achieving a continuous loop ("bottom to top" in this version, but this can be reversed or otherwise modified without departing from the disclosure).

[0094] As can be best seen from the exploded view of FIG. 2, a top frame 130, such as a spider, may also be provided. The top frame 130 may be positioned above the top end of the fixed bed 122, e.g., below and adjacent to the lid 114. The top frame 130 may serve to organize structures extending from the lid 114 into or adjacent to the fixed bed 122, such as one or more probes 132 in communication with port P. In one embodiment, the probes or other structures extending from the lid may contact or be attached to a portion of the top frame 130. In another embodiment, the probes or other structures may be spaced from the top frame to avoid contact with the top frame. These probes 132, if present, may be used to sense conditions within and / or obtain samples from the bioreactor 100, and particularly the fixed bed 122, to determine cell growth characteristics, if desired, without removing the lid 114.

[0095] 3 illustrates one embodiment of a matrix material for use as a structured fixed bed 122 in a bioreactor of the present disclosure, particularly a spiral bed. In some embodiments, one or more cell immobilization layers 122a may be adjacent to one or more optional spacer layers 122b, which may include a woven or nonwoven mesh structure. In some embodiments, the layering may be optionally repeated multiple times to achieve a stacked or layered configuration.

[0096] The mesh structure included in the spacer layer 122b forms a tortuous path for cells (see cells L suspended or trapped within the material of the stationary layer 122a in FIG. 3A, and cell culture may form part of the invention herein) and fluids to flow through the channels thus created when layered between the two stationary layers 122a. By using this type of arrangement, cell uniformity is maintained within the structured fixed bed. In some embodiments, other spacer structures that form such tortuous paths can be used.

[0097] As shown in FIGS. 3, 3A, and 3B, the structured fixed bed 122 may be spirally or concentrically wound around an axis or core (e.g., a conduit or tube 128 provided in multiple components). In some embodiments, the layers of the structured fixed bed are tightly wound, but may also be loosely wound. In some embodiments, the diameter of the open center for receiving the tube 128, the length and / or amount of layers will ultimately define the size of the assembly or matrix. In some embodiments, the thickness of each layer 122a, 122b may be between 0.1 and 5 mm, 0.1 and 10 mm, or 0.001 and 15 mm.

[0098] In some embodiments, other structures that form such tortuous paths can be used. For example, FIG. 3D shows that one or more immobilized cell layers 122a can be adapted to form a structured fixed bed 122. The one or more layers 122a provide a tortuous flow channel (arrow B) from a straight or regular inflow (arrow A) without the use of additional spacer layers (although such spacer layers can be used if desired). This can be achieved, for example, by providing layers of woven fibers or filaments 123, 125 that disrupt the flow.

[0099] 3E shows that such a result can be achieved by using a woven or non-woven material as the cell immobilization layer 122a. This can be achieved by forming this layer 122a as a mesh-like arrangement (e.g., 3D printing) with openings 127 through which the liquid can pass and return again, forming tortuous channels that also promote homogeneity and serve to further shear or break up any air bubbles present in the liquid. This function can be achieved with or without the addition of a spacer layer present.

[0100] The orientation of the structured fixed bed 122 may be different than that shown in the bioreactor 100 shown in Figures 1, 1A, and 2, where the flow is arranged vertically (from bottom to top in the example provided - see arrows V and R). For example, as shown in Figure 3F, the bioreactor 100 may include a chamber 120 with a structured fixed bed 122 formed of one or more horizontally arranged layers of material. The one or more layers may include a woven or mesh-like material, as shown in Figures 3D and 3E, but may also include one or more cell immobilization layers 122a (three are shown, but any number may be present) sandwiched between adjacent spacer layers 122b, as shown in Figure 3F (the vertical spacing is exaggerated for illustrative purposes). Thus, the flow is arranged side-to-side (left to right or right to left), and the layers of material (such as spacers) provide channels to create a meandering flow (arrow B) from a straight or regular inflow (arrow A). Pumping action may be provided by an agitator or other pump at the inlet end of the chamber 120, with a return path provided at the outlet end, as shown diagrammatically by path R. If desired, additional spacer layers may extend between the cell immobilization layers 122a.

[0101] In another possible embodiment, referring to FIG. 3G, the structured fixed bed 122 includes a three-dimensional (3D) monolithic matrix 124 in the form of a scaffold or lattice formed of a plurality of interconnected units or objects 124a having a surface for cell adhesion. Preferably, the matrix includes a tortuous path through which fluids and cells pass during use. In these or other embodiments, the matrix may be in the form of a 3D array, lattice, scaffold, or sponge. In either case, the matrix 124 may be essentially disposable to avoid the costs and complexities associated with cleaning according to bioprocessing standards.

[0102] 1, 1A, 4, and 4A, the bioreactor 100 of this first exemplary embodiment can include a support for supporting a fixed bed. In one form, the support can include a vessel 140 for housing an agitator, such as an impeller 118, in an interior compartment of the housing 112. The vessel 140 can be adapted to receive fluid through a central opening 142 and expel fluid radially outwardly through one or more openings 144 (e.g., four at 90 degree intervals) as a result of the movement (rotation) of the agitator, such as the impeller 118.

[0103] The vessel 140 may further include one or more outward protrusions that act as positioners to center or evenly space the vessel from the inner wall of the housing 112, but are not attached to the vessel. For example, the vessel 140 along the top may include one or more radially extending arms 140a. These arms 140a may be adapted to align or center the vessel within the housing 112 when the vessel is placed on a surface, e.g., on the floor, of the housing 112 of the bioreactor 110. The arms 140a may be on the vessel 140, but the arms may be attached to the inner wall of the housing 112 and extend toward the vessel, but are not attached to the vessel, allowing for easy removal.

[0104] 4A and 4B show that the impeller 118 is configured to rotate around a support in the form of a tubular post 148. In one embodiment, the tubular post 148 may be attached to the bottom of the housing 112. In another embodiment, the tubular post 148 may be removable from the housing 112. The top of the post 148 may include one or more vanes 150 and connect to a conduit 151. The conduit 151 may include flexible tubing and may be used to supply gas to the vessel 140 or as an outlet to remove fluids therefrom. In the latter case, the bottom of the post 148 adjacent the floor may be serrated or provided with openings 148a to allow for fluid flow. As shown in FIG. 1A, the conduit 151 may be connected at the opposite end or top end to the lid 114 and in fluid communication with one of the ports P in the lid.

[0105] 4B, a lower end of the post 148 can be disposed within a central opening 118a of the impeller 118. The post 148 includes a first or upper stop, such as a flange 148b, that limits the distance that an associated conduit 151 can travel when coupled to the upper end of the post. A second, lower stop, such as a flange 148c, may provide an upper limit for the travel of the impeller 118. These stops are considered optional and may take shapes other than a flange as shown.

[0106] A bearing assembly 162 can support the impeller 118 to facilitate low friction rotation. In one example, the bearing assembly 162 can include a base 162a including a race 162b for receiving a bearing 164, such as a ball bearing, a cylindrical bearing, a bushing material, or other bearing element adapted to facilitate relative movement between the impellers. As can be appreciated, the impeller 118 can include a similar but inverted race 118a that forms a compartment when connected to the base 160a, such as by a snap-fit ​​engagement. Referring to FIG. 4C, an example of a snap-fit ​​engagement between a protrusion, shown as a retractable protrusion 119 of the impeller 118, and a groove in the post 148 is shown.

[0107] The impeller 118 includes a compartment with a cover 118b that houses one or more magnets 118c. The cover 118b may be removable or may be permanently fixed in place to seal the compartment with the one or more magnets.

[0108] Base 162a may also include an opening 162c for receiving a portion of post 148, which allows post 148 to reach the floor of bioreactor 100 in container 140. Base 160a may also include an opening 160d for allowing fluid to enter base 160a and be drawn into post 148, such as when negative pressure is applied to form a drain, or to allow fluid to be expelled therefrom when fluid is provided under pressure to post 148 (such as via conduit 151).

[0109] 5 and 6, a fixed bed assembly 149 may be inserted into the interior compartment of the housing 112, and is shown disassembled in FIG. 5 and assembled in FIG. 6. The fixed bed assembly 149 may include a support 152 for supporting the fixed bed 122 (either the entire fixed bed or a portion thereof, e.g., where multiple portions of the fixed bed are arranged in a stacked configuration). The support 152 includes a one-piece or unitary structure including a central member, shown as annular portion 154, that forms a hollow annular wall corresponding to the non-perforated conduit or tube 128 of FIG. 1A, and around which, in one example, the material that forms the fixed bed 122 is spirally wrapped.

[0110] The support 152 may further include an outer portion, such as a support frame 156 for supporting the fixed bed 122 from below in the illustrated arrangement. The support frame 156 may have an extension that extends radially outward from the annular portion 154, through which fluid may flow, for example, upwardly toward the lower (inlet) end of the fixed bed 122. The support frame 156 is located at the bottom of the annular portion 154 and provides a level at which a user can wrap the fixed bed 122 around the annular portion 154 or in other locations.

[0111] As shown, the support frame 156 comprises multiple radially extending portions including arms 156a connected to a rim or peripheral ring 156b. In other embodiments, the support frame 156 may include an annular shelf or disk that may include one or more openings. In some embodiments, the support frame 156 may include a mesh or screen. In each of these embodiments, the support frame 156 may serve the dual function of supporting the fixed bed 122 while simultaneously allowing fluid to flow through the peripheral chamber to the fixed bed.

[0112] The outer diameter of the support frame 156 corresponds to the inner diameter of the housing 112, such that the two structures are directly adjacent to, and may touch, but are not connected to one another (so that the fixed bed assembly 149 can be freely inserted into and removed from the interior compartment of the housing 112). As such, the support frame 156 can function as a positioner or spacer to position or center the fixed bed assembly 149 within the housing 112.

[0113] In another embodiment, the support frame 156 can serve as a base or reference point for placing the fixed bed 122 on the support 152. For example, if the fixed bed 122 is in the form of a sheet or fabric that is wrapped around the annular portion 154 during use, the support frame 156 can serve as a level on which at least one end of the fabric may rest during the wrapping process. Specifically, if the fixed bed 122 is of the type shown in FIG. 3 (or a similar material or combination of materials that can be wrapped or wrapped around a central core), the support 152 is placed on a surface with the support frame 156 facing down, the fabric or other material that forms the fixed bed 122 is placed so that one end of the material contacts the support frame 156, and the material that forms the fixed bed 122 is wrapped around the annular portion 154 using the support frame 156 as a guide.

[0114] Alternatively, if the fixed bed 122 is pre-fabricated or pre-formed prior to placement on the support 152 (e.g., an annular disk or series of disks) that is simply inserted over the annular portion 154, the support frame 156 can provide a stop or floor upon which the pre-fabricated or pre-formed fixed bed 122 rests.

[0115] The annular portion 154 also includes a separator 158 that divides the interior of the annular portion 154 into two (or more) portions 158a, 158b. These portions 158a, 158b may be of the same size and shape or may be of different sizes and shapes. In any case, these portions 158a, 158b may receive, guide, and hold in place any tubes or conduits within the interior compartment, such as a conduit 151 for directing fluid flow to or from a tubular post 148 associated with the vessel 140. In one embodiment, the separator 158 may be adapted to position the tubes or conduits away from the inner wall of the annular support. In this manner, a tube adapted to sample or remove a portion of the liquid within the bioreactor may be held away from the inner wall to prevent it from siphoning or removing the liquid from a falling film of liquid flowing down the inner wall.

[0116] A seal such as an O-ring 160 may be provided to seal the annular portion 154 with an adjacent structure such as another support 152 when the stack is formed. As a result, stacking multiple fixed bed assemblies 149 within the housing may form a substantially fluid impermeable or non-porous conduit or tube 128. This may include sealing with the vessel 140 when present within the bioreactor 100. This may be accomplished by a peripheral seal in the form of an O-ring 159 to seal between the supports 152. In particular, the seal or O-ring 159 may seal the peripheral ring 156b with the inner wall of the housing 112 (see FIGS. 1A and 9A, which show such an O-ring 159 fitting into a recess in the ring 156b and also in contact with the top surface of the fixed bed 122 and the inner wall of the housing 112).

[0117] When formed into an optional stack, the fixed bed supports 152 may be adapted to interlock, which for purposes of this disclosure means to connect without the need for welding or adhesives to facilitate easy assembly and disassembly. The interlocking pieces may be connected in a manner that limits at least one degree of movement between the interlocking pieces. This may include limiting relative rotation between the interlocking pieces and / or limiting horizontal or vertical movement between the interlocking pieces. For example, they may include one or more protrusions received in one or more receptacles to prevent relative movement.

[0118] Specifically, as shown in Figure 6, the support 152 may include a protrusion 154a, such as on the top end of the annular portion 154. Similarly, the support 152 may include a corresponding receiver 154b on the underside or elsewhere on the annular portion 154. When two adjacent supports 152 are stacked, the protrusion 154a and receiver 154b interlock to align the parts and prevent relative rotation. As can be appreciated, the positions of the protrusion 154a and receiver 154b can be reversed with similar effect.

[0119] Returning to FIG. 4, the container 140, which serves as a support for the fixed bed 122 within the housing 112, may include a corresponding projection 140d. Similar to projection 154a, this projection 140d may interface with a receiver 154b on the fixed bed support 152. In this manner, relative alignment and retention is achieved for adjacent fixed bed supports 152, as well as for additional fixed bed supports (which may include any number) forming a stack within the housing 112, as shown in FIG. 1. A seal, such as an O-ring 157, may provide a seal between the fixed bed support 152 and the container 140, which may also include a seating shelf 140c for receiving and engaging the support (see FIG. 4A).

[0120] 7, 7A and 8, further details of the upper frame 130 are shown. The upper frame 130 may include an inner member in the form of a central ring 130a connected to an outer member in the form of a ring 130c. The connection may include a number of arms 130b extending radially between the rings 130a, 130c. As can be seen, each pair of adjacent arms 130b defines an opening to allow fluid to pass therethrough, such as from a lower outlet end of an underlying fixed bed 122 associated with the support 152.

[0121] The middle ring 130a may have a shape and diameter that corresponds to the shape and diameter of the annular portion 154. Similarly, the outer ring 130c may correspond to the inner diameter of the housing 112. An O-ring 157 provides a seal between the middle ring 130a and the upper end of the adjacent fixed bed support 152 (see exploded view in FIG. 2).

[0122] As best shown in Figures 8, 9, and 9A, the top frame 130 may also include features to ensure proper placement or positioning and may further serve to provide a retention function. For example, the outer ring 130c may also include a positioning feature in the form of a receiver 130d to mate with a corresponding positioning feature on the lid 114, such as a protrusion 114a (or receiver) to ensure proper alignment. The top frame 130 may also include a receiver 130e (or protrusion) to engage with a protrusion 140b of the next adjacent support 152 of the fixed bed. In this manner, the lid 114 may secure the top frame 130, the support 152, and the vessel 140 (which also functions as a support) in an interlocking manner to facilitate assembly and disassembly of the bioreactor 100.

[0123] The top frame 130 may be dimensioned to have sufficient height / depth to hold a volume of liquid above the fixed bed 122 before exiting the fixed bed 122 and flowing into the chamber 126 formed by the non-perforated conduit or tube 128 established by the annular portion 154 of the support 152. The fluid wells or pockets thus created provide a stable environment to receive probes / sensors that may be placed in the central chamber 126 and may be subject to turbulence or require further insertion to access the liquid therein (the corresponding chamber may not be completely filled). As can be seen in FIG. 7A, these wells or pockets formed by the top frame 130 may vary in size or volume or have the same size and volume depending on the application.

[0124] 7A and 8 together, the number of openings formed by the arms 130b corresponds to the number of structures passing from the lid 114 to the fixed bed 122, e.g., only one structure (probe) occupying each opening. Thus, as can be seen in FIG. 7A, multiple openings along a portion of the upper frame 130 may receive samplers or probes 132, while other openings may not receive such structures. To facilitate insertion and alignment during manufacturing of the bioreactor 100, the upper frame 130 may include indicia, such as arrows 130f, to identify the proper location of the samplers / probes 132, etc., and ensure alignment with the corresponding ports P of the lid 114.

[0125] Another example of a bioreactor 200 is shown in Figures 10 and 10A. In this version, the bioreactor 200 is similar in structure and arrangement to the larger bioreactor 100 described above, but in a smaller and simplified configuration. The structural elements and terminology used to describe the bioreactor 200 may be the same as or overlap with the structural elements, terminology, and reference numbers of the bioreactor 100 described above, but may differ in some respects, as outlined in the following description (such differences are applicable to all disclosed embodiments).

[0126] In one example, the bioreactor 200 may include a housing 212 in which a fixed bed assembly 249 (not shown, but note the peripheral chamber 220 in which the fixed bed may reside, as further outlined below) including a support 252 for the fixed bed may be placed. The housing 212 may be a one-piece or monolithic structure, such as a container in the form of a bucket or pot. In such a case, the housing 212 may be manufactured using injection molding techniques such that there are no seams, thereby eliminating exposure to leaks and contamination compared to multiple piece constructions. In some applications, the housing 212 may be translucent or transparent. In other applications, the housing 212 may be opaque and formed of any material, although inexpensive plastics are preferred, especially when made as disposable structures.

[0127] The support 252 can define a peripheral chamber 220 between an inner surface of a wall of the housing 212 and an outer surface of a wall of the support 252. As shown, the housing 212 can be cylindrical and the support 252 can be annular. As such, the peripheral chamber 220 can also be annular in shape and have a fixed bed disposed therein (the fixed bed can take any shape, including those described and illustrated herein).

[0128] The support 252 may include one or more positioners in the form of protrusions 234. As shown, the protrusions 234 may comprise radial arms (which in a shortened form may be considered tabs) that extend toward the inner wall of the housing 212 and may engage but not connect with the inner wall of the housing 212 to facilitate separation. Alternatively, the protrusions 234 may be part of the inner wall of the housing 212 and extend from the inner wall of the housing 212 toward the support 252, but are not attached to the support 252, further ensuring the desired spacing between these components.

[0129] In one embodiment, the bioreactor may include an agitator, such as a magnetic impeller or stir bar 218. The agitator may be disposed within a base chamber 216 formed by the support 252 and a bottom wall 252a of the floor or base of the housing 212. As shown, the bottom wall 252a of the interior compartment of the support 252 may include a central opening 242. The central opening 242 may fluidly connect the central chamber 226 of the support 252 with the chamber 216. The chamber 216 may further include one or more sidewalls with openings or apertures that form a fluid pathway with the peripheral chamber 220. The support 252 may include a sidewall of the chamber 216.

[0130] Thus, during operation, a stirrer such as stir bar 218 may be rotated by an externally applied force via a non-contact (e.g., magnetic) coupling. The stirring draws fluid into the central chamber 226, through the central opening 242, and into the base chamber 216. The fluid is then forced out of that opening, passes over or near one or more projections 234, and into the peripheral chamber 220, which contains the fixed bed. Fluid exiting the peripheral chamber 220 passes over or above the top of the support 252 back into the central chamber 226, and finally flows back into the base chamber 216 for recirculation. One or more vanes 250 or baffles may also be optionally included in the central chamber 226 to prevent the formation of vortices.

[0131] The bioreactor 200 may further include removable threads on the lid 214 for sealing the open top of the housing 112. A seal, such as an O-ring 260, may extend between the lid 214 and the housing 212. Instead of threads on the lid, the lid 214 may be secured to the housing 212, such as by fasteners F (screws, bolts, clamps, clips, tabs, etc.), a fitted lip, or other attachment mechanism for removably securing the lid in place, thereby allowing the bioreactor 200 to be easily opened while still ensuring that it is sealed to prevent contamination during use.

[0132] One advantage of easily separating the lid 214 and the housing 212 is that it allows easy access to the interior of the bioreactor 200, such as before, during, or after cell culture. As one specific example, an easily openable lid 214 provides the advantage of allowing the user to monitor cell density (homogenization of colony formation) within the fixed bed. In the specific context of DoE studies, an easily openable lid 214 allows, among other things, the simultaneous inoculation of multiple bioreactors 200 and then serially "sacrificing" one bioreactor at a time to assess cell density (e.g., one per day) and establish cell kinetic growth patterns. This easy-open feature facilitates such DoE studies in ways that a more permanent connection between the lid and the housing would not allow, and allows access to the fixed bed structure to study uniformity of cell distribution and direct access to the cells trapped therein (even in non-sterile conditions).

[0133] The lid 214 may be provided with one or more sensor holders 214b. These sensor holders 214b may include one or more disposable sensors for sensing parameters of the bioreactor 200. In another embodiment, the sensor holder 214b may be adapted to receive a reusable sensor for sensing a parameter of the bioreactor 200.

[0134] The lid 214 may further include one or more connectors 214c for connecting to conduits or tubes in the support 252, such as for supplying culture medium or other additives to the bioreactor 200. An additional connector 214d may be for connecting to an extraction tube T. One or more removable caps may seal the one or more connectors. The lid 214 may optionally include one or more sampling ports 214e.

[0135] 10A, the bioreactor 200 (or bioreactor 100 or other disclosed versions) may include an external temperature regulator. This regulator may take the form of a heating (or cooling) jacket, such as a blanket 262. A heat conductor, such as a metal (e.g., aluminum) vessel 264, may facilitate heat exchange between the regulator and the bioreactor 200. In one embodiment, the blanket 262 and vessel 264 are part of a system, rather than part of the single-use bioreactor 200.

[0136] 11, 12, 13, 14, 15 and associated portions, as outlined below, another example of a bioreactor 300 is shown. As in the above examples, bioreactor 300 may be similar in structure and arrangement to bioreactor 100 and / or bioreactor 200 described above. The structural elements and terminology used to describe bioreactor 300 may be the same as or overlap with structural elements, terminology, and reference numbers of bioreactors 100 and / or 200 described above, but may differ in some respects, as outlined in the following description (such differences being applicable to all disclosed embodiments).

[0137] The bioreactor 300 may include a housing 312 that includes a support 352. In one example, the housing 312 may be a one-piece, unitary, or monolithic vessel such as a canister, bucket, pot, or the like. In such a case, the housing 312 may be manufactured using injection molding techniques to eliminate seams and the associated occurrence of leaks or contamination. In some applications, the housing 312 may be translucent or transparent. In other applications, the housing 312 may be opaque and manufactured of any material, although plastic is preferred when disposability is desired.

[0138] 11A and 11B, the support 352 can define a peripheral chamber 320 between an inner surface of the housing 312 and an outer surface of the support 352. As shown, the housing 312 can be circular in cross section and the support 352 can be annular in shape. As such, the peripheral chamber 320 can also be annular in shape. The peripheral chamber 320 can be adapted to receive a fixed bed 322. The fixed bed 322 can be any type of fixed bed mentioned herein above.

[0139] As can be seen in FIG. 11, as well as in FIG. 12 and FIG. 12A, the support 352 can be positioned within the housing 312 by one or more positioners, such as protrusions or arms 346. These arms 346 may extend radially outward from the bottom of the support 352. In one embodiment, three such arms 346 are spaced approximately equidistant from one another around the circumference of the support 352. These arms 346 serve to center the support 352 and keep the distance between the support 352 and the wall of the housing 312 constant, thereby at least partially defining the size of the peripheral chamber 320. In another aspect, the arms 346, as best seen in FIGS. 11 and 15A, can support the fixed bed 322 (shown transparent for clarity in FIG. 11) from below while allowing fluid to flow through the peripheral chamber and the fixed bed 322 therein.

[0140] The housing 312 may include a receiver 348 adapted to engage at least one arm 346. The receiver may be located at a bottom of the housing 312 to correspond to the position of the arm 346 on the support 352. The receiver 348 may be in the form of a shelf, ledge, bracket, pocket, slot, recess, or other element adapted to receive and / or support and support the arm 346 or arms 346, and thereby the support 352, within the body of the housing 312. The receiver 348 may also take the form of a circumferential lip extending along the inner surface of the housing 312. Alternatively, the positions of the arm 346 and receiver 348 may be reversed, with the arm connected to the housing 312 and the receiver 348 connected to the support 352, but remaining removably connected to one another.

[0141] In one embodiment shown in FIG. 12A , the receiver 348 may comprise a plurality of separate receivers, such as a plurality of shelves or ledges, spaced apart from one another and adapted to engage the plurality of arms 346. The receiver 348 may include one or more stops 349 adapted to engage the arms 346 and limit the arms 346 from rotating upon engagement. For example, the stops 349 may include one or more side walls associated with the receiver 348. As such, the receiver 348 may form a pocket or recess adapted to receive the arms 346 and prevent the support 352 from rotating within the housing 312. In such a case, the arms 346 may function to (1) maintain a space between the support 352 and the wall of the housing 312 (e.g., to keep the dimensions of the fluid chamber 320 constant), (2) maintain the support 352 at a desired height within the housing 312, and / or (3) prevent rotational movement of the support 352 within the housing 312.

[0142] In another embodiment, the positioner or arm 346 can serve as a base or reference point for the positioning of the fixed bed 322 on the support 352. For example, if the fixed bed 322 is in the form of a sheet or fabric that is wrapped around the support 352 during use, the arm 346 can serve as a level on which at least one end of the fabric rests during the wrapping process. Specifically, if the fixed bed 322 is of the type shown in FIG. 3 (or a similar material or combination of materials that can be wrapped or wrapped around a central core), the support 352 is placed on a surface with the arm 346 facing down and the remainder of the support 352 facing up, and the fabric or other material forming the fixed bed 322 is positioned so that one end of the material contacts the arm 346, and the material forming the fixed bed 322 can be wrapped around the annular portion of the support 352 using the arm 346 as a guide.

[0143] Alternatively, if the fixed bed 322 is pre-fabricated or pre-formed prior to placement on the support 352 (e.g., an annular disk or series of disks) that is simply inserted into the annular portion of the support 352, the arm 346 can provide a stop or floor upon which the pre-fabricated or pre-formed fixed bed 322 can rest.

[0144] In one embodiment, the bioreactor 300 may include an agitator such as a magnetic impeller 318. The magnetic impeller 318 may be in the form of a "drop-in" rotatable non-contact magnetic impeller 318, thereby forming a centrifugal pump within the bioreactor. In one embodiment, the impeller 318 includes one or more magnetic caps that may be attached (e.g., glued) to ensure that the magnets are sealed to the impeller. The agitator may also be in the form of an impeller mechanically coupled to a base, an external pump forming part of a fluid circulation system, or other device for causing fluid circulation within the bioreactor. As shown, the impeller 318 may include a body including curved walls or vanes adapted to draw in and expel fluid radially outward therefrom.

[0145] The magnetic impeller 318 can be disposed within a base chamber 316. In one aspect, the base chamber 316 can be at least partially defined by a bottom wall of the support 352 and a bottom wall of the housing 312. The base chamber 316 can be in fluid communication with an outer chamber 320. In one aspect, the arm 346 extends between the base chamber 316 and the outer chamber 320.

[0146] The holder holds the impeller 318 in place, which in the illustrated example takes the form of a post 370. The post 370 may be adapted to support and maintain the position of the impeller 318 within the base chamber 316. The post 370 may include a base 372 adapted to support at least a portion of the impeller 318 from below. The post 370 may include a race, which may be adapted to receive a bearing 374 to facilitate rotation of the impeller 318. The bearing 374 may include one or more ball bearings, cylindrical bearings, bushing material, or other bearing elements adapted to facilitate relative movement between the impeller 318 and the post 370. In one embodiment, the impeller 318 may include a race or a portion of a race for the bearing 374, as shown in FIG. 13 (see also FIG. 4B for bioreactor 200, but equally applicable to bioreactor 300). In one aspect of the present disclosure, post 370 is tubular or hollow so that it can function as a conduit for introducing and withdrawing fluids into and from bioreactor 300, particularly via chamber 316.

[0147] 13, the support 352 may include a central opening 342. The central opening 342 may connect the central chamber 326 of the support 352 to the base chamber 316. A post may be adapted to pass through the central opening 342 of the support 352, thereby allowing the impeller 318 to be positioned and centered relative to the support 352.

[0148] The post 370 may include a stop 378 to limit the insertion distance through the central opening 342. For example, the stop 378 may include a shoulder, lip, detent, protrusion, or other element that engages the support 352 to prevent the post 370 from passing further through the central opening 342. As shown, the stop 378 includes a first portion of the post 370 having a diameter larger than the diameter of the central opening 342. A second portion of the post 370 has a diameter smaller than the diameter of the central opening 342 to allow it to pass through the central opening 342 and into the central chamber 326.

[0149] The stop 378 can be positioned a distance from the base 372 to allow the height of the impeller 318 to fit therebetween. In one example, as shown, the distance from the base 372 to the stop 378 is sufficiently greater than the height of the impeller 318 to suspend the impeller 318 therebetween and provide at least some clearance between the impeller and the bottom wall of the support 352. In one embodiment, the post 370 extends below the bottom of the impeller 318 to suspend the impeller 318 above the floor of the housing 312 when the bioreactor 300 is fully assembled.

[0150] 12A and 13, the lower wall of the support 352 may include one or more fluid openings 380. Each fluid opening 380 may be adapted to pass fluid from the central chamber 326 to the impeller chamber 316 for distribution to the fluid chambers 320. The fluid openings 380 may be equally spaced and distributed around the central opening 342. As shown, the bioreactor 300 includes three fluid openings separated from each other by separators 382, ​​however, any number of fluid openings may be present and any means of providing such openings in the support 352 may be utilized so long as uniform fluid flow is promoted. The separators 382 may extend from a peripheral portion of the support 352 to a central body 384 of the support 352 and may receive the posts 370.

[0151] During operation, the impeller 318 rotates and can draw fluid from the central chamber 326 through the fluid openings 380 and into the chamber 316. Fluid can be pushed into and out through the peripheral chamber 320 between the support 352 and the housing 312. After the fluid rises through the peripheral chamber 320 (which may include a fixed bed 322), the fluid may pass through or through the top of the support 352 and back into the central chamber 326 for recirculation. Although not shown in this bioreactor embodiment, one or more vanes or baffles can prevent the fluid from forming vortices during recirculation (see, e.g., vanes 260 of bioreactor 200).

[0152] The support 352 may be a unitary structure, such as one that can be injection molded. As can be seen from the above, this unitary support 352 can simultaneously function as a pump housing or vessel, a fixed bed support, a vortex prevention structure, form a central chamber for receiving a flow (such as a falling film from chamber 220), and prevent movement of all associated components within the housing 312.

[0153] 11 and 12, and with reference to FIG. 15, the housing 312 may include one or more ports 360 to allow for measurements of characteristics of the bioreactor 300. For example, the ports 360 may be adapted to house or hold sensors adapted to gather information about the bioreactor and / or measure characteristics of the bioreactor, such as, but not limited to, pH, dissolved oxygen (DO), temperature, cell density, flow rate, metabolite levels, media or media component levels, and other parameters. In one aspect, the sensors may be optical sensors.

[0154] In one embodiment, the port 360 may be positioned to communicate with the peripheral chamber 320, as shown in Figure 14. More specifically, the port 360 may be positioned at the top of the peripheral chamber 320. Even more specifically, the port 360 may be positioned within the peripheral chamber 320 at a location above the level of the fixed bed 322. Positioning the port 360 in this location places the port, and thus the sensor, in a location within the fluid that would have passed through the fixed bed 322 under normal flow conditions.

[0155] The port 360 may include a support section, such as a tube or shelf, to support at least a portion of the weight of the sensor. In one embodiment, the port 360 may include a cylindrical hole that allows the sensor direct access to the interior of the bioreactor 300. In another embodiment, the port 360 may be sealed from the interior of the bioreactor but allow a non-contact sensor, such as an optical sensor, to sense a parameter inside the bioreactor. The port 360 may include a lock to secure the sensor in place relative to the port. The lock can maintain a seal between the interior of the bioreactor 300 and the outside environment. For example, the port 360 may include a luer lock or other component to allow the sensor to sense a parameter inside the bioreactor 300 while maintaining the sterility of the bioreactor 300.

[0156] The bioreactor 300 may further include a lid 314 for sealing the bioreactor 300. The lid 314 may be removably secured to the housing 312, such as by a threaded connection, as shown in FIG. 14. Alternatively, the lid 314 may be attached to the housing 312 via one or more fasteners, such as screws, bolts, clamps, clips, tabs, fit lips, or other attachment mechanisms that allow the bioreactor 300 to be easily opened. The housing 312 and lid 314 combined may comprise a container that houses the remaining elements of the bioreactor.

[0157] One advantage of using an easy-open connection between the lid and the housing is that it allows easy access to the interior of the bioreactor 300, e.g., during and after an experiment is performed. For example, in the case of a fixed-bed bioreactor, an easy-open lid has the advantage that it may facilitate monitoring of cell density within the fixed bed (homogenization of colony formation) by direct measurement. In the context of DoE studies, an easy-open lid allows for the simultaneous inoculation of multiple bioreactors 300 and then sequentially "sacrificing" one bioreactor at a time (e.g., one per day) to establish kinetic growth patterns of cells over time. This easy-open feature facilitates such DoE studies in a way that is not possible with a more permanent connection between the lid and the housing.

[0158] A seal, such as an O-ring 315, may establish a fluid-tight connection between the lid 314 and the housing 312. In some embodiments, the O-ring 315 and / or the portion of the lid 314 or housing 312 adapted to contact the O-ring 315 may be crinkled to facilitate a grip between the elements. In some aspects, the O-ring 315 and / or the portion of the lid 314 or housing 312 adapted to contact the O-ring 315 may be smooth to facilitate a proper seal between the elements.

[0159] 11 and 11A, the lid 314 may include one or more connectors or ports P. These ports P may be adapted to connect to one or more conduits, such as for adding or removing medium or other additives, or products, or waste products, to or from the bioreactor 300. In some circumstances, one or more removable caps including appropriate seals may be provided to seal the port or ports. These ports may include one or more of a first port 390a for adding medium, a second port 390b for removing medium, a third port 390c for adding gas, a fourth port 390d for removing gas, a fifth port 390e for inserting or holding a probe, such as a temperature probe, and a sixth port 390f for adding a pH adjuster, such as a base.

[0160] In some embodiments, port 392 allows a user to drain bioreactor 300. Port 392 can be connected to a conduit 394, as shown in FIGS. 11B and 14. In one embodiment, conduit 394 can be fluidly connected to post 370. In such an embodiment, post 370 can comprise a tube or other hollow space to which conduit 394 can be connected. Post 370 can include a base 372 with an opening for draining the bioreactor 300 or, when operated in reverse, for introducing fluids into bioreactor 300.

[0161] The conduit 394 may serve the additional function of maintaining the relative positions between the parts of the bioreactor 300. Specifically, the conduit 394 extends along the post 370 to prevent it from being pulled out of the central opening 342 of the support 352. For example, with reference to FIG. 14, the lower end of the conduit 394 may be positioned along the post 370, such as at a contact point with the support 352 surrounding the central opening 342. In some embodiments, a connector or other support (not shown) may connect the conduit 394 to the post 370, such as a collar. In other embodiments, the conduit 394 is sized to prevent the conduit from dislodging by an interference or friction fit. As such, the conduit 394 may trap or pinch the bottom wall of the support 352 between the conduit 394 and the stop 378 of the post. This maintains the relative positions of the conduit 394, the post 370, and the impeller 318, preventing these elements from dislodging after assembly.

[0162] To facilitate assembly of the bioreactor 300, the length of the conduit 394 may be longer than the height of the bioreactor 300 or longer than the distance between the bottom of the support 352 and the lid 314 (in the assembled state). For example, the conduit 394 may be 10 to 60% longer than the height of the bioreactor 300. In one embodiment, the height of the bioreactor 300 is 50 mm and the length of the conduit 394 may be 55 to 80 mm. In this way, the post 370 can pass the impeller 318 by the bearing 374. The post 370 can extend through the central opening 342 of the support 352 until the stopper 378 contacts the underside of the support 352. The conduit 394 can be connected to the post 370 from the top to prevent vertical movement relative to the support 352.

[0163] As can be appreciated, the assembly including the conduit 394, support 352, post 370, bearing 374, and impeller 318 can be inserted into the housing 312 as a single unit. This can occur simultaneously with or prior to inserting the fixed bed 322 into the housing 312. Once inserted into the housing 312, the other end of the conduit 394 can be connected to the port 392 and the lid 314 can be attached to the housing 312.

[0164] In other embodiments, once the conduit 394, support 352, post 370, bearing 374, and impeller 318 are inserted into the housing 312, the excess length of the conduit 394 may be extended through a hole or opening in the lid 314 in which the port 392 is located. The excess length of the conduit 394 may be cut to a length within about 10 mm of the level of the lid 314. The free end of the conduit 394 may be attached to the port 392, which may then be pressed into the hole or opening or secured to the lid 314 using an adhesive (e.g., a UV adhesive), welding, or other fastening means, etc.

[0165] 15A and 15B, the bioreactor 300 may be adapted to maintain the position of the support 352 within the bioreactor 300. As shown, a depending portion of the lid 314, such as a vertical leg 398, may extend downwardly from the lid 314. The leg 398 may be coupled to or disposed adjacent to the support 352. The leg 398 may also be of a length corresponding to the distance from the lid 314 to the top of the annular wall of the support 352, particularly when the lid is fully seated on the housing 312 as a result of the threaded connection used in the illustrated embodiment.

[0166] Thus, when the bioreactor 300 is assembled with the lid 314 attached to cover the open top of the housing 312, the legs 398 may be adjacent to and in contact with the support 352. In either case, this prevents the support from moving vertically a significant distance or from floating within the bioreactor 300. As shown, the legs 398 may include a substantially flat lower end for engaging the support 352. However, the legs 398 may include one or more extensions, fingers, grips, clips, brackets, or other structures for engaging the support 352 and preventing the support 352 from rising vertically within the bioreactor 300. The legs 398 may also be connected to the support 352 instead of engaging the lid 314.

[0167] In other embodiments, instead of or in addition to the legs 398, an upper frame as shown in FIG. 8 and described above can be positioned between the lid 314 and the support 352 to maintain the position of the support 352 within the housing.

[0168] As described in the previous embodiment, the bioreactor 300 may be equipped with a temperature controller, such as a heating (or cooling) blanket and / or heat conductor as described herein. Similarly, the bioreactor 300 may be used in the same manner and for similar purposes as described herein with respect to the bioreactor 200. In another aspect, the bioreactor 300 may be similar in construction to the bioreactor 100 described above, but on a smaller scale. The more similar the construction of the bioreactor 300 and the larger bioreactor 100, the more likely it is that parameters optimized or determined in the small scale bioreactor will produce similar results for said process in the larger bioreactor 100.

[0169] 16 illustrates, in flow chart form, exemplary steps for assembling the bioreactor 100 according to a first embodiment of the present disclosure. If not pre-assembled, the method may include step 400 of attaching a magnet to the body of the impeller 118. Step 402 may include connecting the post 148 and bearing to the impeller 318 and vessel 140. The next step 404 may include placing an O-ring on the vessel 140 and placing it in the housing 112. If present, the conduit 151 connects to the vessel 140 in step 406.

[0170] In step 408, the method further includes forming a fixed bed assembly 149, which, if spiral or wound, may include winding the fixed bed material onto the support 152 and placing an O-ring 157 onto the vessel 140. In step 410, the fixed bed assembly 149 may be placed within the housing 112 and coupled to the vessel 140. If multiple stacks are desired, in step 414 an O-ring 159 may be placed onto the fixed bed 122 of the first fixed bed assembly and steps 408, 410, 412 may be repeated as necessary.

[0171] In step 414, an O-ring 157 is associated with the top fixed bed assembly 149 in the stack to form a seal, and the top frame 130 couples with this fixed bed assembly 149. In step 416, a sampler or probe can be inserted in a desired orientation through the top frame 130 into the top fixed bed assembly 149. If present, a conduit 151 can be connected to the lid 114 and coupled and sealed with the top frame 130, as shown in step 418.

[0172] For bioreactors 200 and 300, similar steps can be performed with modifications as necessary based on different configurations (e.g., the illustrated embodiment of bioreactors 200, 300 provides only one fixed bed, so O-rings and repeated assembly steps can be omitted). In any of the disclosed methods, steps may be implemented or performed by different people or parties and may be performed in a different order than shown. Additionally, not all steps need be performed and the order of steps may vary.

[0173] In a further embodiment, referring to FIG. 17, it may be advantageous to provide a system S that includes multiple bioreactors 500, such as any of the bioreactors disclosed herein, simultaneously. In one aspect, a single controller may control aspects of each of the multiple bioreactors. Such simultaneous control may allow for efficient operation or application of one or more different parameters to a given process to optimize conditions for that process. For example, this simultaneous control may allow for control of different cell culture conditions in different bioreactors, including but not limited to pH, DO, temperature, agitation speed, flow rate, etc.

[0174] The system S may allow for testing and / or optimization of operating parameters of a given process in the bioreactor 500. Once the desired operating parameters have been determined, it may be desirable to scale up the process from the small scale bioreactor 200 to a bioreactor capable of carrying out the optimized or desired process with the determined parameters. As such, in a further embodiment of the present disclosure, the small scale bioreactor 200, 300 may be similar in configuration to the large scale bioreactor 100. The more similar the configuration of the small scale bioreactor 200, 300 and the large scale bioreactor 100, the more likely it is that parameters optimized or determined in the small scale bioreactor will result in similar results for said process in the large scale bioreactor 100.

[0175] The bioreactors 500 are connected to a single controller 502 that forms part of a system S, which is shown with eight bioreactors but may include any number of bioreactors greater than one. The controller 502 may include a computer, microprocessor, mobile device, or other control means adapted to monitor and / or regulate conditions within the bioreactor 500. In one embodiment, there may be one or more sensors to monitor one or more environmental conditions or parameters within a given bioreactor. One or more multiplexers allow signals, such as sensor signals and / or control signals, to be sent through a single connector, such as a wire. This may reduce the amount of elements required to monitor (or send and receive control signals for) environmental conditions or parameters within the system.

[0176] The system S is modular and one or more bioreactors 500 can be interconnected to form a system. For example, the system may include a single controller 502 connected to two bioreactors 500. The two bioreactors can function individually or as a pair of connected bioreactors, and additional single bioreactors may be included. In one aspect, one or more additional pairs of bioreactors may be included. The manifold M may be connected to multiple bioreactors 200. The illustrated embodiment of FIG. 16 shows four pairs of bioreactors 500, but the system may include more or fewer bioreactors or pairs of bioreactors.

[0177] The controller 502 may be adapted to monitor and / or control various process parameters in each bioreactor 500. For example, the controller 502 may be adapted to control one or more of temperature, pH, DO, agitation speed, or flow rate in a given bioreactor 500. In one embodiment, the controller 502 is adapted to individually control one or more parameters in at least one bioreactor, which parameters are different from the same parameters in another bioreactor. In a further embodiment, the controller 502 may be adapted to individually control all parameters in each of multiple bioreactors 500 in the system. Thus, a single controller 502 can be adapted to control different sets of parameters in each bioreactor 500. This allows multiple parallel process conditions to be run simultaneously. Such parallel process execution allows multiple parameters to be tested simultaneously, such as optimizing conditions for a given process. One advantage of running such parallel processes is that parallel bioreactors can be run using different parameters for DoE (design of experiments) and can guide the user in scaling up a given culture into a larger bioreactor or acquiring data in an efficient manner.

[0178] Running multiple processes simultaneously can save time, but may not be efficient when run on a large scale. Therefore, in a further embodiment of the present disclosure, each bioreactor 500 can include a small volume bioreactor, such as a bioreactor with a volume of 300 mL or less. In some examples, the working volume of the bioreactor can be any of 250 mL, 200 mL, 150 mL, 100 mL, 80 mL, 50 mL, or less. The use of such small volume bioreactors 500 in the system S allows for efficient use of laboratory space and consumable resources such as media. Furthermore, such small bioreactors 500 will necessarily be smaller in size than larger reactors, and therefore may be less expensive to manufacture and therefore less expensive to use than larger suspension reactors. The use of multiple small bioreactors 500 can thus facilitate efficient process development and DoE studies.

[0179] In further embodiments, the bioreactor 500 may be a disposable bioreactor that may include probes or other sensors for measuring its parameters. In one embodiment, such probes or other sensors may be disposable along with the bioreactor 500. In another embodiment, the bioreactor 500 may be adapted to accept reusable probes or sensors in a sterile manner. The probes or sensors may include one or more sensors for sensing cell density, optical density, pH, DO, temperature, agitation rate, flow rates (including flow rates in the bioreactor, including inflow into the bioreactor, outflow from the bioreactor, and during mixing), or other desired parameters associated with cell culture.

[0180] In another aspect, the use of small-scale bioreactors allows the development and testing of processes that can be efficiently scaled up to larger reactors. Conversely, small-scale bioreactors may be useful for scaling down processes in large-scale bioreactors to understand or characterize the process more efficiently. This ability to scale up and / or scale down may be particularly important in the field of viral vectors, as developers and producers work closely together to build large-scale production capacity in a short period of time.

[0181] To allow for scale-up and / or scale-down between reactor sizes, the miniature bioreactor should be representative of the larger one in terms of structure and / or function. In the case of fixed-bed reactors, the miniature bioreactor can have the same height and / or fixed-bed compaction as the larger one. For similar fixed-bed heights, the fixed-bed diameter of the smaller bioreactor can be smaller to achieve a lower surface area. One way to allow for scalability of fixed-bed reactors is to maintain such structural similarities, which, together with similar oxygenation and mixing strategies, allows for direct process scale-up / down between reactors of different sizes. EXAMPLES

[0182] In a non-limiting example illustrating scalability between small and large bioreactors, two test runs performed in the small bioreactor were used and the results compared to known results in the large bioreactor. The bioreactor conditions used are listed in Table 1.

[0183] [Table 1]

[0184] As a first step, adherent HEK293 cells from a cryopreserved cell bank were thawed. Cells were pre-cultured in T-flasks and passaged every 3 to 4 days (20,000 cells / cm) using DMEM medium (4.5 g / L glucose / L) supplemented with 5% calf serum and 1% antibiotic-antimycotic. 2 and harvested at mid-exponential growth phase).

[0185] The cells were further expanded in Cell Factories (Nunc) before being inoculated into a mini-bioreactor. The cells were then inoculated into a 0.5 ml 2 A smaller bioreactor (Applicant's scale X TM 20,000 cells / cm2 in a culture vessel (e.g., bioreactor) 2 The cells were inoculated with 0.17 ml / cm2 of 0.1% fetal bovine serum and kept in batch mode for 2 to 4 h to allow them to adapt to the new environment, after which they were transferred to a recirculation loop (0.17 ml / cm2) containing growth medium (DMEM (4.5 g / L glucose) supplemented with 5% fetal bovine serum). 2 ) was used to initiate the growth phase. Supernatant samples were taken daily to evaluate glucose and lactate profiles. Harvesting was performed at the end of the experiment to evaluate cell density within the fixed bed.

[0186] Before removing the fixed bed for cell counting, the bioreactor was emptied and rinsed with DPBS solution containing 5 mM EDTA. The fixed bed was then removed from the bioreactor. 2 of surface area were taken at different locations of the fixed bed (north, south, west, east, top and bottom, both inside and outside, see Fig. 18) to estimate cell density and uniformity.

[0187] After each test run, the density and viability of suspended cells from fixed-bed samples were determined by trypan blue exclusion using a Thoma hemocytometer. 2 Biomass estimation of PET samples was performed by acid cell lysis followed by crystal violet staining and cell nuclei counting. The average cell densities reached were 617,032 and 673,932 cells / cm for run 1 and run 2, respectively. 2and the results are summarized in Table 2.

[0188] [Table 2]

[0189] The uniformity of cell distribution was demonstrated through two runs of the mini-bioreactor by cell counts at various locations on the fixed bed. These cell count results are shown below in Table 3. At the end of the culture, the maximum difference observed compared to the cell density across the fixed bed was low, only slightly exceeding 15% at one location.

[0190] [Table 3]

[0191] Additionally, as shown in Figure 19, the pH and dissolved oxygen (DO) trends maintained good stability throughout the entire run of the mini-bioreactor. There were expected peaks and changes in pH and DO during phase chances (e.g., equilibration and inoculation), but overall the levels remained remarkably stable throughout the run. pH adhered perfectly to the setpoint and stabilized between 7.2 and 7.4. DO never reached below 100%, indicating that the cell growth oxygenation limit of the bioreactor was never approached.

[0192] Additionally, the various metabolites measured in the small bioreactors during the test runs were compared to metabolites present during similar runs in larger reactors (e.g., Scale-X Hydro (2.4 m2), Carbo (10 m2) products of the applicant). As shown in FIG. 20, the trends in glucose consumption between the various reactors are comparable during similarly timed runs. Additionally, lactate production in the various reactors also showed similar trends.

[0193] The small-scale bioreactor 500 can be easily adapted with a fixed bed structure up to 0.5 m 2A successful proof of concept has been achieved showing that it is possible to create a small-scale commercial bioreactor of 1000m2. Testing has demonstrated that the same performance as larger bioreactors in this range can be reliably achieved in the small-scale version. Data on cell growth, cell distribution and metabolite behavior are comparable to the same process run in the larger bioreactor. Demonstrating that direct scalability can be maintained opens up the possibility of new small-scale systems for more efficient and lower-cost process development, process optimization and scale-down studies. The currently envisaged range of bioreactors is 1000m2 with a growth surface of 2.4m2. 2 600m from 2 However, this study shows the prospect of expanding the range by keeping the minimum size to about one-fifth of the larger versions, reducing the development costs, operations and time associated with running miniature bioreactors.

[0194] In summarizing various aspects to which this disclosure may pertain, the following items are identified and can be arranged in any combination:

[0195] Item 1. A device for culturing cells, comprising: a bioreactor including a housing having a wall forming an internal compartment; A fixed bed for culturing cells; a fixed bed support for the fixed bed, the fixed bed support adapted to be removably disposed within the interior compartment of the housing; one or more positioners for uniformly spacing the fixed bed support from the wall of the housing; An apparatus comprising:

[0196] Item 2. The apparatus of item 1, wherein the fixed bed support comprises a central portion and a peripheral portion extending radially outward from the central portion and including one or more positioners, the peripheral portion having an outer diameter corresponding to the inner diameter of the housing.

[0197] Item 3. The device of item 2, wherein the peripheral portion comprises one or more protrusions.

[0198] Item 4. The device of item 2 or item 3, wherein the peripheral portion comprises an outer ring connected to the central portion via one or more protrusions.

[0199] Item 5. The device of any of items 2 to 4, wherein the peripheral portion comprises an annular disk-shaped surface having a plurality of openings.

[0200] Item 6. The device of any of items 2 to 5, wherein the peripheral portion comprises a mesh or screen.

[0201] Item 7. The apparatus according to any of items 2 to 6, wherein the peripheral portion is adapted to support the fixed bed from below.

[0202] Item 8. The device according to any of items 2 to 7, wherein the peripheral portion is adapted to allow fluid to pass therethrough.

[0203] Item 9. The device of any of items 2 to 8, wherein the central portion includes a separator forming a plurality of spaces within the central portion for accommodating the conduits within the housing.

[0204] Item 10. The apparatus of item 9, wherein the plurality of spaces are different sizes.

[0205] Item 11. The device of any of items 9 to 10, wherein at least one of the conduits is adapted to direct a flow of fluid to or from the interior of the central portion.

[0206] Item 12. The device of item 11, wherein the separator is configured to position at least one of the conduits adapted to transmit a fluid flow away from the inner wall of the central portion to prevent contact between the conduit and a falling film of liquid flowing down the inner wall.

[0207] Item 13. An apparatus according to any one of items 2 to 12, wherein a central portion of the support forms a vessel housing an agitator.

[0208] Item 14. The apparatus of item 13, wherein the agitator comprises an impeller adapted to rotate about an agitator support adapted to receive and hold a fixed bed support.

[0209] Item 15. The apparatus of item 14, wherein the agitator support comprises a tubular post that connects to a flexible drain tube connected to the lid of the bioreactor.

[0210] Item 16. The apparatus of item 15, wherein the peripheral portion of the fixed bed support includes one or more protrusions in the form of a plurality of radially extending arms.

[0211] Item 17. The apparatus of item 16, wherein a plurality of radially extending arms are connected to a rim having an outer diameter corresponding to an inner diameter of the housing.

[0212] Item 18. The apparatus of item 16, wherein a plurality of radially extending arms engage and support the fixed bed.

[0213] Item 19. The device of any of items 16 to 18, wherein the housing includes a receiving portion for receiving at least one of the one or more protrusions.

[0214] Item 20. The apparatus according to any of items 2 to 19, wherein the central portion and the peripheral portion of the fixed bed support have a single integral structure.

[0215] Item 21. The apparatus of any of items 1 to 20, wherein the fixed bed comprises one or more layers of woven or nonwoven material wrapped around a central portion of the fixed bed support.

[0216] Item 22. The apparatus of any of items 1 to 21, further comprising a seal for sealing between the inner wall of the housing and the fixed bed support.

[0217] Item 23. An apparatus according to any one of items 1 to 22, wherein the fixed bed comprises a plurality of fixed bed sections, and the fixed bed support further comprises a plurality of linked support sections for supporting each of the plurality of fixed bed sections.

[0218] Item 24. The device of item 23, wherein each interlocking support portion is adapted to interlock with an adjacent support portion.

[0219] Item 25. The apparatus of item 23 or item 24, further comprising a first seal for sealing adjacent interlocking support portions to one another.

[0220] Item 26. The device of item 25, further comprising a second seal for sealing each of the plurality of portions with an inner wall of the housing.

[0221] Item 27. The apparatus of any of items 1 to 26, further comprising an upper frame for placement above the fixed bed.

[0222] Item 28. The apparatus of item 27, wherein the upper frame is of sufficient height to form at least one pocket that allows fluid exiting the upper end of the fixed bed to accumulate therein, and a sensor that senses a characteristic of the fluid in the at least one pocket.

[0223] Item 29. The apparatus of item 27 or item 28, wherein the upper frame is adapted to engage a lid of a bioreactor.

[0224] Item 30. The apparatus of any of items 27 to 29, wherein the upper frame forms a plurality of pockets, the plurality of pockets having different volumes.

[0225] Item 31. The apparatus of any of items 27 to 30, wherein the upper frame is adapted to receive or align one or more samplers for sampling the fixed bed.

[0226] Item 32. The device of any of items 1 to 31, further comprising a lid removably connected to the housing.

[0227] Item 33. The apparatus of item 32, wherein the lid is adapted to hold the fixed bed vertically in position within the housing.

[0228] Item 34. The apparatus of item 32 or item 33, wherein the lid includes a depending portion for engaging a fixed bed or a fixed bed support.

[0229] Item 35. The device of any of items 32 to 34, wherein the lid is adapted for threaded engagement with the housing.

[0230] Item 36. The device of any of items 32 to 35, further comprising a gasket between the lid and the housing.

[0231] Item 37. The apparatus of any of items 1 to 36, further comprising a port in the wall of the housing above the upper end of the fixed bed when the fixed bed is disposed within the housing.

[0232] Item 38. The device of any of items 1 to 37, wherein the housing comprises a unitary rigid structure forming an internal compartment.

[0233] Item 39. An apparatus for culturing cells, a housing and a lid that together define a container having an interior compartment; An apparatus comprising: an assembly for placement within the internal compartment, the assembly including a fixed bed adapted for culturing cells, the assembly adapted to interface with the container to maintain position of the fixed bed within the internal compartment.

[0234] Item 40. The apparatus of item 39, wherein the assembly comprises an upper portion adapted to couple with a lid.

[0235] Item 41. The apparatus of item 40, wherein the upper portion comprises an upper frame for placement above the fixed bed.

[0236] Item 42. The apparatus of item 41, wherein the upper frame is of sufficient height to form at least one pocket in which fluid exiting the upper end of the fixed bed can accumulate, and a sensor for sensing a characteristic of the fluid in the at least one pocket.

[0237] Item 43. The apparatus of item 41 or item 42, wherein the upper frame includes a recess for engaging a protrusion extending from the lid of the bioreactor.

[0238] Item 44. The apparatus of any one of items 41 to 43, wherein the upper frame forms a plurality of pockets, each of the plurality of pockets having a different volume.

[0239] Item 45. An apparatus according to any of items 41 to 44, wherein the fixed bed comprises a plurality of fixed bed sections, each fixed bed section being associated with one of a plurality of supports adapted to connect with adjacent supports.

[0240] Item 46. The apparatus of any of items 41 to 45, wherein the upper frame is adapted to couple to at least one of the plurality of supports.

[0241] Item 47. An apparatus according to any of items 45 to 46, wherein each of the plurality of supports comprises a central portion and a peripheral portion adapted to allow fluid to reach the fixed bed, the outer diameter of the peripheral portion corresponding to the inner diameter of the housing.

[0242] Item 48. The device of any of items 45 to 47, further comprising at least one O-ring between at least two of the plurality of supports or between the lid and at least one of the plurality of supports.

[0243] Item 49. The apparatus of any of items 45 to 48, wherein the lid is adapted to apply downward pressure to the assembly when the lid is attached to the housing.

[0244] Item 50. The apparatus of item 49, wherein the downward pressure is sufficient to maintain the at least one O-ring in place without adhesive.

[0245] Item 51. The apparatus according to any of items 39 to 50, wherein the assembly comprises a lower portion for accommodating the agitator, the lower portion being adapted to couple with at least one support for supporting the fixed bed.

[0246] Item 52. An apparatus for culturing cells, a bioreactor including a housing having a wall forming an internal compartment; A fixed bed for culturing cells; and a support for a fixed bed adapted to be removably positioned within the internal compartment, the support at least partially defining a chamber containing the agitator and including one or more centering projections extending toward a wall of the housing.

[0247] Item 53. The device of item 52, wherein the protrusion engages the fixed bed.

[0248] Item 54. The device of item 52 or item 53, wherein the housing includes one or more receivers for receiving the one or more protrusions.

[0249] Item 55. The device according to any of items 52 to 54, wherein engagement between the one or more projections and the one or more receivers is adapted to prevent rotation of the support within the housing.

[0250] Item 56. An apparatus for culturing cells, a housing having a wall defining an interior compartment; a fixed bed for culturing cells within the inner compartment; one or more probes extending into an interior compartment adjacent to the fixed bed or into the fixed bed; and an upper frame for holding the fixed bed and covering the fixed bed for organizing one or more probes.

[0251] Item 57. The apparatus of item 56, wherein the upper frame includes one or more indicia for indicating the position or orientation of the one or more probes.

[0252] Item 58. The apparatus of items 56 or 57, wherein the one or more probes are attached to the upper frame.

[0253] Item 59. An apparatus for culturing cells, An integrated housing; at least one fixed bed for culturing cells; and a plurality of fixed bed supports adapted to be coupled together for placement within the integral housing.

[0254] Item 60. The apparatus of item 59, wherein each of the plurality of fixed bed supports comprises an annular portion and a support frame extending radially outward from the annular portion.

[0255] Item 61. The apparatus of item 60, wherein the support frame has an outer diameter corresponding to the inner diameter of the housing.

[0256] Item 62. The apparatus of item 60 or item 61, wherein the support frame comprises a generally planar extension.

[0257] Item 63. The apparatus of any of items 60 to 62, wherein the support frame is adapted to allow fluid to pass therethrough.

[0258] Item 64. The apparatus of any of items 60 to 63, wherein the support frame extends from the bottom of the annulus.

[0259] Item 65. The apparatus of any of items 60 to 64, wherein the support frame comprises a plurality of radially extending arms connected to a peripheral ring.

[0260] Item 66. The apparatus of any of items 60 to 65, wherein the support frame comprises a mesh or screen.

[0261] Item 67. An apparatus according to any of items 60 to 66, wherein the support frame is adapted to support at least one fixed bed from below.

[0262] Item 68. An apparatus according to any of items 60 to 67, wherein the support frame is adapted to serve as a base for placing the fixed bed on the fixed bed support.

[0263] Item 69. An apparatus according to any of items 59 to 68, wherein each of the fixed bed supports includes a projection or receiver for connecting with a corresponding projection or receiver on an adjacent fixed bed support.

[0264] Item 70. An apparatus for culturing cells, a bioreactor including a housing having a wall forming an internal compartment; A fixed bed for culturing cells; an annular fixed bed support for supporting the fixed bed, the fixed bed support adapted to be removably disposed within the interior compartment of the housing to define a peripheral chamber between the annular fixed bed support and the housing; an impeller for circulating a fluid through a fixed bed in a peripheral chamber; an impeller support extending at least partially through the impeller to center the impeller within the housing; The apparatus, wherein the impeller is attached to the impeller support by a snap-fit ​​connection.

[0265] Item 71. A method of producing a fixed bed bioreactor, comprising connecting one or more fixed bed supports within an integral housing.

[0266] Item 72. The method of item 71, wherein the step of coupling comprises interconnecting a first fixed bed support to a second fixed bed support.

[0267] Item 73. The method of any of items 71 to 72, further comprising disposing an impeller within a portion of the first fixed bed support.

[0268] Item 74. The method of any of items 71 to 73, wherein the step of connecting includes connecting the second fixed bed support to a lid covering the housing.

[0269] Item 75. The method of any of items 71 to 74, further comprising forming a first seal between the first fixed bed support and the second fixed bed support.

[0270] Item 76. The method of item 75, further comprising forming a second seal between the second fixed bed support and the housing.

[0271] Item 77. The method of any of items 71 to 76, further comprising wrapping the fixed bed around each of the fixed bed supports.

[0272] Item 78. A bioreactor for culturing cells, comprising: a housing having a wall defining an interior compartment; A plurality of fixed beds for culturing cells; a plurality of annular fixed bed supports, each of the plurality of fixed bed supports adapted to support at least one of the respective plurality of fixed beds, each of the plurality of fixed bed supports comprising: An annular portion; a support frame extending radially from the annulus, an outer diameter of the support frame sized to correspond to an inner diameter of the wall of the housing, the support frame configured to support at least one of the plurality of fixed beds from below such that fluid flows through the support frame. a plurality of annular fixed bed supports, the plurality of fixed bed supports being configured to be interconnected to form peripheral chambers between the plurality of annular fixed bed supports and a wall of the housing and to form a central chamber within the annulus; a lid for connecting to the housing and sealing the plurality of fixed beds and the plurality of fixed bed supports within the interior compartment; a plurality of probes extending into at least one of the interior compartments adjacent to or within the fixed bed; an upper frame overlying the plurality of fixed bed supports and defining a plurality of pockets within which fluid may accumulate from upper ends of the plurality of fixed beds, at least one of the plurality of probes adapted to sense a property of the fluid within a respective one of the plurality of pockets; an impeller for circulating fluid within the bioreactor; A vessel for housing an impeller, a plurality of openings adapted to allow fluid to flow from within the vessel to the peripheral chamber; a container comprising: a container; and a plurality of positioners in the form of radially extending arms extending therefrom adapted to position the container within the housing and space the container from a wall of the housing; The bioreactor, wherein the upper frame is adapted to couple to at least one of the plurality of annular fixed bed supports and to couple to the lid to prevent relative rotation therebetween.

[0273] Item 79. A bioreactor for culturing cells, comprising: a housing having a wall defining an interior compartment; a removable fixed bed for culturing cells; a removable fixed bed support adapted to support the fixed bed, the fixed bed support being annular in shape and including a plurality of radially outwardly extending arms, the radially extending arms defining an outer diameter sized to correspond to an inner diameter of a wall of the housing to locate and center the fixed bed support within the housing, the plurality of arms adapted to support the fixed bed from below; the fixed bed support defines a peripheral chamber between an outer wall of the fixed bed support and the housing, and defines a central chamber within the fixed bed support; The fixed bed is adapted to be disposed within the peripheral chamber; the housing includes one or more receivers within a wall of the housing for receiving at least one of the plurality of arms, the one or more receivers adapted to support the fixed bed support within the interior compartment and prevent relative rotation of the fixed bed support within the housing; a lid for connecting to the housing and sealing the fixed bed and the fixed bed support within the interior compartment; at least one probe extending into the interior compartment at a location within the peripheral chamber and above the fixed bed; an impeller adapted to rotate on an impeller support and for circulating a fluid within the bioreactor, the impeller being disposed within a chamber formed between a lower portion of the fixed bed support and a floor of the housing and adapted to circulate a fluid from a central chamber of the fixed bed support outwardly to a peripheral chamber and upwardly through the fixed bed therein; a discharge tube connected to the impeller support for discharging liquid from the bioreactor.

[0274] As used herein, the following terms have the following meanings.

[0275] As used herein, "a," "an," and "the" refer to both singular and plural referents unless the context clearly indicates otherwise. By way of example, "a compartment" refers to one or more compartments.

[0276] As used herein, "about," "substantially," or "approximately" referring to a measurable value such as a parameter, amount, time duration, and the like, is meant to encompass variations of the specified value and of no more than + / -20%, preferably no more than + / -10%, more preferably no more than + / -5%, even more preferably no more than + / -1%, and even more preferably no more than + / -0.1%, to the extent that such variations are appropriate for practice in the disclosed embodiments, provided that the value to which the "about" modifier refers is itself specifically disclosed.

[0277] As used herein, "comprise," "comprising," "comprises," and "comprised of" are synonymous with "include," "including," "includes," or "contain," "containing," or "contains," and are inclusive or open-ended terms, e.g., which specify the presence of what follows the components, and do not exclude or preclude the presence of additional, unlisted components, features, elements, materials, or steps that are known in the art or disclosed herein.

[0278] While various embodiments have been shown and described herein, it will be apparent to one of ordinary skill in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and alternatives will occur to one of ordinary skill in the art without departing from the scope of the present disclosure. For example, although the bioreactor is shown in a vertical orientation, it can be used in any orientation. In any of the foregoing embodiments, any or all of the components of the bioreactor 100, 200, 300 may be provided as disposable or "single-use" components. This eliminates the need for cleaning or resterilization, making them inexpensive to manufacture and use. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of protection under applicable law, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. 1. An apparatus for culturing cells, comprising: a bioreactor including a housing having walls forming an interior compartment; a fixed bed for culturing cells; a fixed bed support for the fixed bed, the fixed bed support adapted to be removably positioned within the interior compartment of the housing; one or more positioners for uniformly spacing the fixed bed support from the wall of the housing; Including, the fixed bed support comprises a central portion and a peripheral portion extending radially outward from the central portion and including the one or more positioners, the peripheral portion having an outer diameter corresponding to an inner diameter of the housing.

2. The device described in claim 1, wherein the peripheral portion has one or more protrusions, and the peripheral portion has an outer ring connected to the central portion via the one or more protrusions.

3. An apparatus as described in either claim 1 or 2, wherein the peripheral portion has an annular disk-shaped surface having a plurality of openings, or the peripheral portion includes a mesh or screen.

4. An apparatus as described in either claim 1 or 2, wherein the central portion includes a separator that forms multiple spaces within the central portion for accommodating conduits within the housing.

5. The device described in claim 4, wherein at least one of the conduits is adapted to direct a fluid flow to or from the interior of the central portion, and the separator is configured to position at least one of the conduits adapted to direct a fluid flow away from an inner wall of the central portion, thereby preventing contact between the conduit and a falling film of liquid flowing down the inner wall.

6. An apparatus as described in either claim 1 or 2, wherein the fixed bed comprises one or more layers of woven or nonwoven material wrapped around the central portion of the fixed bed support, or the fixed bed is contained within a peripheral chamber of the bioreactor and formed of one or more horizontally arranged layers of material, or the fixed bed comprises a three-dimensional (3D) monolithic matrix in the form of a scaffold or lattice formed of a plurality of interconnected units or objects having a surface for cell adhesion.

7. An apparatus as described in either claim 1 or 2, further comprising a seal for sealing between the inner wall of the housing and the fixed bed support.

8. An apparatus as described in either claim 1 or 2, wherein the fixed bed has a plurality of fixed bed portions, and the fixed bed support further includes a plurality of connecting support portions for supporting each of the plurality of fixed bed portions.

9. The device described in claim 8, further comprising a first seal for sealing adjacent connecting support portions from each other, and a second seal for sealing each of the plurality of portions with the inner wall of the housing.

10. An apparatus as described in either claim 1 or 2, further comprising an upper frame for positioning above the fixed bed, the upper frame being of sufficient height to form at least one pocket in which fluid exiting the upper end of the fixed bed can accumulate, and a sensor for sensing the characteristics of the fluid in the at least one pocket.

11. The method of claim 1, further comprising:

3. The apparatus of claim 1, wherein the lid includes a depending portion for engaging the fixed bed or the fixed bed support.

12. The device of claim 11, further comprising a gasket between the lid and the housing.

13. An apparatus as described in either claim 1 or 2, wherein the housing has an integral rigid structure forming the internal compartment.

14. A method for producing a fixed-bed bioreactor, comprising: coupling one or more fixed bed supports into an integrated housing, said coupling step including interconnecting a first fixed bed support to a second fixed bed support and coupling said second fixed bed support with a lid covering said housing; The method further includes wrapping a fixed bed around each of said fixed bed supports.

15. A system for testing and / or optimizing operating parameters for culturing cells, comprising: The system comprises: - a plurality of devices according to any of claims 1 and 2, each device comprising a bioreactor; a controller adapted to monitor and / or control various process parameters in the bioreactors, said process parameters being selected from temperature, pH, dissolved oxygen (DO), agitation rate, or flow rate, and adapted to individually control all process parameters in each of a plurality of bioreactors in the system; Including, the system.

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