Apparatus and method for perfusion culture

Abstract

A bioreactor vessel includes: a first compartment configured to receive a suspension including cells and a cell culture medium for use in a cell processing operation; a second compartment for receiving an overflow of the cell culture medium from the first compartment; and an overflow section separating the first compartment from the second compartment, the overflow section being configured to maintain a level of the cell culture medium in the first compartment.

Description

Technical Field

[0001] Embodiments of the present invention generally relate to bioprocessing equipment, systems, and methods, and more particularly to a bioreactor vessel and a perfusion method for the bioreactor vessel. Background Art

[0002] A variety of containers, devices, components, and unit operations are known for performing biochemical processes and / or biological processes and / or manipulating the liquids and other products of such processes. Such biological processes can be used, for example, in the manufacture of cell immunotherapies (such as chimeric antigen receptor (CAR) T cell therapy, which redirects a patient's T cells to specifically target and destroy tumor cells). As is known in the art, the manufacture of cell immunotherapies such as CAR T cell therapy can involve the extraction, activation, genetic modification, culture, and expansion of cells in one or more bioreactor vessels.

[0003] Recent advances in the manufacture of cell immunotherapies have enabled the automation of many bioprocess steps. For example, the activation, genetic modification, and / or expansion of cell populations can be performed in an automated or semi-automated manner without significant human operator intervention. U.S. Provisional Application No. 62 / 736,144 discloses an example of a functionally closed automated system for cell culture (and particularly for use in the manufacture of CAR T cell therapy), which is hereby incorporated by reference in its entirety. As disclosed therein, the transfer and handling of fluids to and from (multiple) bioreactor vessels (including the addition and removal of various cell cultures, inocula, media, reagents, wash buffers, etc.) in precise volumes, rates, times, and durations is an important consideration in many bioprocess operations, including the production of cells and cell-derived products for a variety of applications.

[0004] Perfusion, also known as continuous cell culture, is a type of fluid transfer process that is commonly used in static culture vessels as well as stirred-tank and shake-flask bioreactors, such as during the cell expansion phase. Perfusion involves providing a steady source of fresh cell culture medium to a bioreactor vessel and constantly removing waste products and / or used (i.e., spent) medium from the bioreactor vessel. Traditionally, medium perfusion has involved using two coordinated pumps, one pump to draw the "used" medium to waste at a certain defined rate and another pump to supply fresh replacement medium at the same rate. For the operation of such a system, it is crucial that the net rate of addition of the two fluids is equal. However, existing systems require the use of some type of reaction control logic to ensure that equal volumes of medium are added and removed from the bioreactor vessel simultaneously, making the system overall more expensive, complex, and difficult to operate than other systems that employ different culture techniques. Additionally, a filter on the perfusion outlet line is typically necessary to ensure that cells remain in the bioreactor vessel during perfusion and to prevent cells from being suctioned into the waste.

[0005] In view of the foregoing, there is a need for a bioreactor vessel that allows perfusion to be performed without the complex sensors and control logic heretofore necessary to ensure that equal volumes of medium are added and removed from the bioreactor vessel, and for a perfusion method that utilizes such a bioreactor vessel and that is simpler than existing methods. Summary of the Invention

[0006] In an embodiment, a bioreactor vessel includes: a first compartment configured to receive a suspension including cells and cell culture medium for use in a cell processing operation; a second compartment for receiving an overflow of the cell culture medium from the first compartment; and an overflow portion separating the first compartment from the second compartment, the overflow portion being configured to maintain a level of the cell culture medium in the first compartment.

[0007] In another embodiment, a bioprocessing system includes a bioreactor vessel having a bottom, a top, and a plurality of sidewalls that define an inner chamber; a weir that extends upwardly from the bottom, the weir and at least one of the plurality of sidewalls defining a first compartment within the inner chamber and a second compartment within the inner chamber, the first compartment for holding a suspension comprising cells suspended in a cell culture medium, the second compartment for receiving an overflow of spent culture medium from the first compartment; an inlet associated with the first compartment; and an outlet associated with the second compartment. The bioprocessing system further includes a first pump in fluid communication with the inlet for pumping additional cell culture medium from a culture medium reservoir through the inlet into the first compartment of the bioreactor vessel. The outlet is configured to allow spent cell culture medium to flow out of the second compartment simultaneously or nearly simultaneously with the pumping of the additional cell culture medium into the first compartment.

[0008] In yet another embodiment, a method for bioprocessing includes the steps of introducing additional cell culture medium into a first compartment of a bioreactor vessel containing a suspension comprising cells suspended in a cell culture medium to cause spent cell culture medium from the first compartment to leave the first compartment simultaneously or nearly simultaneously, wherein a substantially constant volume is maintained within the first compartment when the additional cell culture medium is introduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention will be better understood by reading the following description of non-limiting embodiments with reference to the accompanying drawings, in which:

[0010] Figure 1 is a schematic illustration of a bioprocessing system according to an embodiment of the present invention.

[0011] Figure 2 is according to an embodiment of the present invention Figure 1 perspective view of the bioreactor vessel of the bioprocessing system.

[0012] Figure 3 is Figure 2 top plan view of the bioreactor vessel.

[0013] Figure 4 is Figure 2 side cross-sectional view of the bioreactor vessel.

[0014] Figure 5 is according to another embodiment of the present invention Figure 1 perspective view of the bioreactor vessel of the bioprocessing system.

[0015] Figure 6 is Figure 5 top plan view of the bioreactor vessel.

[0016] Figure 7 is Figure 5 a side cross-sectional view of a bioreactor vessel.

[0017] Figure 8 is a cross-sectional view of a bioreactor vessel of a bioprocessing system according to another embodiment of the present invention Figure 1 of.

[0018] Figure 9 is a cross-sectional view of a bioreactor vessel of a bioprocessing system according to another embodiment of the present invention Figure 1 of. DETAILED DESCRIPTION

[0019] Exemplary embodiments of the present invention will be described in detail below, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference characters used in all the drawings refer to the same or similar parts.

[0020] As used herein, "fluidly coupled" or "in fluid communication" means that components of a system are capable of receiving or transferring fluid between the components. The term fluid includes gases, liquids, or combinations thereof. As used herein, "operatively coupled" means a connection that can be direct or indirect. The connection is not necessarily a mechanical attachment. As used herein, "weir" means a wall that extends upward from the bottom surface of the bioreactor vessel but does not extend all the way to the top surface of the bioreactor vessel such that fluid is allowed to flow over or spill over the top edge of the weir. The term "overflow section" is used herein to refer to any structure that allows fluid to passively transfer from one compartment or region of the bioreactor to another compartment or region (or a region external to the bioreactor), and may include a weir or wall, an outlet, or a port. As used herein, "static bioreactor vessel" means a bioreactor vessel in which cells are processed under static conditions (i.e., without significant movement of the bioreactor vessel itself or agitation or shaking of the contents therein).

[0021] Although embodiments of the present invention are described herein in connection with the manufacture of biotherapeutic applications such as cell therapy and the manufacture of monoclonal antibodies, the present invention is not limited in this regard. In particular, it is contemplated that the bioreactor vessels of the present invention can be used in any bioprocessing operation (such as, for example, cell culture, cell processing, and / or cell expansion).

[0022] Embodiments of the present invention are directed to a bioreactor vessel that allows removal of spent cell culture medium and control of the depth of the medium and cell suspension without using a perfusion filter and / or active control logic that coordinates input and output pumps. In an embodiment, a bioreactor vessel includes: a first compartment configured to receive a suspension including cells suspended in a cell culture medium for use in a cell processing operation; a second compartment for receiving an overflow of fluid from the first compartment; and a weir that laterally separates the first compartment from the second compartment. When additional cell culture medium is added to the first compartment, the spent medium overflows over the top of the weir and into the second compartment to maintain a constant volume of fluid in the first compartment.

[0023] Reference Figure 1 , shows a schematic illustration of a portion 10 of a bioprocessing system configured for continuous cell culture or perfusion. The bioprocessing system can be generally constructed according to any of a variety of bioprocessing systems known in the art (such as the bioprocessing system disclosed in U.S. Provisional Application No. 62 / 736144). In particular, the bioprocessing system at least includes a bioreactor vessel 100 having an inlet 102 and an outlet 104, the inlet 102 being configured to be fluidly coupled to a medium source 12 via a first fluid delivery line 16, and the outlet 104 being configured to be fluidly coupled to a waste or collection reservoir 14 via a second fluid delivery line 18. A first pump 20 along the first fluid delivery line 16 is used to pump fluid (e.g., medium) from the medium source 12 through the inlet 102 into the bioreactor vessel 100, and a second pump 22 along the second fluid delivery line 18 is used to pump fluid from the bioreactor vessel 100 to the waste or collection reservoir 14. For example, the portion 10 of the bioprocessing system can be constructed and utilized during perfusion, whereby fresh cell culture medium is pumped from the medium reservoir 12 to the bioreactor vessel 100 using the first pump 20, and the used / spent medium is removed from the bioreactor vessel 100 and pumped to the waste 14 using the second pump 22.

[0024] Now turning to Figures 2 to 4, showing a more detailed view of the bioreactor vessel 100 according to an embodiment of the present invention. In an embodiment, the bioreactor vessel 100 is a static bioreactor vessel having a bottom 106, a plurality of sidewalls 108, and a top 110 that define an inner chamber 112 therebetween. The inner chamber 112 is configured to receive a cell population for use in a biological processing operation such as, for example, cell culture, cell processing, and / or cell expansion. In an embodiment, at least one of the bottom 106, top 110, and sidewalls 108 is formed of a gas-permeable, liquid-impermeable material. In an embodiment, the bottom 106 of the vessel 100 is formed of a gas-permeable material. The gas-permeable material allows oxygen and / or other gases used during the cell culture process to diffuse through the vessel wall and into the cell culture medium within the inner chamber 112. Correspondingly, carbon dioxide or other gases diffuse through the wall and out of the vessel 100.

[0025] As Figures 2 to 4 further shown in, in an embodiment, the bioreactor vessel 100 includes one or more walls or weirs 114 that extend upwardly from the bottom 106 of the bioreactor vessel 100 and define an overflow space 116 within the inner chamber 112. The (s) weir(s) 114 have a height that is less than the depth or height of the bioreactor vessel 100 such that the top edge of the (s) weir(s) 114 is spaced from the top 110 of the bioreactor vessel 100. As Figure 4 best shown in, the overflow space 116 is laterally isolated from the remainder of the inner chamber 112, but is otherwise in fluid communication with the inner chamber 112 due to the fact that the weir does not extend all the way to the top 110 of the bioreactor vessel 100. Thus, the weir 114 defines a first region or compartment 118 and a second region or compartment (i.e., the overflow space 116) within the inner chamber 112 and above the bottom surface 106. The first region or compartment 118 is configured to receive a cell population for performing a biological processing operation such as, for example, cell culture, cell processing, and / or cell expansion. The second region or compartment is for receiving the spent culture medium from the first compartment 118, as discussed below.

[0026] In an embodiment, the weir 114 may be generally circular in plan view and define a generally cylindrical overflow space or compartment 116. However, other configurations are possible without departing from the broader aspects of the present invention. For example, the weir 114 may have almost any peripheral shape as long as the weir defines an overflow space that is laterally isolated from the remainder of the inner chamber 112. In an embodiment, the weir 114 may extend laterally across the bioreactor vessel 100 from one side to the opposite side. In any such configuration, and as Figure 2 and Figure 3Best illustrated, the outlet 104 is in fluid communication with the second compartment 116 (e.g., the outlet 104 may be located in the bottom 106 of the bioreactor vessel 100 and surrounded by a weir 114). In other words, the outlet 104 is associated with or located within the second compartment 116. As such, the inlet 102 is located in either the bottom 106 of the bioreactor vessel 100 or one of the sidewalls 108 of the bioreactor vessel 100, and is associated with or located within the first compartment 118. For example, in one embodiment, the inlet 102 is adjacent to the bottom 106 and located in the sidewall 108 of the bioreactor vessel 100. In an embodiment, the inlet 102 is positioned between about 2 millimeters and about 30 millimeters from the bottom 106 of the bioreactor vessel 100. In an embodiment, the inlet 102 is positioned approximately 1 centimeter from the bottom 106 of the bioreactor vessel 100.

[0027] In an embodiment, the area and / or volume of the first compartment 118 (which holds a suspension comprising a population of cells suspended in a cell culture medium) is significantly larger than the area and / or volume of the second compartment 116. By minimizing the area of the second compartment 116 relative to the first compartment 118, the area within the bioreactor vessel 100 available for cell processing can be maximized.

[0028] Specific reference Figure 4 , bioprocessing operations such as perfusion can be performed using the bioreactor vessel 100. In a manner known in the art, a suspension comprising a population of cells 120 suspended in a cell culture medium 122 is added to the bioreactor vessel 100, such as through the inlet 102. The suspension thus remains in the first compartment 118. As illustrated therein, the volume of the suspension contained within the first compartment 118 is defined by the height of the weir 114.

[0029] Prior to initiating perfusion of the cell culture, the cells 120 may be allowed to sediment onto the bottom 106 by gravity, as shown in Figure 4 . Once the cells 120 have sedimented, perfusion of the cells within the first compartment 118 can be performed by adding fresh medium through the inlet port 102 adjacent to the bottom 106 of the first compartment 118 using a first pump 20 operating at a predetermined first rate (although it is envisioned that fresh medium could also be supplied by gravity drip). When the height of the fluid within the first compartment 118 reaches the height of the weir 114, adding more fresh medium causes some of the used / spent medium near the top of the first compartment 118 to overflow the weir 114 and enter the second compartment 116, as indicated by the arrow AAs illustrated. In the case where the fluid level in the first compartment 118 is at its maximum value (i.e., at a depth corresponding to the height of the weir), adding any volume of additional cell culture medium at any rate will cause an equal volume of fluid (i.e., used cell culture medium) to overflow the weir 114 at the same rate and enter the second compartment 116. The used culture medium that overflows into the second compartment 116 can then be discharged from the bioreactor vessel 100, such as under gravity, through the outlet 104.

[0030] However, in other embodiments, the bioprocessing system 10 can employ a second pump 22 to draw the used culture medium out of the bioreactor vessel 100 from the second compartment 116. The second pump 22 can operate at the same or a different rate than the rate of the first pump 20. Since the weir 114 determines the maximum volume of fluid within the first compartment 118, the second pump 22 does not need to operate at the same time or at the same rate as the first pump 20 in order to maintain a constant volume within the first compartment 118 (i.e., it does not need to be linked to the process / perfusion control logic). In particular, as described above, when the first compartment 118 is at maximum capacity, adding additional cell culture medium to the first compartment 118 will automatically cause an equal volume of used culture medium to overflow into the second compartment 116, thereby maintaining a constant volume within the first compartment 118. This is in contrast to existing systems that require careful coordination between the inlet pump and the outlet pump to maintain a substantially constant working volume within the bioreactor vessel during perfusion. In fact, as noted above, if desired, the second pump 22 can be completely omitted from the system.

[0031] Removing the used culture medium from the top of the first compartment 118 (passively by gravity) minimizes the risk of aspirating cells when pulling the medium to waste, thereby eliminating the need for a perfusion filter to retain cells. This is especially true when using low flow rates typically associated with perfusion, such as about 1 volume per day (e.g., for a 500 mL culture volume, 1 volume per day of perfusion would be 500 mL / day, or less than about 0.5 mL / min). Additionally, since the cells will sediment by gravity, adding fresh culture medium near the bottom 106 adjacent to the cells 120 provides the cells 120 with the quickest access to fresh nutrients while simultaneously or almost simultaneously displacing the used / expended medium upward and over the weir 114 and into the second compartment 116, where the medium can then be removed from the bioreactor vessel 100 by gravity or by employing the second pump.

[0032] Now turning to Figures 5 to 7 FIG. 200, a bioreactor vessel according to another embodiment of the present invention is illustrated. The bioreactor vessel 200 is generally similar in construction to Figures 2 to 4bioreactor vessel 100, and includes a bottom 206, a plurality of sidewalls 208, and a top 210 that define an inner chamber 212 therebetween. Similar to bioreactor vessel 100, at least one of the bottom 206, top 210, and sidewalls 208 of bioreactor vessel 200 may be formed of a gas-permeable material. Bioreactor vessel 200 also includes one or more walls or weirs 214 that extend upwardly from the bottom 206 of bioreactor vessel 200 and divide the inner chamber into a first compartment 218 for receiving a population of cells and a culture medium and a second compartment 216 that defines an overflow space, the purpose of the overflow space having been described above. However, as Figure 5 and Figure 6 illustrated therein, instead of the first compartment and the cell culture therein surrounding the overflow space of the second compartment, the first compartment 218 having the cell culture is surrounded by the second overflow compartment 216 on its outer periphery. In such an embodiment, bioreactor vessel 200 may include a fluid passage, such as a tube 219, that fluidly connects the inlet 202 to the interior space of the first compartment 218. As Figure 5 and Figure 6 shown therein, the outlet 204 or discharge port is associated with the second compartment 216, i.e., is located within the second compartment 216.

[0033] As in the embodiment described above, the (plural) weirs 214 have a height that is less than the depth or height of bioreactor vessel 200 such that the top edge of the (plural) weirs 214 is spaced from the top 1210 of bioreactor vessel 200. As Figure 7 best shown therein, the overflow space 216 is laterally isolated from the remainder of the inner chamber 212, but is otherwise in fluid communication with the inner chamber 212 due to the fact that the weirs 214 do not extend all the way to the top 210 of bioreactor vessel 200.

[0034] In use, in a manner known in the art, such as through the inlet 202 and the tube 219, a suspension comprising a population of cells 120 suspended in a cell culture medium 122 is added to bioreactor vessel 100. The suspension thus remains in the centrally located first compartment 218. As Figure 7 illustrated therein, the volume of the suspension contained within the first compartment 218 is defined by the height of the weirs 214. Before starting to perfuse the cell culture, the cells 120 may be allowed to sediment onto the bottom 206 by gravity, as Figure 7As shown. Once the cells 120 have settled, perfusion of the cells within the first compartment 218 can be performed by adding fresh culture medium through an inlet port 202 adjacent to the bottom 206 of the first compartment 218 using a first pump 20 operating at a predetermined first rate (although it is also envisioned that the fresh culture medium can be supplied by gravity drip). When the height of the fluid within the first compartment 218 reaches the height of the weir 214, adding more fresh culture medium causes some of the used / spent culture medium near the top of the first compartment 218 to overflow the weir 214 and enter the second compartment 216, as indicated by the arrow B shown. In the case where the fluid level in the first compartment 218 is at its maximum (i.e., at a depth corresponding to the height of the weir), adding any volume of additional cell culture medium at any rate will cause an equal volume of fluid (i.e., spent cell culture medium) to overflow the weir 214 at the same rate and enter the second compartment 216. The spent culture medium that overflows into the second compartment 216 can then be discharged from the bioreactor vessel 100 through the outlet 104, such as under gravity or by the operation of a second pump 22, as described above.

[0035] Although the embodiments of the bioreactor vessels described herein describe such vessels as static culture vessels, it is contemplated that such bioreactor vessels can equally be used as shaken bioreactor vessels. In the case where the bioreactor vessel is intended to be agitated and / or shaken during a cell processing operation, the bioreactor vessel can employ a membranous filter that encapsulates the top of the compartment containing the cells. For example, as Figure 7As illustrated, the cell retention membrane 230 can extend entirely above the first compartment 218 to prevent the cells 120 contained in the first compartment 218 from spilling out during shaking or agitation. In an embodiment, the cell retention membrane 230 can allow the culture medium 122 to pass therethrough, but prevent the cells 120 from passing. In other embodiments, the membrane 230 can be positioned lower, adjacent to the cells 120, which physically traps the cells 120 at the bottom of the first compartment. In still other embodiments, the cells 120 can be retained at the bottom of the bioreactor vessel (and its first compartment) using surface binding techniques (e.g., using adherent cells, binding the cells directly to the bottom surface, or binding to microcarriers that are much larger in mass than a single cell, etc., to make the cells less likely to remain in suspension). In this regard, a variety of bioreactor vessel configurations and techniques can be employed to retain the cells in the first compartment of the bioreactor vessel, thereby allowing perfusion to be performed using passive "overflow" into the second compartment without sucking the cells into the waste. In embodiments of the invention described herein without using a retention filter, it is well-suited for static culture, where the cells are retained at the bottom of the bioreactor vessel by gravity or biochemical adhesion. As discussed above, for cultures in which the cells are in a homogeneous suspension, as discussed above, a retention filter can be employed to retain the cells during shaking, stirring, agitation, etc.

[0036] In some embodiments, the (multiple) weirs may be capable of being selectively adjusted such that the depth, height, and / or volume of the suspension within the first compartments 118, 218 can vary. For example, in Figures 2 to 4 an embodiment, the weir 114 can be configured as a tube or sleeve that is selectively extendable and retractable within the inner chamber 112. In such an embodiment, one or more sealing elements such as O-rings can be utilized to prevent the culture medium or cells from leaking out of the bioreactor vessel through the interface between the bottom of the bioreactor vessel and the tube or sleeve. The use of a height-adjustable weir allows the culture medium to be conserved throughout the cell culture process, thereby generally reducing the operating cost of the bioreactor system 10. For example, at the start of the cell expansion phase, the weir 114 can be retracted to a low position adjacent to the bottom 106 of the bioreactor vessel 100. As perfusion is performed and the cell population increases, the weir 114 can be extended or raised to allow a larger volume of culture medium to be accommodated within the first compartment 118 to support cell growth.

[0037] In addition, in typical practice, the culture is fed in batches (meaning additional fresh medium is added without removing the used medium) until the maximum desired culture volume is reached. This is usually done for convenience, but has the drawback that the cells are suspended with their own metabolic waste. By adjusting the height of the weir during the early stages of the culture, volume increase can be allowed to keep the cell density within an optimal range. In addition, adjusting the weir height can extend beyond the initial culture period and through the culture duration. In current practice, once the final volume is reached, perfusion is started and then increased over time as the cell density increases, in an attempt to keep fresh medium supplied to the cells. This can result in extremely high cell densities (e.g., >25e6 / mL), which may be detrimental to cell health. In contrast, by allowing the volume to increase continuously by changing the height of the weir or the overflow outlet (described below), the culture can be maintained at a better cell density (e.g., 2e6 cells / mL).

[0038] In both cases described above, the absolute rate of perfusion will increase as the number of cells increases, and in that way, the cost of medium usage will be comparable. However, in the latter case, the perfusion rate can be maintained at a fixed relative rate (e.g., 1 volume per day). In static culture, allowing the relative perfusion rate to increase may be detrimental to the goal of preventing cells from being washed away by the perfusion flow, and thus raising the weir as the number of cells increases can be beneficial for retaining cells.

[0039] In addition to the above discussion regarding the peripheral shape of the weir and the ability to raise and lower the weir, in embodiments, the weir can be configured such that the volume of the first compartment (e.g., the first compartment 118) increases at a rate greater than linear as the height of the weir increases. For example, in Figure 1 an embodiment, the weir 114 can have a funnel shape or angled sidewalls such that as the weir is raised, the volume of the first compartment 118 increases at a rate greater than linear. In particular, a central weir having a funnel shape or angled walls (similar to the weir shown in Figure 2 ) allows the volume of the laterally adjacent first compartments 118 to increase more and more with each incremental raise of the weir (i.e., each successive incremental raise of the weir causes the volume of the first compartment 118 to increase more than each previous increment).

[0040] Moving on to Figure 8, in an embodiment, a bioreactor vessel 300 according to yet another embodiment of the present invention is shown. The bioreactor vessel 300 includes a bottom 306, a plurality of sidewalls 308, and a top 310 that define an inner chamber 312 therebetween. The bioreactor vessel 300 further includes an inlet / port 302 located in one of the sidewalls 308 adjacent to the bottom 306. However, it is contemplated that in some embodiments, the inlet 302 may be located in the bottom 306 or the top 310 of the bioreactor vessel 300. As Figure 8 shown, the bioreactor vessel 300 further includes an outlet / port 304 in one of the sidewalls 308 that is spaced a predetermined distance from the bottom 306. Similar to the weir in the embodiments described above, the height of the outlet 304 defines the maximum fluid height and fluid volume within the bioreactor vessel 300.

[0041] When used during a perfusion process, a suspension comprising cells 120 suspended in a cell culture medium 122 is added to the inner chamber of the bioreactor 300. In the manner described above, additional cell culture medium is added to the bioreactor vessel 300, such as using a first pump 20. As additional cell culture medium is added, the spent medium exits the vessel 300 passively (with or without the use of a separate second pump) through the outlet. Thus, as Figure 8 illustrated, the height of the outlet 304 can be used to maintain the fluid volume at a given value while only controlling the inflow rate of the additional medium. In an embodiment, a perfusion filter may be utilized at the outlet or in the outlet pipe to retain the cells 120 within the bioreactor vessel.

[0042] Although Figure 8 the outlet 304 in the sidewall 308 of the bioreactor vessel 300 is shown, it is contemplated that the outlet may also be provided in the top of the vessel and used in combination with a siphon that extends downwardly towards the bottom 306 of the bioreactor vessel 300, and the distal end of the siphon can be selectively adjusted to a desired distance from the bottom 306. In such an embodiment, the distance of the distal end of the siphon from the bottom 306 defines the maximum medium height within the bioreactor vessel 300. These concepts can be applied to stirred tank bioreactors, enabling perfusion to be performed without active control or monitoring of the removal of the spent medium.

[0043] Finally turning to Figure 9 , in an embodiment, a bioreactor vessel 400 according to yet another embodiment of the present invention is shown. The bioreactor vessel 400 is generally similar to Figure 8bioreactor vessel 400, and includes a bottom 406, a plurality of sidewalls 408, and a top 410 that define an inner chamber 412 therebetween. The bioreactor vessel 400 further includes an inlet / port 402 located in one of the sidewalls 408 adjacent to the bottom 406. However, it is contemplated that in some embodiments, the inlet 402 may be located in the bottom 406 or the top 410 of the bioreactor vessel 400. As Figure 9 shown, the bioreactor vessel 400 further includes a plurality of vertically stacked outlet ports or overflows 420, 422, 424 in one of the sidewalls 408 or in a wall 421 that divides the interior space into a first compartment 416 for cell culture and a second outer jacket or compartment 419, and the outlet ports or overflows 420, 422, 424 are spaced at different distances from the bottom 406. Although Figure 9 three outlet ports are shown, any number of stacked outlet ports may be employed without departing from the broader aspects of the invention. Each outlet 420, 422, 424 may be constructed with valves 426, 428, 430 (e.g., programmable valves).

[0044] In operation, the valves 426, 428, 430 may be continuously opened or closed to control the height of the culture medium within the bioreactor vessel 400. For example, during the first stage of cell culture, the lowest valve 426 may be opened such that the height of the lowest outlet port 420 defines the maximum culture medium depth. During the second stage of cell culture, for example, when the cell population has increased, the lowest valve 426 may be closed and the valve 428 above it may be opened. During this stage, the height of the outlet port 422 defines the maximum culture medium depth (which is now greater than the maximum culture medium depth when the valve 426 was open). As discussed above, by selectively opening and closing the valves 426, 428, 430 associated with each outlet 420, 422, 424, the depth of the culture medium and the volume of the culture within the bioreactor vessel 400 can be easily adjusted. Once the culture medium overflows into the second compartment or jacket 419, the culture medium can be passively discharged through the outlet 404 or pumped to waste.

[0045] In another embodiment, instead of using vertically stacked valves (each positioned at a different distance from the bottom of the bioreactor vessel and opened or closed depending on the desired height of the culture medium within the bioreactor vessel), it is contemplated that the bottom of the bioreactor vessel may be lowered relative to a fixed outlet (or a single outlet) to selectively increase the depth and volume of the culture medium within the bioreactor vessel.

[0046] As described above, the bioreactor vessel of the present invention is much simpler than existing systems, which typically require some type of reaction control logic to ensure that equal volumes of culture medium are added and removed from the bioreactor vessel simultaneously during perfusion (e.g., during cell expansion). In contrast to such systems, the bioreactor vessel of the present invention allows fluid (i.e., spent culture medium) to be passively removed by gravity from the top of the suspension using a weir / wall, overflow tube / exit, or siphon located at a predetermined height. As discussed above, this passive removal of spent culture medium minimizes the risk of aspirating cells and largely eliminates the need for a perfusion filter to retain cells. Additionally, in embodiments where additional fresh culture medium is added at the bottom of the bioreactor vessel where the cells settle, the cells can quickly and immediately access fresh nutrients as the spent culture medium shifts upward over the weir or exits the outlet. In addition to the above, the depth of the suspension (i.e., cells and culture medium) is controlled by the height of the weir or the position of the outlet. Specifically, once the weir height or outlet height is set, the culture medium depth is set. This is in contrast to existing systems where the culture medium height is typically controlled by carefully balancing the culture medium inflow rate / volume relative to the culture medium outflow rate / volume, which is a much more complex procedure that requires almost constant monitoring and adjustment. Thus, the embodiments of the present invention described herein allow perfusion to be implemented using relatively uncomplicated and inexpensive equipment, thereby eliminating the need for complex and expensive control logic to balance culture medium inflow and culture medium outflow.

[0047] While the embodiments described above disclose a single culture area within the bioreactor vessel (i.e., a single first compartment for receiving a suspension containing cells and culture medium), the present invention is not limited in this regard. Specifically, it is contemplated that any number of compartments may be formed within the bioreactor vessel around a central overflow chamber defined by a weir. Each of the compartments may be supplied with fresh culture medium at a desired rate, and the overflow chamber is common to each of the culture compartments. This multi-compartment form may be on the scale of a standard microplate or may be an array of larger bioreactors.

[0048] In an embodiment, a bioreactor vessel includes: a first compartment configured to receive a suspension including cells and a cell culture medium for use in a cell processing operation; a second compartment for receiving an overflow of the cell culture medium from the first compartment; and an overflow portion separating the first compartment from the second compartment, the overflow portion being configured to maintain a level of the cell culture medium in the first compartment. In an embodiment, the bioreactor vessel further includes an inlet associated with the first compartment and configured to direct a supply of additional cell culture medium to the first compartment. In an embodiment, the bioreactor vessel further includes an outlet associated with the second compartment and configured to allow spent cell culture medium to flow out of the bioreactor vessel. In an embodiment, the overflow portion has a height less than a depth of the bioreactor vessel. In an embodiment, a maximum volume of the suspension within the bioreactor vessel is defined by the height of the overflow portion. In an embodiment, the height of the overflow portion can be selectively adjusted to allow adjustment of a depth of the suspension in the first compartment. In an embodiment, the bioreactor vessel includes a bottom, a top, and a plurality of sidewalls, the bottom, the top, and the plurality of sidewalls defining an inner chamber containing the first compartment and the second compartment. In an embodiment, the inlet is located in the bottom of the bioreactor vessel. In an embodiment, the inlet is positioned adjacent to the bottom of the bioreactor vessel, in one of the plurality of sidewalls of the bioreactor vessel. In an embodiment, the bioreactor vessel further includes a cell retention membrane enclosing at least a portion of the first compartment, the cell retention membrane allowing the cell culture medium to overflow from the first compartment into the second compartment while retaining the cells in the first compartment. In an embodiment, the overflow portion is in the form of a weir configured to allow the cell culture medium to overflow from a top of the weir into the second compartment.

[0049] In another embodiment, a bioprocessing system includes a bioreactor vessel having a bottom, a top, and a plurality of sidewalls that define an inner chamber; a weir that extends upwardly from the bottom, the weir and at least one of the plurality of sidewalls defining a first compartment within the inner chamber and a second compartment within the inner chamber, the first compartment for holding a suspension comprising cells suspended in a cell culture medium, the second compartment for receiving an overflow of spent culture medium from the first compartment; an inlet associated with the first compartment; and an outlet associated with the second compartment. The bioprocessing system further includes a first pump in fluid communication with the inlet for pumping additional cell culture medium from a culture medium reservoir through the inlet into the first compartment of the bioreactor vessel. The outlet is configured to allow spent cell culture medium to flow out of the second compartment while or substantially simultaneously with pumping additional cell culture medium into the first compartment. In an embodiment, the system further includes a second pump in fluid communication with the outlet for pumping spent cell culture medium out of the bioreactor vessel. In an embodiment, the weir has a height less than the depth of the bioreactor vessel. In an embodiment, the maximum volume of the suspension within the bioreactor is defined by the height of the weir. In an embodiment, the height of the weir is selectively adjustable to allow adjustment of the depth of the suspension within the first compartment. In an embodiment, the bioreactor vessel includes a cell retention membrane that encapsulates at least a portion of the first compartment, the cell retention membrane allowing cell culture medium to overflow from the first compartment into the second compartment while retaining the cells within the first compartment.

[0050] In yet another embodiment, a method for bioprocessing includes the steps of introducing additional cell culture medium into a first compartment of a bioreactor vessel containing a suspension comprising cells suspended in a cell culture medium to cause spent cell culture medium to leave the first compartment while or substantially simultaneously, wherein a substantially constant volume is maintained within the first compartment when the additional cell culture medium is introduced. In an embodiment, the bioreactor vessel includes a first compartment and a second compartment separated by a weir, wherein introducing the additional cell culture medium into the first compartment causes the spent cell culture medium to flow over the top of the weir and into the second compartment. In an embodiment, the method may further include the steps of actuating a first pump to introduce the additional cell culture medium into the first compartment and actuating a second pump to remove spent cell culture medium from the bioreactor vessel, wherein the first pump operates at least one of at a different time and / or at a different rate than the second pump while maintaining a substantially constant volume within the first compartment. In an embodiment, the method may further include the step of adjusting the height of the weir within the bioreactor vessel to adjust the volume of the suspension within the first compartment.

[0051] As used herein, an element or step recited in the singular and preceded by the word "a" or "an" should be understood as not excluding a plurality of the elements or steps, unless expressly stated to the contrary. In addition, a reference to "one embodiment" of the present invention is not intended to be construed as excluding the existence of additional embodiments that also incorporate the recited features. Further, unless expressly stated to the contrary, an embodiment that "comprises," "includes," or "has" one or more elements having a particular property may include additional such elements that do not have that property.

[0052] This written description uses examples to disclose several embodiments of the invention, including the best mode, and also enables one of ordinary skill in the art to practice embodiments of the invention, including making and using any device or system and performing any incorporated method. The patentable scope of the present invention is defined by the claims, and may include other examples that occur to one of ordinary skill in the art. Such other examples are intended to be within the scope of the claims if such other examples have structural elements that are not different from the literal language of the claims, or if such other examples include equivalent structural elements that are not materially different from the literal language of the claims.

Claims

1. A bioreactor vessel, comprising: A first compartment configured to receive a suspension comprising cells and cell culture medium for use in a cell processing operation; A second compartment configured to receive an overflow of the cell culture medium from the first compartment; An inlet associated with the first compartment and configured to direct a supply of additional cell culture medium to the first compartment; An outlet associated with the second compartment and configured to allow spent cell culture medium to flow out of the bioreactor vessel; And An overflow portion separating the first compartment and the second compartment, the overflow portion configured to maintain the level of the cell culture medium in the first compartment, Wherein at least one of the bottom, top, and / or plurality of side walls of the bioreactor vessel is formed of a gas-permeable, liquid-impermeable material, Wherein the first compartment abuts each of the plurality of side walls, and wherein the second compartment is completely surrounded by the first compartment and spaced apart from the plurality of side walls, and Wherein the height of the overflow portion can be selectively adjusted to allow adjustment of the depth of the suspension in the first compartment.

2. The bioreactor vessel according to claim 1, wherein: The height of the overflow portion is less than the depth of the bioreactor vessel.

3. The bioreactor vessel according to claim 2, wherein: The maximum volume of the suspension within the bioreactor vessel is defined by the height of the overflow portion.

4. The bioreactor vessel according to claim 3, wherein: The overflow portion has an outer perimeter that is generally conical or funnel-shaped.

5. The bioreactor vessel according to claim 3, wherein: The bottom, the top, and the plurality of side walls define an inner chamber containing the first compartment and the second compartment.

6. The bioreactor vessel according to claim 5, wherein: The inlet is located in the bottom of the bioreactor vessel.

7. The bioreactor vessel according to claim 5, wherein: The inlet is positioned adjacent to the bottom of the bioreactor vessel, in one of the plurality of side walls of the bioreactor vessel.

8. The bioreactor vessel according to claim 1, wherein, Further comprising: A cell retention membrane encapsulating at least a portion of the first compartment, the cell retention membrane allowing cell culture medium to overflow from the first compartment into the second compartment while retaining the cells in the first compartment.

9. The bioreactor vessel according to claim 1, wherein: The overflow portion is in the form of a weir configured to allow the cell culture medium to overflow from the top of the weir into the second compartment.

10. A biological treatment system, comprising: A bioreactor vessel having: A bottom, a top, and a plurality of side walls, the bottom, the top, and the plurality of side walls defining an inner chamber; A weir extending upwardly from the bottom, the weir and at least one of the plurality of side walls defining a first compartment within the inner chamber and a second compartment within the inner chamber, the first compartment for holding a suspension comprising cells suspended in cell culture medium, the second compartment for receiving an overflow of spent culture medium from the first compartment; An inlet associated with the first compartment; And An outlet associated with the second compartment; And A first pump in fluid communication with the inlet for pumping additional cell culture medium from a culture medium reservoir to the first compartment of the bioreactor vessel through the inlet; Wherein the outlet is configured to allow spent cell culture medium to flow out of the second compartment simultaneously or almost simultaneously with pumping the additional cell culture medium to the first compartment, Wherein, at least one of the bottom, the top, and / or the plurality of side walls of the bioreactor vessel is formed of a gas-permeable and liquid-impermeable material, Wherein, the first compartment abuts each of the plurality of side walls, and wherein, the second compartment is completely surrounded by the first compartment and spaced apart from the plurality of side walls, and Wherein, the height of the weir can be selectively adjusted to allow adjustment of the depth of the suspension in the first compartment.

11. The biological treatment system according to claim 10, wherein Further comprising: A second pump, which is in fluid communication with the outlet for pumping the spent cell culture medium out of the bioreactor vessel from the second compartment.

12. The biological treatment system according to claim 10, wherein: The height of the weir is less than the depth of the bioreactor vessel.

13. The biological treatment system according to claim 12, wherein: The maximum volume of the suspension in the bioreactor is defined by the height of the weir.

14. The biological treatment system according to claim 10, wherein: The bioreactor vessel includes a cell retention membrane that encapsulates at least a portion of the first compartment, and the cell retention membrane allows the cell culture medium to overflow from the first compartment into the second compartment while retaining the cells in the first compartment.

15. A method for biological treatment, comprising the following steps: In a first compartment of a bioreactor vessel containing a suspension comprising cells suspended in a cell culture medium, additional cell culture medium is introduced to cause the spent cell culture medium from the first compartment to leave the first compartment simultaneously or almost simultaneously; Wherein, when the additional cell culture medium is introduced, a substantially constant volume is maintained in the first compartment, Wherein, at least one of the bottom, the top, and / or the plurality of side walls of the bioreactor vessel is formed of a gas-permeable and liquid-impermeable material, Wherein, the bioreactor vessel includes a first compartment and a second compartment separated by a weir, Wherein, the first compartment abuts each of the plurality of side walls, and wherein, the second compartment is completely surrounded by the first compartment and spaced apart from the plurality of side walls, and Wherein, the method further comprises the step of: adjusting the height of the weir in the bioreactor vessel to adjust the volume of the suspension in the first compartment.

16. The method according to claim 15, wherein: Introducing the additional cell culture medium into the first compartment causes the spent cell culture medium to flow over the top of the weir and into the second compartment.

17. The method according to claim 16, wherein Further comprising the following steps: Actuating a first pump to introduce the additional cell culture medium into the first compartment; And Actuating a second pump to remove the spent cell culture medium from the bioreactor vessel; Wherein, the first pump operates at at least one of a different time and / or a different rate from the second pump while maintaining the substantially constant volume in the first compartment.

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