Reinforced bioreactor component structural system for cell culture

Abstract

The present invention relates to a bioprocess bag 118 having a bag wall defining an enclosed volume for holding biomaterial. The bag wall has at least one inlet port 142 and at least one outlet port 146. The bioprocess bag 118 also includes a tubular structure 400 having a first open end proximate the bag wall and a second distal end, the tubular structure extending into the enclosed volume, the tubular structure 400 including a reinforced portion 402 proximate the first open end.

Description

[Technical Field]

[0001] The present invention relates to novel bioprocess bags for cell culture processes having a reinforced internal structure. More specifically, the present invention relates to improved bioprocess bags and related structures that are reinforced and include an improved tubing structure. [Background technology]

[0002] Cell therapy is a new but rapidly expanding field in biotechnology that involves the administration of autologous or allogeneic cells to exert a therapeutic effect in vivo. Cell therapy involves many essential steps from cell collection to cell infusion into the patient. Cell culture for cell therapy is performed in a clean room environment. Cell culture and clean rooms have many mandatory regulations, such as particle size and count within the clean room, the number of patient samples handled at one time, the number of instruments per suite, and requirements for a sterile environment.

[0003] Conventional cell culture systems are stand-alone, require large spaces, and cannot handle multiple patient samples simultaneously. The procedures for cell culture for cell therapy require a lot of human intervention, which may contaminate the cell culture and damage the growth of cells, especially the culture of small cells.

[0004] Disposable bioreactor or fermenter bags have been developed for cell therapy. The use of disposable bioreactor bags in cell culture reduces batch changeover time by eliminating time-consuming cleaning and equipment validation, thereby resulting in higher throughput. Figure 1 shows a disposable bioreactor system described in U.S. Pat. No. 9,109,193, entitled "Continuous Perfusion Bioreactor System." Figure 1 is described in detail in U.S. Pat. No. 9,109,193 but will be briefly described herein. One aspect includes an apparatus 100 including a vessel 114. In the illustrated embodiment, the vessel 114 is a reusable support structure (e.g., a stainless steel tank) that surrounds and contains a container 118. The apparatus 100 can optionally include an environmental containment enclosure 120 that surrounds a portion of the vessel 114. The container 118 may be configured as a collapsible bag (e.g., a polymeric bag). Collapsible bag 118 may be fluid-tight to allow bag 118 to contain liquid 122. This liquid 122 may contain reactants (e.g., certain solids), media, and / or other components necessary to carry out a desired process, such as a chemical, biochemical, and / or biological reaction. Collapsible bag 118 may be configured so that liquid 122 remains in substantial contact only with the collapsible bag and not with support vessel 114 during use, thereby allowing the support structure to be reused without cleaning.

[0005] As shown in FIG. 1 , optional inlet and outlet ports 142 and 146 may be formed in the container 118 and / or the reusable support structure 114 to facilitate convenient introduction and removal of liquids and / or gases from the container. These ports may be located at any suitable location relative to the container 118. For example, in certain devices including spargers, the container may include one or more gas inlet ports located in a bottom portion of the container. Tubing may be connected to the inlet and / or outlet ports to form, for example, supply and withdrawal lines, to introduce and remove liquids from the container. One or more connections 164 may be located at the top of the container 118 or at other suitable locations. Connection 164 may include openings, tubes, and / or valves for adding or withdrawing liquids, gases, and the like from vessel 118, each of which may optionally include a flow sensor and / or filter (not shown). Optionally, connection 164 may be in fluid communication with gas inlet port 165 and gas outlet port 165.

[0006] For systems including multiple spargers, the control system 134 may be operatively associated with each of the multiple spargers and may be configured to operate the multiple spargers independently of one another. The support structure 114 and / or vessel 118 may also, in some embodiments, include one or more ports 154, which may be used for sampling, analysis (e.g., determining pH and / or the amount of dissolved gases in the liquid), or other purposes. These ports may be aligned with one or more access ports 156 in the optional surrounding containment enclosure 120.

[0007] In some embodiments, the apparatus 100 may include one or more connection ports 180 for interconnecting the interior of the reusable support structure 114 (e.g., gap 132) with the interior of a second apparatus. Additionally, or alternatively, the apparatus may include one or more connection ports 182 adapted to connect the interior of the container 118 (interior 56) with the interior of a second apparatus. The ports may facilitate the transfer of material from interior 56 to the second apparatus or other suitable container (e.g., a sealed bag). Such transfer may be achieved, for example, by pumping the material through tubing (e.g., by peristaltic pumping or by applying positive pressure to an inlet), by use of gravity, and / or by application of a vacuum.

[0008] Apparatus 100 may optionally include a mixing system, such as an impeller 151 positioned within vessel 118, which may be rotated (e.g., about a single axis) using a motor 152, which may be external (or internal) to the vessel. This mixing system may be controlled by control system 134. Optionally, the vessel and / or support structure may include a utility tower 150. This utility tower 150 may be provided to facilitate interconnection of one or more devices, controls, and / or electronics (e.g., sensor electronics, electronic interfaces, and pressurized gas controls) housed within the vessel and / or support structure with one or more pumps, or other devices. Such devices may be controlled using control system 134.

[0009] Figure 2 shows another disposable bioreactor system described in U.S. Patent No. 7,629,167, filed June 6, 2005, and entitled "Disposable Bioreactor System and Method." Figure 2 is described in detail in U.S. Patent No. 7,629,167, but will be briefly described herein. As shown, bioreactor 200 includes one or more ports 202 that can be used to add or withdraw gases and / or fluids to or from the bioreactor. A harvest or drain port 204 is typically provided at the bottom of the bag so that gravity can be used to direct the contents outside the bioreactor. Probes and / or sensors 206 may be integral with the sides of the bioreactor, thereby making the sensors and / or probes disposable as well. In one embodiment of the present invention, the sensors / probes may be optical probes that provide an output in a visual manner. Therefore, the sensor / probe port 206 may be used to visually monitor the status of the sensor / probe.

[0010] Portions of the mixing system may be integral with the bioreactor. Specifically, as shown in FIG. 2, the portion of the mixing system contained within the bioreactor may include mixing system portion 208, i.e., the impeller plate and impeller hub. The impeller plate is connected to the motor's drive system to power the impeller and also provides a seal between the motor and the interior of the bioreactor. Some embodiments of the present invention provide one or more sophisticated mixing systems that provide an inexpensive method for providing agitation to the bioreactor contents. Such mixing systems may utilize materials such as HDPE (high-density polyethylene) and / or other gamma-irradiatable biocompatible plastics. One or more components of the mixing system may be manufactured by machining a block of material, or may be molded and / or cast.

[0011] Figure 3 shows another disposable bioreactor system described in U.S. Patent No. 9,550,969, entitled "Flexible Bag for Cell Culture." Figure 3 is described in more detail in U.S. Patent No. 9,550,969, but will be briefly described here. As shown, the inflatable bioreactor bag for cell culture 1 includes a top sheet 2 and a bottom sheet 3 of flexible material joined together to form two edges 4 and two side edges 5, with one or more baffles 6 extending from the bottom sheet 3 in an area of the bottom sheet 3 where the shortest distance to the two edges 4 (i.e., the nearest edges) is greater than about one-quarter of the shortest distance D between the two edges 4. The bag is generally rectangular, in that the shortest distance to one of the edges 4 (the nearest edges) is never greater than D / 2 for any point on the bottom sheet 3. Therefore, the baffle 6 may extend from the bottom sheet 3 in a region where the shortest distance to the edge 4 is between about one-quarter and about one-half of the shortest distance D between the edges 4, i.e., between D / 4 and D / 2.

[0012] The bag 1 may be pivotally mounted on a base 9 about a movable axis 7 generally parallel to the edge 4. The movable axis 7 may be located below the bag 1, and the bag may be mounted on a support 8, e.g., with the distance between the protrusion of the movable axis 7 on the bottom sheet 3 and each edge 4 generally equal to D / 2. A suitable pivotable support attached to the movable axis may be, for example, a WAVE Bioreactor System (GE Healthcare). The flexible material of the top sheet 2 and the bottom sheet 3 may be a polymeric material, such as a plastic film or laminate having a thickness, for example, in the range of 50 to 500 microns. The laminate may comprise, in addition to one or more polymeric materials, bather layers, which may be, for example, polymers or inorganic oxides or inorganic metals. In particular, the top sheet 2 may be transparent to ensure visibility into the bag. The top sheet 2 and the bottom sheet 3 are defined in a predetermined position during use of the bag, i.e., the top sheet 2 lies above the bottom sheet 3 during use. The top and bottom sheets may also be distinguished in that ports 11 are preferably located in the top sheet 2, providing a smooth outer surface of the bottom sheet 3 suitable for resting on a support 8. The side edges 5 may be longer than the end edges 4.

[0013] The advantage of the central location of the baffle 6 is that when the bag is partially filled with cell suspension, inflated, and swung around the movable shaft 7, all of the cell suspension passes repeatedly near the baffle. This increases the intensity of the agitation and improves gas exchange at the air-liquid interface, while the agitation is gentle enough not to damage the delicate cells.

[0014] In some embodiments, at least one of the baffles 6, e.g., two baffles, is tubular. One advantage of this is that the baffle can be made flexible enough to facilitate packaging and storage of the collapsed bag before use, yet rigid enough to withstand hydrodynamic forces during bag operation. A further advantage is that the tubular structure allows material to be transported into or out of the bag through the baffle. The tubular baffle can be made, for example, from an elastomeric material that allows for collapse of the structure during bag packaging, but is elastic enough to provide full recovery of the open tubular shape when the bag is filled and / or inflated. The elastomeric material can be, for example, cross-linked silicone rubber or other vulcanized rubber material. When a tube with thick walls and / or high stiffness is used, the tubular baffle can act as a column, keeping the top and bottom sheets of the bag separated before and during bag inflation. Summary of the Invention [Problem to be solved by the invention]

[0015] There is a need for an improved bioreactor or fermenter bag that can withstand sterilization, packaging, shipping, installation, and use. [Means for solving the problem]

[0016] The present invention includes aspects relating to improved bioprocess bags and related structures that include reinforced and improved tubing features. In one aspect, the present invention relates to an improved bioprocess bag comprising: a bag wall defining an enclosed volume for holding biomaterial, the bag wall including at least one inlet port and at least one outlet port; and a tubing structure having a first open end proximate the bag wall and a second distal end, the tubing structure extending into the enclosed volume and including a reinforced portion proximate the first open end. The reinforced portion may include a pattern of raised portions relative to an outer tubing surface, the raised portions contacting each other when the tubing structure is bent. This contact may prevent crimping of the tubing structure during transportation, installation, or operation.

[0017] In another aspect, the present invention relates to a bioprocess bag comprising: a bag wall defining an enclosed volume for holding a biomaterial, the bag wall having at least one inlet port and at least one outlet port; and a tubular structure extending into the enclosed volume, the tubular structure having a first open end proximate the bag wall and a second closed end distal to the bag wall, the tubular structure comprising a predominantly cylindrical inner wall and a notch extending through at least a portion of the length of the inner wall, the notch defining a channel that allows air to escape when a probe is inserted into the tubular structure.

[0018] These aspects of the bioprocess bag may include other features used in mixers, bioreactors, and other related applications. These features may include impellers, heaters, and gas outlets. The tubing may be adapted to accept a probe, such as a probe having a thermocouple or resistance temperature detector (RTD). Alternatively, or in addition, the tubing may be adapted to accept a sparging wand. It is envisioned that any number of tubing structures according to embodiments of the present invention may be used together or in combination with other tubing structures within a single bioprocess bag. The tubing may comprise a generally cylindrical inner wall and a notch extending at least a portion of the length of the inner wall. The notch may be adapted to define a channel that allows air to escape when a probe is inserted into the tubing.

[0019] In one aspect, the present invention includes a reinforced probe housing, the reinforced probe housing comprising: a distal portion having a first outer diameter and a first inner diameter defining a thickness, the first inner diameter adapted to receive a probe, the reinforced probe housing having a closed end proximate the distal portion; and a reinforced portion sharing the same inner diameter as the distal portion, the outer diameter of the reinforced portion including raised portions that provide reinforcement through contact with each other when the reinforced probe housing is bent, the reinforced probe housing having an open end proximate the reinforced portion, the open end adapted to receive the probe. The reinforced probe housing may be included in a bioprocess bag or may be used alone. The probe may be adapted to receive a thermocouple or a resistance temperature detector (RTD). [Brief explanation of the drawings]

[0020] [Figure 1] 1 shows an example of a prior art disposable large scale bioreactor system. [Figure 2]1 shows an example of a prior art disposable bioreactor bag. [Figure 3] 1 shows an example of another prior art disposable bioreactor bag. [Figure 4] 1 illustrates a thermowell tube having a thermowell probe inserted therein, according to an embodiment of the present invention. [Figure 5] FIG. 1 shows an enlarged perspective view of a thermowell tube according to an embodiment of the present invention. [Figure 6] 1 illustrates a thermowell tube according to an embodiment of the present invention that is curved. [Figure 7] 1 illustrates a cross-sectional view of a portion of a thermowell tube according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] The inventors have discovered that several internal structures of bioreactor or fermenter bags are susceptible to damage, perceived damage resulting from sterilization and packaging, and further damage to these elements can occur during shipping, installation, or use. Sterilizing the bag by gamma irradiation can, in some cases, exacerbate the problem by welding adjacent pieces of material together or hardening the material into a curved shape. For components internal to the bag, these problems are sometimes exacerbated by the inability to inspect the interior of the bag while maintaining sterility and not damaging the bag structure. Perceived damage to an internal component can result in the entire bag being deemed unsuitable for use. The tubing structure may be adapted to accept a probe or sparging wand, such as a thermocouple or resistance temperature detector (RTD). Alternatively, or in addition, the tubing structure may be adapted to introduce or withdraw materials from the bioprocess bag.

[0022] Those internal bioreactor bag structures most susceptible to or perceived damage of this type are the tubular structures inside the bag, which can become bent, crimped, or folded at some point in the supply chain.

[0023] One particular structure susceptible to damage is the thermowell tube found in existing bioreactor bags. An example of such a thermowell is described in U.S. Pat. No. 6,599,012, entitled "Thermowell Adapter," which is incorporated by reference for its disclosure of thermowell structures. The thermowell tube is a straight piece of tubing made from a plastic material that allows the thermowell to be inserted into the bioreactor bag without destroying the seal of the bioreactor bag. In many cases, the thermowell is made from a molded plastic or rubber material. A rigid insert is used to prevent the tube from collapsing and self-sealing internally during gamma irradiation. In some cases, the rigid support can break, puncturing the tube. Without such support, the tube can kink and seal during gamma irradiation. The temperature sensor 405, when inserted, can puncture the tube.

[0024] In one embodiment shown in FIG. 4, thermowell tube 400 includes tip portion 401 and reinforced portion 402. Tip portion 401 may have a length of approximately 1 inch (approximately 2.54 cm). However, tip portion 401 may be longer or shorter depending on the needs of the user. The tip portion of thermowell tube 400 is substantially thinner than reinforced portion 402 and is designed to allow improved heat flow from the interior portion of a bioreactor (not shown) to thermowell distal end 401 when inserted into thermowell tube 400 as shown in FIG. 4. Reinforced portion 402 is defined by rib portions 402a equally spaced apart by separation portions 402b. Reinforced portion 402 provides greater structural stability for thermowell tube 400.

[0025] The thermowell tube 400 may also include an optional base portion 403 that is attached to a thermowell flange 404, which can be used to form a seal with a bioreactor bag (not shown). Alternatively, the thermowell tube can be molded directly into the bioreactor bag. The base portion provides additional structural support for the thermowell when it is inserted into the thermowell tube 400.

[0026] FIG. 5 shows an enlarged perspective view of a thermowell tube 400. The distal end portion 401 of the thermowell tube 400 has a shape that maximizes thermal exposure of a thermowell (not shown) when inserted into the thermowell tube 400. The rib portion 402a is preferably provided in a cylindrical configuration and is separated by a separation portion 402b, which can have the same geometric shape as the distal end portion. In one embodiment, the rib portion 402a is designed to have a length 406, and the separation portion 402b is designed to have a length 407. In the cylindrical configuration, the rib portion protrudes outward to a height 408 defined by the difference between the outer diameter of the rib portion 402a and the outer diameter of the separation portion 402b. The thermowell tube 400 has an inner diameter 409 that is adapted to fit snugly over the outer diameter of a thermowell probe (not shown).

[0027] The reinforcement portion 402 may be defined by a variety of geometries to achieve one or more objectives of the present invention. For example, in one variation, the height 408 of the rib portion 402a may gradually increase in a continuous or stepped manner in the direction from the tip portion 401 to the base portion 403. In some cases, the gradual increase in height 408 may be a more continuous transition from the outer diameter of the tip portion 401 to the base portion 403. Various other reinforcement geometries are within the scope of the present invention.

[0028] FIG. 6 shows a thermowell tube having a tip portion 401 and a base 403 connected to a flange 404, bent to illustrate the operation of the reinforcement portion in accordance with an embodiment of the present invention. Rib portions 402a toward the inside of the bend 410 may be configured to come into contact with each other to resist further deformation of the thermowell tube 400. In a preferred embodiment, the contact between multiple rib portions 402a reinforces the overall structure when the tube is bent. This reinforcement protects the thermowell tube's internal conduit 409 from crushing, crimping, or other deformation. In this embodiment, the length 407 of the separation 402b between adjacent rib portions 402a increases toward the outside of the bend 411.

[0029] FIG. 7 shows a cutaway of thermowell tube 400 detailing the inner diameter of thermowell tube 400, according to an optional embodiment of the present invention. Thermowell tube 400, as described above, has an inner diameter 409 that accommodates a cylindrical thermowell probe (not shown). In this embodiment, the inner wall of thermowell tube 400 is provided with a notch 413, and the inner diameter increases from length 412 to length 414. The notch forms an internal channel that will allow the passage of air during sensor insertion and removal. Notch 413 allows air to escape from the interior of thermowell tube 400 when a thermowell probe is inserted into thermowell tube 400. It will be understood that the exact geometry of the notch can be varied to achieve one or more objectives of the present invention. The notch may also be designed to extend the entire length of the inner diameter of the thermowell cavity from the edge of the thermowell tube 400 or the edge of the optional thermowell base 403 to the end of the thermowell tip portion 401. Alternatively, the notch may extend over a defined portion of the inner diameter of the thermowell tube 400. In one aspect, the notch 413 extends beyond the reinforced portion and terminates at some point in the tip portion 401. Alternatively, the notch 413 extends to the edge of the reinforced portion 402. In yet another aspect, the notch terminates at a point within the reinforced portion 402.

[0030] It should be understood that the reinforcement section 402 described above for the thermowell tubing 400 can be applied to any other structure within the bioreactor bag or fermenter bag. The ribbed structure on the tubing can be applied to other applications for disposable bioreactor bags where the tubing may kink and block fluid flow. These areas include internal tubing for fluid transfer, such as internal sparge tubing or internal dip tubes that feed fluid directly into the bulk process fluid. This can be used at the top of the bioreactor for the discharge filter line, which needs to be curved to be contained and supported within the heater while providing a drain path for condensate returning to the bioreactor. Any additional lines supported across and draped over the rigid bar can benefit from a small section of curved ribbed tubing. This prevents tubing from becoming bloated, which can cause flow restrictions and high pressures. As with the thermowell tubing, these sections may be molded directly into the bag or may be attached to the bag using a flange as shown with the thermowell above.

[0031] It should be appreciated that the reinforced tube structure of the present invention is particularly beneficial when added to a single-use bioreactor. The ribs along the tube, along the molded thermowell tube 400, prevent kinking or self-sealing due to gamma irradiation. When used with a thermowell probe, the thermal sensor portion of the probe will be within less than 1 inch (less than 2.54 cm) of the thermowell tube tip portion 401, and the thermowell tube 400 will have a thin wall to promote a fast heat transfer response. An internal channel, or notch 413, will allow air passage during insertion and removal of the temperature sensor 405.

[0032] The reinforced tubing structure of the present invention, particularly the reinforced thermowell tubing, may be applied to any disposable bioprocess bag, particularly those systems manufactured from flexible materials such as plastic. Any of the disposable bags shown in Figures 1-3 may be modified to include a reinforced internal tubular structure using embodiments of the present invention. Additionally, any of the bags shown may be modified to include a reinforced thermowell tubing in accordance with aspects of this invention.

[0033] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited in this application, including all referenced patents and patent applications, are specifically and entirely incorporated herein by reference where permissible. It is intended that the specification and examples be considered exemplary with the true scope and spirit of the invention being determined by the following claims. [Explanation of symbols]

[0034] 1 bioreactor bag, bag 2 Upper seat 3 Bottom Sheet 4 Edge 5 Side edge 6 baffles 7 Movable axis 8. Support 9. Base 11 ports 56 Inside 100 devices 114 Support structure, support vessel 118 Containers, Collapsible Bags, Bioprocess Bags 120 Surrounding containment enclosure 122 Liquid 132 Gap 134 Control System 142 Inlet port 146 Outlet Port 150 Utility Tower 151 Impeller 152 Motor 154 port 156 Access Port 164 Connection 165 Gas inlet port 180 connection port 182 connection port 200 Bioreactor Port 202 204 Drain port 206 Sensor / Probe Port, Sensor / Probe 208 Part 400 tube structure, thermowell tube 401 Tip, thermowell distal end 402 Reinforcement parts, rib parts 403 base, thermowell base 404 flange, thermowell flange 405 Temperature Sensor 409 Internal conduit, inner diameter 410 Curved section 411 Curved section 413 Notch

Claims

1. A bag wall defining an enclosed volume for holding biomaterial, the bag wall having at least one inlet port (142) and at least one outlet port (146); a tube structure (400) extending into the enclosed volume, the tube structure (400) having an open end proximal to the bag wall and an end farther away (hereinafter referred to as the "distal end"), the tube structure (400) having a reinforced portion (402) proximal to the open end; In a bioprocess bag (118) comprising: The bioprocess bag (118), wherein the reinforcing portion (402) comprises a ribbed structure.

2. the distal end is a closed end; 10. The bioprocess bag (118) of claim 1, wherein the tubular structure is adapted to receive a probe (206) for measuring one or more properties of the biomaterial.

3. The bioprocess bag (118) of claim 2, wherein the probe (206) comprises a thermocouple or a resistance temperature detector (RTD).

4. The bioprocess bag (118) of any one of claims 1 to 3, wherein the tubing structure (400) is adapted to receive a sparging wand.

5. the rib structure comprises a pattern of raised portions (402a) relative to the outer tube surface; The bioprocess bag (118) of any one of claims 1 to 4, wherein the raised portions are in contact with each other when the tubular structure (400) is bent.

6. 6. The bioprocess bag (118) of claim 5, wherein the raised portion contact prevents crimping of the tubular structure (400).

7. 7. The bioprocess bag (118) of claim 5 or 6, wherein the height of the raised portion (402a) increases continuously or stepwise in a direction from the distal end toward the open end of the tubular structure (400).

8. The bioprocess bag (118) of any one of claims 1 to 7, wherein the tubular structure (400) comprises a mostly cylindrical inner wall and a notch (413) extending along at least a portion of the length of the inner wall.

9. 10. The bioprocess bag (118) of claim 8, wherein the notch (413) defines a channel that allows air to escape when a probe (206) is inserted into the tubular structure (400).

10. The bioprocess bag (118) of any one of claims 1 to 9, wherein the bag wall comprises one or more of an impeller (151), a heater, and / or a gas outlet.

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