Large bioreactor system and method

Abstract

A large bioreactor system comprising: a stainless steel large bioreactor having at least one valve assembly; and a sterile connector assembly coupled to the at least one valve assembly of the bioreactor. The perfusion device includes an alternating tangential filter assembly with an autoclaved valve assembly coupled to a sterile connector assembly, and the sterile connector assembly includes one of a three-clamp sterile connector or a hose assembly. A disposable feed container includes a sterile connector assembly.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority and filing date of U.S. Provisional Patent Application No. 63 / 404,033, filed on September 6, 2022. The entire contents of that provisional application are hereby expressly incorporated herein by reference. Technical Field

[0003] The present disclosure relates generally to large-scale bioreactor systems and, more particularly, to the integration of perfusion devices and / or disposable feed containers with large-scale bioreactors and the associated pressure management. Background Art

[0004] The use of perfusion in cell culture bioreactors allows for improved performance compared to traditional fed-batch cell culture processes. Perfusion allows for the continuous addition of nutrients to the cell culture as well as the continuous removal of metabolic byproducts. In fed-batch mode, the tank volume limits the cell culture process and all byproducts are contained within the bioreactor until harvest.

[0005] Traditionally, perfusion has been applied to small bioreactors, e.g., with working volumes up to 2000 L. When the perfusion device is mounted in the traditional position near the bottom of the side of a stainless steel large bioreactor, the pressure achieved by the perfusion device on the large bioreactor is larger due to the increased height of the liquid (static head) above the perfusion device. In addition, the perfusion device containing disposable components is particularly sensitive to pressure due to the low pressure rating of the disposable components. Summary of the invention

[0006] According to a first aspect, a large bioreactor system includes: a stainless steel large bioreactor having at least one valve assembly; and a sterile connector assembly coupled to the at least one valve assembly of the bioreactor. A perfusion device including an alternating tangential filtration (ATF) assembly is coupled to the sterile connector assembly, the alternating tangential filtration assembly having an autoclaved valve assembly, and the sterile connector assembly includes one of a three-clamp sterile connector or a hose assembly.

[0007] According to a second aspect, a large bioreactor system includes: a stainless steel large bioreactor having a side; and an autoclave valve assembly coupled to the side of the bioreactor. At least one sterile connector is coupled to the autoclave valve assembly, and an irradiated disposable perfusion device is coupled to the at least one sterile connector.

[0008] According to yet another aspect, a large bioreactor system includes: a stainless steel large bioreactor having at least one valve assembly; and a disposable adapter assembly including a Y-connector assembly coupled to the at least one valve assembly. A plurality of disposable perfusion devices are connected to the disposable adapter assembly, so that a plurality of perfusion units can be coupled to the bioreactor without having to steam-in-place the bioreactor when coupling the plurality of disposable perfusion units.

[0009] According to yet another aspect, a large bioreactor system includes a stainless steel large bioreactor and at least one stainless steel transfer plate having a plurality of inputs coupled to the bioreactor. A plurality of disposable feed containers are coupled to the at least one stainless steel transfer plate at the working level of the bioreactor. Another aspect is that the stainless steel large bioreactor can be mounted in a recess in the floor to facilitate easy access to the probe belt for work. Another aspect is that a fully enclosed stainless steel large bioreactor can be mounted in an uncontrolled space and work only at the probe belt and local attachment plates. When so configured, the size of the controlled space is reduced, making the operating cost of the manufacturing facility housing the bioreactor lower.

[0010] According to yet another aspect, a method of integrating at least one disposable perfusion device with a stainless steel large-scale bioreactor includes making one of the following connections: (1) connecting a connector assembly to at least one valve assembly of the stainless steel large-scale bioreactor; or (2) connecting an autoclave valve assembly to a higher elevation of the side of the stainless steel large-scale bioreactor to reduce pressure from the hydrostatic head. The method further includes making one of the following connections: (1) connecting the autoclave valve assembly of the ATF assembly of the disposable perfusion device to the connector assembly; or (2) connecting an irradiated disposable perfusion device to the autoclave valve assembly. The method further includes managing the pressure of the disposable perfusion device via at least one pressure sensor of the disposable perfusion device, and automatically reducing one or more of the flow rate or pressure in the disposable perfusion device by a control system when the pressure is detected to be greater than a safety limit pressure.

[0011] In some embodiments of any aspect, the stainless steel large-scale bioreactor is configured to hold a volume greater than 2,000L, such as a volume in the range of greater than 2,000L to 20,000L, such as a volume in the range of 10,000L to 20,000L. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] It is believed that the present disclosure will be more fully understood from the following description in conjunction with the accompanying drawings. Some of the drawings may be simplified by omitting selected elements in order to more clearly illustrate other elements. Unless otherwise expressly described in the corresponding written description, such omission of elements in some drawings does not necessarily indicate the presence or absence of a particular element in any example embodiment. Moreover, all drawings are not necessarily drawn to scale.

[0013] Figure 1 is a schematic diagram of a steam sterilizable in place (SIP) bioreactor (BRX) having a valve assembly of a stainless steel large-scale bioreactor system according to one aspect of the present disclosure, showing how the SIP bioreactor can be steam sterilized in place without a priming device or feed container connected to the valve;

[0014] Figure 2 yes Figure 1 A schematic diagram of an online steam sterilization bioreactor, wherein a sterile connector assembly of the online steam sterilization bioreactor is connected to a valve assembly of the online steam sterilization bioreactor;

[0015] Figure 3 yes Figure 2 Another schematic diagram of an online steam sterilization bioreactor, wherein the connector assembly is coupled to a valve assembly, the valve assembly can be steam sterilized online, regardless of the operation of the online steam sterilization bioreactor;

[0016] Figure 4 yes Figure 2 or Figure 3 Schematic diagram of an online steam sterilizable bioreactor having a disposable (SU) perfusion device or a reusable perfusion device including an alternating tangential filtration (ATF) assembly coupled to a connector assembly using a sterile connector;

[0017] Figure 5 yes Figure 4 Close-up view of the perfusion device and associated valve assembly after being cleaned off-line (COP) and assembled prior to autoclaving in preparation for connection to an online steam sterilization bioreactor;

[0018] Figure 6 is another large-scale bioreactor system according to another aspect of the present disclosure, wherein one of a plurality of autoclave valve assemblies of the large-scale bioreactor system is coupled to a stainless steel large-scale bioreactor and an irradiated disposable perfusion device is aseptically coupled to the autoclave valve assembly using a sterile connector;

[0019] Figure 7is another large-scale bioreactor system according to another aspect of the present disclosure, the large-scale bioreactor system comprising a hose assembly coupling at least one valve assembly of a stainless steel large-scale bioreactor to an autoclaved perfusion device, such as a disposable perfusion device or a reusable autoclaved perfusion device including an ATF assembly;

[0020] Figure 8 is a schematic diagram of a CIP bioreactor having at least one valve assembly, wherein the CIP bioreactor is capable of online cleaning of the at least one valve assembly using the CIP bioreactor;

[0021] Fig. 9 is another large-scale bioreactor system according to another aspect of the present disclosure, the large-scale bioreactor system comprising a stainless steel large-scale bioreactor having at least one valve assembly and a Y-assembly coupled to the at least one valve assembly to enable use of multiple perfusion devices on a single port;

[0022] Fig.10 is a schematic diagram of a plurality of Y-shaped assemblies coupled to a plurality of valve assemblies, so that a plurality of disposable feed containers can be coupled to the Y-shaped assemblies and ultimately to a stainless steel large bioreactor without having to perform in-line steam sterilization of the valve assemblies multiple times;

[0023] Fig.11 is a schematic diagram of a large-scale bioreactor system having a stainless steel large-scale bioreactor, one of a plurality of autoclave valve assemblies of the large-scale bioreactor system coupled to a top portion of the stainless steel large-scale bioreactor and an irradiated perfusion device aseptically coupled to at least one of the autoclave valve assemblies;

[0024] Fig.12 is a schematic diagram of a stainless steel large-scale bioreactor of any large-scale bioreactor system of the present disclosure, the stainless steel large-scale bioreactor being at least partially disposed in a pit to enable efficient operation at a probe belt and a transport plate; and

[0025] Fig.13 is a schematic diagram of a stainless steel large-scale bioreactor with a transfer plate coupled to the bioreactor allowing multiple feed vessels to be coupled to the stainless steel bioreactor at a working level. DETAILED DESCRIPTION

[0026] In general, a large-scale bioreactor system is disclosed. The large-scale bioreactor system includes: a stainless steel large-scale bioreactor having at least one valve assembly; a sterile connector assembly coupled to at least one valve assembly of the bioreactor; and a disposable or reusable perfusion device coupled to the sterile connector assembly. The disposable perfusion device includes a perfusion filter assembly with an autoclave valve assembly coupled to the sterile connector assembly. When so configured, a new perfusion device can be installed while the stainless steel large-scale bioreactor is undergoing cell culture by repeating in-line steam sterilization of the sterile connector assembly or by using a sterile connector valve assembly.

[0027] Reference now Figures 1 to 5 , depicts a large-scale bioreactor system 10 ( Figure 4 ). Figure 1 As depicted, the large bioreactor system 10 includes a stainless steel large bioreactor 12 having at least one valve assembly 14. In these examples, the stainless steel large bioreactor 12 is an online steam sterilization SIP bioreactor BRX with a large capacity. For example, in one example, the large bioreactor 12 is configured to accommodate a volume of fluid up to 10,000L or more. The at least one valve assembly 14 of the online steam sterilization large bioreactor 12 enables the online steam sterilization large bioreactor 12 to be completely sterilized before the start of perfusion. Specifically, at least one valve assembly 14 includes a first valve 16, a second valve 18, a third valve 20 disposed downstream of each of the first valve 16 and the second valve 18, and a fourth valve 21. Steam (such as cleaning steam CS) flows into the third valve 20, flows upward into the first valve 16 and the second valve 18, and flows to the port 24. In addition, after flowing into the third valve 20, the steam also flows downward into the fourth valve 21 and passes through the steam trap 22, in which the clean steam condensate CSC is released, for example, to sterilize the bioreactor 12 and create a sterile environment for steam sterilization. In one example, when the SIP cycle ends, the condensate (such as clean steam condensate CSC) is discharged from the steam trap 22. In addition, as understood by ordinary technicians, during cleaning, the process waste PW flows out of the third valve 20 and the fourth valve 21 and is emptied into the drain pipe. After use, the online steam sterilization bioreactor 12 is cleaned online and the online steam sterilization sterilization process is performed again before any further use. At least one valve assembly 14 also includes a port 24, which is used to connect to a sterile connector valve assembly 26 (such as Figure 2The depicted autoclaved sterile connector valve assembly 26) or another device, as explained in more detail below.

[0028] Now refer to Figure 2 , the sterile connector valve assembly 26 is coupled to at least one valve assembly 14. Specifically, and in the present example, the sterile connector valve assembly 26 is coupled to the port 24 of at least one valve assembly 14 and includes a first valve 28 and a second valve 30 disposed downstream of the first valve 28. A steam trap 32 is further disposed downstream of the second valve 30. In the present example, the autoclaved sterile connector valve assembly 26 also includes a three-clamp sterile connector 34 coupled to the first valve 28. The sterile connector valve assembly 26 enables a disposable perfusion device or a reusable perfusion device to be aseptically connected to a large bioreactor 12. The three-clamp sterile connector 34 is configured to be coupled to a perfusion device, as explained more below. As will be understood, and in one example, the three-clamp sterile connector 34 and the first valve 28 and the second valve 30 can be pre-assembled and autoclaved, and then connected to at least one valve assembly 14 for, for example, steam sterilization.

[0029] Reference now Figure 3 , again depicting the large-scale bioreactor 12, the large-scale bioreactor having: at least one valve assembly 14; and a sterile connector valve assembly 26, which is coupled to the at least one valve assembly 14. However, in this embodiment, the sterile connector valve assembly 26 is sterilized independently of the steam-in-place sterilization of the large-scale bioreactor 12. Specifically, for example, if during a cell culture run in the large-scale bioreactor 12 it is determined that the perfusion device requires repair and / or replacement, operation of the large-scale bioreactor 12 can continue. This is because the sterile connector valve assembly 26 is capable of steam sterilizing, for example, the first valve 28 and the second valve 30 and all other portions of the sterile connector valve assembly 26 to provide a sterile connector assembly for coupling to a repaired and / or new perfusion device.

[0030] Reference now Figure 4 , depicts a schematic diagram of a large-scale bioreactor system 10 according to one aspect of the present disclosure. More specifically, a stainless steel large-scale bioreactor 12 is depicted having: at least one valve assembly 14; and a sterile connector valve assembly 26 coupled to the at least one valve assembly 14. In addition, a perfusion device 36, which may be a reusable perfusion device or a disposable perfusion device, is coupled to a three-clamp sterile connector 34 of the sterile connector valve assembly 26. As shown in FIG. Figure 4 and Figure 5, the perfusion device 36 includes an alternating tangential filtration (ATF) assembly 38 with an autoclave valve assembly 40 operably coupled to the sterile connector 34 of the sterile connector valve assembly 26. When so configured, the large-scale bioreactor system 10 enables the perfusion device 36 (such as a reusable perfusion device or a disposable perfusion device) to be connected to the stainless steel large-scale bioreactor 12 without the need for additional steam sterilization.

[0031] Also like Figure 5 As depicted, the autoclave valve assembly 40 of the perfusion device 36 includes a first valve 42 and a second valve 44 disposed downstream of the first valve 42. In this example, the autoclave valve assembly 40 is cleaned off-line (COP) and assembled prior to autoclaving in preparation for connection to the online steam sterilization bioreactor 12. Generally, COP means that systems and equipment (such as the autoclave valve assembly 40 in this example) are one or more of disassembled, relocated, or specially processed for cleaning and sterilization. In other words, COP is defined as a method of cleaning equipment items by removing the equipment items from their operating area and taking them to a designated cleaning station for cleaning. In addition, as Figure 4 As depicted, the sterile connector 46 is operably coupled to the first valve 42 and directly coupled to the triple-clamp sterile connector 34 of the sterile connector assembly 26. The sterile connector assembly 26 allows for connection between the disposable perfusion set 36 and the stainless steel large-scale bioreactor 12 and provides the ability to re-steam sterilize on subsequent perfusion sets during a single run of the bioreactor 12. In other words, in this configuration, the perfusion set 36 and / or a new perfusion set can be installed while the stainless steel large-scale bioreactor 12 is currently culturing cells, for example, by repeated online steam sterilization or by using the sterile connector assembly 26.

[0032] Now refer to Figure 6 , depicting another large bioreactor system 100. Similar to Figures 1 to 5 The large-scale bioreactor system 10 further includes a stainless steel large-scale bioreactor 112 having a side 113 and a first valve assembly 114 coupled to the side 113 of the stainless steel large-scale bioreactor 112, the first valve assembly being, for example, an autoclave valve assembly 114. The autoclave valve assembly 114 includes a first valve 116, a second valve 118 downstream of the first valve 116, a third valve 120 downstream of the first valve 116 and the second valve 118, and a fourth valve 121 downstream of the third valve 120. Similarly similar to Figures 1 to 5In the valve assembly 14 of the system 10 of the present invention, steam (e.g., clean steam CS) flows into the third valve 120, flows upward to the first valve 116 and the second valve 118, and flows to the port 134. In addition, after flowing into the third valve 120, the steam flows downward through the fourth valve 121 and flows through the steam trap 122 to sterilize the bioreactor 112 and, for example, produce a sterilization process of online steam sterilization before further use. However, in this large bioreactor system 100, only at least one sterile connector 134 is connected to the autoclave valve assembly 114 (e.g., the first valve 116 of the autoclave valve assembly 114) instead of being connected to the second valve assembly, similar to Figures 1 to 5 The same autoclave valve assembly 114 can be connected to a factory assembled and irradiated perfusion device 136 via at least one sterile connector 134 of the autoclave valve assembly 114, which can be, for example, a disposable perfusion device or a reusable perfusion device. More specifically, as Figure 6 As depicted, the irradiated perfusion device 136 includes a first sterile connector 138 coupled to at least one sterile connector 134. The irradiated perfusion device 136 also includes a second sterile connector 140 that is configured to couple to a second autoclave valve assembly (not shown), such as a second valve assembly, that is also configured to couple directly to the side 113 of the stainless steel large bioreactor 112 on a different valve assembly similar to the valve assembly 114. As a result, multiple perfusion devices can be aseptically coupled to the stainless steel large bioreactor 112 without the need for additional steam sterilization.

[0033] Reference now Figure 7 , depicts a schematic diagram of another large-scale bioreactor system 200 of the present disclosure. For example, similar to Figure 4 The large-scale bioreactor system 10 of the present invention comprises a stainless steel large-scale bioreactor 212 having a side wall 213 and at least one valve assembly 214 disposed on the side wall 213. Figure 4 Large-scale bioreactor systems 10, Figure 7 The same valve assembly 214 of the large bioreactor 200 can achieve the connection of the autoclaved perfusion device and can achieve online cleaning of the valve assembly 214, but without using a sterile connector (such as Figure 4 Instead, the hose assembly is used as an alternate connector to connect the stainless steel large bioreactor 212 to the autoclaved perfusion device, as explained more below.

[0034] More specifically, and as Figure 7As depicted, the stainless steel large bioreactor 212 of the large bioreactor system 200 is also an online steam sterilization bioreactor with a large capacity. For example, in one example, the large bioreactor 212 is configured to accommodate a fluid volume of up to or greater than 10,000 L. The at least one valve assembly 214 of the online steam sterilization large bioreactor 212 enables the online steam sterilization stainless steel large bioreactor 212 to be completely sterilized at the beginning of perfusion. Specifically, and similar to Figure 4 At least one valve assembly 14 of the bioreactor system 10 of the present invention includes a first valve 216, a second valve 218, and a third valve 220 disposed downstream of each of the first valve 216 and the second valve 218. As depicted, a fourth valve 221 may also be disposed downstream of the third valve 220. Steam (such as clean steam CS) also flows into the third valve 220, flows upward to the first valve 116 and the second valve 118, and flows to the port 224. In addition, after flowing into the third valve 220, the steam also flows downward into the fourth valve 221 and flows through the steam trap 222 to sterilize the bioreactor 212 and create a sterile environment for steam sterilization. In addition, as will be understood by a person of ordinary skill, the process waste PW may also flow out of the third valve 220 and the fourth valve 221 during cleaning and be emptied into the drain. After use, the online steam sterilization bioreactor 212 is cleaned online, and the sterilization process of online steam sterilization is performed again before any further use. The at least one valve assembly 214 also includes a port 224 for coupling to a hose assembly 235, which in turn is coupled to an irrigation device or another device, as explained more below.

[0035] The hose assembly 235 includes a hose body 237 having a first end 237A and a second end 237B. The first end 237A is removably coupled to the port 224 of the at least one valve assembly 214, and the second end 237B is coupled to an autoclave filling device 236, which is functionally equivalent to, for example, Figure 4 The perfusion device 36 of the system 10, but without the disposable connector, such as Figure 5The autoclave perfusion device 236 may include a disposable perfusion device or a reusable perfusion device and still fall within the scope of the present disclosure. The autoclave perfusion device 236 includes an ATF assembly 238 with an autoclave valve assembly 240 that is coupled to a second end 237B of the hose body 237. The autoclave valve assembly 240 of the autoclave perfusion device 236 includes a first valve 242 and a second valve 244 disposed downstream of the first valve 242. The second end 237B of the hose body 237 is operably coupled to a connector of the first valve 242. The hose assembly 235 allows connection between the autoclave device 236 and the stainless steel large bioreactor 212 and provides the ability to re-steam sterilize on subsequent perfusion devices during a single run of the stainless steel large bioreactor 212.

[0036] More specifically, and with reference now to Figure 8 , the hose assembly 235 can be removed from the connection with the perfusion device 236 and connected to another port of the plurality of ports 224 on the same large bioreactor 212. The online cleaning valve assembly 250 allows cleaning of the at least one valve assembly 214, which enables another perfusion device (such as a disposable perfusion device) to be aseptically connected to the stainless steel large bioreactor 212 via the hose assembly 235. In this example, the online cleaning steam supply of the online cleaning valve assembly 250 flows upward into the third valve 220 and the second valve 218 of the at least one valve assembly 214, flows upward through the first valve 216, reaches the port 224 and flows through the hose assembly 235. In addition, the online cleaning steam supply also flows downward through the fourth valve 221 downstream of the third valve 220. In this way, each of the first valve 216, the second valve 218, the third valve 220 and the fourth valve 221 and the port 224 of the at least one valve assembly 214 are cleaned. Although the present description relates to at least one valve assembly 214, it should be understood that the at least one valve assembly 214 may include multiple valve assemblies (not shown), and the online cleaning steam supply of the online cleaning valve assembly 250 may also flow through each valve and port of another valve assembly in the multiple valve assemblies, thereby allowing cleaning of the multiple valve assemblies.

[0037] Reference now Fig. 9, depicts another schematic diagram of another large-scale bioreactor system 300 of the present disclosure. The large-scale bioreactor system 300 includes a stainless steel large-scale bioreactor 312 having a side 313, and at least one valve assembly 314 coupled to the side 313, and an autoclave valve assembly 326 coupled to the at least one valve assembly 314. In one example, the stainless steel large-scale bioreactor 312 is also an online steam sterilization bioreactor with a large capacity. For example, in one example, the large-scale bioreactor 312 is configured to accommodate a fluid volume of up to or greater than 10,000 L. The at least one valve assembly 314 of the online steam sterilization large-scale bioreactor 312 enables the online steam sterilization large-scale bioreactor 312 to be completely sterilized at the beginning of perfusion. Specifically, and similar to Figure 4 At least one valve assembly 14, for example, at least one valve assembly 314 includes a first valve 316, a second valve 318, a third valve 320 disposed downstream of each of the first valve 316 and the second valve 318, and a fourth valve 321 disposed downstream of the third valve 320, as depicted. Steam (also such as clean steam CS) flows into the third valve 320 and flows upward into the second valve 318 and the first valve 316 and the port 324 to sterilize the at least one valve assembly 314 and the bioreactor 312. In addition, after flowing through the third valve 320, the steam also flows downward through the fourth valve 321 and flows through the steam trap 322 to sterilize the bioreactor 312 and create a sterile environment for steam sterilization. In addition, as understood by ordinary technicians, the process waste PW flows out of the fourth valve 321 during cleaning and is emptied into the drain pipe. After use, the online steam sterilization bioreactor 312 is cleaned online and the sterilization process of online steam sterilization is performed again before any further use. The at least one valve assembly 314 also includes a port 324 for coupling to an autoclave valve assembly 326 , and a sterile connector 334 .

[0038] Unlike the other bioreactor systems 10, 100, and 200, the bioreactor system 300 includes an adapter assembly 360, such as a disposable adapter assembly and / or a Y-assembly, which is configured and / or coupled to a sterile connector 334, also such as Fig. 9 When so configured, in the present example, the Y-assembly 360 is coupled to the at least one valve assembly 314 via the sterile connector 334 .

[0039] The adapter assembly 360 (e.g., Y-assembly 360) includes a connector 361 directly coupled to the sterile connector 334 (which is ultimately coupled to the port 324), a pair of tubes 362 extending outwardly from the connector 361, a first sterile connector 364 coupled to one of the tubes 362, and a second sterile connector 366 coupled to the other tube 362. Each of the first sterile connector 364 and the second sterile connector 366 of the Y-assembly 360 is configured to be coupled to a reusable or disposable perfusion device, such as any of the aforementioned perfusion devices 36, 136, and 236. This enables multiple perfusion devices to be operably coupled to the stainless steel large bioreactor 312 via the port 324 of the valve assembly 314, for example, without having to perform in-line steam sterilization of the stainless steel large bioreactor 312 when multiple reusable perfusion devices or disposable perfusion devices are coupled. In another example, the adapter assembly 360 (e.g., Y-assembly 360) is a first Y-assembly 360, and a second Y-assembly (not shown) can be coupled to the first Y-assembly. When so configured, coupling one or more additional Y-assemblies to the first Y-assembly 360 enables more than two reusable or disposable perfusion devices and multiple reusable or disposable perfusion devices to be operably coupled to the stainless steel large bioreactor 312, and also when multiple disposable perfusion devices are coupled, it is not necessary to perform online steam sterilization of the stainless steel large bioreactor 312. Likewise, a greater number of perfusion devices can be connected to the large bioreactor 312, and each device does not require a different reactor port.

[0040] Reference now Fig.10 , in another example, a large-scale bioreactor system 300 is depicted and includes a stainless steel large-scale bioreactor 312, which in one example is configured to accommodate a fluid volume of up to or greater than 10,000 L. In this embodiment, at least one valve assembly of the large-scale bioreactor system 300 includes at least one valve assembly 380, which is configured to couple to a transfer plate (at Fig.10(not depicted), the transfer plate is installed locally or remotely from the stainless steel large bioreactor 312 for the purpose of connecting and transferring feed liquid contained in a disposable container to the stainless steel large bioreactor 312. The valve assembly 380 is configured to enable steam sterilization of the transfer line 385 and connect to the disposable feed container. Steam from the large bioreactor 300 passes through at least one transfer line, such as the transfer line 385 in one example, and reaches the three-clamp port 324 and the sterile connector assembly 334, and passes through the first valve 381, the second valve 382, ​​the third valve 383 and the fourth valve 384, and goes down to the steam trap 322. In this way, the entire pipeline is sterile all the way to the sterile connector 334. Similarly, cleaning of the pipeline is accomplished by supplying an online cleaning (CIP) solution through the first valve 381 and the second valve 382 and through the transfer line 385 to the large bioreactor 300. As shown Fig.10 As depicted, the ports 324 of the first valve 381 and the second valve 382 are connected to a disposable adapter, such as a sterile connector 334, designed to allow direct exposure to clean steam at sterilization temperature and pressure. As further depicted, the disposable adapter (such as a sterile connector assembly 334) is then connected to a disposable manifold 370. In this example, each disposable manifold 370 includes eight or more arms 371 with an inlet 372, each of which is configured to be connected to a feed container 373, such as a disposable feed container. In another example, the disposable manifold 370 may include more than or less than eight arms 371, such as four or six arms, and still fall within the scope of the present disclosure. When configured in this way, multiple feed containers can be operably connected to a stainless steel large bioreactor 312 without additional steam sterilization and cleaning. When the stainless steel large bioreactor 312 is at work, one or more additional feed containers 373 can be connected using a sterile connector 372. Furthermore, one or more additional disposable manifolds 370 may be connected to the inlet 372 via a sterile connector on the first disposable manifold assembly 370 to further increase the number of feed containers 373 that may be connected.

[0041] Refer again Fig.10If the sterile connector to be employed cannot tolerate direct exposure to clean steam at sterilization temperature and pressure, the alternate configuration enables the sterile connector to be isolated from the steam. For example, one or more valve assemblies 390A and 390B can be prepared by coupling one or two valves, such as valve 391A of valve assembly 390A and first valve 391B and second valve 393B of valve assembly 390B (these valves can be any valves with a small retention volume on the side to be steam sterilized) to a sterile connector (such as sterile connector 392A or sterile connector 392B) and autoclaving. Each of valve assembly 390A and valve assembly 390B can be inserted into a valve assembly (such as a sterile connector 392A or a sterile connector 392B) that can be mounted on a local or remote transmission board by, for example, coupling with a three-clamp connector. Fig.10 At least one valve assembly 380A) is depicted in FIG. From this point on, cleaning, steam sterilization and operation are combined with the above Fig. 9 The same as described above. By using a disposable manifold assembly with a sterile connector 392, a larger inner diameter, less shear, and higher flow rate than can be achieved using a sterile connector that can be directly exposed to clean steam at sterilization temperature and pressure is provided. However, since the main transfer line 385 and at least one valve assembly 380, 380A are the same, either type of sterile connector can be used and similar multiple feed containers 373 can be connected. Alternatively, when a higher flow rate or less shear is required, a single connection to the feed container 373 can be used.

[0042] Reference now Fig.11 , depicted again Figure 6 The bioreactor system 100 of the present invention includes all of the same features of the bioreactor system 100, except for the position of the valve assembly 114 relative to the stainless steel large-scale bioreactor 112. Specifically, the valve assembly 114 is mounted higher on the side 113 of the stainless steel large-scale bioreactor 112, which in turn allows the perfusion device 136 to be mounted higher on the side 113 of the stainless steel large-scale bioreactor 112. In particular, in this embodiment, the side 113 of the bioreactor 112 includes a bottom end 113A and a top end 113B, and the valve assembly 114 is coupled to the side 113 of the bioreactor at a position closer to the top end 113B of the side 113 than to the bottom end 113A, as shown in FIG. Fig.11 When so configured, the height of the liquid L disposed above the perfusion device 136 is reduced, and thus the pressure is reduced.

[0043] Typically, the pressure achieved by a disposable perfusion device (such as the perfusion device 36, 136, 236) is greater on a stainless steel large-scale bioreactor 12, 112, 212, 312 due to the increased liquid height (static head) above the perfusion device 36, 136, 236 when installed in a typical location near the bottom side (such as the bottom end 113A) of the stainless steel large-scale bioreactor 12, 112, 212, 312. For example, due to the low pressure rating of the disposable components included in the perfusion device, the perfusion device including these disposable components is particularly sensitive to larger pressures. Therefore, by placing the valve assembly 114 and thus Fig.11 The perfusion device 136 is installed near the top end 113B of the bioreactor 112, and the pressure is reduced.

[0044] Still reference Fig.11 , the perfusion device 136 (such as a disposable perfusion device) includes at least one pressure sensor 180 and may include additional pressure sensors. In this example, the perfusion device 136 (such as an RFT disposable perfusion device) includes a first pressure sensor 180, a second pressure sensor 182, and a third pressure sensor 184. In addition, the perfusion device 136 also includes a circulation pump 186 disposed upstream of the first pressure sensor 180 and a permeate pump 188 disposed downstream of the third sensor 184.

[0045] like Fig.11As further depicted in FIG. 1 , a control system 190 for monitoring the pressure of at least one perfusion device 136 is communicatively coupled to the disposable perfusion device 136 via a network such as a wireless network 192 or a wired connection 193. Although the control system 190 is operably coupled to the perfusion device 136, the control system 190 may also be optionally coupled to any of the other aforementioned perfusion devices 36, 236 and still fall within the scope of the present disclosure. In one example, the control system 190 includes a computing device 194 having a memory 195, a processor 196, and a network interface 197. It should be understood that the computing device 194 may also include any known inputs, receivers, and transmitters. In addition, the control system 190 further includes an alarm 198. When so configured, the control system 190 monitors the pressure at one or more of the first pressure sensor 180, the second pressure sensor 182, and the third pressure sensor 184. If the pressure approaches a predefined safety limit, the alarm 198 will be activated to notify the user, so that the bioreactor system 100 can be investigated and evaluated. If the pressure monitored by the control system 190 is closer to the predefined safety limit, the control system 190 will automatically reduce the flow rate of the fluid and thus reduce the pressure to maintain a safe operating pressure for the bioreactor system 100. If the pressure monitored by the control system 190 reaches the safety pressure limit, the control system 190 will stop the circulation pump 186 to prevent damage to the perfusion device 136.

[0046] Reference now Fig.12 Any of the aforementioned large stainless steel bioreactors 12, 112, 212, 312 can be disposed in the pit 402, such as Fig.12 In addition, the valve assembly 14, 114, 214, 314 is then mounted near the bottom end 13A, 113A, 213A, 313A of the side 13, 113, 213, 313 of the stainless steel large bioreactor 12, 112, 212, 312 and against the bottom weld 404 of the body of the bioreactor 12, 112, 212, 312. This allows the disposable perfusion device 36, 136, 236 to be positioned higher, minimizing the hold-up volume between the perfusion device 36, 136, 236 and the stainless steel large bioreactor 12, 112, 212, 312. Alternatively, by adjusting the depth of the pit, the pit 402 can be used to position any of the perfusion devices 36, 136, 236 higher on the side 13, 113, 213 of the bioreactor 12, 112, 212, thereby also reducing pressure.

[0047] Reference now Fig.13, depicts another schematic diagram of another large-scale bioreactor system 500 of the present disclosure. The large-scale bioreactor system 500 includes a stainless steel large-scale bioreactor 512 having a side 513 with a bottom end 513A and a top end 513B. In addition, at least one stainless steel transfer plate 520 having a plurality of input ends 522 is coupled to the side 513 of the stainless steel large-scale bioreactor 512 at the top end 513B of the side 513. Further, at a working level 526 of the stainless steel large-scale bioreactor 512, a plurality of disposable feed containers 524 are coupled to one or more of the plurality of input ends 522 of the transfer plate 520. Although in Fig.13 One stainless steel transfer plate 520 is depicted in the drawings, but it should be understood that multiple stainless steel transfer plates may alternatively and / or additionally be coupled to the bioreactor 512 and still fall within the scope of the present disclosure. When so configured, this prevents a user from having to climb a stairwell each time something is connected to the bioreactor 512. Additionally, this allows for connection to disposable feed containers within a smaller clean space without positioning the entire bioreactor 512 within the clean space, for example, a portion of the bioreactor is located in a controlled non-classified (CNC) space. This also enables a user to work from a clean, controlled space, operating the bioreactor 512 from a clean, controlled space without having to approach the top of the bioreactor 512.

[0048] Furthermore, it should be understood that all components of any one or more of the aforementioned bioreactor systems 10, 100, 200, 300, and 500 that require steam sterilization can be steam sterilized at the same time as the associated bioreactor 12, 112, 212, 312, 512 is steam sterilized. In this scenario, the steam source is from the bioreactor tank 12, 112, 312, 412, 512, and the steam flows out through the valve and flows to the steam trap depicted in the figure. For example, in Figure 2 In the bioreactor system 10, steam can flow out of the bioreactor 12 through the first valve 16 and the second valve 18, flow to the third valve 20 and the fourth valve 21 and flow to the steam trap 22. Figure 6 In the bioreactor system 100 of FIG. 1 , steam can also flow out of the bioreactor 112, flow through the first valve 116, the second valve 118, the third valve 120, and the fourth valve 121 and flow to the steam trap 122. In a similar manner, in Fig. 9In the bioreactor system 200, steam can again flow out of the bioreactor 212 into the first valve 216, the second valve 218, the third valve 220, and the fourth valve 221 and flow to the steam trap 222. Similarly, in the bioreactor system 300, steam can also flow from the bioreactor 312 into the first valve 316 and the second valve 318, flow through the third valve 320 and the fourth valve 321, and flow to the steam trap 322.

[0049] In view of the above, it should be understood that any one or more of the aforementioned bioreactors 12, 112, 212, 312 can be integrated with any one or more of the aforementioned perfusion devices, such as a reusable perfusion device or a disposable perfusion device 36, 136, 236, according to one or more of the following methods. Specifically, and according to one example, the method of integrating at least one disposable perfusion device 36, 136, 236 with the stainless steel large-scale bioreactor 12, 112, 212, 312 includes making one of the following connections: (1) connecting a connector assembly 26 (such as a sterile connector assembly 26) to at least one valve assembly 14, 214 of the stainless steel large-scale bioreactor 12, 112, 212, 312; or (2) connecting an autoclave valve assembly 116 to the side 113 of the stainless steel large-scale bioreactor 112. In addition, the method further includes performing one of the following connections: (1) connecting the autoclave valve assembly 40, 240 of the ATF assembly of the disposable perfusion device 36, 236 to the connector assembly 26; or (2) connecting the irradiated perfusion device 136 to the autoclave valve assembly. Moreover, the method further includes managing the pressure of the perfusion device 36, 136, 236 via at least one pressure sensor of the perfusion device 36, 136, 236, and automatically reducing one or more of the flow rate or pressure in the perfusion device 36, 136, 236 through the control system 190 when the pressure is detected to be greater than the safety limit pressure.

[0050] In one example, the method includes coupling the sterile connector assembly to at least one valve assembly 14, 214 of the stainless steel large bioreactor 12, 212, and coupling the autoclave valve assembly 40 to the sterile connector assembly 26, wherein the sterile connector assembly 26 includes one of a three-clamp connector assembly or a hose assembly. In another example, the method includes coupling the autoclave valve assembly 114 to the side 113 of the stainless steel large bioreactor 112 and coupling the perfusion device 136 to one of the plurality of autoclave valve assemblies 114. Although only one autoclave valve assembly 114 is depicted, it should be understood that two or more autoclave valve assemblies 114 can be coupled to the perfusion device 136 and still fall within the scope of the present disclosure.

[0051] In yet another example, the method includes coupling a connector assembly 360 to at least one valve assembly 314 of a stainless steel large bioreactor 312, the connector assembly 360 comprising a Y-shaped connector assembly. In addition, the method further includes coupling an autoclave valve assembly 40 of an ATF assembly of at least one perfusion device 36 to the connector assembly 360, wherein the at least one perfusion device 36 or feed container 373, 524 comprises a plurality of perfusion devices or feed containers connected to the Y-shaped connector assembly. This enables a plurality of perfusion devices or feed containers to be coupled to the bioreactor 12, 112, 212, 312 without having to perform online steam sterilization of the bioreactor 12, 112, 212, 312 when coupling the plurality of perfusion devices or feed containers.

[0052] The above description describes various bioreactor systems and methods of integrating at least one disposable perfusion device and / or at least one feed container with a stainless steel large-scale bioreactor. It should be understood that the systems and methods of the present disclosure include several advantages. For example, the described systems and methods enable the connection of a perfusion device to a large-scale bioreactor (e.g., a bioreactor with a capacity greater than 2,000 L), replacement of a perfusion device or feed container during a cell culture run, and pressure management of a perfusion device while creating a steam sterilized sterile environment. In addition, the same valve assembly at the bioreactor is capable of connecting factory-assembled and irradiated disposable perfusion devices and autoclaved perfusion devices, and the valve assembly of the bioreactor can be cleaned in-line without the use of, for example, a sterile connector.

[0053] Although the foregoing systems and methods and elements thereof have been described with respect to exemplary embodiments, they are not limited thereto. The detailed description is to be construed as exemplary only and does not describe every possible embodiment of the invention, as describing every possible embodiment would be impractical, if not impossible. Many alternative embodiments may be implemented using current technology or technology developed after the filing date of this patent, which still fall within the scope of the claims defining the invention.

[0054] It should be understood that the legal scope of the present invention is defined by the words of the claims set forth at the end of this patent. The appended claims should be interpreted broadly to include other variations and embodiments of the appended claims that may be made by those skilled in the art without departing from the scope of equivalents of the devices, systems, methods, and elements thereof.

Claims

1. A large-scale bioreactor system comprising: a stainless steel large-scale bioreactor having at least one valve assembly; a sterile connector assembly coupled to at least one valve assembly of the bioreactor; as well as A perfusion device or reusable alternating tangential filtration (ATF) assembly having an autoclave valve assembly coupled to the sterile connector assembly including one of a three-clamp sterile connector or hose assembly.

2. The bioreactor system of claim 1, wherein: The perfusion device is a disposable perfusion device, the disposable perfusion device further comprises at least one pressure sensor, and the bioreactor system further comprises a control system coupled to the disposable perfusion device for monitoring the pressure of the disposable perfusion device via the at least one pressure sensor.

3. The bioreactor system of claim 1, wherein: The perfusion device or reusable ATF assembly is a disposable perfusion device or reusable ATF assembly further comprising at least one pressure sensor, and the bioreactor system further comprises a control system coupled to the disposable perfusion device or the reusable ATF assembly for monitoring the pressure of the disposable perfusion device or the reusable ATF assembly via the at least one pressure sensor.

4. A bioreactor system according to claim 2 or claim 3, wherein: The control system further includes an alarm configured to be activated when the control system detects that the pressure is greater than a safety pressure limit, and the control system is configured to automatically reduce one or more of the flow rate or pressure in the disposable perfusion device or the reusable ATF assembly in response to detecting that the pressure is greater than the safety pressure limit.

5. The bioreactor system according to any one of claims 1 to 3, wherein: The bioreactor has a body with a side having a top end, wherein at least one valve assembly of the bioreactor and one or more of the autoclave valve assembly are coupled to the side of the bioreactor proximate a top portion of the bioreactor.

6. A bioreactor system as claimed in any one of claims 1 to 5, the bioreactor having a body and a bottom portion, wherein: The bioreactor is configured to be at least partially disposed in a pit, and the at least one valve assembly of the stainless steel large-scale bioreactor and one or more of the autoclave valve assemblies are coupled against a bottom weld of a bottom portion of the bioreactor.

7. The bioreactor system according to any one of claims 1 to 6, wherein: The sterile connector assembly is a sterile connector valve assembly with a three-clamp sterile connector, which enables a new perfusion device or ATF assembly to be connected to the bioreactor while the stainless steel large bioreactor is undergoing cell culture by repeated online steam sterilization of the sterile connector assembly.

8. The bioreactor system according to any one of claims 1 to 7, wherein: The sterile connector assembly is a hose assembly that connects at least one valve assembly of the bioreactor to the perfusion device or ATF assembly, wherein the hose assembly includes a hose body having a first end and a second end, the first end being operably connected to the at least one valve assembly, and the second end being operably connected to the perfusion device or ATF assembly.

9. A large-scale bioreactor system comprising: Stainless steel large bioreactor with sides; an autoclave valve assembly coupled to a side of the bioreactor; at least one sterile connector coupled to the autoclave valve assembly; as well as An irradiated disposable infusion set is coupled to the at least one sterile connector.

10. The bioreactor system of claim 9, wherein: The autoclave valve assembly is a first valve assembly, and the bioreactor system further comprises: a second autoclave valve assembly configured to be coupled to a side of the bioreactor adjacent to the first valve assembly; and at least one sterile connector configured to be coupled to the second autoclave valve assembly.

11. A bioreactor system according to claim 9 or claim 10, wherein: The irradiated disposable perfusion set further comprises at least one pressure sensor, and the bioreactor system further comprises a control system coupled to the irradiated disposable perfusion set for monitoring the pressure of the disposable perfusion set via the at least one pressure sensor.

12. The bioreactor system of claim 11, wherein: The control system further includes an alarm configured to be activated when the control system detects that the pressure is greater than a safe pressure limit, and the control system is configured to automatically reduce one or more of the flow rate or pressure in the disposable perfusion device in response to detecting that the pressure is greater than the safe pressure limit.

13. The bioreactor system according to any one of claims 9 to 12, wherein: The bioreactor has a body with a side having a top end, wherein the autoclave valve assembly is coupled to the side of the bioreactor proximate the top end of the bioreactor.

14. A bioreactor system as claimed in any one of claims 9 to 13, the bioreactor having a main body and a bottom end, wherein: The bioreactor is configured to be at least partially disposed in a pit, and the autoclave valve assembly is coupled against a bottom weld of a bottom portion of the bioreactor.

15. A large-scale bioreactor system comprising: a stainless steel large-scale bioreactor having at least one valve assembly; a disposable adapter assembly comprising a Y-shaped connector assembly coupled to the at least one valve assembly; as well as A plurality of disposable perfusion devices are connected to the disposable adapter assembly, enabling a plurality of perfusion units to be coupled to the bioreactor without having to perform in-line steam sterilization of the bioreactor when coupling the plurality of disposable perfusion units.

16. The bioreactor system of claim 15, further comprising a disposable manifold coupled to one of the disposable adapter assembly or the at least one valve assembly, the disposable manifold comprising any of two, three, four, five, six, seven, or eight arms, each arm comprising an inlet for coupling to a disposable perfusion unit, such that the plurality of disposable perfusion devices can be operably coupled to the stainless steel large bioreactor.

17. The bioreactor system of claim 15, wherein: The Y-shaped connector assembly is a first Y-shaped connector assembly, and a plurality of Y-shaped connector assemblies are coupled to the first Y-shaped connector assembly such that the plurality of disposable perfusion devices can be operably coupled to the stainless steel large bioreactor.

18. The bioreactor system according to any one of claims 15 to 17, wherein: The disposable perfusion set further comprises at least one pressure sensor, and the bioreactor system further comprises a control system for monitoring the pressure of at least one disposable perfusion set of the plurality of disposable perfusion sets via the at least one pressure sensor.

19. The bioreactor system of claim 18, wherein: The control system further includes an alarm configured to be activated when the control system detects that the pressure is greater than a safe pressure limit, and the control system is configured to: in response to detecting that the pressure is greater than the safe pressure limit, automatically reduce one or more of the flow rate or pressure in at least one of the plurality of disposable perfusion devices.

20. The bioreactor system of any one of claims 15 to 19, wherein: The bioreactor has a body with a side having a top end, wherein at least one valve assembly of the bioreactor is coupled to the side of the bioreactor proximate the top portion of the bioreactor.

21. The bioreactor system of any one of claims 15 to 20, wherein: The bioreactor has a body and a bottom portion, wherein the bioreactor is configured to be at least partially disposed in a pit and at least one valve assembly of the stainless steel large bioreactor is coupled against a bottom weld of the bottom portion of the bioreactor.

22. A large-scale bioreactor system comprising: Stainless steel large bioreactor; at least one stainless steel transfer plate having a plurality of inputs coupled to the bioreactor; as well as A plurality of feed containers are coupled to the at least one stainless steel transfer plate at a working level of the bioreactor.

23. The bioreactor system of claim 22, wherein: The at least one stainless steel transfer plate is a first stainless steel transfer plate, and wherein the bioreactor system further comprises one or more additional stainless steel transfer plates, each of the additional stainless steel transfer plates being configured to couple to an additional plurality of feed containers.

24. The bioreactor system of any one of claims 22 or 23, wherein: The plurality of feed containers further include at least one pressure sensor, and the bioreactor system further includes a control system for monitoring the pressure of at least one feed container among the plurality of disposable feed containers via the at least one pressure sensor, wherein the at least one feed container is at least one disposable feed container.

25. The bioreactor system of claim 24, wherein: The control system further includes an alarm, which is configured to be activated when the control system detects that the pressure is greater than the safety pressure limit. The control system is configured to automatically reduce one or more of the flow rate or pressure in at least one of the multiple disposable feed containers in response to detecting that the pressure is greater than the safety pressure limit.

26. The bioreactor system of any one of claims 22 to 25, wherein: The bioreactor has a body with a side having a top end and at least one valve assembly coupled to the side of the bioreactor proximate the top end of the bioreactor.

27. The bioreactor system of any one of claims 22 to 26, wherein: The bioreactor has at least one valve assembly and a body having a bottom portion, wherein the bioreactor is configured to be at least partially disposed in a pit and the at least one valve assembly of the stainless steel large bioreactor is coupled against a bottom weld of the bottom portion of the bioreactor.

28. The bioreactor system of any one of claims 1 to 27, wherein: The stainless steel large-scale bioreactor has a volume of greater than 2,000 L.

29. The bioreactor system of any one of claims 1 to 28, wherein: The stainless steel large-scale bioreactor has a volume in the range of greater than 2,000L to 20,000L.

30. A method of integrating at least one perfusion device with a stainless steel large-scale bioreactor, the method comprising: One of the following couplings is performed: (1) coupling the connector assembly to at least one valve assembly of the stainless steel large-scale bioreactor; or (2) coupling the autoclaved valve assembly to the side of the stainless steel large-scale bioreactor; Connecting one of the following: (1) connecting the autoclave valve assembly of the alternating tangential filtration (ATF) assembly of the perfusion device to the connector assembly; or (2) connecting the irradiated perfusion device to the autoclave valve assembly; as well as The pressure of the irrigation device is managed via at least one pressure sensor of the irrigation device, and when a pressure greater than a safety limit pressure is detected, one or more of the flow rate or the pressure in the irrigation device is automatically reduced by a control system.

31. The method of claim 30, wherein: One of the following connections is made: (1) connecting a connector assembly to at least one valve assembly of a stainless steel large-scale bioreactor; or (2) connecting an autoclaved valve assembly to the side of the stainless steel large-scale bioreactor; including connecting an aseptic connector valve assembly to at least one valve assembly of the stainless steel large-scale bioreactor.

32. The method of claim 31, wherein: Making one of the following connections: (1) connecting the autoclave valve assembly of the ATF assembly of the perfusion device to the connector assembly; or (2) connecting the irradiated perfusion device to the autoclave assembly includes connecting the autoclave valve assembly of the perfusion device to the connector assembly, wherein the connector assembly includes one of a three-clamp connector assembly or a hose assembly.

33. The method of claim 32, wherein: One of the following couplings is performed: (1) coupling a connector assembly to at least one valve assembly of a large stainless steel bioreactor; or (2) coupling an autoclave valve assembly to a side of the large stainless steel bioreactor includes coupling an autoclave assembly to a side of the large stainless steel bioreactor.

34. The method of claim 33, wherein: One of the following couplings is performed: (1) coupling the autoclave valve assembly of the ATF assembly of the pouring device to the connector assembly; or (2) coupling the irradiated pouring device to the autoclave valve assembly includes coupling the irradiated pouring device to the autoclave assembly.

35. The method of claim 30, wherein: Making one of the following connections: (1) connecting the connector assembly to at least one valve assembly of the stainless steel large-scale bioreactor; or (2) connecting the autoclaved valve assembly to the side of the stainless steel large-scale bioreactor includes connecting the connector assembly to at least one valve assembly of the stainless steel large-scale bioreactor, the connector assembly comprising a Y-shaped connector assembly.

36. The method of claim 35, wherein: One of the following connections is made: (1) connecting the autoclave valve assembly of the ATF assembly of at least one perfusion device to the connector assembly; or (2) connecting the irradiated perfusion device to the at least one sterile connector includes connecting the autoclave valve assembly of the ATF assembly of at least one perfusion device to the connector assembly, wherein the at least one perfusion device includes a plurality of perfusion devices connected to the Y-shaped connector assembly, so that the plurality of perfusion devices can be connected to the bioreactor without having to perform online steam sterilization of the bioreactor when connecting the plurality of perfusion devices.

37. The method of any one of claims 30 to 36, further comprising coupling at least one stainless steel transfer plate having a plurality of inputs to the stainless steel large bioreactor.

38. The method of any one of claims 30 to 37, further comprising coupling one or more of the at least one valve assembly of the stainless steel large-scale bioreactor and the autoclave valve assembly to a side of the stainless steel large-scale bioreactor.

39. The method of any one of claims 30 to 38, wherein: The stainless steel large-scale bioreactor has a volume of greater than 2,000 L.

40. The method of any one of claims 30 to 39, wherein: The stainless steel large-scale bioreactor has a volume in the range of greater than 2,000L to 20,000L.

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