Disposable flexible sprayer

Abstract

A multi-layer flexible sparger (100) having a bottom film layer (102), a middle film layer (106), and a top film layer (110), a first internal grid (104) disposed between the bottom film layer and the middle film layer, a second internal grid (114) disposed between the middle film layer and the top film layer, and a port capable of delivering a gas to the multi-layer flexible sparger disposed between the top film layer and the bottom film layer, wherein the middle film layer includes a bore and the top film layer includes a bore.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 175,696, filed April 16, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The embodiments disclosed herein relate to apparatus for the biological treatment of biological fluids. More specifically, the apparatus includes aeration devices used in containers or vessels, such as bioreactors, for example, disposable stirred tank bioreactors with a volume between 50 and 5000 liters. Background Technology

[0003] Traditionally, fluids such as biological materials have been handled in systems using stainless steel containers or vessels. These containers are sterilized after use so that they can be reused. The sterilization process is expensive, cumbersome, and sometimes ineffective. To provide greater flexibility in the manufacturing process and reduce the time required for effective regeneration of equipment, manufacturers have begun using disposable sterilized containers and / or bioreactors (e.g., collapsible bags) that are used once and then discarded. Examples of the use of these disposable or single-use bags are in systems for mixing two or more components, where at least one component is liquid and the other is liquid or solid, and the bag has mixing elements to ensure the contents are mixed as uniformly as possible. Examples of disposable containers are bioreactors or fermenters where cells are in suspension or on microcarriers, and the container has a mixing system for circulating liquids, gases, and in some cases, cells within the container.

[0004] Many conventional mixing bags, typically ranging in size from three (3) liters to smaller and fifty (50) liters or larger (i.e., 5000 liters), are shaped to resemble cylinders, with the bottom of the bag optionally tapered to mimic the shape of the stainless steel cans that disposable bags are replacing. Cylindrical bioreactors are capable of efficiently mixing the contents within the bag. Typically, the bag houses a mixer for mixing or circulating the contents, such as a magnetically coupled impeller housed within the bag and a magnetic motor located outside the bag, which remotely rotates the impeller. The container may also house one or more aeration devices (e.g., gas ejectors) through which air bubbles are introduced into the container contents. The contents are typically biopharmaceuticals or other biofluids. Such fluids typically include cell culture media and adjuvants. The container contains gases such as air, oxygen, carbon dioxide, nitrogen, etc. Ejectors used with 50-5000 liter bioreactors supply air through the bottom of the bioreactor. Due to the pressure of the fluid, ranging from 50 to 5000 liters, the injector must have a check valve or use high pressure to generate the back pressure required to prevent liquid from flowing back into the injector during aeration.

[0005] Aeration of biofluids within bioreactors is typically used to support the oxidation of cell cultures via a backup device. However, achieving high levels of oxygenation (measured as kLa) using high gas flow rates results in high-velocity and very small bubbles, which can lead to cell shear stress and induce cell death; where kLa is the gas delivery coefficient, for example, a measure of the bioreactor's ability to deliver oxygen into the culture. High-velocity and small bubbles can cause undesirable bioprocess product loss or changes in product quality; however, high kLa is necessary, for example, to achieve high cell densities during perfusion. Therefore, high kLa can be achieved by using jets tailored to specific flow rate requirements to distribute the gas flow and maximize gas delivery.

[0006] Past attempts at injection devices have included perforated ejectors formed from membranes or molds. These devices are designed to control bubble size and / or outlet gas velocity. However, for membrane ejectors, the flexibility of the membrane material results in a lack of controlled pressure gradients throughout the ejector region, leading to lower oxygen delivery, a wider bubble size distribution, and leakage. Molded devices are expensive and bulky, potentially occupying significant space within a single-use bioreactor and damaging the bioreactor's interior during transport. Another prior art flexible ejector, comprising two membrane layers, lacks uniform air distribution; that is, the airflow exits from the highest point of the ejector and is not distributed throughout the entire ejector region, for example, only in discrete pockets.

[0007] As mentioned above, the bubble size of the aeration gas is important. For example, in bioreactor applications, there exists a balance to manage the number and size of bubbles so that mass transfer from the gas-liquid phase or vice versa is sufficient for the process while preventing negative culture effects such as significant shear stress or foaming. Generally, smaller bubble sizes are more efficient in transporting gas from bubbles to liquids or biofluids due to increased surface area. However, smaller bubbles, compared to larger bubbles, pose a greater potential for cell damage because their size is similar to that of cells, and they have the potential to promote foam buildup on liquid surfaces. Similarly, in bioreactor / bioprocessing operations, it is important to create and maintain a generally homogeneous environment for the contents of the container (e.g., cells in a culture). It is undesirable to have zones and / or gradients within a bioreactor, i.e., differences in mixing (pH, nutrients, and dissolved gases), shear stress, temperature, etc. Some cell culture processes may require the highest possible mass transfer capacity, while others may require a specific bubble size large enough to prevent damage to sensitive cells. To date, there is no jetting device suitable for balancing bubble size with shear stress and foam generation.

[0008] Therefore, providing a container or bioreactor, such as a disposable or single-use container or bioreactor for biofluids, wherein one or more jetting devices help optimize cell culture growth performance and viability by providing a small, flexible jetting device that can balance multiple competing aspects of bubble size, shear force, foaming, air distribution and other biological treatment conditions. Summary of the Invention

[0009] Embodiments of a multi-layer flexible injector with drilling capability are disclosed, comprising three membrane layers and two mesh layers, substantially as shown in at least one of the accompanying drawings and / or described in conjunction with at least one of the accompanying drawings, as set forth more fully in the claims. The novel and inventive features of this disclosure and the details of exemplary embodiments thereof will be more fully understood from the following description and accompanying drawings. Some embodiments of this disclosure include a multi-layer flexible injector having: a bottom membrane layer, an intermediate membrane layer, and a top membrane layer; a first inner mesh disposed between the bottom membrane layer and the intermediate membrane layer; a second inner mesh disposed between the intermediate membrane layer and the top membrane layer; and a port capable of delivering gas to the multi-layer flexible injector disposed between the top membrane layer and the bottom membrane layer, wherein the intermediate membrane layer includes a borehole and the top membrane layer includes a borehole. Some embodiments of this disclosure include a multi-zone, multi-layer flexible injector having: a bottom membrane layer, an intermediate membrane layer, and a top membrane layer; a first internal grid disposed between the bottom membrane layer and the intermediate membrane layer and bonded to the bottom membrane layer and the intermediate membrane layer; a second internal grid disposed between the intermediate membrane layer and the top membrane layer and bonded to the intermediate membrane layer and the top membrane layer; and a port capable of delivering gas to at least two injection zones within the multi-zone, multi-layer flexible injector, disposed between the top membrane layer and the bottom membrane layer, wherein the intermediate membrane layer includes a borehole, and the top membrane layer includes a borehole.

[0010] In some embodiments, the multilayer flexible drilled ejector is a disposable ejector. Some embodiments include an intermediate membrane layer containing a small number of small drill holes that obstruct airflow and create back pressure between the intermediate and bottom membrane layers. Compared to the top layer, the intermediate layer has a smaller number of drill holes (e.g., between 1% and 50% of the number of drill holes in the top layer) and / or smaller drill holes. In some embodiments, the intermediate layer includes as many holes as 5-25% of the number of holes in the top layer. In some embodiments, the intermediate layer includes as many holes as 10-20% of the number of holes in the top layer. Furthermore, for example, drill holes with diameters between 5 micrometers and 1000 micrometers facilitate gas distribution and back pressure on the intermediate layer. Embodiments of this disclosure include an intermediate membrane layer with a number and size of drill holes that obstruct airflow and create back pressure between the intermediate and bottom membrane layers. Furthermore, for example, drill holes with diameters between 5 micrometers and 1000 micrometers facilitate gas distribution and back pressure on the intermediate layer. The top membrane layer contains drill holes designed for specific outlet gas velocities and bubble sizes. A mesh layer exists between the top and middle membrane layers to provide support for the membrane and physical separation of the membrane layers, thereby distributing the airflow across the entire injection area.

[0011] The top membrane layer contains perforations designed for specific outlet gas velocities and bubble sizes. A mesh layer exists between the top and intermediate membrane layers to provide support and physical separation of the membranes, thereby distributing the airflow across the entire injection area. The top and intermediate membrane layers are combined into segments that restrict airflow to specific areas regardless of the injector's orientation. At least three membrane layers are combined in the periphery of the injector and in the central region of the injector. The embodiments of the injectors disclosed herein demonstrate a substantially equal distribution of gas flow rate throughout the injector, independent of its orientation. The embodiments of the injectors disclosed herein provide uniform bubble sizes independent of gas flow rate. The embodiments of the injectors disclosed herein advance the art because the injector designs of this invention broaden the window of optimal performance, providing high kLa for increased cell densities, ease of fabrication, and flexibility for integration into existing bioreactors and vessels. Some embodiments include the use of multiple injectors within a single bioreactor. Some embodiments include means for switching between injectors with different pore sizes and / or kLa characteristics. This device includes a control scheme that utilizes a manifold and a microprocessor-controlled bioreactor and mass flow controller to optimize and vary the gas flow range. In some embodiments, a microprocessor-controlled bioreactor and mass flow controller are used to obtain desired kLa characteristics and are further combined with multiple injectors capable of producing different bubble sizes. These advancements, and others embodied herein, will become clear from the following description, claims, and drawings. Details of the various advantages, aspects, novel and inventive features of this disclosure and exemplary embodiments thereof will be more fully understood from the following description and drawings. Therefore, the embodiments of this disclosure, briefly summarized above, can be described in more detail by referring to the accompanying drawings to understand the features disclosed herein in detail. However, it should be noted that the drawings illustrate only typical embodiments of this disclosure and should therefore not be considered as limiting its scope, as the described embodiments may allow the use of other equally effective bags, biocontainers, membranes, and / or materials. It should also be understood that elements and features of one embodiment may be found in other embodiments without further description, and where possible, the same reference numerals have been used to refer to comparable elements common to the drawings. As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these embodiments pertain. Attached Figure Description

[0012] Figure 1 A side exploded perspective view of a multilayer flexible injector according to some embodiments of the present disclosure is depicted;

[0013] Figure 2 A top perspective view of a multi-layer flexible injector with flanges according to some embodiments of the present disclosure is depicted;

[0014] Figure 3 Some embodiments according to this disclosure are depicted. Figure 2 Side view of a multi-layer flexible injector;

[0015] Figure 4 Some embodiments according to this disclosure are depicted. Figure 2 A top view of a multi-layer flexible injector;

[0016] Figure 5 A side view of a multilayer flexible injector placed inside a bioreactor according to some embodiments of the present disclosure is depicted;

[0017] Figure 6 The following are depictions of some embodiments according to this disclosure. Figure 5 The top view of the bottom inner surface of the bioreactor, taken by line 6-6, shows the impeller and two multi-layer flexible injectors.

[0018] Figure 7 A top view of a multi-layer injector with selectable positioning adjustment tabs according to some embodiments of the present disclosure is depicted;

[0019] Figure 8A Depicting Figure 7 Top perspective view of a multi-layered injector;

[0020] Figure 8B Depicting Figure 7 A close-up view of the borehole in the top membrane layer of the multilayer injector;

[0021] Figure 9 Depicting Figure 7 Exploded view of a multi-layer injector;

[0022] Figure 10 A top view of a multi-zone, multi-layer injector with selectable positioning adjustment tabs according to some embodiments of the present disclosure is depicted; and

[0023] Figure 11 Depicting Figure 10 Exploded view of a multi-zone, multi-layer injector. Detailed Implementation

[0024] The term "membrane" as used in this disclosure refers to any flexible material capable of fusing with another flexible membrane, including but not limited to polymer sheets, composite materials, laminates, single-layer and / or multi-layer polymer materials. These membranes may further include substrates, which may include plastic meshes, woven fabrics, nonwoven fabrics, knitted fabrics, and / or metal foils, as well as other flexible structures and materials. Membranes may include, for example, polyolefin materials, such as low-density polyethylene, linear low-density polyethylene, medium-density polyethylene, high-density polyethylene, ultra-high-density polyethylene, polypropylene, and other polyolefins. In some embodiments, the flexible membrane includes a laminated membrane structure having a low-melting-point material within an outer high-melting-point polymer. Furthermore, in some embodiments, the flexible membrane includes a laminated membrane structure in which the low-melting-point material surrounds a high-melting-point woven, knitted, or nonwoven material. In some embodiments, any or all of the bottom membrane, intermediate membrane, or top membrane includes any of the membranes described in WIPO International Bureau Publication WO2020101848A1, which is incorporated herein by reference in its entirety. In some embodiments, one or more of these membranes are substantially similar to those sold by EMD Millipore Corporation (Burlington, Massachusetts, USA). or Membranes. The membranes discussed herein can be multilayer membranes, comprising one or more layers of polyethylene, ethylene vinyl acetate, ethyl vinyl alcohol, and other materials. In some embodiments, any or all of the bottom membrane, intermediate membrane, and / or top membrane comprises a substrate, which is a mesh, woven fabric, nonwoven fabric, knitted fabric, or other structure made of, for example, nylon, polyamide, and other abrasion-resistant materials, and various bonding layers, such as polyurethane, may be provided between these layers.

[0025] The term "biocontainer" is broadly defined as any flexible container or vessel capable of holding fluid within an internal volume or region, which may take the form of a two-dimensional, three-dimensional, and / or multifaceted bag or bioreactor. In some embodiments, a baffle is incorporated into the biocontainer or bioreactor, wherein the baffle is capable of disrupting eddies formed within the liquid when the liquid is mixed by a mixer (e.g., an impeller).

[0026] Figure 1 An exploded side perspective view of a multi-layer flexible injector 100 according to some embodiments of the present disclosure is depicted. Figure 1As depicted, the multilayer flexible injector includes a bottom membrane layer 102 without injection holes, which serves as a support base, and a first inner grid 104 disposed adjacent to the bottom membrane layer 102. The first inner grid 104 supports the bottom membrane layer 102 and is a means for distributing airflow throughout the injection area (discussed in more detail below). In some embodiments, the first inner grid 104 distributes airflow throughout the entire injection area. The multilayer flexible injector also includes a second membrane 106 disposed between the bottom membrane layer 102 and a top membrane layer 110, the top membrane layer 110 having a plurality of boreholes 112. In other words, the second membrane layer 106 is an intermediate membrane layer. The intermediate membrane layer 106 also includes a plurality of boreholes 112, and in some embodiments, the number of boreholes 112 is typically less than the number of boreholes 112 in the top membrane layer 110. In some embodiments, there are approximately 10 to 50,000 boreholes in both the intermediate membrane layer 106 and the top membrane layer 110. In some exemplary embodiments, the intermediate membrane layer 106 includes 20 to 200 boreholes. In some exemplary embodiments, the top membrane layer 110 includes 1,000 to 36,000 boreholes. It will be understood that the boreholes can be formed by any suitable process (e.g., using a pore-forming agent or various lasers). Because the intermediate membrane layer 106 includes fewer boreholes 112, it is able to generate back pressure on the airflow. This back pressure promotes a uniform distribution of gas or airflow over the injection area. In some embodiments, the flexible injector 100 includes a second inner grid 114 disposed between the intermediate membrane layer 106 and the top membrane layer 110. The thicknesses of the first and second inner grids 104 can also be varied to affect injection performance. Without being theoretically limited, it is believed that a thicker grid can maintain further separation between the bottom membrane layer 102 and the intermediate membrane layer 106, resulting in better injection performance. For example, it is believed that a thicker grid can promote a uniform airflow distribution. Similarly, a thicker mesh is believed to further separate the intermediate membrane layer 106 and the top membrane layer 110, resulting in better jetting performance. In some embodiments, the top membrane layer 110 includes perforations 112 designed for specific outlet gas velocities and bubble sizes. It is also believed that perforations 112 in the membrane layer provide enhanced consistency compared to nonwoven or membrane structures within the ejector, thus providing constant and predictable bubble sizes. In some embodiments, the intermediate membrane layer 106 and the top membrane layer 110 are segmented to restrict gas flow to specific areas. All three membrane layers (i.e., the bottom membrane layer 102, the intermediate membrane layer 106, and the top membrane layer 110) are combined at the periphery and center of the flexible ejector. The multilayer flexible ejector 100 further allows vertical gas flow into the bioreactor regardless of the orientation of the multilayer flexible ejector 100 within the bioreactor (not shown). In other words, the gas or air flow remains uniform across the flexible ejector regardless of orientation.In some other embodiments, the flexible injector 100 does not have an intermediate membrane layer 106. In other words, the multilayer flexible injector 100 includes a bottom membrane layer 102, a top membrane layer 110 with perforations 112, and a grid 114 disposed therebetween. In some embodiments, the perforations 112 in the intermediate membrane layer 106 are sized to have a diameter ranging from 10 micrometers to 800 micrometers. In some embodiments, the perforations 112 in the top membrane layer 110 have a diameter ranging from 10 micrometers to 800 micrometers. In some embodiments, the perforations 112 in the top membrane layer 110 have a diameter less than 100 micrometers. All embodiments of the flexible injector herein include a port for supplying air or another gas into the flexible injector. This port may be located on the top of the flexible injector (e.g., at the center of the top membrane layer), on the bottom of the flexible injector (e.g., at the center of the bottom membrane layer), or on the side of the flexible injector. The flexible injector may include a generally circular shape, or, for example, a generally circular shape with a flange adjacent to a circular region (e.g., a prank seat). In some embodiments, the port supplies air or gas to the flexible injector via a flange.

[0027] Figure 2 A top perspective view of a flexible injector 200 having a flange 202 according to some embodiments of the present disclosure is depicted. The flexible injector 200 is generally circular, while the flange 202 can have any suitable shape. As drawn, the flange 202 is rectangular. The flange 202 includes a port 204 extending from the upper portion of the flange 202. In some embodiments, the port 204 may extend from the lower portion of the flange 202. When the port 204 is connected to a gas supply such as air or oxygen, the port 204 delivers air into the flexible injector 200. In some embodiments, the port 204 includes a connector, such as a barb 206 for connection to a tube (not shown), a connector, or a sterile connector. In some embodiments, the port 204 is connected to an intermediate membrane layer 106 and / or a top membrane layer 110.

[0028] In some embodiments, the first and / or second internal mesh comprises a woven or extruded mesh, embossed and / or porous membrane or membrane with small opening regions (regions leading to the airflow) as an intermediate layer to generate back pressure, such that air is distributed through all openings in the intermediate layer. In some embodiments, flow pockets are created by sealing multiple layers of the membrane together, wherein air or airflow is directed to specific areas of the flexible ejector, thereby achieving a substantially uniform distribution of airflow within the flexible ejector. This results in consistent bubble size and bubble velocity on the surface of the flexible ejector regardless of the ejector's azimuth, gas flow rate, or head pressure. For example, the flexible ejector 200 also includes flow pockets 208. As shown, there are eight flow pockets 208. Flow pockets 208 are formed by bonding, for example, by ultrasonic welding, thermal welding, adhesives, and other methods known in the art for bonding plastic films. In some embodiments, high-frequency / radio frequency (RF) bonding is used to form the bond. In some embodiments, the RF bonding process is a two-step RF bonding process. In some embodiments, the RF bonding process is a three-step RF bonding process. RF welding involves placing plastic material between two opposing metal plates. Pressure is applied to these plates, and thus to the plastic, while RF waves are transmitted through the plates, generating heat that fuses the plastic together. The joint 210 is formed precisely inside the periphery of the flexible injector 200. A central joint 212 is formed at the center, where all layers are bonded together. Eight radial legs 214 extend from the central joint 212 and the peripheral joint 210. The peripheral joint 210 is formed through a portion including the bottom membrane layer 102, the intermediate membrane layer 106, and the top membrane layer 110. A flow bag 208 is formed by sealing the top membrane layer 110 and the intermediate membrane layer 106. Alternatively, a point joint 216 is formed in each flow bag 208 by combining the intermediate layer 106 with a second inner mesh 114.

[0029] Figure 3 Some embodiments according to this disclosure are depicted. Figure 2An exploded side view of the cross-section of a multilayer flexible injector 200. As shown, a bottom membrane layer 102, an intermediate membrane layer 106, and a top membrane layer 110 are present in the multilayer flexible injector 200. A port 204 is depicted as being joined to a flange. The port 204 is a barb 206 type port and includes a shoulder 218 for attachment to the intermediate membrane layer 106 and the top membrane layer 110. Because the port 204 extends between the intermediate membrane layer 106 and the bottom membrane layer 102, air or another gas is also supplied between them. A first inner mesh 104 and a second inner mesh 114 are also shown. In some embodiments, as shown, the first inner mesh 104 and the second inner mesh 114 are smaller than the diameter of the flexible injector 200, such that the bottom membrane layer 102, the intermediate membrane layer 106, and the top membrane layer 110 can be joined at a peripheral joint 210. In some embodiments, the first inner mesh 104 and the second inner mesh 114 have substantially the same diameter and thus form the peripheral joint 210. Figure 3 (Not shown in the image)

[0030] Figure 4 Some embodiments according to this disclosure are depicted. Figure 2 A top view of a multi-layer flexible injector 200. This top view depicts the multi-layer flexible injector 200, which has radial legs 214 in each of the eight flow bags 208 and around the peripheral joint 210 and central joint 212 regions of the outer boundary. Point joints 216 are shown, with one point joint per flow bag 208. Ports 204 are shown located within flanges 202. A joint 222 is also shown, positioned between the top membrane layer 110 and the intermediate membrane layer 106, and completing two of the flow bags 208. In some embodiments, an optional joint is arranged between two opposite sides of the joint 222, further supporting the multi-layer flexible injectors 100, 200.

[0031] Figure 5 A flexible ejector (e.g.,) arranged within a bioreactor 300 according to some embodiments of the present disclosure is depicted. Figure 2A side view of the multi-layer flexible injector 200. Some embodiments of this disclosure include a bioreactor system having a bioreactor and multi-layer flexible injectors 100, 200 as described above. The multi-layer flexible injectors 100, 200 may be disposed on the bottom inner surface of the bioreactor 300. Furthermore, the multi-layer flexible injectors 100, 200 may be disposed on the bottom inner surface 308 of the bioreactor 300, at or off-center from the geometric center of the bioreactor (as shown). In some embodiments, the multi-layer flexible injectors 100, 200 are attached to the bottom inner surface 308 of the bioreactor 300 or float freely within the bioreactor 300. In some embodiments, a plurality of multi-layer injectors 100, 200 are arranged within the bioreactor 300. For example, two (e.g., Figure 6 (As shown) up to eight multi-layer ejectors. Any of the bioreactors described herein (e.g., bioreactor 300) can have a working volume 310 of 50 liters to 3000 liters. In some embodiments, the working volume 310 of bioreactor 300 is 200 liters to 2000 liters. As shown, bioreactor 300 also includes a port 304 for conveying or removing gas or liquid from the working volume 310. A baffle 302 is shown. An impeller 306 for mixing liquids within the working volume 310 is also shown.

[0032] Figure 6 The following are depictions of some embodiments according to this disclosure. Figure 5 Line 6-6 shows a close-up top view of the bottom inner surface 308 of the bioreactor 300, which reveals two multi-layer flexible injectors 100 and 200 and an impeller 306. The multi-layer flexible injectors 100 and 200 show boreholes 112 and ports 204. Port 204 can be supplied by port 304 or other ports located outside the bioreactor 300.

[0033] Figure 7A top view of a multilayer injector 400 with selectable positioning adjustment tabs according to some embodiments of the present disclosure is depicted. The multilayer injector 400 includes radial legs 414 adjacent to each of eight flow bags 448 and surrounding an outer boundary, specifically peripheral joints 442 and central joints 413a, 413b regions. Radial legs 414 are joint regions. Joints are formed within two or more layers of the multilayer injector 400. In some embodiments, the radial legs 414 are joints that bond all layers of the multilayer injector 400 together. The multilayer injector 400 includes a borehole 450 (discussed below) at least in the top layer. The borehole 450 may also be located in intermediate layers (discussed below). In some embodiments, the borehole 450 has a size between 5 micrometers and 1000 micrometers, or any size in between. For simplicity, the borehole 450 is depicted as being located in one of the eight flow bags 448. It should be understood that the borehole 450 may be present in all of the flow bags 448. It should also be understood that the boreholes 450 may have different sizes within the same flow bag 448. In some embodiments, the total pore area in the intermediate membrane layer, as a function of the number and size of the boreholes 450, is smaller than the total pore area in the top membrane layer, thereby generating back pressure.

[0034] As shown, the multilayer injector 400 has an arcuate periphery. In some embodiments, central joints 413a, 413b form a central aperture 444. The optional central aperture 444 can be used to position, connect, releasably attach, or otherwise attach the multilayer injector 400 to a column within, for example, a bioreactor (not shown). A port 404 is shown located within a flange 401. The flange 401 is formed by one or more layers forming the multilayer injector 400. A joint 427 between all five layers is also shown. As shown below, the joint 427 does not necessarily include material from all five layers. For example, as described below, in some embodiments, a window 417 is present in some layers. Furthermore, in some embodiments, an adjustment tab 421 is optionally disposed within the multilayer injector 400.

[0035] Figure 8A Depicting Figure 7A top perspective view of the multilayer injector 400. In some embodiments, the first inner mesh 408 and the second inner mesh 416 have a diameter smaller than the diameter of the flexible injector 400, such that the bottom membrane layer 402, the middle membrane layer 406, and the top membrane layer 410 can be bonded together along the periphery. In some embodiments, as shown, the first inner mesh 408 and the second inner mesh 416 have the same diameter as the bottom membrane layer 402, the middle membrane layer 406, and the top membrane layer 410. As shown, the first inner mesh 408, the second inner mesh 416, the bottom membrane layer 402, the middle membrane layer 406, and the top membrane layer 410 include central holes 444a, 444b, and 444c to form a central hole 444 of the multilayer injector 400. The multilayer injector 400 may optionally include a variety of adjustment tabs on the periphery of the multilayer injector. For example, the bottom film layer 402, the middle film layer 406, and the top film layer 410 may each include optional adjustment tabs 403, 407, 409, and 411, which can be used to position the layers during manufacturing (e.g., joining, welding, etc.). Similarly, the first inner grid 408 and the second inner grid 416 may also include adjustment tabs. Figure 8B Depicting Figure 7 A close-up view of a borehole in the top membrane layer of the multilayer injector. For simplicity, a borehole 450 is shown in one section of the top membrane layer 410. It should be understood that any combination of all eight sections or more sections may have a borehole 450. Furthermore, as mentioned above, the intermediate membrane layer 406 may include boreholes 450 in all sections or any combination thereof (not shown in this view).

[0036] Figure 9 Depicting Figure 7An exploded view of the multilayer injector. The bottom membrane layer 402 includes a flange 401 and an adjustment tab 403. As can be seen, the diameter of the hole 444b in the intermediate membrane layer 406 is smaller than the diameter of the hole 444c in the first grid layer 408. The larger diameter of the hole 444c contributes to a stronger bond between the bottom layer 402 and the intermediate layer 406. An adjustment tab 419, which may optionally include two holes, can be used to position the top membrane layer 410 during manufacturing or to attach to columns or other features of the bioreactor bag. The first grid layer 408 also includes a central bonding region 413c, a central hole 444c, and an adjustment tab 405. The intermediate layer 406 is also attached with a port 404. It should be understood that during bonding, the port 404 may be bonded to the intermediate layer 406 before being bonded to other layers (e.g., the top layer 410), or alternatively, the port 404 may be bonded (e.g., RF soldered) to all layers simultaneously. Alternatively, the first mesh layer 408 may also include a window 417. The window 417 is a cutout in the first mesh layer 408, thereby allowing for easier bonding of the bottom film layer 402 to the intermediate layer 406. The intermediate film layer 406 includes an adjustment tab 407, a central bonding region 413b, and a hole 444b. A drill hole 450 located in a portion of the intermediate film layer 406 is also shown, although it should be understood that all portions of the intermediate film layer 406 may include drill holes 450. As shown, the drill hole 450 is shown as being located in a segment of the top film layer 410. It should be understood that any combination of all eight segments or more segments may have drill holes 450. Furthermore, as described above, the intermediate film layer 406 may include drill holes 450 in all segments or any combination thereof.

[0037] Port aperture 415b allows port 404 to pass through it during assembly. The second mesh layer 416 includes an adjustment tab 409 and radial legs 414. As shown, the radial legs 414 are cutouts in the second mesh layer 416. The second mesh layer may optionally include a peripheral cutout 438, which can facilitate bonding with adjacent layers (e.g., intermediate membrane layer 406 and top membrane layer 410). The top membrane layer includes a perforation 450 that delivers gas to the biofluid during biological treatment. The top membrane layer 410 also includes a central junction region 413a surrounding the column aperture 444a and the port aperture 415a. The top membrane layer 410 also includes an adjustment tab 419 having two holes for positioning or anchoring to the bioreactor. The top membrane layer 410 also includes an adjustment tab 411 and an optional adjustment tab 421. Furthermore, the adjustment tab 421 may include an optional slot 446 for pipe management, i.e., a gas supply pipe connected to port 404. Gas delivered to the multilayer ejector 400 via port 404 travels around the first mesh layer 408 between the bottom membrane layer 402 and the intermediate membrane layer 406. From there, the gas can travel through the perforations 450 in the intermediate membrane layer 406 into the eight flow bags 448, and through the perforations 450 in the top membrane layer 410 into the fluid inside the bioreactor bag.

[0038] Figure 10A top view of a multi-zone, multi-layer injector 500 with selectable positioning adjustment tabs according to some embodiments of the present disclosure is depicted. The multi-zone, multi-layer injector 500 is similar to the multi-layer injector 400 as described above. The multi-zone, multi-layer injector 500 is four of the multi-layer injectors 400 having monolayers. In other words, the multi-zone, multi-layer injector 500 has similar materials, layers, and features to the multi-layer injector 400. The multi-zone, multi-layer injector 500 includes a central port 504 surrounded by a central region 513, which feeds gas into thirty-two bags 448, eight bags 448 in each of the four injection zones. As shown, the central port 504 is located on the first mesh layer 408. However, this is for convenience. In practice, the central port 504 is positioned on the first mesh layer 408 during assembly. The central port 504 may be attached to the intermediate membrane layer 406 by heat fusion or other bonding. Alternatively, the five layers can be stacked, with a central port 504 provided, and the entire assembly is joined, for example, by RF welding. As can be seen, the central region 513 also includes four joints 517, which are formed above the window (similar to window 417 discussed above). The multi-zone, multi-layer injector 500 may also optionally include one or more adjustment tabs 421 for piping management. Each of the four injection zones may also optionally include a central aperture 444 for connection to a bioreactor bag. It should be understood that any reasonable number of injection zones can be used within the injector. For example, two, three, four, five, six, seven, or eight injection zones.

[0039] Figure 11 Depicting Figure 10An exploded view of the multi-zone, multi-layer injector 500. In some embodiments, as described above, the diameters of the first inner mesh 408 and the second inner mesh 416 are smaller than the diameter of the multi-zone, multi-layer injector 500, such that the bottom membrane layer 402, the middle membrane layer 406, and the top membrane layer 410 can be bonded along the periphery, these three layers being bonded to any other layer (e.g., the first inner mesh 408 and the third inner mesh 416). In some embodiments, as shown, the first inner mesh 408 and the second inner mesh 416 have the same diameter as the bottom membrane layer 402, the middle membrane layer 406, and the top membrane layer 410. As shown, the first inner mesh 408, the second inner mesh 416, the bottom membrane layer 402, the middle membrane layer 406, and the top membrane layer 410 include a central aperture 513, which can be used to receive a port 504. One or all of the apertures 413 can be used to releasably connect the injector 500 to a bioreactor. As described above, the multi-zone, multi-layer flexible injector 500 may include boreholes 450 with diameters ranging from 10 micrometers to 800 micrometers in the intermediate membrane layer 406. The multi-zone, multi-layer flexible injector 500 may also include boreholes 450 with diameters ranging from 10 micrometers to 800 micrometers in the top membrane layer 410. The multi-layer flexible injector 500 may include boreholes 450 in the intermediate membrane layer 406 that are larger than the boreholes 450 in the top membrane layer 410. The multi-layer flexible injector 500 may include boreholes 450 with diameters ranging from 50 micrometers to 800 micrometers in the intermediate membrane layer 406 and boreholes 450 with diameters ranging from 20 micrometers in the top membrane layer 410. The multi-zone, multi-layer flexible injector 500 may include an intermediate membrane layer 406 and a top membrane layer 410, with the intermediate membrane layer 406 including 80-800 boreholes and the top membrane layer 410 including 4000-144000 boreholes. In some embodiments of the multi-zone, multi-layer flexible injector 500, the total pore area in the intermediate membrane layer 406, as a function of the number and size of the boreholes 450, is smaller than the total pore area in the top membrane layer 410, thereby generating back pressure. The multi-zone, multi-layer flexible injector 500 also includes a junction surrounding the periphery of the multi-layer flexible injector 500.

[0040] Furthermore, depending on the bioreactor's tilt, pressure requirements, and / or perforation configuration, some embodiments of the flexible ejector can be designed with different numbers of cross sections and cross-sectional shapes. In some embodiments, the placement of a woven or extruded mesh, embossed, and / or porous membrane or membrane between two bonded sheets of a laser- or needle-perforated membrane, a woven or extruded mesh, or a membrane-like structure allows for a uniform distribution of gas flow, maximizing gas transport for high kLa. The size of the bubble generated by the flexible ejector can be controlled using more or fewer open areas (areas leading to the gas flow) between the mesh and / or membrane layers. Furthermore, the bubble size can be controlled by employing different shapes, such as cross, slotted, and / or bent shapes or contours of open areas. The pattern and spacing (density) of the open areas can be adjusted to optimize the kLa required for the gas demand in the bioreactor. Gas velocity has been identified as a significant factor in kLa. The pattern and spacing of the open areas of the mesh or perforated membrane are determined by gas velocity calculations performed for a range of flow rates. Gas velocities calculated based on the pattern and spacing of the open areas and the maximum flow rate allow for scalable solutions from bioreactor sizes of 50-2000 L while maintaining constant velocity and maximum flow rate in the bioreactor system.

[0041] The total spray area of ​​a flexible ejector can be varied to accommodate specific flow requirements (e.g., cell density driven), thereby producing a consistent bubble velocity across a range of air or gas flow rates. In some embodiments, multiple ejectors (or a single flexible ejector comprising multiple segments) may be manufactured from a single set of diaphragms. In some embodiments, one or more segments of the flexible ejector are not used at low gas flow rates. Partial sealing maintains separation between the spray segments at low flow rates and ruptures at higher gas flow rates, thereby allowing a consistent bubble velocity across a range of gas flow rates by increasing the total spray area.

[0042] Some embodiments of this disclosure described herein include a device for switching between ejectors with different orifice sizes and kLa performances, depending on the gas flow range / requirements. This device utilizes a computerized bioreactor control platform by leveraging novel mass flow controllers, novel manifolds, and novel control schemes. Multiple ejectors with different bubble sizes produce different kLa performances. Having multiple options for kLa performance allows for precise control of specific cell lines. Flexible ejector designs allow for optimization of the shape and placement of the bottom of the bioreactor bag to improve kLa. Some embodiments of flexible ejectors allow for proper aeration of fluid samples while creating a homogeneous environment without negatively impacting the fluid contents of the vessel via shear forces or significant foaming.

[0043] Gas velocity was determined to be a significant factor in kLa. The pattern and spacing of the opening regions of the mesh or perforated membrane were determined by gas velocity calculations performed over a range of flow rates. In some cell lines, high bubble velocities can be a cause of shear stress, and it is recommended to keep gas velocities below the operating range of 30 m / s. To ensure cell safety for most cell lines and to achieve the highest possible performance, some embodiments of flexible ejectors were designed around a constant gas velocity at the maximum flow rate of the bioreactor system. For higher-performance flexible ejectors, a lower flow rate, rather than the system's maximum flow rate, can be used to determine the shear limit.

[0044] In the formula, m represents meters and s represents seconds.

[0045] Within the aforementioned gas velocity equation, the ejector orifice area is defined by multiplying the area of ​​each borehole by the number of boreholes defined by the spacing and pattern of the boreholes in the ejection region. Calculating the gas velocity and defining the borehole pattern based on a constant 30 m / s at the system's maximum flow rate offers several advantages, such as determining predictable kLa performance and scalability strategies. For example, using velocity calculations to implement scalability, rather than scaling up by changing borehole and bubble sizes, allows for the selection of the number and size of boreholes for each scale based on performance limits and a constant velocity. This has proven successful in adapting the dimensions of the borehole ejector.

[0046] curve Figure 1 Comparative data for four 200-liter flexible injectors according to embodiments of the present disclosure are shown.

[0047]

[0048] curve Figure 2 Comparative data for flexible injectors for 200-liter and 2000-liter bioreactors according to embodiments of this disclosure are shown. As defined herein, the maximum flow rate of the system is plotted relative to kLa. For the 200-L bioreactor system, a range of 0-50 SLPM is shown, for example, 50 SLPM is 100% of the maximum flow rate. As can be seen, for both the 200-L and 2000-L scales, the performance curves of injectors with higher performance at high power (i.e., the best case) and injectors with lower performance at low power (i.e., the worst case) are substantially similar. This results in predictable and scalable kLa across different sizes. For other bioreactor sizes, scaling can also be achieved by varying the number of orifices and the injection area, rather than by increasing the number of injectors, each with a constant area. For larger bioreactors, the flow rate can be increased, for example, >1000L, or, alternatively, the number of flexible injectors can be increased to match the specified flow rate.

[0049]

[0050] curve Figure 3 Comparative data for a novel multilayer flexible injector to a molded injector according to embodiments disclosed herein are shown, where flow rates (standard liters per minute (SLPM)) are plotted relative to kLa in a 200L bioreactor. Flexible injectors can compromise performance due to inadequate air distribution and inability to properly reduce the amount of material injected. However, it should be noted that embodiments of the novel multilayer flexible injector described herein exhibit performance comparable to or superior to molded injectors, where the flexible injector is easier to manufacture and package. Furthermore, some embodiments include boreholes having dimensions between 5 micrometers and 1000 micrometers or any size therebetween. In some embodiments, the borehole diameter is between 20 micrometers and 800 micrometers. In some embodiments, the borehole has any diameter between 20 micrometers and 150 micrometers. In some embodiments, the borehole has any diameter between 70 micrometers and 150 micrometers. In some embodiments, the borehole has any diameter between 150 micrometers and 500 micrometers. In any flexible injector, the number of boreholes can be selected by maintaining a constant air velocity. Furthermore, the size of the borehole depends on the shear forces experienced by any cell in the biological process. Specifically, a 5-micron borehole generates greater shear forces. Therefore, shear-sensitive cells can be better treated using a flexible jet with, for example, a 20-micron borehole. Two 20-micron curves show 45 kLa at 20 SLPM, while two 150-micron curves show approximately 30 kLa at 20 SLPM.

[0051]

[0052] curve Figure 4 An updated analysis of the bubble size in the flexible prototype is presented. It was found that the bubble size (in micrometers) remains constant within the standard deviation regardless of the flow rate (as measured by vvm). For example, 1 vessel volume (vvm) per minute (L / L / m) means 1 liter of air passes through 1 liter of medium in 1 minute. Therefore, the bubble size is known, remains constant, and is predictable.

[0053]

[0054] It should also be understood that another advancement compared to other injectors is the determination of how many orifices to use, based on maintaining a constant opening area, regardless of whether larger or smaller boreholes are used, and thus determining scalability. Furthermore, for larger bags or bioreactors, such as 2000L, more injectors can be used, for example, four, five, or six injectors, in contrast to using a single injector with a larger orifice.

[0055] curve Figure 5 This is a process that allows for the selection of multiple orifices (from 0 to 8000 orifices) in a flexible injector design based on an airflow velocity limit of 30 m / s for various orifice sizes. For different embodiments of the flexible injector, the orifice size varies between 10 micrometers and 800 micrometers. From left to right, the curves shown represent diameters of 800 micrometers, 150 micrometers, and 20 micrometers, respectively.

[0056]

[0057] curve Figure 6 This is a process for specifying the number of injectors available for a 200L and 2000L bioreactor at a given airflow rate. One injector can be used for a 200L bioreactor with an airflow rate of 30 m / s. To maintain a constant velocity of 30 m / s, four injectors are used at the 2000L scale, or the injection area is increased to quadruple the number of orifices. In some embodiments, multiple injectors are used, for example, 2-8 injectors.

[0058]

[0059] Several operating modes may exist in biological treatment processes. For example, injection into a bioreactor (e.g., a single-use bioreactor) may include a continuous gas flow mode, a formulation mode, or a feedback control loop via software and a microprocessor, manual operation of the flow rate, and / or specification of a specific injector using valve assemblies. It should be further understood that some biological treatments may include two or more of these injection modes.

[0060] All ranges described herein include the ranges in between and may include or exclude endpoints. Optionally included ranges are integer values ​​in between (or including a primary endpoint), within the stated order of magnitude or the next smaller order of magnitude. For example, if the lower limit is 0.2, optional included endpoints could be 0.3, 0.4, ... 1.1, 1.2, etc., and 1, 2, 3, etc.; if the upper limit is 8, optional included endpoints could be 7, 6, etc., and 7.9, 7.8, etc. Unilateral boundaries (e.g., three or more) also include consistent boundaries (or ranges) starting from an integer value on the stated order of magnitude or one order of magnitude lower. For example, 3 or more includes 4 or 3.1 or more.

[0061] In this patent specification, references to “one embodiment,” “some embodiments,” “one or more embodiments,” “some embodiments,” or “an embodiment” indicate that a feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. Therefore, throughout the patent specification, the appearance of phrases such as “in one or more embodiments,” “in some embodiments,” “in one embodiment,” “in some embodiments,” or “in one embodiment” does not necessarily refer to the same embodiment. Nevertheless, it should be understood that any feature described herein can be incorporated into any embodiment disclosed herein. All publications of patent applications and patents and other non-patent references cited in this patent specification are incorporated herein by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be fully set forth in accordance with the descriptions above in connection with the publications and references. Any patent application claiming priority to this application is also incorporated herein by reference in the manner described above with respect to the publications and references.

Claims

1. A multi-layer flexible injector, comprising: Bottom membrane layer, middle membrane layer, and top membrane layer; A first internal grid is disposed between the bottom film layer and the intermediate film layer; A second internal grid is disposed between the intermediate film layer and the top film layer; and It is capable of delivering gas to a port of the multilayer flexible injector, the port being disposed between the top membrane layer and the bottom membrane layer. The intermediate membrane layer includes boreholes, and the top membrane layer includes boreholes.

2. The multi-layer flexible injector as described in claim 1, wherein, The multi-layer flexible injector also includes a junction between the bottom membrane layer and the middle membrane layer.

3. The multi-layer flexible injector as described in claim 1, wherein, The multi-layer flexible injector also includes a junction between the top membrane layer and the middle membrane layer.

4. The multi-layer flexible injector as described in claim 1, wherein, The multi-layer flexible injector also includes a flange for accommodating the port.

5. The multi-layer flexible injector as described in claim 1, wherein, The total borehole area in the intermediate membrane layer is lower than the total borehole area in the top membrane layer, resulting in back pressure.

6. The multi-layer flexible injector as described in claim 1, wherein, The diameter of the boreholes in the intermediate film layer ranges from 10 micrometers to 800 micrometers.

7. The multi-layer flexible injector as described in claim 1, wherein, The diameter of the boreholes in the top membrane layer ranges from 10 micrometers to 800 micrometers.

8. The multi-layer flexible injector as described in claim 1, wherein, The boreholes in the intermediate membrane layer are larger than those in the top membrane layer.

9. The multi-layer flexible injector as described in claim 1, wherein, The boreholes in the intermediate membrane layer are smaller than those in the top membrane layer.

10. The multi-layer flexible injector as claimed in claim 1, wherein, The number of boreholes in the intermediate membrane layer is less than the number of boreholes in the top membrane layer.

11. The multi-layer flexible injector as claimed in claim 1, wherein, The boreholes in the intermediate membrane layer are between 50 micrometers and 800 micrometers, and the boreholes in the top membrane layer are 20 micrometers.

12. The multi-layer flexible injector as claimed in claim 1, wherein, The intermediate membrane layer includes 20 to 200 boreholes, and the top membrane layer includes 1,000 to 36,000 boreholes.

13. The multi-layer flexible injector as claimed in claim 1, wherein, The multi-layer flexible injector also includes a connecting portion surrounding the periphery of the multi-layer flexible injector.

14. The multi-layer flexible injector as claimed in claim 1, wherein, The multi-layer flexible injector also includes a bonding portion surrounding the periphery of the multi-layer flexible injector, the bonding portion bonding the top film layer, the middle film layer and the bottom film layer.

15. The multi-layer flexible injector as claimed in claim 1, wherein, The multi-layer flexible injector also includes a central joint at the center of the multi-layer flexible injector, the central joint being formed from the top membrane layer through the intermediate membrane layer and including the bottom membrane layer.

16. The multi-layer flexible injector as claimed in claim 1, wherein, The multi-layer flexible injector also includes at least two radial joints extending from the central joint to the peripheral joint and from the top membrane layer and the middle membrane layer, wherein two flow bags are generated.

17. The multi-layer flexible injector as claimed in claim 1, wherein, The drilled holes include cross-shaped, slotted, and / or bent profiles.

18. The multi-layer flexible injector as claimed in claim 1, wherein, The multi-layer flexible injector also includes at least eight radial joints extending from the central joint to the peripheral joints and from the top membrane layer and the middle membrane layer, wherein eight flow bags are generated.

19. The multi-layer flexible injector as claimed in claim 1, wherein, The multi-layer flexible injector also includes a point joint in at least one flow bag.

20. The multi-layer flexible injector as claimed in claim 1, wherein, The port is suitable for attachment to a pipe or connector.

21. The multi-layer flexible injector as claimed in claim 20, wherein, The connector is a sterile connector.

22. The multi-layer flexible injector as claimed in claim 1, wherein, The port capable of delivering gas to the multilayer flexible injector is located between the top membrane layer and the middle membrane layer.

23. A bioreactor system, comprising: Bioreactor; and The multi-layer flexible injector as described in claim 1 is disposed therein.

24. The bioreactor system of claim 23, wherein, The bioreactor system also includes multiple of the aforementioned multi-layer flexible injectors.

25. The bioreactor system of claim 23, wherein, The working volume of the bioreactor is between 50 liters and 5000 liters.

26. The bioreactor system of claim 23, wherein, The working volume of the bioreactor is between 200 liters and 2000 liters.

27. A multi-zone, multi-layer flexible injector, comprising: Bottom membrane layer, middle membrane layer, and top membrane layer; A first internal grid is disposed between the bottom film layer and the intermediate film layer and is bonded to the bottom film layer and the intermediate film layer; A second internal grid is disposed between the intermediate film layer and the top film layer and is bonded to the intermediate film layer and the top film layer; and The port is capable of delivering gas to at least two injection zones within the multi-zone, multi-layer flexible injector, the port being disposed between the top membrane layer and the bottom membrane layer, wherein the intermediate membrane layer includes a borehole and the top membrane layer includes a borehole.

28. The multi-zone, multi-layer flexible injector as described in claim 27, wherein, The multi-zone, multi-layer flexible injector includes four injection zones.

29. The multi-zone, multi-layer flexible injector as described in claim 28, wherein, The port is located at the center of the four injection zones.

30. The multi-zone, multi-layer flexible injector as described in claim 27, wherein, The diameter of the boreholes in the intermediate film layer ranges from 10 micrometers to 800 micrometers.

31. The multi-zone, multi-layer flexible injector as described in claim 27, wherein, The diameter of the boreholes in the top membrane layer ranges from 10 micrometers to 800 micrometers.

32. The multi-zone, multi-layer flexible injector as described in claim 27, wherein, The boreholes in the intermediate membrane layer are larger than those in the top membrane layer.

33. The multi-zone, multi-layer flexible injector as described in claim 27, wherein, The boreholes in the intermediate membrane layer are between 50 micrometers and 800 micrometers, and the boreholes in the top membrane layer are 20 micrometers.

34. The multi-zone, multi-layer flexible injector as described in claim 27, wherein, The intermediate membrane layer includes 80 to 800 boreholes, and the top membrane layer includes 4,000 to 144,000 boreholes.

35. The multi-zone, multi-layer flexible injector as described in claim 34, wherein, The boreholes in the intermediate membrane layer are between 50 micrometers and 800 micrometers, and the boreholes in the top membrane layer are between 10 micrometers and 800 micrometers.

36. The multi-zone, multi-layer flexible injector as described in claim 35, wherein, The intermediate membrane layer includes boreholes ranging from 50 micrometers to 800 micrometers, and the top membrane layer has boreholes of 20 micrometers.

37. The multi-zone, multi-layer flexible injector as described in claim 27, wherein, The multi-zone, multi-layer flexible injector also includes a connecting portion surrounding the periphery of the multi-layer flexible injector.

38. The multi-zone, multi-layer flexible injector as described in claim 27, wherein, The total borehole area in the intermediate membrane layer is lower than the total borehole area in the top membrane layer, resulting in back pressure.

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