Biomolecular production systems and methods

Abstract

This invention provides a simple, modular biomolecule production system suitable for the production of viral particles, proteins, and gene therapy products for clinical and therapeutic purposes. [Solution] The present invention provides a bioreactor cabinet configured for integration into a biomolecule production system. In this configuration, it is preferable that the bioreactor cabinet is movable and suitable for receiving a bioreactor. The bioreactor cabinet is equipped with a bioreactor docking station. The bioreactor cabinet is also equipped with a connector, preferably on the side wall of the bioreactor cabinet, which can transmit power, signals, and / or data when paired with a biomolecule production system such as a bioreactor chamber of the system. The present invention also relates to a biomolecule production system and a method for producing biomolecules.

Description

[Technical Field]

[0001] This invention relates to the field of production (manufacturing field) of viral vaccines, antibodies, or gene therapy products, and discloses a system and method thereof. [Background technology]

[0002] Biomolecules are becoming increasingly important in the field of pharmaceuticals. Antibody-based therapies and RNA, DNA, and gene therapies are seen as potential solutions for many diseases that are currently incurable. Furthermore, outbreaks of viral infections such as SARS, SARS-CoV-2, and MERS have increased the demand for vaccine manufacturing facilities.

[0003] Traditional methods for producing and purifying biomolecules from cultured cells are monotonous, laborious, time-consuming, and excessively costly. Furthermore, they often require large-scale equipment, negatively impacting the effectiveness of production, the total time required, and the overall cost.

[0004] To obtain products suitable for clinical administration, there is a need for methods to rapidly and efficiently produce biomolecules. Furthermore, there is a need for systems that are simple in structure, require minimal space, and can be easily transported and installed, for example, in benches or laminar flow cabinets.

[0005] The object of the present invention is to solve at least some of the above problems. More specifically, it is to provide a simple and modular biomolecule production system suitable for the production of viral particles, proteins, and gene therapy products that can be used for clinical and therapeutic purposes.

[0006] The present invention provides a system and solution for the production and / or purification of biomolecules, such as cells, DNA, RNA, proteins, peptides, and viral products, that can be produced at low cost while maintaining high quality conditions. According to the present invention, biomolecules can be produced under GMP conditions. A second objective is to provide an ergonomic, highly accessible, user-friendly, flexible, and preferably modular system. Another objective of the present invention is to provide a method that can reduce the amount of operating processes while maintaining a high yield of biomolecules, significantly reduce operating expenses (OPEX), and achieve a high level of containment. [Overview of the project]

[0007] The present invention provides a solution to one or more of the above-mentioned problems. To this end, the present invention provides a bioreactor cabinet as described in claim 1. More specifically, the present invention provides a bioreactor cabinet, preferably wheeled (in other words, movable), suitable for receiving a bioreactor, which is configured for integration into a biomolecular production system. The bioreactor cabinet is provided with a bioreactor docking station. The bioreactor cabinet is preferably provided with a connector on its side wall that transmits power, (signal) and / or data when paired with a biomolecular production system such as a bioreactor chamber of the biomolecular production system.

[0008] A preferred embodiment of the bioreactor cabinet is shown in any of the claims. This bioreactor cabinet is compact, requires minimal space, and is easy to transport.

[0009] In a second aspect, the present invention provides a system according to claim 24. More specifically, the present invention provides a biomolecule production system. The system is - At least one or more process chambers having one or more purification devices or filtration devices that can purify or filter the biomolecules of the cell extract, and - A bioreactor chamber configured to receive a bioreactor, and a connector provided in the bioreactor chamber for performing power transmission, (signal) transmission, and / or data transmission when paired with a bioreactor cabinet having a bioreactor.

[0010] The present invention also provides a biomolecule production system according to claim 36. More specifically, the present invention provides a biomolecule production system. The system includes - A process chamber having one or more purification devices or filtration devices that can purify or filter the biomolecules of the cell extract, - A downstream chamber, and - A bioreactor chamber suitable for receiving a bioreactor, provided between the process chamber and the downstream chamber, fluidly connected to one or more chambers, and provided with a connector for performing power transmission, (signal) transmission, and / or data transmission when paired with a bioreactor cabinet.

[0011] In a final aspect, the present invention discloses a method according to claim 39. More specifically, the present invention provides a method for producing a protein, a virus or virus particle, or a product for gene therapy. The method includes - Providing a bioreactor in a bioreactor cabinet, preferably a mobile / wheeled bioreactor cabinet, that docks to the bioreactor chamber of the biomolecule production system, - Purifying the extract from the bioreactor in a processing chamber located on the side of the bioreactor chamber to produce a biomolecule extract.

[0012] In one embodiment, the biological molecule extract is further concentrated by a concentrator provided in the bioreactor chamber.

[0013] In yet another aspect, the present invention discloses a method for docking a bioreactor cabinet to a chamber of a biological molecule production system. In this method, a connector is used to connect between the cabinet and the system, and power transmission, (signal) transmission, and / or data transmission are performed when they are paired.

[0014] According to the systems and methods according to the present disclosure, biological molecules such as virus products can be produced at low cost while maintaining high-quality requirements. Furthermore, biological molecules can be produced under GMP conditions, and a system that is highly ergonomic, accessible, and user-friendly can be provided. Finally, by adopting the methodology of the present disclosure, the yield of biological molecules can be increased while keeping the work process volume small, and the operating cost (OPEX) can be significantly reduced, achieving a high level of containment.

Prior Art Documents

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Brief Description of the Drawings

[0016] [Figure 1A-C] Figures 1A, 1B, and 1C are diagrams showing different embodiments of the bioreactor cabinet according to the present disclosure. [Figure 1D-F] Figures 1D, 1E, and 1F are cross-sectional views showing an embodiment of the bioreactor cabinet according to the present disclosure. [Figure 2] Figure 2 is a front view showing an embodiment of the system according to the present disclosure. [Figure 3] Figure 3 is a top view of a system according to one embodiment of the present disclosure, including dimensions of different elements. [Figure 4A] Figure 4A is a front view of a system according to one embodiment of the present disclosure, including the dimensions of the entire casing. [Figure 4B] Figure 4B shows a rear view and a front view illustrating a system according to one embodiment of the present disclosure. [Figure 5] Figure 5 is a detailed front view showing a system having a front window according to one embodiment of the present disclosure. [Figure 6A-B] Figures 6A and 6B show one embodiment of the system according to this disclosure, which has a collection container and a TFF. [Figure 7] Figure 7 is a perspective view showing a first embodiment of the bioreactor according to this disclosure. [Figure 8] Figure 8 is a perspective view showing the bioreactor in Figure 7, including several enlarged views. [Figure 9A-C] Figures 9A, 9B, and 9C show substrate materials used when constructing a structured fixed bed for culturing cells in one of the disclosed bioreactors. [Figure 10] Figure 10 shows a modular version of one embodiment of the bioreactor according to this disclosure. [Figure 11] Figure 11 is a cross-sectional view showing one embodiment of the bioreactor according to this disclosure. [Figure 12] Figure 12 is a cross-sectional view showing the base portion of the bioreactor in Figure 11. [Figure 13] Figure 13 is a cutaway top view showing the middle section of the bioreactor in Figure 11. [Figure 14] Figure 14 is a cutaway top view showing the middle section of the bioreactor in Figure 11. [Figure 15.15AB] Figures 15, 15A, and 15B are various drawings illustrating a third embodiment of the bioreactor according to this disclosure. [Figure 16] Figure 16 is a cross-sectional view showing the bioreactor in Figure 15. [Figure 17] Figure 17 is a cross-sectional view showing the bioreactor in Figure 15. [Figure 18] Figure 18 shows the process flow in one embodiment of the system according to this disclosure. [Figure 19] Figure 19 shows one possible embodiment of the level sensor according to this disclosure. [Figure 20] Figure 20 shows one possible embodiment of the pressure sensor according to this disclosure. [Figure 21] Figure 21 shows two possible embodiments of the flow meter according to this disclosure. [Figure 22] Figure 22 shows one possible embodiment of the foam capture device according to the present invention. [Figure 23] Figure 23 shows a container for a fluid stirring device having a gas supply pipe according to one embodiment of the present disclosure. [Figure 24] Figure 24 shows another embodiment of a container according to one aspect of the present disclosure. [Figure 25] Figure 25 shows another embodiment of a bioreactor equipped with a gas supply pipe according to one aspect of the present disclosure. [Figure 26-27] Figures 26 and 27 show another embodiment of the bioreactor shown in Figure 25. [Figure 28-30] Figures 28 to 30 show embodiments of a flow extender according to one aspect of the present disclosure. [Figure 31] Figure 31 shows one embodiment of a lift that transports various parts of a system according to one aspect of this disclosure. [Figure 32-33] Figures 32 and 33 are cross-sectional views showing yet another embodiment of the bioreactor according to this disclosure. [Figure 34] Figure 34 is a schematic diagram of a filter unit according to one embodiment of the present invention. [Modes for carrying out the invention]

[0017] The present invention relates to apparatus, systems, and methods for manufacturing or producing biomolecules such as proteins, RNA, DNA, virus particles, viral vectors, viral vector vaccines, and antibodies.

[0018] Unless otherwise defined, all terms used in disclosing this invention, including technical and scientific terms, have the meanings that are ordinarily understood by those skilled in the art in which this invention pertains. For reference, definitions may be provided for each term to better understand the gist of this invention.

[0019] The following terms used in this specification have the meanings set forth below.

[0020] As used herein, singular expressions (a, an, the) include both singular and plural forms unless the context explicitly states otherwise; for example, “one section” refers to one section or two or more sections.

[0021] The word "approximately" used before a measurable value such as a parameter, a quantity, or a period of time, encompasses the stated numerical value, or a numerical value within a range of ±20% or less, preferably ±10% or less, more preferably ±5% or less, even more preferably ±1% or less, and even more preferably 0.1% or less, from that numerical value, insofar as it is appropriate for carrying out the invention disclosed herein. The numerical value itself to which the modifier "approximately" is attached is also specifically included.

[0022] As used herein, “comprise,” “comprising,” “comprises,” and “comprised of” are synonymous with “include,” “including,” or “contain,” “containing,” and are comprehensive, unrestricted terms that specifically refer to what follows them, such as components, and do not exclude or preclude the existence of previously known or additional, undescribed components, features, elements, parts, or processes.

[0023] Furthermore, terms such as “first,” “second,” and “third” used herein, including in the claims, are used to distinguish between similar elements and, unless otherwise specified, do not necessarily indicate a continuous or chronological order. These terms are interchangeable under reasonable circumstances, and embodiments of the present invention described herein can be operated in any order other than those described herein or illustrated herein.

[0024] The numerical range defined by the endpoint is described, and this range includes not only all integers and fractions contained within it, but also the endpoint itself.

[0025] Throughout this specification, the specific expressions “weight %”, “weight percent”, “%wt”, or “wt%” refer to the relative weight of each component based on the total weight of the preparation, unless otherwise specified.

[0026] The terms "one or more" or "at least one" are self-evident, and to clarify, these terms refer in particular to any one of the members in question, or any two or more of the members in question, for example, to members ≥3, ≥4, ≥5, ≥6, or ≥7, and in some cases to all of the members in question.

[0027] Unless otherwise specified, all terms used in disclosing this invention, whether technical or scientific, have the meanings that any person skilled in the art in which this invention pertains would ordinarily understand. For the sake of clarity, the definitions of terms used herein are provided solely to aid in understanding the gist of this invention.

[0028] Throughout this specification, the terms “one embodiment” or “a certain embodiment” refer to a specific feature, structure, or characteristic described in relation to that embodiment, meaning that the specific feature, structure, or characteristic described is encompassed in at least one embodiment of the present invention. Throughout this specification, the specific phrases “one embodiment” or “a certain embodiment” do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in a manner preferred by one or more embodiments, as can be understood from this disclosure to those skilled in the art. In addition, some embodiments described herein have only some of the features included in other embodiments, but features of different embodiments can be combined within the scope of the present invention to constitute other different embodiments, as can be understood by those skilled in the art. For example, in the claims, any of the embodiments described in the claims can be used in combination.

[0029] A "biomolecule" refers to any biological substance that is produced in a bioreactor. Examples of biomolecules include viruses, virus-like particles, viral products, gene therapy products, viral vectors, DNA, RNA, proteins such as antibodies, carbohydrates, lipids, nucleic acids, metabolites, and peptides.

[0030] "Genetic therapy products" refer to therapeutic products that possess nucleic acids that treat or prevent diseases and disorders such as genetic diseases and genetic disorders.

[0031] "Viral gene therapy products" refer to viral products in which a portion of the genetic material of a virus is replaced with therapeutic nucleic acid, and the therapeutic nucleic acid is introduced into the patient's cells using the virus. Numerous viruses, including retroviruses, adenoviruses, herpes simplex viruses, vaccinia, and adeno-related viruses, are used in human gene therapy. An "antibody" refers to any monoclonal or polyclonal immunoglobulin molecule, antigen-binding immunoglobulin fragment, or immunoglobulin fusion protein derived from human or other animal cell lines. Examples include natural and genetically modified forms such as humanized antibodies, human antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, transplanted antibodies, and in vitro-generated antibodies. Commonly known natural immunoglobulins include IgA (dimer), IgG, IgE, and IgG and IgM (pentamers).

[0032] A "virus" or "virion" is a microscopic (approximately 20-300 nm in diameter) infectious pathogen that replicates only within the cells of living hosts, such as bacteria, plants, and animals, and consists of RNA or DNA in the cell nucleus, a protein membrane, and, in more complex types, a surrounding membrane.

[0033] A “bioreactor” refers to any device or system that supports a biologically active environment for culturing cells or microorganisms to produce, for example, biological products or biomolecules. This includes cell stacks, roller bottles, shakers, flasks, agitated tank suspension bioreactors, high-density cell-structured or unstructured fixed-bed bioreactors, and batch reactors.

[0034] "Purification" refers to substantially reducing the concentration of one or more target impurities or contaminants relative to the concentration of the target biomolecule.

[0035] Tangential flow filtration (TFF) refers to a membrane filtration method in which a fluid is forced to flow through a space partitioned by one or more perforated membranes. Molecules small enough to pass through the pores are filtered out into the filtrate (osmote), while molecules too large to pass through the pores remain in the "retaining liquid." The name "tangential flow" specifically refers to the fact that, unlike so-called "dead-end filtration" where the direction of fluid flow is roughly perpendicular to the membrane, the direction of fluid flow is roughly parallel to the membrane.

[0036] As used herein, "viral infection" refers to the process in which a virus enters a cell and replicates within that cell.

[0037] "Cell culture sample," "culture sample," and "sample" are synonymous and refer to unpurified cell cultures obtained from cell culture in a bioreactor. Cultured cells or proliferating cells are also called host cells.

[0038] "Serial" or "in-line" refers to connecting devices or units so that the effluent from one unit or device is sent directly to the next unit or device without any intermediate storage.

[0039] As used herein, “docking” means stably connecting two elements so that, for example, these elements can constitute either a recipient or a connector. In this disclosure, docking is applied between a bioreactor cabinet and a biomolecule production system, or between the bioreactor itself and the bioreactor cabinet.

[0040] In a first embodiment, the disclosure relates to a bioreactor cabinet configured to be incorporated into a biomolecular production system. The bioreactor cabinet is preferably a wheeled (or optionally mobile) bioreactor cabinet that receives a bioreactor, and the bioreactor cabinet may be provided with a bioreactor docking station, and the bioreactor cabinet may be provided with a connector on its side wall that can transmit power, (signal) transmission and / or data when paired with, for example, the bioreactor chamber for the biomolecular production system.

[0041] In one embodiment, the disclosure relates to a bioreactor cabinet configured to be incorporated into a biomolecular production system. The bioreactor cabinet is preferably a wheeled bioreactor cabinet that receives a bioreactor, and the bioreactor cabinet may be equipped with a bioreactor docking station, and a connector may be provided on the wall of the bioreactor cabinet, preferably on its side wall, which, when paired with the bioreactor chamber, can transmit power, (signal) signals and / or data from the biomolecular production system.

[0042] In one embodiment, the bioreactor cabinet is docked into the bioreactor chamber of the biomolecular production system by connecting means having connectors capable of transmitting power, (signal) transmission, and / or data transmission. In another embodiment, additional connections are added to physically fix the bioreactor cabinet to the bioreactor chamber.

[0043] In yet another embodiment, the bioreactor cabinet is integrated into the production system. The advantage of designing a separate bioreactor cabinet that can be integrated into the production system is that the bioreactor can be easily installed by one or two operators outside the production system. Furthermore, all manifold connections of the production system can be safely accessed when removing the bioreactor cabinet.

[0044] In one embodiment, the bioreactor cabinet can be designed to be movable. Such a design allows for the inclusion of a structure or component in the bioreactor cabinet that allows it to be moved or transported. Examples of transport means include, but are not limited to, manually controlled and / or electronically controlled means, any means preferred in the art, such as wheels, tracks, or rolls. Alternatively, or in addition, in yet another embodiment, the bioreactor cabinet can be provided with a suitable structure to which a lift or hoisting device can be attached for later transport of the bioreactor cabinet. In a preferred embodiment, the bioreactor cabinet is equipped with wheels.

[0045] The bioreactor cabinet can be constructed from any material that is preferred in the art, such as metal alloys, metals, or plastics. In one embodiment, the bioreactor cabinet is constructed from a material such as aluminum or stainless steel. In a particularly preferred embodiment, the bioreactor cabinet is constructed from a material made of stainless steel.

[0046] In some embodiments of this disclosure, the bioreactor cabinet is equipped with one or more handles, making it easy to operate. The presence of handles(s) allows the operator to move the bioreactor cabinet by pushing or pulling the handles. The handles(s) can be of various sizes and can be installed at different locations on the outer casing of the bioreactor cabinet. In some embodiments, a single handle is provided along the entire length of the wall of the bioreactor cabinet. In other embodiments, two or more handles are provided on the right and left sides of the front wall of the bioreactor cabinet.

[0047] In another embodiment, these handles are adjustable, detachable, and / or retractable. The handle(s) may be made of any material preferred in the art of the invention, such as plastic, aluminum, steel, or metal alloys. In a particularly preferred embodiment, the handle(s) are made of stainless steel. Stainless steel is highly corrosion-resistant and maintains its strength at high temperatures.

[0048] In a preferred embodiment, the handle is connected to the front wall of the bioreactor cabinet. In this position, the operator can push and manipulate it precisely, especially when docking the bioreactor cabinet to a biomolecular production / purification system. In one embodiment, a metal bar is used as the handle. This metal bar is spring-loaded and has two rods at both ends, which can be pulled toward the center of the metal bar. In one embodiment, the metal bar can be lowered to a lower position by pulling from both ends toward the center of the metal bar. Lowering the handle facilitates operations (such as sampling and docking the bioreactor cabinet to the system).

[0049] For docking, the bioreactor cabinet is equipped with a connector that allows it to transmit power, signals, and / or data when paired with a biomolecular production system and, in some cases, one or more magnetic or other types of coupling (e.g., radio frequency (RF) technology). Both the bioreactor cabinet and the system can communicate with each other and transfer data.

[0050] In one embodiment, the connector can connect a bioreactor cabinet to the bioreactor chamber of a production system by a connecting portion and a receiving portion. The connecting portion can be located in the bioreactor cabinet, while the receiving portion can be located inside the bioreactor chamber of the system.

[0051] In one embodiment, a modular connector system can be used as the connector, and power and signal contacts, Ethernet, optical fiber, coaxial connectors, hydraulic couplings, pneumatic couplings, or thermal couplings can be housed together in a small frame or housing. This modular connector system can be configured according to the specific connection requirements. In a preferred embodiment, these connectors are waterproof. In one embodiment, a male connector on a bioreactor cabinet connects to a female connector on a production system. To ensure accurate connection between the male and female connectors, the female connector may be equipped with a centering pin. In another embodiment, the connector is equipped with an electronic eye to ensure accurate connection. In yet another embodiment, the connector is equipped with a magnetic element to ensure accurate connection.

[0052] In yet another embodiment, the bioreactor cabinet is docked to the system by a connector and a receiving part of the system, tightly connecting them to prevent the bioreactor cabinet from detaching from the system during biomolecule production. These connectors and receiving parts can be any systems, such as mechanical or magnetic systems, that are suitably used in this art. A breakaway function can be incorporated to detach the bioreactor cabinet from the system.

[0053] In one embodiment, the bioreactor cabinet comprises both a connector capable of transmitting power, (signal) transmission, and / or data when paired with a biomolecular production system, and a magnetic connector for docking the bioreactor cabinet to the biomolecular production system. In one embodiment, the magnetic connector may be made of a permanent magnet. In another, more preferred embodiment, the magnet is an electromagnet, which generates a magnetic field through electric current. The advantage of an electromagnet over a permanent magnet is that the magnetic field can be rapidly changed by controlling the amount of current. The present invention uses a magnet, more specifically an electromagnet, which enhances the safety of the system, thereby preventing unauthorized docking of the bioreactor cabinet to or removal from the production system. The system may consist of corresponding magnetic parts that can interact with the magnet of the bioreactor cabinet. In one embodiment, a magnetically connected powered part is provided in the system, and a magnetically connected stainless steel part is provided at the rear of the bioreactor cabinet. In one embodiment, these two parts are blocked when powered. In one embodiment, these two components are blocked when subjected to a force of 1000N.

[0054] In one embodiment, the connector and one or more magnetic connections to the bioreactor cabinet are located on different walls. In a preferred embodiment, the connector and magnetic connections are located on the same wall of the bioreactor cabinet. In either case, docking is facilitated.

[0055] In some embodiments, the connector and one or more magnetic connections are located on the wall of the bioreactor cabinet opposite the front wall. By pushing the bioreactor cabinet forward using a handle on the front wall of the bioreactor cabinet, the connector and magnetic connections can be easily aligned with their corresponding parts on the opposite side of the production system.

[0056] In a preferred embodiment, the bioreactor docking station is located inside the bioreactor cabinet and is preferably protected from the external environment by the walls of the bioreactor cabinet. As a result, the bioreactor cabinet not only serves as a means of transporting the bioreactor but also as a protective shield that shields the bioreactor from harmful accidents such as collisions with other objects during transport.

[0057] In some embodiments, the docking station of the bioreactor cabinet has a detachable height adjustment device, which allows the bioreactor to be positioned within the docking station.

[0058] Such a detachable height adjuster functions as a support for mounting the bioreactor. This detachable height adjuster can be removably attached to the bioreactor cabinet and / or docking station, and its dimensions can be changed according to the size and dimensions of the bioreactor. In one embodiment, the detachable height adjuster allows the bioreactor to be positioned regardless of its dimensions, so, for example, the top of the bioreactor can be perfectly aligned with the top surface of the bioreactor cabinet. For example, the bioreactor cabinet is 600m 2 This bioreactor cabinet can be designed to be suitable for bioreactors of specific sizes, such as bioreactors with an internal culture surface of 200m². 2When used in conjunction with a bioreactor, a height adjustment device can be used to accommodate the small bioreactor. In this case, the bioreactor and its outlet, provided on its surface, can be ergonomically operated. The height adjustment device can be formed into any shape suitable for use, such as a square, rectangle, or circle. In one embodiment, the height adjustment device is a cylindrical or disc-shaped element. The surface of the height adjustment device does not deform the device. The height adjustment device can be made of any material suitable in the art, such as plastic, aluminum, steel, or metal alloy. Stainless steel is a material that is highly corrosion-resistant and maintains its strength at high temperatures.

[0059] In one embodiment, the bioreactor cabinet has a bioreactor support plate on its upper surface, the support plate having a recess into which the bioreactor is inserted. This recess improves the ergonomic effectiveness of the bioreactor cabinet and system. In some embodiments, the bioreactor support plate is a grid-like plate.

[0060] The presence of these openings in the support plate allows for airflow circulation within the bioreactor cabinet and further improves the appearance of the bioreactor inside the cabinet. In some embodiments, the plate has one or more pieces. In preferred embodiments, the support plate has two pieces. The support plate can be made of any corrosion-resistant material that is preferred in the art.

[0061] In a preferred embodiment, the bioreactor cabinet has a bioreactor located within a bioreactor docking station. The bioreactor cabinet is designed to house the bioreactor along with its heating support and agitation motor. With this design, the three elements (bioreactor, heating support, and agitation motor) can be easily and quickly installed or removed by a single operator. In addition, a holding tray can be provided in the bioreactor cabinet and connected to the main holding tray of the bioreactor chamber. A leak detector can be installed in this holding tray. The holding tray collects any potential leaks from the process. In one embodiment, the bioreactor cabinet has two or more holding trays. In another embodiment, the different holding trays in the bioreactor cabinet are at different levels and can be connected to each other by piping. In another embodiment, one or more of the holding trays have a maximum capacity of 85L.

[0062] In some embodiments, positioning means can be provided on one or more walls of the bioreactor cabinet, thereby aligning the bioreactor cabinet with the biomolecular production system and further enhancing the docking of the bioreactor cabinet into the system. In preferred embodiments, positioning means can be provided on multiple walls of the bioreactor cabinet, thereby aligning the bioreactor cabinet with the biomolecular production system and further enhancing the docking of the bioreactor cabinet into the system.

[0063] This positioning mechanism guides the bioreactor cabinet and helps in precisely positioning it within the biomolecular production system.

[0064] The positioning means can consist of any type of element that helps guide the bioreactor cabinet into the system and dock it therewith. In one embodiment, the positioning means is a circular or cylindrical element on a rotating axis, such as a wheel or gear, incorporated into a socket in the side wall of the bioreactor cabinet.

[0065] In yet another embodiment, the positioning means is a pair of wheels, each of which has a rotation axis, in which case each pair has two wheels, and the position of the rotation axis of the first wheel is perpendicular to the rotation axis of the second wheel.

[0066] In yet another embodiment, each side wall of the bioreactor cabinet has, for example, two pairs of wheels located on the opposite side of the side wall, each positioned along or near the corner of the side wall. The presence of wheels on both sides of the side wall allows the front and rear of the bioreactor cabinet to be precisely guided into the bioreactor chamber.

[0067] In one embodiment, the bioreactor chamber further has one or more receiving elements which receive the bioreactor cabinet within the bioreactor chamber. In another embodiment, the bioreactor chamber further has guide members which guide the bioreactor cabinet into the bioreactor chamber.

[0068] In one embodiment, the bioreactor cabinet is further equipped with an inclinometer to confirm that the bioreactor is in a horizontal plane. In yet another embodiment, this inclinometer is installed in the bioreactor cabinet to confirm that the bioreactor is in a horizontal plane. In yet another embodiment, a maximum deviation angle of the horizontal state limit is set, and a warning is issued if the bioreactor cabinet is not in a perfectly horizontal position. In one embodiment, the inclinometer is determined by a wear-free semiconductor sensor element.

[0069] Conventional bioreactors are designed with fixed dimensions (especially height), making them difficult and costly to transport to remote locations (particularly developing countries) where cell culture is needed for treatment. Due to this fixed nature, older bioreactors cannot accommodate a variety of applications.

[0070] Another challenge exists, concerning the ability to maximize cell density in a given area. Conventional proposals for bioreactors utilize fluidized beds. While such fluidized beds have a high cell proliferation effect and several advantages, they result in a large bioreactor space volume required to form them. Although it is also important to easily scale up bioreactors using unstructured, fluidized beds, bioreactors can achieve the desired cell proliferation, but the current demand in this field is for bioreactors that can be used under various operating conditions (e.g., sterilization hoods, cabinets, or isolators when cleanup is restricted).

[0071] Therefore, there is a high demand for improved bioreactors that are easy to transport and assemble, especially in remote locations, and / or can easily accommodate various sizes and configurations, and / or can easily accommodate different applications. In some embodiments of the present invention, this disclosure relates to a manufacturing system and production of biopharmaceuticals. In particular, it relates to a manufacturing system and method for cells, viruses, cell-derived preparations, or virus-derived preparations. In some embodiments, high-density cell culture can be achieved using the bioreactor of the present invention. For example, densities of at least 2,000,000 cells / ml, at least 5,000,000 cells / ml, at least 10,000,000 cells / ml, at least 20,000,000 cells / ml, at least 40,000,000 cells / ml, at least 60,000,000 cells / ml, or at least 100,000,000 cells / ml can be achieved. In some embodiments, densities of 3 million cells / ml, 2 million cells / ml, or 2.5 million cells / ml can also be achieved. In some embodiments, the total capacity of the bioreactor is at least 1 L, at least 10 L, at least 30 L, at least 40 L, or at least 50 L. In some embodiments, the total capacity of the bioreactor can be set to a maximum of 2500 L, a maximum of 200 L, a maximum of 150 L, a maximum of 100 L, or a maximum of 75 L. Here, the total capacity of the bioreactor refers to the total volume of liquid that can be introduced into the bioreactor until it is full. In some embodiments, the bioreactor is equipped with a drainage system for excess products and fluids.

[0072] In some embodiments, the diameter of the bioreactor may be approximately 50-60 cm. In some embodiments, the diameter or height of the bioreactor may be set to approximately 5 cm or more, approximately 10 cm or more, approximately 15 cm or more, approximately 20 cm or more, approximately 25 cm or more, approximately 30 cm or more, approximately 35 cm or more, approximately 40 cm or more, approximately 45 cm or more, approximately 50 cm or more, approximately 60 cm or more, approximately 70 cm or more, approximately 80 cm or more, approximately 90 cm or more, or approximately 100 cm or more. In some embodiments, the diameter of the cover or lid that can be used in conjunction with the bioreactor may be approximately 2 cm or more, approximately 4 cm or more, approximately 5 cm or more, approximately 6 cm or more, approximately 8 cm or more, approximately 10 cm or more, approximately 12 cm or more, approximately 15 cm or more, approximately 20 cm or more, approximately 25 cm or more, approximately 30 cm or more, or approximately 50 cm or more. In some embodiments, the overall height of the bioreactor may be approximately 20-50 cm.

[0073] In some embodiments, the bioreactor can be a perfusion bioreactor, a corrugated bioreactor, a cylindrical bioreactor, a bag bioreactor, a mobile bed bioreactor, a packed bioreactor, a fibrous bioreactor, a membrane bioreactor, a batch bioreactor, a continuous bioreactor, or a combination thereof. In some embodiments, the bioreactor can be made of or composed of a suitable material, such as stainless steel, glass, aluminum, or plastic. In some embodiments, the bioreactor can analyze its products.

[0074] Access to the bioreactors disclosed herein can be provided via a lid or a door. In some embodiments, the access mechanism for the bioreactor may consist of, for example, a lock / key mechanism, a passcode punch pad, a card swipe, a transponder reader, a fingerprint scanner, a retinal scanner, a sensor, an automated identification / data capture method such as radio frequency identification (RFID), a QR code, biometrics (such as an iris recognition system or a facial recognition system), a magnetic stripe, optical character recognition (OCR), a smart card, voice recognition, or other access mechanisms. In some embodiments, this access mechanism restricts access to the bioreactor itself. In other embodiments, the access mechanism restricts access to a bioreactor cabinet or bioreactor chamber containing the bioreactor. In yet another embodiment, the access mechanism restricts access to a process controller that controls the bioreactor and / or the entire production system.

[0075] In some embodiments, the bioreactor disclosed herein may be used in conjunction with a process controller. In some embodiments, the biomolecular production system may be used in conjunction with one or more process controllers. In some embodiments, one or more process controllers are configured to control both the bioreactor and the biomolecular production system. In some embodiments, the process controller is configured to control the operation of the bioreactor and / or the biomolecular production system and may be used in conjunction with multiple sensors, a local computer, a local server, a remote computer, a remote server, or a network. In some embodiments, the bioreactor and / or the biomolecular production system may be used in conjunction with one or more sensors, such as temperature sensors (thermocouples, etc.), flow sensors, gas sensors, level sensors, and other sensors. In some embodiments, the process controller is configured to control various aspects of the production process and is coupled to sensors installed in the bioreactor and / or biomolecular production system to control the temperature, volume flow rate, or gas flow rate flowing into the bioreactor and / or biomolecular production system in real time. In some embodiments, the process controller is divided into two parts: a programmable logic controller (PLC) and a supervisory control and data acquisition unit (SCADA). A PLC is the intelligence of a system, coupled with sensors and actuators. A PLC only holds data, not power. SCADA is crucial for visualization, data historianship, and audit trails. The SCADA system operates on a server that stores data historians and supports visualization. In some embodiments, a client network can be directly connected to the server for remote access. In some embodiments, the process controller is equipped with a human-machine interface (HMI) such as a display. Examples include computer monitors, smartphone apps, tablet apps, and analog displays, which users can access to check the system status (based on sensors installed in the system) and control the system with various actuators such as pumps, valves, heaters, and agitators. In some embodiments, the process controller is equipped with input devices such as a keyboard, detachable smart tablet, keypad, mouse, or touchscreen, so that users can input control parameters and control the operation of the bioreactor. In some embodiments, the process controller can control access to the bioreactor.

[0076] In some embodiments, the bioreactors disclosed herein can be used in combination with sensors that monitor different parameters. In some embodiments, the sensors can be electrically connected. In other embodiments, wireless sensors can be used as sensors. In other embodiments, the bioreactor can use both electrically connected and wireless sensors. In some embodiments, these sensors can be installed in any compartment of the bioreactor disclosed herein. In some embodiments, gas sensors (such as oxygen, nitrogen, or carbon dioxide sensors), pH sensors, temperature sensors, cell density sensors, liquid level sensors, and dissolved oxygen (DO) sensors can be used as these sensors disclosed herein. In some embodiments, the sensors disclosed herein can be used to measure biomass, i.e., cell density, dissolved oxygen partial pressure, oxygen content, pH value, temperature, pressure, flow rate, liquid level, and the concentration of nutrients such as lactate, ammonium, carbonate, glucose, or any metabolites to be metabolized that reflect cell density. In some embodiments, cell density (biomass density) can be measured by electrical impedance analysis or electrical impedance spectroscopy using a measuring electrode device. In some embodiments, the bioreactor according to this disclosure may be used in conjunction with sensors for measuring culture parameters. In some embodiments, certain sensors disclosed herein may be in contact with the culture medium in the bioreactor. In some embodiments, culture parameters may include, among other things, the concentration of nutrients such as dissolved oxygen partial pressure, pH, temperature, optical density, lactate, ammonium, carbonate, glucose, or any metabolites to be metabolized that reflect cell density. In some embodiments, the sensor unit installed in the bioreactor (e.g., a pH probe) may be a disposable sensor, while the sensor unit that does not come into contact with the bioreactor (e.g., a pH sensor transmitter) may be reusable. In some embodiments, the bioreactor of the present invention may use a regulation loop according to the parameters disclosed herein.In some embodiments, the regulation loop can adjust, for example, the amount of oxygen injected, the amount of dissolved oxygen consumed by the cells, and the circulation rate of the culture medium according to the value of the dissolved oxygen partial pressure present, and can inject CO2 according to the pH value obtained by a sensor or other form of regulation commonly used in this culture field. In some embodiments, the cells are exposed to dissolved oxygen concentrations of 300 mM (160 mmHg partial pressure) or less, less than 200 mM, or in the range of 20 mM to 150 mM. In some embodiments, cells are exposed to approximately 0%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 78%, 80%, 90%, or 100% nitrogen, and / or approximately 0%, 1%, 5%, 10%, 21%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% oxygen. In some embodiments, cells can be exposed to pure oxygen or an oxygen-enriched atmosphere. In one embodiment, a sampling device can be connected to the lid of the bioreactor.

[0077] In some embodiments, the bioreactor disclosed herein may be used in conjunction with a heating and / or cooling device for heating and / or cooling the culture medium. In some embodiments, one or more, or all, of the vessels in the biomolecular production system may be used with a heating and / or cooling device. In some embodiments, the heating device may be an electrical element, an electrical coil, or other heating means commonly used in the field of cell culture, such as a thermostat-controlled double jacket. In some embodiments, the heating means is a heating plate. In some embodiments, the heating device consists of seven elements. In another embodiment, each element is equipped with a temperature sensor. In yet another embodiment, each element is equipped with a temperature limiter. In a more preferred embodiment, the temperature limiter is set to 110°C. In some embodiments, the cooling device may be any suitable cooling device, such as a Peltier element. In some embodiments, the bioreactor has, with respect to the culture medium and gas used, at least one inlet for introducing gas and at least one outlet for recovering the culture medium contained in the bioreactor. In some embodiments, the gas or gaseous mixture and the culture medium mixture may be supplied by the same supply line. In one embodiment, the bioreactor inlet is used in conjunction with a pump to pump fluid into the bioreactor. In another embodiment, the bioreactor outlet is used in conjunction with a pump to pump fluid to the outside of the bioreactor. In another embodiment, one or more pumps in the system are Watson-Marlow peristatic pumps.

[0078] In one embodiment, the pH inside the bioreactor is adjusted by a base adjustment kit. In one embodiment, this base adjustment kit has two parts: a bag containing the base, and a transfer unit that connects to the bioreactor and optionally fills the bag. In one embodiment, the bag is hung on a hook. In one embodiment, the bag has a 5L disposable bag. In a preferred embodiment, the bag is attached to the bioreactor using a sterile connection. In one embodiment, the base is attached to the bioreactor by a pump such as a Watson-Marlowe peristaltic pump.

[0079] In some embodiments, the circulation of the culture medium in the bioreactor can be carried out by a stirrer. In some embodiments, a rotary, non-contact magnetic impeller, a bladed or screw system, or an external circulation system can be used as the stirrer. In some embodiments, the stirrer can consist of a disc blade turbine, a curved blade turbine, an open-blade fluid foil axial impeller, a turbine impeller with pitched blades, or an impeller consisting of three blades. In a preferred embodiment, the bioreactor is composed of a magnetic stirrer with five magnets. In a more preferred embodiment, the magnetic stirrer controls a propeller provided in the bioreactor. In yet another embodiment, the propeller is a disposable propeller. In some embodiments, the agitator flow rate can be set to less than approximately 0.01 liters / min, less than approximately 0.05 liters / min, less than approximately 0.1 liters / min, less than approximately 0.5 liters / min, less than approximately 1 liter / min, less than approximately 2 liters / min, less than approximately 5 liters / min, less than approximately 10 liters / min, less than approximately 15 liters / min, less than approximately 20 liters / min, less than approximately 50 liters / min, less than approximately 100 liters / min, more than approximately 150 liters / min to 160 liters / min, less than approximately 180 liters / min, less than approximately 200 liters / min, or less than approximately 250 liters / min. In preferred embodiments, the agitator flow rate is controlled by an HMI.

[0080] In some embodiments, the bioreactors disclosed herein have a fixed bed. In some embodiments, this fixed bed is a structured fixed bed (i.e., a bed that is easily replicated, homogeneous as a whole, and substantially fixed, with a non-random orientation and not randomly unstructured, and that satisfies these qualities and can take on various sizes or shapes). In some embodiments, the structured fixed bed is constructed by stacking substrate disks. For the substrate layers of disks, the first or second side of one substrate layer is stacked toward the first or second side of an adjacent substrate layer. In some embodiments, the structured fixed bed extends spirally around a tubular section. In some embodiments, the structured fixed bed can circulate culture medium and cells while ensuring a large cell growth surface within a small volume. In some embodiments, the structured fixed bed may consist of a mesh or a mesh structure. In some embodiments, the mesh structure or mesh may consist of a network or web-like pattern of filaments, wires, or threads. In some embodiments, this network can constitute pores, openings, or perforations formed in three-dimensional tissue. In some embodiments, the structured bed can constitute winding pathways for cells and cell culture media. In some embodiments, these winding pathways or channels generate turbulence, facilitating the uptake of cells and cell culture media into the structured bed. In some embodiments, the mesh structure is a cell immobilization structure. In some embodiments, the mesh structure forms a spacer layer or spacer portion of the cell and culture media flow. In some embodiments, the mesh structure performs two functions: cell immobilization and spacer layering.

[0081] In some embodiments, the use of spacer layers facilitates the formation of winding pathways. In some embodiments, the structured bed can have one or more cell-fixing layers that have a surface on which cells attach and proliferate, and one or more cell-fixing layers that form a cell-fixing section. In some embodiments, one or more spacer layers are adjacent to the cell-fixing layers. In some embodiments, the spacer layers can have a structure that forms a spacer portion. In some embodiments, the spacer portion allows cells and culture media to pass through open but winding pathways. In some embodiments, the configuration or properties of the spacer layer can be selected so that the spacer layer creates winding, open pathways and cells and cell culture media move parallel to the surface of the spacer and the cell-fixing layers. In some embodiments, the winding pathways or channels formed by the spacer portion create turbulence that promotes the entry of cells and cell culture media into the fixation layer.

[0082] In some embodiments, the spacer layer may consist of a mesh or a mesh structure. In some embodiments, the mesh structure or mesh may consist of a network or web-like pattern of filaments, wires, or threads. In some embodiments, this network may constitute pores, openings, or perforations formed in a three-dimensional structure. In some embodiments, the spacer layer and / or cell immobilization layer of the spacer portion and immobilization portion may be formed from biocompatible polymers such as polyester, polyethylene, polypropylene, polyamide, plasma-treated polyethylene, plasma-treated polyester, plasma-treated polypropylene, or plasma-treated polyamide. In some embodiments, the spacer layer or cell immobilization layer may consist of silica, polystyrene, agarose, styrenedivinylbenzene, polyacrylonitrile, or latex. In some embodiments, these layers may be hydrophilic or hydrophobic. In some embodiments, these layers are hydrophilic. In some embodiments, the cell immobilization portion may be woven or nonwoven. In some embodiments, the cell immobilization portion and the spacer portion may be arranged alternately. In some embodiments, the alternating arrangement portions may be arranged alternately in a vertical position or alternately in a horizontal position. In some embodiments, the cell-immobilizing portions may be layered or arranged alternately in a vertical or horizontal position. In some embodiments, one or more layers may be connected. In some embodiments, one or more cell-immobilizing layers may be stacked on one or more spacer layers (or vice versa). In some embodiments, the structured bed disclosed herein may be tightly or loosely rolled into structures such as helical structures or monolithic structures or structures of different shapes, or may be configured in the form of upper and lower layers with fluids flowing parallel or perpendicular to the surface of the layers.

[0083] In some embodiments, the fixed bed growth surface has an area of ​​1 m². 2 ~2m 2 , 7-30m 2 , 150~600m 2 , 2,400m 2It is sufficient if it can be changed within the range of different sizes (height or diameter) of the bioreactor. As described above, for a plurality of fixed beds, one, two, three, four or more fixed beds can be stacked. In certain embodiments, the fixed bed growth surface has an area of, for example, 200 m 2 or, alternatively, 600 m 2 and may be.

[0084] In some embodiments, for the impeller speed, when a pressure drop occurs, it can be adjusted to keep it small and maintain a constant linear speed from the bottom to the top or from the top to the bottom of the bioreactor. In such cases, the shear stress on the cells can be kept constant regardless of the overall size of the bioreactor. In some embodiments, a sparger can also be used in combination. In some embodiments, it is desirable to interrupt the operation of the impeller during sparger treatment to prevent air bubbles from mixing into the fixed bed.

[0085] In some embodiments, one or more bioreactor components exhibit flexibility. In some embodiments, one or more bioreactor members exhibit rigidity. In some embodiments, one or more bioreactor members are made of polycarbonate. In some embodiments, one or more bioreactor members are made of rigid polycarbonate. In some embodiments, the bioreactor vessel is made of polycarbonate. In some embodiments, one or more bioreactor members are formed by injection molding.

[0086] In a certain embodiment, a bioreactor described in PCT / EP2018 / 086394, which is incorporated herein by reference in its entirety, is provided in the system. Briefly described, a modular bioreactor is provided that uses one or more structured fixed beds to facilitate manufacturing and use in a state where excellent cell culture results are achieved in terms of homogeneity and reproducibility even during expansion or contraction.

[0087] In some embodiments, the modular bioreactor has a base portion having a first chamber, an intermediate portion forming at least a portion of a second outer chamber that receives a fixed bed and at least a portion of a third inner chamber that returns the fluid flow from the second outer chamber back to the first chamber, and a cover portion covering the intermediate portion. The fixed bed may be formed of a structured fixed bed, and the intermediate portion may be composed of a tubular member. In this case, the structured fixed bed extends spirally around the tubular member. Alternatively, the intermediate portion may be composed of the inner wall of the fixed bed. In any embodiment, the intermediate portion may have a plurality of intermediate members, each corresponding to a structured fixed bed.

[0088] In some embodiments, at least one of the multiple intermediate members is perforated to allow fluid to flow from a first structured fixed bed below at least one intermediate member to a second structured fixed bed above at least one intermediate member. In some embodiments, each of the multiple intermediate members is tubular, and each structured fixed bed consists of a spiral bed wrapped around a tubular intermediate member. Perforated supports can be provided relative to the structured fixed beds.

[0089] In some embodiments, the intermediate section may be further combined with a tubular casing that forms the periphery of a modular bioreactor. This tubular casing forms a space for heating, cooling, or insulating the bioreactor. The intermediate section may consist of multiple intermediate members, each connected to the others.

[0090] In some embodiments, the intermediate section has piping that engages with at least one intermediate member and forms the inner wall of an outer second chamber that receives a fixed floor. This piping engages with a first intermediate member below it and a second intermediate member above it. The second intermediate member may have an opening that forms a fluid film along a third inner chamber. Supports, such as vertical rods, can be provided to support the second intermediate member from the first intermediate member.

[0091] In some embodiments, the cover portion consists of a cap having multiple ports. In some embodiments, the cover portion consists of a removable cap. The outer diameter of the removable cap may be less than the outer diameter of the intermediate portion. The outer diameter of the removable cap may be greater than or equal to the outer diameter of the intermediate portion. At least one of the ports may be equipped with a screw-type metal insert. The outer diameter of the cover portion may be equal to or greater than the outer diameter of the intermediate portion.

[0092] The intermediate portion can be composed of an intermediate member that is at least partially located within the base. The intermediate member may further include a flow disruptor that obstructs the fluid flow.

[0093] The base portion may include another chamber radially outward from the first chamber, which is in fluid contact with a second outer chamber having a fixed floor. This further chamber may be partially formed by an upright wall having a plurality of openings that transmit fluid from the first chamber to this chamber.

[0094] In some embodiments, a stirrer is provided at the base. The intermediate section moves left and right to align with an external drive device, and the stirrer can be suspended inside the first chamber to align with the external drive device.

[0095] In some embodiments, a container housing an agitator is provided. In some embodiments, this container has a central inlet and multiple radially oriented outlets. A flow divider can be associated with the central inlet. In any embodiment, the agitator can be composed of multiple curved blades as an independent component separate from any bioreactor.

[0096] In some embodiments, multiple flow disruptors are provided to divide the fluid flow into the third inner chamber into multiple streams. Rings may be provided corresponding to the multiple flow disruptors. In some embodiments, one or more conduits are provided into which gas can flow into the space behind one of the streams. One or more conduits may be connected to a structure having multiple flow disruptors. For example, the first conduit may be connected to the structure, or both the first and second conduits may be connected to the structure. Alternatively, the first and second conduits may not be connected to the structure.

[0097] An apparatus according to yet another aspect of the present disclosure is a cell culture apparatus. This apparatus comprises a modular bioreactor, the bioreactor having a base portion that is detachably connected to both a central column and / or an outer casing. The outer casing and the central column together form a cell culture compartment.

[0098] In feasible embodiments, each of a plurality of stacked structured fixed beds is wrapped around a central column. The central column comprises first and second pipes connected to each other, with the first structured fixed bed of the plurality of structured fixed beds wrapped around the first pipe, and the second structured fixed bed of the plurality of structured fixed beds wrapped around the second pipe. In some embodiments, the central column comprises first and second pipes that engage with a perforated support extending between at least two of the plurality of structured fixed beds.

[0099] In any embodiment, the structured fixed bed can consist of a cartridge that is inserted into or removed from a second outer chamber or compartment.

[0100] Another embodiment of the present disclosure is a cell culture bioreactor. This bioreactor may consist of a base member having a first chamber configured to receive a stirrer for agitating a fluid. Optionally, a detachable first central column may be attached to the base member, which forms at least part of a second outer chamber for culturing cells and a third inner chamber for returning the fluid flow from the second outer chamber back to the first chamber.

[0101] In this embodiment, or in any embodiment, the second outer chamber has a first structured fixed bed. In this embodiment, or in any embodiment, the first structured fixed bed has a spiral bed and can wrap around or enclose the first central column. The second central column can also form at least a portion of the second outer chamber and has a second structured fixed bed perpendicularly spaced apart from the first structured fixed bed. A perforated support can be provided between the first and second structured fixed beds.

[0102] In each embodiment, the second structured outer chamber has an unstructured floor.

[0103] A further embodiment of the present invention relates to a bioreactor for culturing fluid-connected cells. This bioreactor has a first chamber having a stirrer for fluid agitation, a second outer chamber having a plurality of stacked beds for culturing cells, and a third inner chamber for returning fluid from the second outer chamber to the first chamber. In another embodiment, the bioreactor has a first chamber having a stirrer for fluid agitation, a second inner chamber having a plurality of stacked beds for culturing cells, and a third inner chamber for returning fluid from the second inner chamber to the first chamber.

[0104] In some embodiments, the bioreactor has a base portion having a first chamber, an intermediate portion forming at least a part of a second outer chamber and at least a part of a third inner chamber, and a cover portion covering the intermediate portion. In this embodiment, and in other embodiments as well, the intermediate portion has a first support that supports a first bed consisting of a plurality of stacked beds. The intermediate portion has a second support that supports a second bed consisting of a plurality of stacked beds, and is configured to be detachably connected to the base portion and the cover portion.

[0105] In some embodiments, the second outer chamber borders the outer wall. The bioreactor may also be used with an outer casing that forms a space against this outer wall, which is used to insulate, heat, or cool the second outer chamber.

[0106] A further embodiment of the present invention relates to a bioreactor for use with the bioreactor platform. This bioreactor is suitable for cell cultures occurring with a fluid. In one embodiment, the bioreactor has a first chamber having a stirrer for fluid agitation, a second outer chamber having at least one bed for culturing cells, and a third inner chamber for returning fluid from the second outer chamber to the first chamber. The second outer chamber may have an outer casing that is in contact with the outer wall and further forms a space with respect to this outer wall, which is a space for insulating, heating, or cooling the second outer chamber. In another embodiment, the bioreactor has a first chamber having a stirrer for fluid agitation, a second inner chamber having at least one bed for culturing cells, and a third outer chamber for returning fluid from the second inner chamber to the first chamber. The third outer chamber may have an outer casing that is in contact with the outer wall and further forms a space with respect to this outer wall, which is a space for insulating, heating, or cooling the third outer chamber.

[0107] In this embodiment, or in other embodiments, at least one floor is a structured fixed floor such as a spiral floor or a layered / laminated floor, but it may also be an unstructured floor. The inner chamber can be formed by at least one pipe (which may be separate from or part of this floor). This at least one pipe can be connected to a first support and a second support that border at least one floor. The first support and the second support may be connected to the outer wall, and the first support and the second support may be perforated in at least part.

[0108] This disclosure relates to a bioreactor having first and second stacked structured beds. The bioreactor may further be used in combination with screens that abut the first and second stacked structured beds. The first and second stacked beds may be structured beds such as spiral beds.

[0109] This disclosure also relates to a bioreactor having a structured fixed bed that forms the central column of the bioreactor. A spiral bed can be used as the structured fixed bed. Since fluid does not permeate the inner surface of the structured fixed bed, the fluid can be returned to the structured fixed bed and a central column can be formed that recirculates, for example, from top to bottom. The bioreactor may be modular, and in some cases, multiple stacked structured fixed beds can be provided with gaps or spaces remaining between each bed in the stack.

[0110] In the case of small bioreactors as described herein, there is a problem in that the inoculant must be concentrated (by means of centrifugation, etc.). Otherwise, the bioreactor will not be able to handle the amount of inoculant (maximum workload - minimum workload < unconcentrated inoculant). Depending on the circumstances, it may be necessary to partially empty the bioreactor in order to minimize the maximum workload.

[0111] Generally, the surface of a bioreactor m 2 A cell quantity of 5,000 to 25,000 cells is required per unit. 600m 2 In the case of a bioreactor, this is 3 x 10 10~15×10 10 This corresponds to the amount of viable cells. This is a considerable amount of cells, equivalent to the amount of cells harvested by 9 or 10 cell factories.

[0112] This disclosure also relates to a method for inoculating a bioreactor without the need to concentrate the inoculum. For this purpose, the method involves inoculating the bioreactor in recirculation mode and changing the inoculum recipient in a later phase of the process.

[0113] In one embodiment, the method comprises the following steps. - A process of filling a bioreactor with culture medium and equalizing the pH of this medium. - A step of preparing an inoculum having a sufficient amount of cells in a cell culture medium volume, without concentrating the inoculum (by means of centrifugation, etc.). For example, 150 ml 2 A bioreactor can be used to prepare 6 liters of inoculum. This inoculum is then placed into an inoculum recipient such as a bag or bottle. - The inoculum recipient is connected to a bioreactor and gently agitated, preferably by any known and suitable means. - A circulating loop is established between the bioreactor and the inoculant recipient, circulating cells along with the culture medium from the recipient to the bioreactor, or vice versa. The circulation continues for a suitable amount of time depending on the volume of the inoculant and the size of the bioreactor. Generally, the circulation time can range from 1 to 10 hours, or from 1 to 4 hours. By this time, viable cells need to have attached to and become fixed to the fixed bed present inside the bioreactor. Non-viable cells will remain in the suspension in either the bioreactor or the recipient. -Once the circulation period is complete, the inoculation recipient is separated from the bioreactor. - The cells grow inside the bioreactor (batch growth), after which a new culture medium free of cells is recirculated, or preferably the recirculation mode is continued in a recirculating medium container such as a disposable container. In one embodiment, the container is a bag. The above method eliminates the need to concentrate the cell inoculation material, and there is no risk of non-viable or non-adherent cells being left behind in the recirculation loop. This method allows for efficient inoculation into small (fixed-bed) bioreactors, ensuring high cell density while minimizing workload. Furthermore, it removes undesirable excipients (such as trypsin) used to collect cells. Failure to remove excipients would negatively impact cell proliferation. In one embodiment, the bioreactor inoculation density is 1 × 10⁻⁶. 3 ~1 × 10 4 cells / cm 2 , comfortable 5×10 3 cells / cm 2 2 x 10 3 ~8×10 3 cells / cm 2 In one embodiment, the cell density at the time of collection is 5 × 10⁻⁶. 4 ~5×10 5 cells / cm 2 , more comfortable 1.5×10 5 cells / cm 2 1 x 10 5 ~4×10 5 cells / cm 2 That is the case.

[0114] One embodiment of the method is as follows:

[0115] - A step of connecting an inoculated recipient containing a predetermined amount of cells in a predetermined volume of culture medium to a bioreactor equipped with a predetermined volume of culture medium. - A step of populating the cells on the fixed bed by circulating the contents of the inoculated recipient to the bioreactor and returning them to the recipient, and - A method for inoculating a fixed-bed bioreactor, comprising the step of separating the recipient from the bioreactor after the completion of circulation.

[0116] The method is used to inoculate a bioreactor present in the system disclosed herein.

[0117] In a preferred embodiment, conditioning can be performed within the bioreactor vessel.

[0118] In another embodiment (using a bioreactor that utilizes recirculation to a culture medium condition adjustment container during the cell proliferation stage), two recirculation steps are utilized. Specifically, the first recirculation is performed at inoculation, and the second recirculation is performed during cell proliferation.

[0119] In some embodiments, a (re)circulation loop for conditioning the culture medium can also be configured. This loop fluidly connects the fixed-bed reactor to the culture medium conditioning vessel, thereby distributing the cell culture medium from the conditioning vessel to the bioreactor. The medium from the culture medium conditioning vessel flows into the bioreactor through an inlet. This inlet is equipped with an injection port for cell inoculation material, initiating inoculation and cell proliferation. The bioreactor vessel is provided with one or more outlets through which the cell culture medium flows out of the vessel. Furthermore, cells or cell products may be discharged through the outlets. One or more sensors can be installed in the line to analyze the effluent from the bioreactor. In some embodiments, the system includes a flow control unit that controls the flow into the bioreactor. For example, the flow control unit receives signals from one or more sensors (such as an O2 sensor) and, based on these signals, regulates the flow to the bioreactor by sending signals to a pump (such as a peristaltic pump) located upstream of the inlet to the bioreactor. Thus, based on a combination of one or more factors measured by the sensors, the pump can control the flow to the bioreactor to determine the desired cell culture state.

[0120] The culture medium conditioning vessel can incorporate sensors and control elements commonly found in bioreactors used in bioprocess fields such as suspension batch, fed-batch, and perfusion culture. Examples include, but are not limited to, DO oxygen sensors, pH sensors, deoxygenators / gas spraying units, temperature probes, and nutrient / base addition ports. The gas mixture supplied to the spraying unit can be controlled by a gas flow controller such as N2, O2, and CO2. A culture medium mixing impeller is incorporated into the culture medium conditioning vessel. All culture medium parameters measured by the sensors listed above are communicated to the culture medium conditioning vessel and can be controlled by a culture medium conditioning control unit capable of measuring and / or adjusting the state of the cell culture medium to the desired level. In one embodiment, the culture medium conditioning vessel is a separate container from the bioreactor vessel, which is advantageous because it allows for conditioning of a separate medium from the one used to culture cells, and then supplying the conditioned medium to the cell culture space.

[0121] Therefore, the system described herein may be provided with means for binding the inoculant recipient to the bioreactor and means for recirculating the inoculant and, optionally, a new conditioned medium to the bioreactor. The inoculant recipient may be located inside any of the three chambers described herein (bioreactor, process chamber, or downstream chamber), but is preferably located inside the bioreactor chamber. In one embodiment, the inoculant is provided in a skid.

[0122] In yet another embodiment, the disclosure also relates to a biomolecule production system having a bioreactor chamber configured to receive one or more purification or filtration devices and a bioreactor capable of purifying or filtering biomolecules from a cell collection. In one embodiment, the bioreactor chamber is provided with a connector capable of transmitting power, (signal) transmission and / or data transmission when paired with a bioreactor cabinet having a bioreactor. In one embodiment, the system is coupled with other modules, such as modules for additional processing, pharmaceuticalization, or packaging.

[0123] In a preferred embodiment, the system can be designed in the form of a package that can pass through a 93cm x 200cm door. Furthermore, the system can be designed for easy maintenance, with each critical component being easily removed by two workers without the use of lifts or other special tools, and the maintenance time for each component being less than two hours.

[0124] The casing enclosing the entire system is the main structure. In a feasible embodiment, its dimensions are 2445 mm × 2496 mm × 950 mm. In some embodiments, the system length can be shortened depending on the number of filters in the process chamber and downstream chamber. In one embodiment, the total casing footprint is approximately 2 m². 2 ~about 10m 2 However, the preferred installation area is approximately 3m². 2 ~about 5m 2 Therefore, a more preferable installation area is approximately 3m². 2 That is the case.

[0125] In another embodiment, the system includes a bioreactor described in PCT / EP2020 / 084317 and US2021 002 4868, which are incorporated herein by reference in their entirety. Briefly, this bioreactor comprises a stirrer that delivers liquid to a fixed cell medium and cell medium bed, the stirrer being installed in a container. In one embodiment, the stirrer is connected to a conduit. In one embodiment, the conduit comprises an injector that delivers bubbles to the container. In one embodiment, the stirrer converts the bubbles generated from the injector into second bubbles of a second size, smaller than the first bubbles, and delivers them along with the liquid to the cell culture bed. Due to their smaller size, the second bubbles flow efficiently into channels formed by the spacer layer and the adjacent cell immobilization layer of the fixed bed (or other available pathways). This further enhances the oxygenation of cells growing in the fixed bed, and there is no need to increase the impeller speed and liquid flow rate. Furthermore, the gas is released into or near the agitator container, resulting in a flow and preventing the formation of harmful air pockets within the bioreactor. As is well known, these air pockets are extremely difficult to remove without interrupting the bioreactor's operation.

[0126] In one embodiment, the system is equipped with control software that can collect, transmit, process, and visualize parameter measurements within the system. Furthermore, the control software can also adjust these parameters. Examples of parameters, though not limited to them, include pH, temperature, dissolved oxygen, volume, nutrients, level, and transmembrane pressure. In one embodiment, the control software can display a warning signal when the system is not operating properly. In one embodiment, the system can be remotely accessed via a network connection. In one embodiment, the user controls the system via a smart tablet connected to the control system.

[0127] The system is a mobile system equipped with wheels or tracks that enable transport.

[0128] In one embodiment, the system is designed to be mounted on a wall. In a preferred embodiment, the system is mounted on wheels so that it can be maintained when biomolecules are not being produced. In some embodiments, brakes and directional locks are attached to the wheels to prevent unnecessary movement of the system.

[0129] In a preferred embodiment, each chamber has a wall sheet or back sheet facing the operating area of ​​the chamber. This wall sheet or back sheet is equipped with one or more pieces of equipment selected from pumps, piping, electrical sockets, and / or manifolds necessary for the chamber to function.

[0130] The backsheet is preferably a vertical metal sheet at the back of the chamber, confining the chamber. In one embodiment, this backsheet supports at least the process equipment and disposable manifold used in the corresponding chamber. In yet another embodiment, the backsheet supports all process equipment (i.e., pumps, valves, sensors) and disposable manifold. In one embodiment, depending on the process performed in the system, different types of modular backsheets, different backsheets with specific hardware, are designed to accommodate compatibility and replacement for future needs and conditions. Multiple backsheets are integrated to meet different process needs. Furthermore, the combination and positioning of these backsheets are adjusted in place at any time to facilitate high-volume manufacturing. For each backsheet, all components such as motors, network cables, and power supplies are designed to be mounted on the back of the sheet, along with all equipment and devices that the operator will access. This design facilitates maintenance, increases accessibility, reduces particles, and enhances safety.

[0131] In some embodiments, the user-handled area of ​​each chamber within the system is protected by a front window. In one embodiment, it is preferable to have a gap in the front window to allow access during operation. In yet another embodiment, the front window can be sealed. In one embodiment, the window opening can be locked by a locking device. In one embodiment, the locking device can be controlled by control software. In one embodiment, the software issues a command to lock the locking device when the process is ready to begin and keeps the locking device locked throughout the entire process. In one embodiment, the left-side door of the chamber is provided with a locking device. In one embodiment, the locking device is an electromagnet consisting of a power supply part and a stainless steel part that is blocked after power is applied. In one embodiment, the two parts block with a force of 1000N. In one embodiment, the window opening is locked by one or more locking devices. In one embodiment, the window opening is locked by three locking devices. In one embodiment, the window is locked when the system is running. In one embodiment, an emergency stop device for the system is activated. Specifically, all operating actuators are stopped within the process chamber, and the process is put into a suspended state when force is applied to the locked windows. Locking the windows and side doors ensures operator safety and the efficiency of the laminar airflow (coming from the top of the chamber).

[0132] In one embodiment, the overall system casing has a front window with a gap of 100-300 mm, starting with 200 mm, along with the workspace. This gap allows air to be exhausted from the process chamber and is accessible during operation. This overall design allows the operator to be positioned in front of the chamber. The window may be made of any material suitable for this art, such as (thermoplastic) plastic or glass. In a particularly preferred embodiment, the window is made of plexiglass, which is lightweight and more shatterproof than glass.

[0133] In one embodiment, the window can be opened for easier operator access. The window can be opened vertically, toward the side of the system or chamber, horizontally, or toward the top of the system. The window can be opened and closed manually or automatically.

[0134] To enable the complete execution of the production or purification task of the target biomolecule, the bioreactor chamber is provided with a bioreactor cabinet. The bioreactor cabinet is connected to the system by a connection system having connecting and receiving components.

[0135] In one embodiment, the bioreactor cabinet and the system are connected, and the bioreactor cabinet is docked to the system, allowing for a secure interconnection between the two, preventing the bioreactor cabinet from detaching from the system during biomolecule production. In a preferred embodiment, a magnetic connection is utilized. An electromagnet can be used as the magnet, generating a magnetic field by electric current. An advantage of electromagnets over permanent magnets is that the magnetic field can be quickly changed by controlling the amount of current. In the present invention, the use of magnets, more specifically electromagnets, enhances the safety of the system because it prevents unauthorized docking or detachment of the bioreactor to the production system. The system can consist of corresponding magnetic components that interact with the magnets of the bioreactor cabinet. In one embodiment, the magnetic connection is controlled by software.

[0136] Furthermore, in order to dock and function, the bioreactor cabinet is provided with a connector that transmits power, signals, and / or data when paired with the biomolecular production system, and preferably a magnetic connection for connecting to the biomolecular production system. In one embodiment, the connector may consist of a connecting part and a receiving part. The connecting part may be located in the bioreactor cabinet, and the receiving part may be located in a recess in the bioreactor chamber. Alternatively, this configuration may be reversed. In one embodiment, the power supply unit of the magnetic connection is located in the system, and the stainless steel part of the magnetic connection is located at the back of the bioreactor cabinet. In one embodiment, once power is turned on, these two parts are blocked. In one embodiment, these two parts are blocked by a force of 1000N.

[0137] In one embodiment, the male connector of the bioreactor cabinet connects to the female connector of the production system. In a preferred embodiment, the female connector may be equipped with a centering pin to ensure accurate connection between the male and female connectors.

[0138] In a preferred embodiment, a modular connector system can be used as the connector, which can combine power and signal contacts, Ethernet, optical fiber, coaxial contacts, hydraulic couplings, pneumatic couplings, and thermal couplings within a small frame or housing. This modular connector system can be configured according to the specific connection requirements. In a preferred embodiment, these connectors are waterproof.

[0139] The bioreactor cabinet will be connected to industrial connectors that are highly reliable and capable of transmitting power, signals, and data in a plug-in configuration.

[0140] The bioreactor cabinet described above is preferred as the bioreactor cabinet. In some embodiments, the system's bioreactor cabinet protrudes from the processing chamber surface. This improves ergonomic conditions for the operator using the system, making it easier to access the various elements within the bioreactor cabinet.

[0141] In one embodiment, the bioreactor cabinet and bioreactor chamber can function as a standalone biomolecular production system. In another embodiment, the bioreactor chamber and bioreactor cabinet are connected to one or more upstream (process chambers) or downstream chambers.

[0142] In one embodiment, the bioreactor cabinet is connected to the bioreactor chamber, and then the bioreactor cabinet and bioreactor chamber are connected to an upstream (process chamber) or downstream chamber for purification or clarification.

[0143] In one embodiment, the bioreactor chamber and process chamber (either for upstream or downstream processing) are pre-assembled. In another embodiment, the bioreactor chamber is connected to the downstream chamber, while in yet another embodiment, the bioreactor chamber / process chamber is connected to the downstream chamber.

[0144] The system and its chamber will be equipped with a device for further purification or filtration of the bioreactor sample. The sample will contain a culture medium originating from the bioreactor, or lysate of cells cultured in the bioreactor. The purification method may consist of one or more of the following: clarification, aggregation, and precipitation of cell debris, lipids, host cell proteins, and DNA, as well as limit filtration, tangential flow filtration, or modification of the chemical state (pH, conductivity, ionic strength, etc.) for the purpose of concentrating the supernatant. Examples of such methods include chromatography in capture mode or flow-through mode, which can be performed in pack mode, monolith mode, membrane mode, or fluidized bed mode. When chromatography is performed in fluidized bed mode, classical media separated by precipitation or centrifugation, or (para)magnetic media separated by an external magnetic field, can be used. It is also possible to combine any of the above methods.

[0145] Such apparatuses are not limited to one or more types of chromatography, but may include affinity chromatography, ion exchange chromatography (such as anionic or cationic), hydrophobic interaction chromatography, size exclusion chromatography (SEC), immunoaffinity chromatography (using columns packed with affinity resins such as anti-IgM resin, protein A, protein G, or anti-IgG resin), or apparatuses combining these. Anion exchange utilizes the charge difference between different products contained in the collected supernatant. Neutral-charged products pass through the anion exchange chromatography column cartridge without stopping, while charged impurities are stopped. Column size can be changed based on the type of protein to be purified and / or the volume of the solution to which the protein should be purified.

[0146] To this end, in one embodiment, the bioreactor chamber is provided with a concentrator such as a TFF, and optionally a fluid collection container that receives the effluent from the concentrator and recirculates it back to the concentrator or to the downstream process. In a preferred embodiment, not only the concentrator such as the TFF but also the fluid collection container is connected to the back seat of the bioreactor.

[0147] In one embodiment, the chamber bioreactor and concentrator are connected by a conduit that facilitates the transport of liquid from the bioreactor to the concentrator. Alternatively, if a container is incorporated into the system, the bioreactor and the container are connected by a conduit that facilitates the transport of fluid from the bioreactor to this intermediate container via an inlet. Furthermore, these containers and concentrators are also connected by a concentrator supply conduit that enables the transport of fluid from the container to the concentrator. In a preferred embodiment, the concentrator is controlled by one or more valves, such as a pinch valve. In one embodiment, liquid is pumped from the collection container to the concentrator. In yet another embodiment, the pump is a disposable pump. In a preferred embodiment, the pump is a disposable diaphragm pump. In one embodiment, the collection container consists of a conduit that allows the fluid to bypass the concentrator. Finally, a conduit that facilitates the transport of liquid from the concentrator to the bioreactor can also be installed. In a preferred embodiment, the collection container has an inlet for small-volume additions. In a preferred embodiment, it has an inlet for CO2. In a preferred embodiment, the collection container has inlets for one or more buffer solutions. In one embodiment, the collection container has one or more level sensors. In a preferred embodiment, the collection container has a conduit for connecting a bubble trap. Foaming during bioprocessing is a major technical challenge to be addressed. The foaming tendency of culture media used in bioreactors causes not only direct depletion and contamination of various microbial cells, but also indirect adverse effects. Specifically, the addition of chemical degassing agents denatures the culture media, which leads to toxicity at the microbial metabolic level and contamination of downstream processing equipment. In one embodiment, the system includes a bubble trap to remove foam from the system.

[0148] In one embodiment, the collection container is provided with one or more handles to facilitate transport of the collection container. In another embodiment, a disposable, recyclable, and / or autoclavable container can be used as an intermediate container. The shape of the container may be any shape known to those skilled in the art and suitable for this purpose.

[0149] In one embodiment, each component and multiple components of the system are installed and / or removed from the system by a lift. In another embodiment, the concentrator (e.g., TFF cartridge), collection container, pump head and necessary piping are transported by a lift. In yet another embodiment, the lift comprises wheels for transporting each component to be installed and a holder for gripping each component. In yet another embodiment, the lift grips the collection container (and connecting components) by positioning the holder between the handles of the collection container.

[0150] In some embodiments, one or more containers, such as system collection containers or bioreactors, are disposable containers. In some embodiments, one or more containers in the system are rigid containers. In some embodiments, one or more containers in the system are flexible containers. In some embodiments, sensors such as level sensors, flow meters, pressure sensors, temperature sensors, and pH sensors are installed in or connected to one or more containers in the system. In some embodiments, these sensors are disposable sensors. Flow meters monitor the fluid non-invasively from the outside via piping. In some embodiments, flow meters are installed behind the filtration pump and used to measure filtrate flow. In some embodiments, flow meters are installed in the permeate conduit and used to measure permeate flow. Pressure sensors protect the system from overpressure. In preferred embodiments, a single pressure sensor is installed downstream of all pumps in the system. In some embodiments, level measurement of the collection container is performed by three capacitive level sensors that measure levels within a 20 cm range. In some embodiments, some of the level sensors are installed in the process chamber on the backsheet and must be operated in contact with the collection container. A level sensor allows for continuous measurement of the liquid level in the collection container. In one embodiment, the system includes one or more pH sensors. In one embodiment, the collection container has a pH sensor. In one embodiment, the pH sensor includes a pH probe and a transmitter that come into contact with the fluid inside the collection container. In one embodiment, the pH probe is a disposable pH probe. In one embodiment, the pH probe is a reusable pH probe. In one embodiment, the transmitter is a disposable transmitter. In one embodiment, the transmitter is a reusable transmitter. In a preferred embodiment, the pH probe is a disposable pH probe and the transmitter is a reusable transmitter. In one embodiment, the gas flow to the bioreactor and collection container is controlled by one or more mass flow controllers (MFCs). In one embodiment, three MFCs are used to control the pH and DO inside the bioreactor using air, CO2, and O2, respectively.In one embodiment, a single MFC is used to control the DO inside the sparger with O2. In another embodiment, a single MFC is used to control the pH inside the collection container with air or CO2. In one embodiment, multiple MFCs are grouped together in an MFC block. In yet another embodiment, the MFC block is located inside the bioreactor cabinet. In one embodiment, the MFC block is located inside the electrical cabinet of the bioreactor chamber.

[0151] The system's concentrator is selected from a number of devices known to those skilled in the art that are suitable for reducing the volume of the liquid containing the target biomolecule. In some embodiments, the concentrator comprises one type of concentrator (such as a tangential flow filter). In some embodiments, the concentrator comprises two or more concentrators (such as a tangential flow filter and a dead-end filter). Most of these devices are based on filtration and / or size exclusion chromatography. In one embodiment, the concentrator is a filtration device, more preferably a microfiltration device, a diafiltration device, or a device combining microfiltration and diafiltration. When a diafiltration device is provided in the system to reduce the volume of the liquid containing the target biomolecule, water and low molecular weight solutes, generally called permeates, pass through the membrane of this device, while macromolecules such as biomolecules are retained in the membrane within the concentrate or by-product (retentate).

[0152] In one embodiment, the TFF is provided with at least one hollow fiber having pores with sufficient porosity to hold almost all of the target biomolecules, while smaller contaminants such as growth media and solutes can pass through the membrane pores. Unlike dead-end filtration, where liquids pass through the membrane or bed but solids are trapped in the filter, in the TFF apparatus, tangential flow can traverse the surface of the filter, so no filter cake forms within the TFF. In another embodiment, the TFF may be provided with a cassette / cartridge from which tangential flow occurs. In yet another embodiment, the TFF is a single-pass tangential flow filtration (SP-TFF). This apparatus is particularly advantageous when purifying proteins such as antibodies. In some embodiments, the TFF has an area of ​​approximately 1000 cm². 2 ~About 2000cm 2 Preferably about 1500 cm 2 It comprises a membrane. The TFF is reusable, can be used again, and / or discarded. In some embodiments, the TFF is plug and play.

[0153] In yet another embodiment, a kit is provided. This kit consists of a TFF cartridge and one or more pre-assembled manifolds. The one or more manifolds preferably have piping, sterilization connectors, and optionally one or more pressure sensors. In one embodiment, the one or more pressure sensors can be discarded.

[0154] As described above, this system is equipped with a conduit for concentrated liquid / by-products that mediates the recirculation of concentrated liquid / by-products (retentate) to the bioreactor input or the container input. The advantage of implementing a TFF device as a concentrator in this system is that the TFF device is suitable for operation in a continuous perfusion process. Therefore, the culture medium volume can be concentrated to a large extent.

[0155] In a preferred embodiment, the collection container with the TFF attached is located in the center of the bioreactor chamber behind the bioreactor. The TFF is connected to the collection container using a support. The collection container-TFF- and TFF pump unit are mounted on the system's background metal sheet. In one embodiment, the collection container and TFF may be installed or removed after the bioreactor cabinet has been removed. In yet another embodiment, a support is formed within the bioreactor chamber to suspend the bag for adding less than 5 liters of reagent.

[0156] In one embodiment, a pump is used to fill the collection container with cells collected from the bioreactor. The manifold connection between the collection container and the TFF to the bioreactor or bubble trap is made aseptically. Benzoase and NaCl are prepared in bags, and these bags are suspended in the process chamber using bag supports (i.e., hooks). In one embodiment, a single pump is used to add these reagents to the collection container. To maintain volumetric accuracy, the bags must be filled with the exact amount of reagents, or the peristaltic pump must be controlled with a flow meter. Homogeneity within the collection container is ensured by a recirculation loop that recirculates the TFF using a TFF pump. Waste from the permeate line is collected in a waste tank / container / bag / line (located outside the system), and the connection to this waste tank / container / bag / line is made aseptically. In a preferred embodiment, the tank / container / bag / line should be equipped with a vent system and / or check valve to prevent backflow. Ideally, a cutting system should be employed. In some embodiments, the product is sent to a device for further purification or filtration after TFF concentration. A bubble detector is installed immediately before these purification or filtration devices to detect when the collection container is empty and to confirm the presence of bubbles or liquid. In some embodiments, the bubble detector is equipped with an ultrasonic sensor. In one embodiment, the collection container and TFF are washed to minimize product loss. In one embodiment, a chasing buffer is added to the collection container and TFF loop, and the chasing buffer in the loop is recirculated for a certain period of time as needed to wash and reduce the amount of product. After washing, process air is sent to the TFF inlet to push the product and minimize dead volume as much as possible. In yet another embodiment, the collection container is drained to reduce the void volume. In some embodiments, a sampling device is connected to the collection container to collect a sample. In yet another embodiment, the sampling device is connected to the top of the collection container.

[0157] The general purpose of a collection container is to obtain a gamma-irradiated "plug-and-play / ready-to-use" solution. In one embodiment, the container is constructed from polypropylene and a holder is used to suspend a TFF from the side. The selected TFF is irradiated with gamma rays to complete a gamma-resistant manifold (collection container + pump + TFF).

[0158] Embodiments of the system according to this disclosure include, firstly, a bioreactor chamber having a bioreactor cabinet having a bioreactor. Processing is performed in the bioreactor to produce biomolecules from cultured cells. The obtained products are optionally connected to the bioreactor chamber and purified in an adjacent process chamber. In one embodiment, the cultured cells are lysed before further processing. In another embodiment, DNA is extracted from the cultured cells before further processing. This process chamber includes one or more purification, clarification, or filtration devices to purify or filter the biomolecules of the cell samples.

[0159] In another embodiment, the disclosure also relates to a biomolecule production system. This system comprises a process chamber having one or more purification, clarification, or filtration devices for purifying, clarifying, or filtering biomolecules from a cell sample, a downstream chamber, and a bioreactor chamber configured to receive a bioreactor. The bioreactor chamber is located between the process chamber and the downstream chamber and is fluidly connected to both chambers. The bioreactor chamber is provided with connectors that transmit power, (signal) transmission, and / or data transmission when paired with a bioreactor cabinet having a connection system that connects to the bioreactor and the bioreactor cabinet. In another embodiment, the bioreactor chamber is not located in the process chamber or the downstream chamber, but is located outside the system.

[0160] In one embodiment, the fluid connection includes one or more intervening manifolds, containers, devices, etc.

[0161] In one embodiment, one or more of the filtration or purification devices are connected to an outlet line, the outlet line being parallel to the filtration or purification device and having a portion that rises vertically and causes a vertical flow in the outlet line.

[0162] In one embodiment, the process chamber includes a deep filtration system. The number of filters in this filtration system can be arbitrarily changed. In one embodiment, one or more walls of the process chamber are freestanding, allowing the process chamber to be expanded and additional filters to be added to the filtration system. Because the filters are located on the side of the system, the design can adequately accommodate the need for a large number of filters. The number of filters directly affects the system dimensions. In one embodiment, the deep filtration system includes one or more high-performance polyethersulfone (PES) liquid filters.

[0163] In one embodiment, the process chamber has the equipment, instruments, and disposable manifolds necessary to carry out a process within the chamber. In another embodiment, the process chamber has the equipment, instruments, and disposable manifolds necessary for the production of biomolecules. In yet another embodiment, the process chamber has the equipment, instruments, and disposable manifolds necessary for gene therapy. In yet another embodiment, the process chamber has the equipment, instruments, and disposable manifolds necessary for the production of antibodies. In yet another embodiment, the process chamber has the equipment, instruments, and disposable manifolds necessary for the production of viral vaccines. In the case of a process chamber, the equipment, instruments, manifolds, and sterile connecting parts must be easily accessible. The workspace is located approximately 90 cm above the ground, allowing the operator to work while standing.

[0164] The processes performed within the process chamber can be divided into one or more modules. In one embodiment, the process chamber has multiple modules. In another embodiment, the hardware corresponding to one or more modules is installed, or at least partially installed, within one or more panels. In yet another embodiment, each module is one (or more) panels on which the hardware is installed. In one embodiment, all panels are fixed to the process chamber. With a modular configuration / panel, it is possible to convert between them according to future demands and needs, and this can be easily accommodated. For each panel, it is preferable to design it so that the operator can access all equipment and devices, and to install all technical components such as motors, network cables, and power supplies on its rear. In one embodiment, the panel forms part of the backend cabinet. In one embodiment, the backend cabinet is a modular design.

[0165] After this first purification step, known as primary clarification, and the subsequent filtration, the collected material is concentrated in a bioreactor chamber. A TFF (Thorough Fibre) can be used as a concentrator. If necessary, the collected material is buffered using diafiltration.

[0166] Diafiltration is a separation process that washes smaller molecules through the membrane, retaining the target molecules in a concentrate. Diafiltration can be used to remove salts and exchange buffers. In other words, the ultramodule can be used to exchange one buffer for another and is a more efficient alternative module for dialysis. Diafiltration can be used to neutralize pH and can be used as a concentration process (to concentrate cell products).

[0167] In yet another embodiment, the system includes a downstream chamber on the side of the bioreactor chamber. In the downstream chamber, the sample is further clarified, if applicable, after the concentration process (and buffer exchange process) in the bioreactor chamber. This process is known as a secondary clarification process.

[0168] In one embodiment, the downstream chamber is fluidly connected to the bioreactor chamber and / or process chamber. In a preferred embodiment, the downstream chamber is fluidly connected to the bioreactor chamber. In another embodiment, the purification or clarification device in the downstream chamber first flows the product into a waste tank before viewing it. After this, the product in the collection container is sent to the device in the downstream chamber until the collection container is empty. In yet another embodiment, the device is washed with a chasing buffer for a specific time. This chasing process is used in conjunction with a collection container washing cycle and TFF. The buffer is prepared outside the system and introduced into the system on the left side with respect to the culture medium. In some embodiments, the buffer is scheduled to be added to the pumps specifically used. In one embodiment, a device (a so-called bioharvest feeder) is provided to transfer the collected material from the process chamber to the collection container and waste container.

[0169] In another embodiment, a device (called a diafiltration buffer device) is provided. This device starts from the diafiltration buffer supply pipe of the process chamber and ends in the collection container of the bioreactor chamber and the secondary clarification system of the downstream chamber, or from the collection container of the bioreactor chamber to the secondary clarification system of the downstream chamber. In one embodiment, this diafiltration buffer device is connected to a buffer supply device. This buffer supply device delivers the buffer to the clarification filter, wetting the filter and preparing it for priming. In one embodiment, this buffer supply device includes four inlet connections and a disposable pump head.

[0170] In another embodiment, a device (referred to as the permeate / waste device) is connected to the TFF permeate and waste container in the downstream chamber. In yet another embodiment, a device (referred to as the bulk bio-outlet device) is connected to a secondary clarification device or a dialysis filtration buffer device to the waste container and transfer bag.

[0171] The present invention also provides an innovative method for venting filters, such as clarification filters. Prior to use, these filters need to be prepared for priming and venting. In the classic method, the filter is sterile vented by adding a priming solution using a pump. This venting continues until the priming solution reaches the vent line (generally at the top of the filter). When the solution is added, the outlet line of the filter is closed. In this closed state, some of the priming solution flows into a bottle connected to the filter. When this solution reaches the vent line, the pump stops and the vent line is closed by a (manual) clamp or pinch valve, etc. At this point, the filter is primed and vented (there is no air inside the filter). Once the outlet line is opened again, the filter can be used. Because this operation is manual, it carries several risks. In fact, if the operator misses the priming solution reaching the vent line, the bottle connected to the vent will be rapidly filled with the priming solution, causing clogging of the bottle's vent. This results in high pressure, excessive pressure, and a risk of leakage. Furthermore, the filter is not transparent, which means that the operator(s) cannot visually determine whether the level inside the filter is high, or to what extent.

[0172] In conclusion, the above operation requires the utmost care and attention, and therefore, at least two operators are necessary. In most cases, these operators will manually adjust the flow rate and perform this process carefully. The operator(s) must immediately respond and stop the pump when the liquid reaches the vent line.

[0173] Automating this process is difficult and risky because the waste manifolds and filters used typically cannot withstand high pressure.

[0174] The present invention aims to provide a solution to the above-mentioned problems. To this end, the present invention vents and primes filters, such as clarification filters, in a novel and innovative way that avoids the above-mentioned risks and solves the above-mentioned problems.

[0175] In the present invention, a line (vertical upward piping) is used that connects to the filter and moves the filter upward, bringing it to the level inside the filter by the principle of a connecting vessel. With this configuration, the outlet line is filled parallel to the filter and reaches the same level (communicating vessel principle). This latter means that the outlet line remains open during priming and venting, preventing excessive pressure. If some of the pressure in the system becomes excessive, the priming solution will be forced into the inside of the outlet line.

[0176] In detail, this method utilizes the following steps: - The vent line (top of the filter) opens (automatic pinch valve) and connects to a sterilization bottle with a vent. An outlet line is provided on the filter, and a portion of the outlet line is raised perpendicular and parallel to the filter(s). - A priming solution is added to the filter by a pump. As this solution fills the filter(s), the vertical section of the outlet line is also filled. Filling continues until the filter is completely filled and the solution reaches the vent line of the filter. The vent line is detected by a liquid sensor such as a bubble sensor. The liquid sensor is preferably located in the vertical section of the outlet line. -Once it reaches the vent line, the vent line is closed (automatic pinch valve). In this process, the filter is primed and vented (no air is present inside), and the filter is now ready for use.

[0177] Depending on the situation, (digital) pressure sensors can be installed and used to shut down the system in case of emergencies or technical problems.

[0178] Using the method described above, the filter becomes immediately usable after the vent line has been closed. This limits manual operation to an absolute minimum, minimizing the risk of undesirable occurrences during priming / venting.

[0179] One embodiment describes a method for venting and priming a filter, such as a clarification filter. In this method, the filter is connected to a liquid outlet line, which is positioned parallel to the filter and has a vertically rising portion, thereby creating a vertical flow in the outlet line. The priming solution is added to the filter by a pump, and the priming solution fills the filter and the vertical portion of the outlet line during priming.

[0180] The filling process continues until the solution reaches the vent line of the filter, after which the vent line preferably closes automatically.

[0181] The above method can be used in the system described herein. For this purpose, an outlet line can be provided in a filter, such as a clarification filter, which has a vertical portion parallel to the filter and is connected to the filter with a liquid. An automatic pinch mechanism is employed to open and close the vent line (located at the top of the filter). A digital liquid detector, such as a bubble sensor, can be placed in the vent line to prevent leaks or to shut down the system in an emergency.

[0182] In embodiments that actively produce biomolecules, the system is paired with a bioreactor cabinet.

[0183] In a preferred embodiment, the bioreactor cabinet is located in the center of the system. This location allows for better operator access and requires less space.

[0184] Pairing with a separate bioreactor cabinet that is detachable from the system improves access to all other elements of the process, as the operator can enter the system when the bioreactor cabinet is removed. Similarly, during manifold installation, the bioreactor cabinet can be removed from the system, and the bioreactor and container can be installed. The bioreactor cabinet can then be reconnected to the system, improving ergonomic constraints.

[0185] In one embodiment, one or more of the flow path, cell culture medium, buffer, waste container, or active pharmaceutical ingredient container are located outside the bioreactor cabinet and connected with standard sterile connectors.

[0186] In a preferred embodiment, the system is mobile. For this purpose, the chamber can be configured with wheels or other elements that allow for movement. With wheels, the system can be moved and transported.

[0187] In one embodiment, the system is designed to be installed within a GMP manufacturing area. Furthermore, the system can be easily transported to a different location for cleaning purposes. In another embodiment, the system is designed to withstand standard VHP decontamination cycles. In yet another embodiment, the system is suitable only for indoor environments and requires maintaining room temperature at ambient temperature.

[0188] In a preferred embodiment of the present invention, the system includes an HVAC system that extracts air from the surrounding area, filters the air, preferably by a HEPA filter, and sends the filtered air to the system chamber.

[0189] An HVAC system is a system that can supply air of an appropriate quality level to the system chamber, thereby enhancing protection for products. In a preferred embodiment, ventilation is provided to ensure that the system chamber meets environmental grade C ISO 7 (as defined in ISO 14644).

[0190] In a preferred embodiment, air is drawn from the surrounding Grade C or D area, filtered through a HEPA (H14), and blown into the system chamber. Exhaust air from the system is achieved through a wind gap at work plan level. The preferred velocity of the airflow (downward flow) inside the system chamber (within the work area) is 0.45 m / s at a position 15 cm from the work plan and backsheet. In a preferred embodiment, the HVAC system consists of three interconnected blocks. Each block has one HEPA (H14) filter with a fan, each HEPA being capable of integrity testing and easily replaceable by a single engineer, with the fan easily replaceable by a single operator and accessible for maintenance. For 100% DOP central connections, integrity testing is considered easy in the art. In some embodiments, each HEPA filter can be monitored by a magnetic indicator. In some embodiments, a membrane diffusion screen is provided above one or more system chambers to generate a homogeneous airflow within these chambers. In some embodiments, the membrane diffusion screen has a stainless steel frame and stainless steel mesh.

[0191] In some embodiments, the HVAC system is located at the top of the chamber. In preferred embodiments, the system is connected around a central column, with the HVAC system located at the top of the column. In preferred embodiments, the airflow flows in only one direction and is guided between the backplate of the technical enclosure and the glass. The glass can be made of any material preferred in the art, such as plexiglass, glass, or polycarbonate (PC).

[0192] In some embodiments, an electrical cabinet is provided at the rear of each chamber in the system. This electrical cabinet houses the electrical components of the equipment located within that chamber.

[0193] The electrical cabinets, which supply power to the equipment and control the process, are integrated into the rear of the system. In preferred embodiments, these electrical cabinets consist of a backsheet, a technical enclosure, and space for circuit boards. In some embodiments, the electrical cabinets are made of stainless steel and are accessible by opening the rear door of the system. The backsheet is fixed to the electrical cabinets and properly secures the equipment and electronic components. In some embodiments, critical components accessible during maintenance work in operation are located on the front of the chamber, while all terminal boxes, wires, and electronic components are located on the rear and are not accessible during operation. In preferred embodiments, the electrical cabinets are removable, replaceable, interchangeable, and / or interchangeable. In some embodiments, the electrical cabinets are modular. In some embodiments, the system comprises at least one electrical cabinet. In yet another embodiment, the system comprises multiple electrical cabinets. In preferred embodiments, the system comprises nine electrical cabinets. In yet another embodiment, each chamber within the system (bioreactor chamber, process chamber, and downstream chamber) comprises three electrical cabinets.

[0194] In yet another aspect, the present invention also relates to using the systems described above for producing biomolecules such as proteins, viruses, viral particles, and gene therapy products.

[0195] In a preferred embodiment, the internal flow path of the system is a fully sealed system consisting of disposable consumable components (bioreactor, filter, TFF membrane, bottle, sampling device, disposable sensor, etc.) interconnected by disposable piping manifolds. In one embodiment, the flow path includes a sampling system. In another embodiment, a collection container is connected to a foam trap. In yet another embodiment, the bioreactor is disposable and includes a pre-assembled disposable manifold for upper and lower liquid bioreactor drains, a liquid sample line, a bubble or foam trap, and a base structure.

[0196] In the final aspect, the present invention also relates to a chain method for producing biomolecules such as proteins, viruses or viral particles, or gene therapy products, using a system according to any of the embodiments described above. The method comprises the step of providing a bioreactor installed in a bioreactor cabinet, preferably a wheeled bioreactor cabinet, which is docked into a bioreactor chamber of a biomolecular production system; producing biomolecular samples by filtering or purifying samples taken from the bioreactor in a processing chamber located on the side of the bioreactor chamber; and further concentrating the biomolecular samples using a concentrator located within the bioreactor chamber.

[0197] In some embodiments, the biomolecular sample is clarified in-line in one or more filters located in the downstream chamber on the side of the bioreactor chamber.

[0198] One possible process flow in one embodiment of this system is intended for the production of biomolecules such as viral particles, for example, vaccines or products for viral gene therapy. For this purpose, cells are cultured in a bioreactor inside a bioreactor cabinet embedded in a bioreactor chamber. Culture media and buffers are supplied to the bioreactor by an external supply bag connected to the bioreactor chamber. Waste generated during the production cycle is led to a waste container, and the bioreactor sample is lysed and transported to a process chamber, where it is filtered using a purification or filtration device. After this process, the product is collected or transferred to the bioreactor chamber, concentrated using a collection container and TFF, and then the concentrated liquid is transported to a purification or filtration device in a downstream chamber. The system is also equipped with an auxiliary chamber in case further upstream or downstream processing is required.

[0199] In a specific embodiment, the present invention provides a system and method for producing a human gene therapy product that introduces genetic material into a patient using a gene therapy product, preferably a human gene therapy product, and more preferably a viral vector.

[0200] In one embodiment, the viral vector may include retroviruses, adenoviruses, herpes simplex viruses, cowpox viruses, lentiviruses, and adeno-associated viruses.

[0201] During the infection phase, the virus is added to the bioreactor. In one embodiment, the virus is added to the bioreactor by a virus infection kit. In one embodiment, the virus infection kit consists of a two-component bottle for the virus infection process and two spare connections. In one embodiment, the virus is added to the bioreactor by a pump, such as a Watson-Marlow peristaltic pump.

[0202] In one embodiment, endonuclease is added to the bioreactor for nucleic acid removal. In another embodiment, endonuclease is added to the bioreactor via an inlet for small-volume addition. In one embodiment, endonuclease is an ideal tool for nucleic acid removal during viral vector and vaccine production. In yet another embodiment, endonuclease is added by an endonuclease apparatus consisting of a 5-liter disposable bottle and special connections. In one embodiment, endonuclease is added to the bioreactor by a pump such as a Watson-Marlow peristaltic pump. In one embodiment, this endonuclease is a benzonase endonuclease, which degrades both DNA and RNA into small, non-base-selective 3-5 base pair (<6kDa) fragments. Using a benzonase endonuclease results in higher viral purification yields, prevents contamination of downstream chromatography and filtration equipment, and has the added benefit of lower feed flow viscosity.

[0203] In one embodiment, the transfer reagent is added to the bioreactor by an transfer device consisting of a disposable bag and two special connections.

[0204] In another embodiment, the biomolecule produced are vaccines such as influenza vaccine, SARS vaccine, MERS vaccine, COVID-19 vaccine, measles vaccine, rabies vaccine, Zika vaccine, polio vaccine, mumps vaccine, and rubella vaccine.

[0205] The present invention will be described in detail below with reference to the attached drawings, which are not intended to be limiting. Description of the drawings

[0206] The present invention is not limited to the embodiments described below and / or shown in the accompanying drawings, and the methods according to the present invention can be implemented in a number of different forms without departing from the scope of the invention.

[0207] The bioreactor cabinet 001 will be described with reference to Figure 1A, which shows one embodiment of the bioreactor cabinet 001. Arrows indicate the positions in the bioreactor cabinet 001 where bioreactors 100, 200, 300, a heating support (not shown), and a stirring motor (not shown) can be installed. Bioreactors 100, 200, and 300 are installed in the bioreactor docking station 044. Furthermore, wheels 005 as a mobile structure are clearly shown in Figure 1A. Two handles 004, 004' are provided to facilitate the operation of the bioreactor cabinet 001. A detachable height adjuster 006 is 200m 2 It can handle bioreactors 100, 200, and 300.

[0208] Figure 1B shows another embodiment of the bioreactor cabinet 001. A handle 004 is attached to the front wall of the bioreactor cabinet 001, making it easier to operate, especially when docking the bioreactor cabinet. Wheels 005 (or other components that provide mobility) are attached to the bioreactor cabinet, making it easy to move. Bioreactors 100, 200, 300, a heating support (not shown) for heating the culture medium in the bioreactors, and a stirring motor (not shown) are installed inside the bioreactor cabinet 001. The detachable height adjuster 006 has a disc-shaped structure, so 200m 2 It can accommodate bioreactors 100, 200, and 300, and can reliably align the top level of the bioreactor 114 with the top level of the bioreactor cabinet 008. 2When using bioreactors 100, 200, and 300, the height adjuster 006 does not need to be used. The connection portion of the connector 009 and the magnetic connection 010 are provided on the wall of the biocabinet 001 facing the front wall 011 of the bioreactor cabinet. As shown in Figure 1B, the connection portion of the connector 009 and the magnetic connection 010 do not necessarily need to be provided on the same wall of the bioreactor cabinet 001, but providing them on the same wall improves the ergonomic characteristics of the bioreactor cabinet 001 and makes it easier to handle. The connection portion of the connector 009 and the magnetic connection 010 are designed to engage with corresponding parts present in the biomolecular production system. In the embodiment shown in Figure 1B, each side wall 012 of the bioreactor cabinet 001 is provided with positioning means at the corners of these side walls. These positioning means are basically a pair of wheels 013. Each pair of wheels has two wheels, and the first wheel is positioned perpendicular to the direction of the second wheel. The bioreactor cabinet has a grid-shaped bioreactor support plate 014 on the top surface of the cabinet 008, and this plate has recesses 015 so that the bioreactor surface 114 can protrude. The bioreactor cabinet 001 can be divided into shelves 016 inside for storing materials and hardware. The bioreactor cabinet of this embodiment has two holding trays 037. The different holding trays 037 of the bioreactor cabinet are at different levels and are connected to each other by piping 038.

[0209] Figure 1C shows a more detailed embodiment of Figure 1B. A single handle 004 is connected to the front wall 011 of the bioreactor cabinet 001, making it easier to operate the bioreactor cabinet 001, especially when docking it. Wheels / casters 005 are attached to the bioreactor cabinet 001, making it easy to move. Alternatively, other components can be used to give the cabinet 001 mobility. The bioreactors 100, 200, 300, a heating support (not shown) for heating the culture medium in the bioreactor, and a stirring motor (not shown) are installed inside the bioreactor cabinet. The detachable height adjuster 006 has a disc-shaped structure, allowing for adjustments of 200m. 2 In addition to accommodating the bioreactor, the top surface level of the bioreactor 114 can be reliably aligned with the top surface level of the bioreactor cabinet 008. The connection portion of the connector 009 and the magnetic connection 010 are provided on the wall of the biocabinet facing the front wall 011 of the bioreactor cabinet. In the embodiment shown in Figure 1C, each side wall 012 of the bioreactor cabinet 001 is provided with positioning means at the corners of these side walls. These positioning means are basically a pair of wheels 013. Each pair of wheels has two wheels, with the first wheel positioned perpendicular to the direction of the second wheel. The bioreactor cabinet has a grid-shaped bioreactor support plate 014 on the top surface of the cabinet, and this plate has recesses 015 so that the bioreactor surface 114 can protrude. The bioreactor cabinet 001 can be divided into shelves 016 inside for storing materials and hardware. The bioreactor has an external casing or housing 112 that forms an internal compartment, and a removable cover or top surface 114 that covers this internal compartment, which may be equipped with openings or ports P with removable covers or caps C for selectively introducing or discharging fluids or gases (by a sparger, etc.), probes, sensors, samplers, etc.

[0210] Figures 1D to 1F show cross-sectional views of embodiments according to the present invention. A handle 004 is connected to the front wall 011 of the bioreactor cabinet 001, making it easier to operate the bioreactor cabinet 001, especially when docking it. Wheels 005 (or other members that provide mobility) are attached to the bioreactor cabinet 001, making it easy to move.

[0211] As shown in Figures 1E to 1F, the bioreactors 100, 200, 300, a heating support (not shown) for heating the culture medium in the bioreactors, and a stirring motor (not shown) are installed inside the bioreactor cabinet 001. The detachable height adjuster 006 has a disc-shaped structure, so 200m 2 In addition to being able to accommodate bioreactors, the top surface level of the bioreactor 114 can be reliably aligned with the top surface level of the bioreactor cabinet 008. The bioreactor cabinet 001 has a grid-like bioreactor support plate 014 on the top surface of the cabinet, and this plate has recesses 015 so that the bioreactor surface 114 can protrude. The bioreactor cabinet 001 can be divided into shelves 016 inside for storing materials and hardware. The bioreactor has an external casing or housing 112 that forms an internal compartment, and a removable cover or top surface 114 that covers this internal compartment, which may be equipped with various openings or ports P for selectively introducing or discharging fluids or gases (by a sparger, etc.), probes, sensors, sample collectors, etc.

[0212] Figure 2 is a front view of a biomolecular system 017 according to one embodiment of the present invention, showing a bioreactor chamber 020 configured to receive bioreactors 100, 200, and 300 within a process chamber 018, a downstream chamber 019, and a bioreactor cabinet 001. Therefore, the system 017 is provided with a recess 021 to accommodate the bioreactor cabinet 001. The bioreactor chamber 020 is essential, while the process chamber 018 and downstream chamber 019 are chambers used as appropriate and can be individually connected to the bioreactor chamber 020. Each bioreactor 100, 200, and 300 has an external casing or housing 112 (not shown) forming an internal compartment, and a removable cover or top surface 114 covering this internal compartment. The user-accessible area of ​​each chamber in the system is protected by a front window 022, and it is preferable that this front window has a gap 023 to allow access during operation.

[0213] Figure 3 is a top view showing a system 017 according to an embodiment of the present invention, having a process chamber 018, a downstream chamber 019, and a bioreactor chamber 020. The bioreactor cabinet 001 of this system 017 protrudes from the process chamber 018 when installed in the system, allowing the operator easy access to the bioreactor cabinet 001. An electrical cabinet 024 can be placed behind each chamber in the system. These electrical cabinets supply power to the equipment and control the process, and consist of a back sheet 025, a technical enclosure 028, and space for a circuit board 029. In this embodiment, the electrical cabinets are made of stainless steel and are accessible by opening the back door of the system. The back sheet 025 is fixed to the front of the electrical cabinet 024, allowing the equipment to be properly secured and electronic components to be kept out of sight. Important components accessible for operational maintenance are located in the front of the chamber, and in this configuration, all terminal boxes, wires, and electronic components are located at the back and are inaccessible during operation.

[0214] Figure 4A is a front view showing system 017 according to an embodiment of the present invention. This system comprises a process chamber 018, a downstream chamber 019, and a bioreactor chamber 020, each having one or more purification or filtration devices capable of purifying or filtering biomolecules from cell samples. The bioreactor chamber is configured to receive bioreactors 100 and 200 into a bioreactor cabinet 001. The bioreactor cabinet 001 is guided to the bioreactor chamber by a guide 036 located in the bioreactor chamber 020 and wheels 013 located in the bioreactor cabinet 001. The overall casing 026 is the main structure of the system. In some embodiments, the length of system 017 can be shortened depending on the number of filters in the process chamber 018 and the downstream chamber 019. The material constituting the overall casing 026 of the system is corrosion-resistant. In the embodiment shown in Figure 4A, the metal elements are made of stainless steel SS316 with a surface roughness (arithmetic mean roughness) Ra ≤ 1.2 μm.

[0215] Figure 4B shows the rear and front views of system 017 shown in Figure 4A. The HVAC system 027 is installed above chambers 018, 019, and 020, ensuring a reliable supply of air of appropriate quality to the system chambers. The rear and sides of the system casing consist of an electrical cabinet 024 and a pneumatic cabinet 030, which are critical electrical and pneumatic components of the system. This facilitates operator access to these electrical and pneumatic components, while eliminating the need for operators to enter the biomolecular production space.

[0216] Figure 5 shows details of a front view of a system according to an embodiment of the present invention having a front window 022. In a preferred embodiment, the front window of the overall casing of the system has a 200 mm gap 023 with a work plane 031. This gap allows exhaust from the process chamber 018 and access during operation. Due to this overall design, the operator can work in front of the chamber. In a particular embodiment, the window can be opened in two different ways (vertically and horizontally).

[0217] Figure 6A shows a preferred embodiment of the system 017 according to the present invention. In this embodiment, the bioreactor 020 is located in the center of the production system 017, and the process chamber 018 and the downstream chamber 019 are located on the side of the production system. The bioreactor chamber 020 can dock a bioreactor cabinet 001 having bioreactors 100, 200, and 300. For this purpose, the bioreactor chamber 020 is provided with a recess (not shown) that can receive the bioreactor cabinet 001. To facilitate docking the bioreactor cabinet, the bioreactor cabinet 001 has a handle 004. The bioreactor cabinet 001 has wheels 005 to facilitate movement. The bioreactors 100, 200, and 300 each have an external casing or housing (not shown) that forms an internal compartment, and a removable cover or top surface 114 that covers this internal compartment. These may be equipped with openings or ports P with removable covers or caps C for selectively introducing or discharging fluids or gases (by a sparger, etc.), probes, sensors, sample collectors, and the like.

[0218] Bioreactor samples taken from the bioreactor port are transported to the process chamber 018, where appropriate piping 039 for fluid transport is provided. The process chamber 018 is equipped with one or more purification or filtration devices 032 for purifying or filtering the biomolecules of the cell samples. The purification or filtration device 032 is provided with an outlet line having a vertical section 502 parallel to the purification or filtration device 032, and the purification or filtration device 032 is safely primed and vented prior to use. A priming solution is supplied via an inlet line 503 connected to the purification or filtration device. For example, a deep filtration system can be used as this filter. The number of filters in the process chamber 018 can be arbitrarily changed depending on the product to be produced. Since the filters are installed on the side of the system, this design offers a high degree of flexibility when it is necessary to attach a very large number of filters. The workspace 031 is set at approximately 90 cm from the ground, so the operator can work while standing.

[0219] The background metal sheet 025 inside the process chamber 018 is designed to allow the operator access to all equipment and devices, and the back 028 houses all technically necessary components such as motors, network cables, and power supplies.

[0220] The bioreactor chamber 020 is equipped with a collection container 033 and a TFF 034 for concentrating the collected material. The collection container 033 and TFF 034 are fluidly connected to each other and are both located in the center of the bioreactor chamber 020 behind the bioreactors 100 and 200. The collection container 033-TFF 034-TFF pump (not shown) is attached to the background metal sheet 025 of the system 017. When the bioreactor cabinet 001 is not docked to the system 017, the collection container 033 and TFF 034 are accessible. Homogeneity within the collection container can be ensured by using a recirculation loop formed by the TFF pump (not shown) via TFF 034. From TFF 034, the concentrated biomolecular material can be transferred to the downstream chamber 019 of the system. Here as well, fluid transfer can be carried out by providing appropriate piping 039. The downstream chamber 019 is located on the side of the bioreactor chamber 020 opposite the process chamber 018. The downstream chamber 019 is added as needed. In the downstream chamber 019, the collected material can be further clarified after the concentration process in the bioreactor chamber 020. The downstream chamber 019 is fluidly connected to the bioreactor chamber 020 and has one or more purification or filtration devices 032 that can purify or filter the biomolecules of the collected cell material. The back sheet 025 of the downstream chamber is provided with the pumps, piping, electrical sockets and / or manifolds necessary for the function of the chamber. All technical components such as motors, network cables and power supplies must be installed on the back of the technical enclosure 028.

[0221] Figure 6B is a view of an embodiment of Figure 6A from a different perspective.

[0222] The description continues with reference to Figure 7, which shows one embodiment of a cell culture bioreactor 100 according to one aspect of the present invention. In some embodiments, the bioreactor 100 has an external casing or housing 112 that forms an internal compartment, and a removable cover or 114 that covers this internal compartment, which may be equipped with openings or ports P with removable covers or caps C for selectively introducing or discharging fluids or gases (by a sparger, etc.), probes, sensors, sample collectors, etc.

[0223] Within the internal compartment formed by the bioreactor housing 112, several compartments or chambers can be provided to supply fluid or gas flow to the entire bioreactor 100. As shown in Figure 8, in some embodiments, a first chamber 116 may be provided at or near the base of the bioreactor 100. In some embodiments, this first chamber 116 may contain an agitator that generates fluid flow within the bioreactor 100. In some embodiments, the agitator may be in the form of a drop-in rotating non-contact magnetic impeller 118, which, as will be further described below, can be housed in or contained within a container (not shown) having multiple openings through which fluid flows in and out.

[0224] In some embodiments, as a result of agitation, the fluid becomes an upward flow (as indicated by arrow A in Figure 8) and flows into the annular chamber 120 along the outer periphery or surrounding portion of the bioreactor 100. In some embodiments, the bioreactor is configured to receive a fixed bed, such as a structured spiral bed 122, which can contain or hold growing cells during operation. As shown in Figure 8, in some embodiments, the spiral bed 122 takes the form of a cartridge, which can be dropped into or positioned in the chamber 120 at the time of use. In some embodiments, the spiral bed 122 can be pre-assembled during factory manufacturing prior to transport.

[0225] In some embodiments, the fluid flowing out of chamber 120 is routed to chamber 124 on one side (upper side) of the spiral bed 122, where it is exposed to a gas (such as oxygen or nitrogen). In some embodiments, the fluid then flows radially inward and into a central return chamber 126. In some embodiments, this return chamber takes the form of a column and can be formed by a non-porous conduit or piping 128 rather than by a central opening in the structured spiral bed. In some embodiments, a continuous loop is created as the return chamber 126 returns the fluid to the first chamber 116 (return arrow R) and recirculates it to the bioreactor 100 (in this embodiment, a continuous loop from the base to the top). In some embodiments, it is also possible to provide sensors such as a temperature probe or sensor T to detect the temperature of the fluid in chamber 126. In some embodiments, sensors (such as a pH sensor, oxygen sensor, dissolved oxygen sensor, and temperature sensor) can also be provided before the fluid flows into (re-into) chamber 116. The sensors and probes described herein may be reusable, single-use, and / or disposable.

[0226] Figure 9A shows one embodiment of a structured fixed bed, particularly a helical bed 122, used in the bioreactor of the present disclosure. In some embodiments, one or more cell immobilization layers 122a are provided adjacent to one or more spacer layers 122b composed of a mesh structure. In some embodiments, the layering process may be performed several times as needed to obtain a layered or stacked configuration. In some embodiments, the mesh structure included in the spacer layer 122b (see cells L in Figure 9B suspended or trapped in the material of the immobilization layer 122a) forms winding pathways for cells, and the cell culture medium can constitute part of the claims, and a fluid can flow between the two immobilization layers 122a during layering. As a result of adopting this configuration, cellular homogeneity is maintained within the structured fixed bed. In some embodiments, other spacer structures may also be used to form such winding pathways. In some embodiments shown in Figure 9A, the structured fixed floor can then be rolled spirally or coaxially along an axis or core (such as a conduit 128 which may be provided in multiple component parts). In some embodiments, multiple layers of the structured fixed floor are tightly wound together. In some embodiments, the diameter of the core, the length of the layers, and / or the quantity ultimately determine the size of the device or substrate. In some embodiments, the thickness of each layer 122a, 122b may be 0.1 to 5 mm, 0.01 to 10 mm, or 0.001 to 15 mm.

[0227] In some embodiments of one aspect of this disclosure, the bioreactor 100 can be designed in a modular manner. In some embodiments, the modular bioreactor can consist of a plurality of discrete modules that interact to form a space suitable for cell culture in a highly predictable manner depending on the homogeneity of the modules during manufacturing. In some embodiments, the modular bioreactor is not limited to a specific shape or form (for example, it may be cylindrical and may have a structured fixed bed or an unstructured bed depending on the application). This is illustrated in Figure 10. In some embodiments, the module can consist of a base portion formed by a base module 130, an intermediate portion formed by an intermediate module 140 (which may be formed from a plurality of stackable modular portions as will be described in detail below), a central module such as a conduit or pipe 128 used as appropriate (which may also be considered part of the intermediate module), and a cover module formed by, for example, a lid or removable cover 114. In some embodiments, these modules may be manufactured individually as separate components, assembled in a manufacturing facility (and transported to the point of use) according to their intended use, or assembled at the final point of use according to their intended use. In some embodiments, the modules of the bioreactor 100 interact to construct a site for cell culture in a high-density manner using a fixed bed, such as a structured or unstructured fixed bed.

[0228] Another embodiment of the bioreactor 200 according to the present invention is shown in Figures 11 to 14. In some embodiments, the bioreactor (whether modular or pre-assembled as a single unit) can consist of a base, an intermediate, and a cover. In some embodiments, the base can consist of base components. In some embodiments, the intermediate can consist of intermediate components 250 and / or 270. In some embodiments, intermediate components 250 and 270 are not the same. In some embodiments, the cover can consist of a cover component 280. Referring to Figure 11, the base component 230 may consist of an outer wall 232 and an inner wall 234, which form a first chamber 216 that receives an agitator (not shown). In some embodiments, the inner wall 234 has an opening 234a through which fluid flows into a second radially outward chamber 220, which borders the outer wall 232 (Figure 12).

[0229] As can be seen from Figure 12, in some embodiments, the inner wall 234 may have a plurality of connectors, such as grooves 236, which engage with corresponding connectors, such as tongues 250a, on the first intermediate part 250, as shown in Figure 13. In some embodiments, the height of the inner wall 234 may be higher or lower than the outer wall 232, as can be seen from Figure 8. Referring to Figure 11, in some embodiments, the first intermediate part 250 can be at least partially fitted into the base part 230.

[0230] In some embodiments, the base component 230 may have connectors around a groove 237 (Figure 11). In some embodiments, the connector or groove 237 is configured to receive a corresponding connector on a second intermediate component 270, which is simply part of the outer wall 262. In some embodiments, multiple fixed floors 274 in a third chamber 224 can be installed inside the intermediate component 270 (a single monolithic fixed floor may also be used, which may be of any size, shape, or form in this embodiment and other embodiments). This can be supported by interposing supports, but gaps G can also be provided between adjacent portions of the fixed floors. These gaps do not have to be used, in which case the upper fixed floor can be placed on top of the lower fixed floor and supported.

[0231] In some embodiments, the structured fixed bed may be helical in shape, as shown in Figures 9, 9A, 9B, and 9C (this helical shape can be implemented in any embodiment of the bioreactor described herein or elsewhere). In the case of a helical bed, it may be wrapped around the inner wall 266, which forms a fifth chamber 228 that returns fluid to the first chamber 216 in the base component 230. The inner wall 266 may be composed of a multi-layered tubular component, as shown. In some embodiments, the height of these multi-layered tubular components can be adjusted according to the number of fixed beds present (for example, one tubular component may be provided for each layered bed) (Figure 11).

[0232] In some embodiments, the cover component 280 or lid component is detachably connected to the second intermediate component 270, forming a fourth chamber 226 where the liquid meets a gas such as air. In some embodiments, the connection between the cover component and the second intermediate component can be made by connectors such as the upper end of the outer wall 262 or a groove 282 that receives any access mechanism disclosed herein. Various ports P may be provided on the lid component or cover component 280 (Figure 11).

[0233] Returning to Figures 11 and 14, further details of the intermediate component 250 are shown. In some embodiments, the intermediate component 250 may comprise a plurality of radially extending supports 254, which, when placed in the adjacent third chamber 224, support a structured fixed bed. In some embodiments, the height H of the supports 254 is sufficiently high so that the fluid generates a sufficient upward velocity, which flows into the third chamber 224 and passes through the entire fixed bed 274 (Figure 11).

[0234] In some embodiments, the annular inner wall 258 can be connected to the inner end of the support 254. The diameter of the inner wall 258 corresponds to the diameter of the inner wall 266 of an intermediate component 270, which can also be connected to the inner wall (by nesting, etc.). In some embodiments, the inner wall 266 can form a flow channel that carries fluid from the fifth chamber 228 to the first chamber 216. In some embodiments, a flow disruptor 260 can be provided in this flow channel so that no vortices are generated in the fifth chamber 228.

[0235] In some embodiments, as can be seen from Figure 11, the flow from one fixed-bed module to the next adjacent fixed-bed module within the cell culture chamber 224 is a continuous flow, i.e., an uninterrupted flow. In some embodiments, the outer chamber 224 can form a continuous flow channel through a multi-layered fixed-bed system that can use structured, unstructured, or other types of fixed-beds. In some embodiments, the continuous, uninterrupted flow through pre-designed and matched fixed-bed modules increases the homogeneity of cell proliferation and other processes, enhances the consistency of cell culture operations, and improves the ability to take measurements or samples from the layered beds. This is not easily achieved when partition plates are present (as opposed to porous supports, as described below). Finally, in the structured-bed embodiments, the construction of the entire bioreactor is less complex and less labor-intensive because the effort required to match the characteristics and features from one fixed-bed module to another is significantly reduced.

[0236] The following explanation will continue with reference to Figures 15 and 16. Figures 15 and 16 are schematic diagrams showing a third embodiment of the bioreactor 300 and are shown as cross-sectional views for clarity. In some embodiments, the bioreactor 300 (whether in a modular design or a design that is pre-assembled as a single unit) consists of an outer housing 331 with a cover 333, both of which may be equipped with various openings or ports that allow for fluid introduction or discharge. In some embodiments, several compartments or chambers are provided within the bioreactor housing 331. These chambers have a first chamber 316 equipped with a “drop-in type rotating non-contact magnetic impeller 318 or a stirrer disclosed herein” that causes fluid flow within the bioreactor 300. As shown in Figure 15A, in some embodiments, the impeller 318 can be housed, captured, or contained within a housing such as a housing or container 318a having a plurality of openings 318b that act as inlets and outlets for fluid to flow in and out (or other forms of stirrers are also available). In some embodiments, a second or external tubular chamber 320, radially outward from the first chamber 316, is agitated to allow fluid to flow in.

[0237] In some embodiments, the fluid then flows upward (as indicated by the arrow in Figure 16) and enters a third annular chamber 324 along the middle outer portion of the bioreactor 300. In some embodiments, this outer portion can be configured to receive a fixed bed such as a structured spiral bed 325, but other configurations are also available. This fixed bed can contain cells that are growing at the time of use. In some embodiments, the spiral fixed bed 325 can take the form of a cartridge that is simply dropped into the chamber 324 at the point of use, but it may also be pre-assembled into the chamber during manufacturing prior to transport.

[0238] In some embodiments, the fluid exiting the third chamber 324 can then be diverted to the fourth chamber 326, where it is exposed to a gas (such as air) before flowing radially inward and into the fifth chamber 328. This fifth chamber has column properties and returns the fluid to the first chamber 316 for recirculation by the bioreactor 310, thus creating a continuous loop. In some embodiments, a temperature probe or temperature sensor T or other sensors described herein can be provided to detect parameters such as the temperature of the fluid flowing directly into the fifth chamber, and a sensor (e.g., a pH sensor or a dissolved oxygen sensor) may also be attached at this location (before the fluid flows into (or re-flows into) the fixed bed 325).

[0239] As can be seen from the cutaway view of Figure 15B, the third chamber 324 may be adjacent to the upper plate 330 and the lower plate 332, which typically have openings or holes through which a fluid, not containing cells, flows into or out of the fixed bed 325. In some embodiments, the lower plate 332 may have a central opening 332a, configured to allow fluid to move from the fifth chamber 328 to the first chamber 316 and recirculate. In some embodiments, the upper plate 330 may have an opening 330a through which fluid can move and flow into the fifth chamber, i.e., the return chamber 328.

[0240] In some embodiments, the support for the upper plate 330 is composed of a hollow, cylindrical pipe 334, although other shapes are also possible. In some embodiments, both ends of this pipe 334 can be fitted into corresponding grooves 330b, 332b in plates 330, 332 (depending on the case, the lower plate 332 can be integrated with the impeller housing or impeller container 318a of the illustrated embodiment). In some embodiments, the support function of the plate 330 can be further enhanced by using a support such as a vertical rod 336. In some embodiments, this vertical rod 336 does not obstruct the fluid flow in the corresponding chamber 328 in any way. In some embodiments, both ends of the rod 336 may be provided in recesses of plates 330, 332, or they may be placed in place by appropriate fastening devices or locking mechanisms (such as locking connections, bolts or adhesives).

[0241] As can be seen from Figure 16 and the accompanying action arrows, as a result of agitation, in some embodiments, the fluid flows outward from chamber 316 and into chamber 320. In some embodiments, the fluid then changes direction, passes perpendicularly through chamber 324, which has a fixed bed, and flows into chamber 328. The fluid then turns inward relative to chamber 328 and returns to the first chamber 316 through opening 332a. In some embodiments, the fluid may be called the culture medium.

[0242] Continuing the explanation with reference to the configuration in Figure 17, in some embodiments, an opening 330c is provided around the upper plate 330, allowing the fluid to flow directly along the inner wall formed by the piping 334. This configuration creates a thin layer or film of fluid that flows through the fifth chamber 328. In some embodiments, this increases the volume of fluid exposed to the gas (air) in the fifth chamber 328 before returning to the first chamber 316. In some embodiments, this configuration allows for stronger oxygen transfer, necessary for larger size or faster cell growth rates, and enables adjustment of process parameters based on the product. In some embodiments, a “waterfall” is formed by initially adding a limited amount of cell culture medium, creating a fluid film and generating a very small overflow. Alternatively, in some embodiments, the “waterfall” is formed by adding cell culture medium and cells, and then, during cell culture on a fixed bed, by drawing out the medium from the corresponding chamber, such as chamber 328 (e.g., using an immersion tube).

[0243] Figure 18 shows a possible process flow in an embodiment of system 017. This process produces biomolecules such as virus particles to manufacture vaccines or viral gene therapy products. For this purpose, cells are cultured in bioreactors 100, 200, and 300 located in a bioreactor cabinet 001 embedded in a bioreactor chamber 020. Culture medium 040 and buffer 041 are supplied to the bioreactors by externally supplied bags connected to the bioreactor chamber. Waste generated during the production cycle is led to a waste container 042. After this, the bioreactor sample is lysed and transported to a process chamber 018, where it is filtered using a purification or filtration device 032. After this step, the product is collected or transported to the bioreactor chamber 020 for concentration using a collection container 033 and TFF 034. The concentrated liquid is then transported to a purification or filtration device 032 in a downstream chamber 019. If further upstream or downstream processing is required, an attached chamber 043 is connected to the system.

[0244] As will be obvious to those skilled in the art, the process flow shown in Figure 18 is illustrative, and other process flow sequences can be used in connection with the present invention.

[0245] Figure 19 shows a feasible embodiment of the level sensor according to this disclosure. Using such a level sensor, the liquid level of a collection container can be continuously measured from the outside.

[0246] Figure 20 shows a feasible embodiment of the pressure sensor according to this disclosure. With such a pressure sensor, excessive pressure will not be applied to the system.

[0247] Figure 21 shows two implementable embodiments of the flow meter according to this disclosure. Such flow meters allow for non-invasive monitoring of fluids from the outside via piping.

[0248] Figure 22 shows a feasible embodiment of the bubble trap according to this disclosure. Using such a bubble trap, bubbles can be removed from an aqueous solution.

[0249] Figure 23 shows a possible embodiment of the container 140. Those skilled in the art will understand that the walls of such a container 140 can be formed independently of the rest of the bioreactor, or they can be integrated with other walls or parts of the bioreactor. For example, in Figure 23, the bottom of such a container is formed by the bottom of the bioreactor. To generate liquid flow, the container 140 may be provided with multiple openings 141, one or more of these openings may be configured as inlets or outlets for liquid inflow and outflow (note the action arrows I (IN) and O (OUT) illustrated in Figure 23). In some embodiments, the liquid leaving the container 140 flows upward into a structured floor, such as a spiral floor, and then returns to the inlet through the openings 141 of the container 140. Those skilled in the art will understand that this operation can be reversed. To ensure bubble stability and proper injection position, piping 142 may be connected along the side walls of the container 140.

[0250] In a bioreactor, when it is desirable to increase the amount of gas transferred to the cell culture medium while the cell culture medium is circulated, according to one aspect of the present disclosure, this can be achieved by introducing a gas flow such as air or oxygen into the bioreactor as described with reference to FIG. 24. Specifically, an injector for injecting a gas (such as sterilized air) is provided, and bubbles are injected into the fluid generated by a stirrer such as an impeller 118 in the container 140 at or near the centrifugal pump position. In some embodiments, the injector can be connected to a gas supply source outside the bioreactor (see, for example, FIGS. 25 and 26), and can be composed of an injector conduit or pipe 142 that can be connected to any wall portion thereof. For example, as shown in FIGS. 25 and 26, this pipe 142 can be connected to the cover 114 of the bioreactor 100. Alternatively, as shown in FIG. 24, the pipe 142 can be connected to the bioreactor 100 at a position along the base of the bioreactor adjacent to the position of the container 140.

[0251] In some embodiments, as shown in FIG. 24, the outlet of the pipe 142 for supplying gas to the liquid of the bioreactor 100 is inside the container 140 or in fluid communication with the inside, and thus will be located within the fluid path between the inlet I and the outlet O. For example, when the pipe 142 is passed through another wall or surface of the bioreactor 100, the outlet of the pipe 142 can also be provided within the central chamber 126. FIG. 25 shows a state where the pipe 142 passes through the cover 114 and enters the lower part of the central chamber of the bioreactor adjacent to the container 140, and FIG. 26 shows a state where the pipe 142 enters the container 140 and the open distal end of the pipe directly sends liquid to or is in direct fluid communication with the internal compartment of the container.

[0252] In any of the embodiments, the oxygen-containing gas (such as air) injected into the bioreactor 100 through the pipe 142 forms bubbles of a relatively large size in the fluid. This is because the pipe simply has an unobstructed open end. Alternatively, it is also possible to use smaller bubbles generated using a device such as a sparger in a dedicated nozzle or a corresponding vibration system. In either case, as a result of positioning the open end of the pipe 142 or the nozzle as described above, larger "macro bubbles" or smaller "micro bubbles" (size 1) are released at a position before they face the turbulent flow and shear action generated by the stirrer (for example, the rotating impeller 118 acting as a stirrer) that forms the pump of the bioreactor 100. This stirring divides the larger macro bubbles into a larger number of bubbles of a smaller size, that is, "micro bubbles", or divides the smaller micro bubbles into even smaller-sized and more numerous bubbles (size 2). Increasing the residence time of the bubbles in the stirrer vessel 140 (if used) results in more bubbles being generated in combination with the speed and design of the stirrer.

[0253] As a result of the rotation of the impeller 118, these micro bubbles will then be carried by the liquid flowing radially (radially) outward and upward in the illustrated bioreactor 100. Those skilled in the art should be able to understand that reversing the pump direction will result in an opposite flow pattern.

[0254] On the premise of a smaller size, it is more preferable for the micro bubbles to enter and pass through the channels formed by the spacer layer 122b and the adjacent cell immobilization layer 122a (or available paths) of the fixed bed 122. This further promotes the oxygenation of the cells growing in the fixed bed, but there is no need to correspondingly increase the speed of the impeller 118 and the resulting liquid flow rate. Furthermore, since the gas is released into or near the stirrer vessel 140, no air pockets harmful to the bioreactor 100 will be generated due to the resulting flow. It is extremely difficult to remove this air pocket unless the operation of the bioreactor is stopped.

[0255] In another embodiment of the present invention, as microbubbles (size 2) pass through the structured fixed bed 122, they divide into smaller and more numerous microbubbles (size 3). For example, this is because the bubbles flow into channels formed within the fixed bed 122 by the spacer layer 122b and / or adjacent cell immobilization layer 122a, etc., and travel through all available winding (or labyrinthine) paths. As these bubbles move, larger bubbles divide into even smaller microbubbles, which then flow out of the fixed bed 122. As a result, gas transfer during residence time in the fixed bed 122 is increased, and the size of the bubbles that have passed through and flowed out of the fixed bed is actually even smaller (and more numerous) than at the time of inflow. This is because shear forces are generated by the bubbles engaging with the mesh of the spacer layer 122b in the illustrated embodiment.

[0256] As shown in Figures 25 and 26, in the illustrated embodiment of the bioreactor 122, the liquid flowing out of the fixed bed 122 proceeds to the central chamber 126, where it flows directly along the inner surface of a wall that can be formed by the piping 128. The liquid level in the central chamber 126 can be lower than the liquid level in the fixed bed 122. A thin layer or “film” of liquid is formed, which (in this embodiment) flows downward as it passes through the central channel 126. In some embodiments, this gas-liquid interface acts to increase the volume of liquid that is exposed to gas (such as air) before returning to the first chamber 116 and finally re-flowing into the fixed bed 122. This is due to the pumping action generated by the agitator. In some embodiments, this can result in enhanced oxygen transfer, which is necessary for larger bioreactors, or otherwise necessary to increase cell growth rates or to adjust process parameters based on the biomolecules produced.

[0257] Figure 27 shows yet another embodiment of the bioreactor 100. This embodiment consists of multiple stacked beds (two layers, but any number of layers are also available). In this bioreactor configuration, a gas injector is provided to supply new gas to a location at the inlet end of each fixed bed 122 of the stack, which can be configured using a corresponding section connected to a gas supply source (e.g., housing 112a) or individual piping 142 passing through manifold 144. The size of the bubbles may be large upon introduction, but once they have passed through the corresponding fixed bed 122, their size actually becomes smaller than their size upon introduction. This is because, in the illustrated embodiment, shear forces are generated by bubbles engaging with the mesh of the spacer layer 122b. Alternatively, to suppress the formation of clouds at the inlet of the fixed bed section, a sparger / nozzle system, possibly in combination with a transducer, may be used to reduce the size of the bubbles.

[0258] Furthermore (or alternatively), the value of the gas transfer coefficient kLa may be increased by extending the distance and time the liquid medium travels in contact with the gas phase before returning to the fixed bed. In one feasible embodiment, the expanded flow pattern can be established by a flow expander (wherein herein, the expander can be made of a suitable material such as a polymer or metal, and means a structure with a large surface area over which the liquid needs to flow in order to increase the residence time in contact with the gas). It is also desirable to use a flow disruptor in conjunction with the flow expander to generate turbulence that minimizes the diffusion layer that normally occurs at the gas-liquid boundary, as described below. Those skilled in the art will understand that flow expanders and / or flow disruptors can be introduced into any fixed-bed bioreactor design, including unstructured packed-bed bioreactors. As shown in Figure 28, the flow expander 150 can be used in a modified form having a series of concentric ledges or steps 158 over which the liquid flows while moving radially or in a waterfall-like manner. This modified form not only extends the residence time of the liquid but also generates some turbulence when the liquid changes direction while flowing along and / or over step 158.

[0259] If the fixed bed 122 is located inside the outer (annular) chamber 120, the expander 150 can also be provided inside the central chamber 126, as shown in Figure 29. In this embodiment, the expander 150 has a labyrinthine structure formed by segmented wall portions 180 that can be spaced apart vertically and circumferentially to generate a winding flow within the film of liquid flowing out of the fixed bed 122 and into the chamber 126 from above. This allows the normally occurring diffusion layer to be broken as intended, and the liquid combines with the gas inside the chambers 124 and 126, thereby improving gas movement. The structure of the flow extender 150 that causes flow disruption can also take the form of pins or forks. As shown in Figure 30, for example, an expander 150 composed of pins 182 or other disruption structures can also be provided in the chamber 120 if the fixed bed 122 is located inside the central chamber 126.

[0260] Figure 31 shows an embodiment of a lift 403 for transporting parts of a system according to one aspect of the present disclosure. This lift includes wheels 401 for transporting parts to be mounted, and a holding device 402 (not shown) for holding each part.

[0261] Figures 32 and 33 show another embodiment of a modular bioreactor 400 with a fixed bed 496. In some embodiments, the base 430 and cover 470 are connected to an outer casing 492, forming a gap or space around the intermediate section 450. In some embodiments, this gap G or space can be used to ensure a heating or cooling effect to control the temperature of the fixed bed corresponding to the intermediate section 450. The gap G or space may simply be the wall of the intermediate region of the bioreactor, which is close to the cell proliferation cells in the fixed bed and is susceptible to temperature fluctuations, being insulated. Such insulation prevents heat applied to the bottom of the bioreactor base 430 from acting on adherent cells in the fixed bed(s) 496.

[0262] Figure 32 shows that sparging can be utilized in a bioreactor, which can be used in any embodiment disclosed herein. In the illustrated configuration, sparging is performed by a sparger 494 provided in the fifth chamber 428. The resulting bubbles flow upward as a backflow against the return fluid flow.

[0263] These drawings also show that the intermediate components 450 can engage with the internal piping 436, although Figure 33 is probably the best example. These piping are fluid-impermeable and form a chamber 428 that returns the flow to the base 430, where agitation occurs, the flow is returned, and (in any of the disclosed embodiments) flows into the fixed bed from below and then upward. For these piping 436, each fixed bed 496 has one piping as shown in the figures, and the two intermediate components 450 engage with each piping 436 (for example, one engages from below and the other from above). It should be noted that in this embodiment and other embodiments, similar functionality can be achieved by making the innermost surface of the fixed bed, such as the innermost lap of the spiral bed, fluid-impermeable or by conditioning it to be fluid-impermeable. For example, coating this innermost surface with a fluid-impermeable or hydrophobic material can retain fluid in the fixed bed(s) and maintain a clear return flow of fluid through the central column formed by the chamber 428.

[0264] Figure 34 is a schematic diagram showing a filter device 500, such as a clarification filter. As can be seen from the figure, the filter has an outlet line 501 with a vertical section 502 parallel to the filter, allowing for safe priming and venting prior to use of the filter device 500. The priming solution is supplied by a pump 504 via an inlet line 503 connected to the filter 500. This filter is also connected to the outlet line 501 with the vertical section 502 as described. A vent line 505 connected to the top of the filter 500 is opened when the priming solution is added. The opening and closing of the vent line 505 can be controlled by an automated pinch valve 506. As will be apparent to those skilled in the art, there are other known options for adjusting the vent line. Depending on the circumstances, automated (pinch) valves 507, 508 may be placed in the inlet 503 and / or in the outlet line 501. A digital liquid detector 509 can be provided in the vent line 505 to monitor the liquid in the vent line. Furthermore, a digital pressure sensor 510 is provided in the inlet line 503 to monitor the pressure within the inlet line 503. A vent 511 is located at the end of the vent line 505, and this vent line 505 terminates inside the bottle 512 to collect any excess priming solution.

[0265] The system shown in Figure 34 can be operated within the system shown in Figures 6A and 6B, and more specifically, within the process chamber 018. As will be apparent to those skilled in the art, the principle can also be applied to other filters that require priming and venting and are present in any of the other chambers of the system. [Explanation of Symbols]

[0266] 001 Bioreactor Cabinet 004, 004' Handle 005 Wheels / Casters 006 Height Adjuster 008 Bioreactor Cabinet 009 Connector 010 Magnetic connection 011 Front wall 012 Side wall 013 Wheel 014 Bioreactor support plate 015 Recess 016 Shelf 017 Biomolecular system 018 Process chamber 019 Downstream chamber 020 Bioreactor chamber 021 Recess 022 Front window 023 Gap 024 Electrical cabinet 025 Backsheet 026 Overall casing 027 HVAC system 028 Technical enclosure 029 Circuit board 030 Pneumatic cabinet 031 Working plane 032 Purification device or filtration device 033 Collection container 034 TFF 036 Guide 037 Holding tray 038 Pipe 039 Pipe 040 Medium 041 Buffer 042 Waste container 043 Attached chamber [[ID=6B]]044 Bioreactor docking station 100 Bioreactor 112 Housing 112a Housing 114 Bioreactor 114 Top surface 114 Cover 116 First chamber 118 Rotating non-contact magnetic impeller 120 Annular chamber 122 Structured Spiral Floor 122a Cell immobilization layer 122b Spacer layer 124 Chambers 126 Return Chamber / Center Chamber 128 Non-perforated conduits or piping 130 Base Module 140 Intermediate Modules / Containers 141 Aperture 142 Piping 144 Manifold 150 Flow expander 158 steps 180 Segmented wall section 182 pins 200 Bioreactors 216 Chamber 1 220 Second Chamber 224 Third Chamber / Cell Culture Chamber 226 Chamber 4 228 Chamber 5 230 Base Components 232 Exterior Wall 234 Interior wall 234a aperture 236 Groove 237 Groove 250 intermediate parts 250a Tang 254 Support 258 Circular inner wall 260 Flow Breaker 262 Exterior Wall 266 Interior wall 270 Intermediate parts 274 Fixed floor 280 Cover parts 282 Groove 300 Bioreactors 316 Chamber 1 318 Rotating Non-Contact Magnetic Impeller 318a Impeller container 318b aperture 320 Second Chamber 324 Third Chamber 325 Structured Spiral Floor 326 Chamber 4 328 Chamber 5 330 Top plate 330a aperture 330b Groove 330c aperture 331 Outer Housing / Bioreactor Housing 332 Lower plate 332a center opening 332b Groove 333 Cover 334 Piping 336 Rods 400 Modular Bioreactors 401 wheels 402 Holding device 403 Lift 428 Chamber 5 430 Base 436 Internal Piping 450 Middle section 470 Cover section 492 Outer casing 494 Sparger 496 Fixed floor 500 filter device Exit Line 501 502 Vertical section 503 Entrance Line 504 Pump 505 Ventline 506 Automated Pinch Valve 507, 508 Automated (pinch) valves 509 Liquid detector 510 Pressure Sensor 511 Vent 512 bottles A Arrow C Cap G Gap H Height I Entrance O exit P port R (Return arrow) T sensor

Claims

[Claim 1] A bioreactor cabinet configured to be incorporated into a biomolecular production system, The bioreactor cabinet is a mobile bioreactor cabinet suitable for receiving a bioreactor, the bioreactor cabinet is provided with a bioreactor docking station, and the side wall of the bioreactor cabinet is provided with connectors that enable power transmission, signal transmission and / or data transmission when paired with a biomolecular production system.

Images