Systems and methods for bio-manufacturing

Abstract

A sterile bioprocessing system for cell culture, the system comprising at least two closed sterile elements configured to contain a fluid wherein: each of the elements comprises at least one sterile tube configured for fluid communication; and all said sterile tubes are made of the same material and are adapted to be welded to each other to allow fluid communication between different sterile elements of the system; and therefore, the system can become a closed sterile system.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480032893.X (22) Application Date 2024.03.13 (30) Priority Data 63 / 490,005 2023.03.14 US (85) PCT International Application Entering National Phase Date 2025.11.14 (86) PCT International Application Application Data PCT / US2024 / 019622 2024.03.13 (87) PCT International Application Publication Data WO2024 / 192060 EN 2024.09.19 (71) Applicant: Olginis Corporation Address: Maryland, USA (72) Inventor: Moran Melon Hardy Basha (74) Patent Agency: Beijing Anxin Fangda Intellectual Property Agency Co., Ltd. 11262 Patent Attorneys Wei Changjin and Zheng Xia (51) Int.Cl. B01L 3 / 00 (2006.01) B29C 65 / 74 (2006.01) C12M 3 / 00 (2006.01) C12N 5 / 0783 (2006.01) (54) Title of Invention System and Method for Biomanufacturing (57) Abstract A sterile bioprocessing system for cell culture, the system comprising at least two closed sterile elements configured to contain fluid, wherein: each of the elements comprises at least one sterile tube configured for fluid communication; and all the sterile tubes are made of the same material and are adapted to be welded to each other to allow fluid communication between the different sterile elements of the system; and thereby enabling the system to be a closed sterile system. Claims (2 pages), Description (13 pages), Drawings (8 pages), CN 121358541 A, 2026.01.16, CN 1 21 35 85 41 A 1. A sterile bioprocessing system for cell culture, the system comprising at least two closed sterile elements configured to contain fluid, wherein: each of the elements comprises at least one sterile tube configured for fluid communication; and all the sterile tubes are made of the same material and adapted to be welded to each other to allow fluid communication between different sterile elements of the system; and thereby enabling the system to be a closed sterile system. 2. The system of claim 1, wherein the sterile tube is at least partially flexible. 3. The system of claim 1, wherein the material of the sterile tube comprises polyvinyl chloride (PVC) or thermoplastic elastomer (TPE). 4. The system of claim 1, wherein each of the sterile tubes initially has a sterile fluid connection to its corresponding element at its proximal end and is sealed at its distal end.5. The system of claim 1, wherein the aseptic element is selected from: rigid containers, flexible containers, at least partially flexible containers, and any combination thereof. 6. The system of claim 1, wherein the elements of the system comprise at least two of the following: at least one culture container; at least one container configured to contain tissue and / or cells; at least one bottle configured to contain tissue and / or cells; at least one syringe; at least one filter bag; at least one filtration device; at least one waste bag; at least one fluid bag; at least one manual pipette or automated pipette; at least one harvest element; at least one washing element; and any combination thereof. 7. The system of claim 1, wherein the cells comprise at least one of the following: tumor-infiltrating lymphocytes (TILs), T cells, CAR-T cells, engineered T cell receptor (TCR) cells, natural killer (NK) cells, CAR-NK cells, natural killer T (NKT) cells, and CAR-NKT cells, and any combination thereof. 8. The system of claim 1, wherein the closed aseptic system is configured to avoid the use of laminar flow hoods and / or any open processes. 9. A method for closing a sterile bioprocessing system for cell culture according to claim 1; the method comprising: aseptically cutting two sterile tubes of the system, providing a cut edge for each sterile tube; and aseptically welding the cut edges of the two sterile tubes to each other, the two sterile tubes being configured to allow sterile fluid communication between their respective elements. 10. The method of claim 9, wherein the cutting step is provided simultaneously. 11. The method of claim 9, further comprising aseptically cutting and aseptically sealing at least one sterile tube, the at least one sterile tube being configured to aseptically disconnect its fluid communication. 12. The method of claim 9, further comprising aseptically cutting and aseptically welding any one of the connecting tubes of the system more than once to allow sequential fluid communication between its respective elements and several different elements of the system via their respective sterile tubes. 13. The method of claim 9, wherein the aseptic cutting and aseptic welding steps are provided via an aseptic welding machine. 14. The method of claim 9, further comprising avoiding the use of laminar flow hoods and / or any open processes. 15. A closed sterile bottle configured for cell bioprocessing, the bottle comprising at least one sterile tube configured for fluid communication; wherein the sterile tube is made of polyvinyl chloride (PVC) or thermoplastic elastomer (TPE), respectively adapted to be welded to any other PVC tube or TPE tube.16. A closed sterile syringe configured for cell bioprocessing, the syringe comprising at least one sterile tube configured for fluid communication; wherein the sterile tube is made of polyvinyl chloride (PVC) or thermoplastic elastomer (TPE), respectively adapted to be welded to any other PVC tube or TPE tube. 17. A method for culturing cells, comprising seeding cells in a sterile bioprocessing system according to claim 1. 18. The method according to claim 17, wherein the cells comprise at least one of the following: tumor-infiltrating lymphocytes (TIL), T cells, CAR-T, engineered T cell receptor (TCR) cells, natural killer (NK) cells, CAR-NK, natural killer T (NKT) cells, and CAR-NKT cells and any combination thereof. 19. The method according to claim 17, further comprising avoiding the use of laminar flow hoods and / or any open processes. Claims 2 / 2 Page 3 CN 121358541 A Systems and Methods for Biomanufacturing Background of the Invention

[0001] The biomanufacturing of commercial-scale living pharmaceuticals must meet the highest regulatory requirements. Modern biomanufacturing processes include automated devices combined with manual processes, carried out in laminar flow hoods to provide sterile working areas, thereby allowing control of infectious splashes or aerosols generated by numerous microbial processes. Bacterial, mycoplasma, and other microbial contamination is one of the major problems in culturing cells intended for cell therapy.

[0002] Therefore, there has long been a need to culture cells in a completely closed system with minimal manual steps, particularly for cell and gene therapy (CGT). Summary of the Invention

[0003] According to some embodiments of the invention, a novel sterile bioprocessing system is provided, configured for cell culture. The system includes at least two closed sterile elements configured to contain fluid, wherein: • each of the elements includes at least one sterile tube configured for fluid communication; and • all of the sterile tubes are made of the same material and adapted to be welded to each other to allow fluid communication between the different sterile elements of the system; and thereby enabling the system to be a closed sterile system.

[0004] According to some embodiments, the sterile tube is at least partially flexible.

[0005] According to some embodiments, the material of the sterile tube includes polyvinyl chloride (PVC) or thermoplastic elastomer (TPE).

[0006] According to some embodiments, each of the sterile tubes initially has a sterile fluid connection to its corresponding element at its proximal end and is sealed at its distal end.

[0007] According to some embodiments, the sterile element is: a rigid container, a flexible container, a container that is at least partially flexible, or any combination thereof.

[0008] According to some embodiments, the system components include at least two of the following: • at least one culture container; as a non-limiting example, a G-Rex® culture container; • at least one container configured to contain tissues and / or cells; • at least one bottle configured to contain tissues and / or cells; • at least one syringe; • at least one filter bag; • at least one filtration device; • at least one waste bag; • at least one fluid bag; according to some embodiments, the fluid is a liquid culture medium for cell culture; • at least one manual pipette or an automated pipette; • at least one harvest element; as a non-limiting example, GatheRex®; • at least one washing element; as a non-limiting example, Lovo®; and • any combination thereof.

[0009] According to some embodiments, the cells include at least one of the following: tumor-infiltrating lymphocytes (TILs), T cells, CAR-T cells, engineered T cell receptor (TCR) cells, natural killer (NK) cells, CAR-NK cells, natural killer T (NKT) cells, and CAR-NKT cells and any combination thereof.

[0010] According to some embodiments, the closed aseptic system is configured to avoid the use of laminar flow hoods and / or any open processes.

[0011] According to some embodiments, a novel method is provided for closing a sterile bioprocessing system for cell culture according to any of the above embodiments; the method includes: · aseptically cutting two sterile tubes of the system, providing a cutting edge for each sterile tube; and · aseptically welding the cutting edges of the two sterile tubes to each other, the two sterile tubes being configured to allow sterile fluid communication between their respective elements.

[0012] According to some embodiments, a cutting step is also provided.

[0013] According to some embodiments, the method further includes aseptically cutting and aseptically sealing at least one sterile tube, said at least one sterile tube being configured to aseptically disconnect its fluid communication.

[0014] According to some embodiments, the method further includes aseptically cutting and aseptically welding any one of the connecting tubes of the system more than once to allow sequential fluid communication between its respective components and several different components of the system via their respective sterile tubes.

[0015] According to some embodiments, the aseptic cutting and aseptic welding steps are provided by an aseptic welding machine.

[0016] According to some embodiments, the method further includes avoiding the use of laminar flow hoods and / or any open processes.

[0017] According to some embodiments, a novel closed sterile bottle is provided, configured for cell bioprocessing, wherein: • the bottle includes at least one sterile tube configured for fluid communication; and • the sterile tube is made of polyvinyl chloride (PVC) or thermoplastic elastomer (TPE), respectively adapted to be welded to any other PVC tube or TPE tube.

[0018] According to some embodiments, a novel closed sterile syringe is provided, configured for cell bioprocessing, wherein: • the syringe includes at least one sterile tube configured for fluid communication

[200] ; and • the sterile tube is made of polyvinyl chloride (PVC) or thermoplastic elastomer (TPE), respectively adapted to be welded to any other PVC tube or TPE tube.

[0019] According to some embodiments, a novel method for culturing cells is provided, the novel method comprising inoculating cells in a sterile bioprocessing system according to any of the above embodiments.

[0020] According to some embodiments, the cells include at least one of the following: tumor-infiltrating lymphocytes (TILs), T cells, CAR-T cells, engineered T cell receptor (TCR) cells, natural killer (NK) cells, CAR-NK cells, natural killer T (NKT) cells, and CAR-NKT cells, and any combination thereof.

[0021] According to some embodiments, the method also includes avoiding the use of laminar flow hoods and / or any open processes. Brief Description of the Drawings

[0022] Subject matter considered to be the invention is particularly pointed out and expressly claimed in the concluding section of the specification. However, the invention (both in terms of organization and operation) and its objects, features and advantages can be best understood together with the following detailed description when read in conjunction with the accompanying drawings, in which: Figure 1 schematically illustrates a bioprocessing closed system according to some embodiments of the invention; Figure 2 illustrates a bioprocessing closed culture vessel according to some embodiments of the invention; Figure 3 illustrates a bioprocessing closed filter bag according to some embodiments of the invention; Figure 4A illustrates a welded connection of two fluid tubes according to some embodiments of the invention; Figure 4B illustrates a sealed and cut fluid tube according to some embodiments of the invention; Figure 5 schematically illustrates a bioprocessing closed bottle according to some embodiments of the invention; Figures 6A and 6B schematically illustrate and demonstrate a bioprocessing closed syringe according to some embodiments of the invention; and Figure 7 schematically illustrates a TIL manufacturing process flow chart.

[0023] Figure 8 schematically illustrates a process of TIL therapy according to one embodiment, which includes TIL manufacturing and administration.

[0024] It will be understood that, for simplicity and clarity, the elements shown in the drawings are not necessarily drawn to scale.For example, for clarity, certain dimensions of the elements may be enlarged relative to other elements. Furthermore, reference numerals may be repeated in the drawings where deemed appropriate to indicate corresponding or similar elements. Detailed Description of the Invention

[0025] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, those skilled in the art will understand that the invention can be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the invention.

[0026] As used herein, in one embodiment, the term “about” refers to ±10%. In another embodiment, the term “about” refers to ±9%. In another embodiment, the term “about” refers to ±9%. In another embodiment, the term “about” refers to ±8%. In another embodiment, the term “about” refers to ±7%. In another embodiment, the term “about” refers to ±6%. In another embodiment, the term “about” refers to ±5%. In another embodiment, the term “about” refers to ±4%. In another embodiment, the term “about” refers to ±3%. In another embodiment, the term “about” refers to ±2%. In another embodiment, the term “about” refers to ±1%.

[0027] The present invention provides a sterile bioprocessing system for cell culture. According to some embodiments, the sterile bioprocessing system is a closed system. According to some embodiments, the sterile bioprocessing system described herein allows for the production of TILs with a reduced risk of contamination and does not include non-sterile processes (such as open operation) or the use of a biosafety cabinet. Thus, the system and related methods described herein allow for the culture of TILs suitable for therapeutic use with less human intervention and exposure to the environment.

[0028] As used herein, the term “closed system” can encompass a system closed to the external environment. In some embodiments, after a tumor biopsy is added to the closed system, the system is not opened to the external environment until the cells are ready for use in a pharmaceutical product. At the end of this process, the cell therapy product is ready for infusion into a patient.

[0029] According to some embodiments, the terms “sterile” and “aseptic” are interchangeable and refer to elements and / or systems free from unwanted microorganisms. According to some embodiments, the components and / or systems are not open to the environment and therefore do not allow external contamination, i.e., do not allow microorganisms to enter the system.

[0030] According to some embodiments, the term "non-sterile" means, for example, that sterility cannot be guaranteed after exposure to the environment, which may potentially lead to the presence of microorganisms.

[0031] According to some embodiments, the phrases “laminar flow cabinet,” “tissue culture hood,” “laminar flow hood,” and “biosafety cabinet” are used interchangeably to refer to a partially enclosed workbench surface designed to prevent contamination by biological samples, semiconductor wafers, or any particulate-sensitive materials. Specification 3 / 13 pages 6 CN 121358541 A

[0032] According to some embodiments, the phrases “open process” or “open manipulation,” as used herein, can encompass processes or steps performed outside the enclosed system described herein. In some embodiments, such processes may expose cell cultures to the external environment.

[0033] Few instruments are specifically designed for cell and gene therapy (CGT) procedures. To provide advanced therapies to patients in the real world, it is necessary to reuse bioprocessing equipment rather than custom-designed technologies. Then, at the end of the process, the patient can receive treatment in a healthcare facility via a medical device.

[0034] According to some embodiments of the invention, new configurations are needed to overcome the technological gap between the bioprocessing worlds, which tend to use thermoplastic elastomer (TPE) tubing (e.g., C-Flex and Tygon) as the standard, compared to the PVC standard widely used in the medical device industry.

[0035] Tube welding is a key method for aseptic work required in closed system manufacturing methods. TPE welding and PVC welding are two different techniques, using their own unique instruments.

[0036] On the one hand, sterile bottles with transfer caps and pre-assembled syringes with tubing and stopcocks for liquid management are only commercially available with TPE tubing. On the other hand, culture containers (G-Rex) and downstream instruments (such as LOVO) are only commercially available with PVC tubing.

[0037] Therefore, incorporating PVC tubing for bioprocessing products is a novel solution.

[0038] According to some embodiments of the invention, and as shown, for example, in FIG1, a novel aseptic bioprocessing system

[100] is provided, which is configured for culturing cells; the system includes at least two closed and sterile elements [110, 121, 122, 141, 142] configured to contain fluid, wherein: • each of the sterile elements includes at least one sterile tube

[200] configured for fluid communication; and • all of the sterile tubes are made of the same material and are adapted to be welded to each other

[210] , as shown in FIG4A for tubes [200A, 200B], to allow fluid communication between the different sterile elements of the system; and thereby enabling the system to be a closed sterile system.

[0039] According to some embodiments of the invention, and as shown, for example, in FIG5, a novel closed sterile bottle

[140] is provided, which is configured for cellular bioprocessing; wherein: • the bottle includes at least one sterile tube

[200] configured for fluid communication; and • the sterile tube is made of a material suitable for welding any one of the sterile communication tubes

[200] of the system

[100] to allow fluid communication between the bottle and any sterile element of the system

[100] ; and thereby enabling the system to be a sterile closed system.

[0040] According to some embodiments of the invention, and as shown, for example, in Figures 6A and 6B, a novel closed sterile syringe

[180] is provided, which is configured for cellular bioprocessing; wherein: • the syringe includes at least one sterile tube

[200] configured for fluid communication; and • the sterile tube is made of a material suitable for welding any one of the sterile communication tubes

[200] of the system

[100] to allow fluid communication between the syringe and any sterile element of the system

[100] ; and thereby enabling the system to be a closed sterile system.

[0041] According to some embodiments, and as shown in FIG1, the system components include at least two of the following: • At least one culture container [121, 122]; as a non-limiting example, a G-rex® culture container; • At least one container [110, 141, 142] configured to contain tissues and / or cells; • At least one bottle [140, 141, 142] configured to contain tissues and / or cells; • At least one syringe

[180] ; • At least one filter bag

[130] ; • At least one filtration device; • At least one waste bag; • At least one fluid bag [151, 152]; according to some embodiments, the fluid is a liquid culture medium for cell culture; • At least one manual pipette and / or an automated pipette; • At least one harvesting element

[160] ; as a non-limiting example, a GatheRex® device; • At least one washing element

[170] ; as a non-limiting example, a Lovo® device; and • any combination thereof.

[0042] According to some embodiments, and as shown in FIG3, for a non-limiting example of the filter bag

[130] , the sterile tube

[200] is in direct communication with the fluid contained in the element.

[0043] According to some embodiments, and as shown in FIG5, for a non-limiting example of the bottle

[140] , the sterile tube

[200] is not in direct communication with the fluid contained in the element.According to such an embodiment, element

[140] further includes at least one of the following for allowing sterile fluid communication: a sterile connector

[410] ; a sterile cap

[420] ; a sterile sealer

[430] ; a sterile tube

[400] that may be made of different materials; the material being a material unsuitable for welding to a closed sterile tube

[200] into the system; and any combination thereof.

[0044] According to some embodiments, cell culture includes cell expansion. According to some embodiments, the cells include at least one of the following: tumor-infiltrating lymphocytes (TILs), T cells, CAR-T, engineered T cell receptor (TCR) T cells, natural killer (NK) cells, CAR-NK, natural killer T (NKT) cells, and CAR-NKT cells, and any combination thereof. According to some embodiments, the cultured cells include TILs. According to some embodiments, the cultured cells include T cells. According to some embodiments, the cultured cells include CAR-T cells. According to some embodiments, the cultured cells include natural killer (NK) cells. According to some embodiments, the cultured cells include T cell receptor (TCR) cells.

[0045] TILs are white blood cells that leave the bloodstream and migrate toward the tumor and attempt to attack it. According to one embodiment, TILs include T cells. According to one embodiment, TILs include B cells. According to one embodiment, TILs include natural killer (NK) cells. According to one embodiment, TILs include macrophages. According to one embodiment, TILs include neutrophils. According to one embodiment, TILs include dendritic cells. According to one embodiment, TILs include mast cells. According to one embodiment, TILs include eosinophils. According to one embodiment, TILs include basophils. According to one embodiment, TILs include plasma cells. According to one embodiment, TILs include mature dendritic cells. According to one embodiment, TILs include antigen-presenting cells (APCs).

[0046] According to one embodiment, TILs include CD45+ cells. According to one embodiment, TILs include CD4+ cells. According to one embodiment, TILs include CD8+ cells. According to one embodiment, TILs include CD163+ cells. According to one embodiment, TILs include CD20+ cells. According to one embodiment, the TIL comprises CD3+ cells. According to one embodiment, the TIL comprises CD138+ cells. According to one embodiment, the TIL comprises CD163+ cells. According to one embodiment, the TIL comprises CD56+ cells. According to one embodiment, the TIL comprises FoxP3+ cells. According to one embodiment, the TIL comprises DC-LAMP+ cells. According to one embodiment, the TIL comprises CD28+ cells. According to one embodiment, the TIL comprises CD69+ cells.

[0047] According to one embodiment, the TIL comprises a combination of different types of lymphocytes.Those skilled in the art will understand that different combinations of lymphocytes have different tumor-killing abilities. According to embodiments, specific combinations of lymphocytes that are particularly effective at killing tumors are disclosed herein.

[0048] According to embodiments, one or more TILs are modified to express a T-cell receptor (TCR) that is antigen-specific to a cancer antigen (e.g., any of the cancer antigens described herein). According to embodiments, the TCR contains antigen specificity against a melanoma antigen. According to embodiments, the antigen includes g100 or MART-1. According to embodiments, one or more TILs are modified to express a chimeric antigen receptor (CAR) that is antigen-specific to a cancer antigen (e.g., any of the cancer antigens described herein). According to embodiments, one or more TILs are modified to express cell growth factors that promote the growth and activation of one or more TILs. According to embodiments, the growth factors include T-cell growth factors, IL-2, IL-7, IL-12, IL-15, or IL-18. According to embodiments, the modified TILs express T-cell growth factors at high levels. The coding sequence for T cell growth factor is readily available in the art, as are the promoters, and the operative linking of the promoter to the coding sequence for T cell growth factor promotes high-level expression. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Methods for modifying TILs are known in the art. For example, TILs can be transduced to express TCRs that are antigen-specific to cancer antigens using transduction techniques described in Morgan et al., Science 314(5796):126-9(2006) and Johnson et al., Blood 114:535-46(2009).

[0049] According to some embodiments, the sterile tube is at least partially flexible.

[0050] According to some embodiments, the material of the sterile tube includes one of the following: polyvinyl chloride (PVC), thermoplastic elastomer (TPE).

[0051] According to some embodiments, the sterile element is: a rigid container, a flexible container, a container that is at least partially flexible, and any combination thereof.

[0052] According to some embodiments, the fluid is primarily a liquid, and fluid movement between different components of the system is provided via direct or indirect communication through at least one of the following: a pump, a syringe, gravity, a manual or automatic pipette, a siphon, the communication vessels law, and any combination thereof.

[0053] According to some embodiments, and as shown in FIG2, for a non-limiting example of the culture container

[120] , each of the sterile tubes

[200] initially has a sterile fluid connection to its corresponding component

[120] at its proximal end

[201] and is sealed at its distal end

[202] .

[0054] According to some embodiments, and as shown in FIG2 and FIG5, for a non-limiting example of the culture container

[120] , at least one component of the system may be in non-direct communication with a non-sterile tube or any other non-sterile component or a non-sterile environment (only the environment is shown); for example, for gas communication. Materials are not suitable for welding to the closed sterile tubes

[200] of the system. According to such an embodiment, fluid communication is achieved via a sterile filter

[310] and optional sterile tubing

[400] , thereby maintaining the system aseptic. According to such an embodiment, the non-sterile tubing (not shown) may be made of different materials.

[0055] According to some embodiments of the invention, a method is provided for enclosing a sterile bioprocessing system for cell culture according to any of the above embodiments; the method comprising: · sterilely cutting two of the sterile tubings of the system, providing cutting edges [203A, 203B] for each sterile tubing; and · sterilely welding the cutting edges [203A, 203B] of the two sterile tubings [200A, 200B] to each other

[210] , as shown in FIG4A, configured to allow fluid communication between their respective elements, and thus avoid any non-sterile connection with the elements of the system, including avoiding the need for the use of a laminar flow hood.

[0056] According to some embodiments, the method further includes aseptically sealing a portion of the sterile tube [200C] along the length of one of the sterile tubes [200C], and then cutting

[220] the sterile tube [200C], as shown in FIG4B, the sterile tube [200C] being configured to aseptically disconnect its fluid communication, wherein the cutting and disconnection is such that: • only one sterile element remains closed; or • both previously connected sterile elements remain closed.

[0057] According to some embodiments, the sealed and cut sterile tube may be a connecting tube of two previous sterile tubes [200A, 200B], as shown in FIG4A, which were previously welded together for fluid communication between their respective sterile elements (elements not shown in FIG4A).According to such an embodiment, after sealing, a cut can be provided such that: • the two separate sterile tubes remain sealed, and thus their respective elements remain closed and sterile; or • only one of the two separate (originally sterile) tubes remains sealed, and thus only its respective element remains sterile, since the sterile system no longer requires the other (originally sterile) element.

[0058] According to some embodiments, the method further includes more than one aseptic cut and aseptic welding of any one of the sterile tubes of the system to allow sequential fluid communication between its respective element and several different elements of the system via their respective sterile tubes, i.e., repeated welding and connection of a particular sterile tube and its respective element, each time welding and connecting to a different element of the system.

[0059] According to some embodiments, the aseptic cutting and aseptic welding steps are provided via an aseptic welding machine; as a non-limiting example, the CompoDock® sterile tube connection system is designed for connection of sterile medical PVC tubes.

[0060] According to some embodiments of the invention, a method is provided for using a bioprocessing closed sterile system

[100] as shown in FIG. 1 according to any of the above embodiments.

[0061] According to some embodiments, for example for the TIL manufacturing process, all procedures are performed using a closed aseptic operation while G-Rex [121, 122] are placed in an incubator.

[0062] According to some embodiments of the invention, a method for culturing cells using a closed aseptic bioprocessing system

[100] as described herein is provided.

[0063] According to some embodiments of the invention, a method for cell expansion using a closed aseptic bioprocessing system

[100] as described herein is provided. According to some embodiments, the method for culturing cells includes seeding cells in the closed aseptic bioprocessing system

[100] as described herein.

[0064] According to some embodiments of the invention, and as shown, for example, in FIG8, a process for a TIL therapy

[800] is provided, the process comprising: obtaining a biopsy of a freshly excised tumor

[802] from a patient

[801] ; processing the tumor sample into fragments

[803] ; placing the fragments in a closed, sterile bioprocessing system; expanding cells using a pre-Rapid Expansion Protocol (pre-REP)

[804] ; harvesting cells from the pre-REP culture; optionally cryopreserving the cells

[805] ; expanding the cells using a Rapid Expansion Protocol (REP)

[806] ; harvesting cells from the REP culture; formulating the harvested cells into a product

[807] ; preparing a final pharmaceutical TIL product for quality control testing

[808] ; and administering the TIL to the patient

[809] .

[0065] According to some embodiments, a method for culturing cells includes: (a) placing fragments of a tumor processed from a resected tumor from a patient into a closed, sterile bioprocessing system described herein; (b) expanding the cells using a pre-rapid expansion protocol (pre-REP); (c) harvesting the cells from the pre-REP culture; (d) expanding the cells using a rapid expansion protocol (REP); and (e) harvesting the cells from the REP culture to obtain TILs.

[0066] According to some embodiments, the method for culturing cells further includes: cryopreserving the cells. According to some embodiments, cryopreserving the cells is performed after the step of expanding the cells using the pre-rapid expansion protocol.

[0067] According to some embodiments, the method for culturing cells further includes: filtering the cells. According to some embodiments, filtering the cells is performed after the step of expanding the cells using the pre-rapid expansion protocol.

[0068] According to some embodiments, the method for culturing cells further includes: supplementing the culture medium with IL-2.

[0069] According to some embodiments, the cultured cells include at least one of the following: tumor-infiltrating lymphocytes (TILs), T cells, CAR-T cells, T cell receptor (TCR) cells, and natural killer (NK) cells. According to some embodiments, the cultured cells include TILs. According to some embodiments, the cultured cells include T cells. According to some embodiments, the cultured cells include CAR-T cells. According to some embodiments, the cultured cells include natural killer (NK) cells. According to some embodiments, the cultured cells include T cell receptor (TCR) cells.

[0070] According to some embodiments, TILs include at least one of the following: T cells, B cells, natural killer (NK) cells, dendritic cells, plasma cells, antigen-presenting cells (APCs), CD4+, CD8+, CD163+, CD20+, CD3+, CD138+, CD163+, CD56+, CD28+, CD69+, FoxP3+, DC-LAMP+ cells, and any combination thereof.

[0071] According to some embodiments, a cell seeding step is provided. Tumor fragments are seeded according to a pre-rapid expansion protocol (REP), i.e., day 0. The tumor fragments suspended in the culture medium are collected into a custom-made 500 ml collection bottle

[141] in a biomass, the collection bottle having a transfer cap with PVC tubing

[200] . The bottle is then connected to the port of a culture vessel (G-Rex®)

[121] via welded tubing

[200] to allow the tumor fragments to be transferred from the bottle to the G-Rex

[121] in a closed system.

[0072] According to an embodiment, TILs are extracted from the tissue. According to an embodiment, TILs are extracted from the excised tumor. According to an embodiment, TILs are extracted from the tumor fragments.According to the implementation plan, TILs are extracted from unablated and untreated tissue. According to the implementation plan, TILs are extracted from biopsies. According to the implementation plan, the biopsies include tumor stroma. According to the implementation plan, the biopsies include tumor. According to the implementation plan, the biopsies include ablated tissue. According to the implementation plan, the biopsies include ablated tumor. According to the implementation plan, the biopsies include resected tumor. According to the implementation plan, the biopsies include the area surrounding the tumor. According to the implementation plan, the biopsies include necrotic tissue. According to the implementation plan, the biopsies include any combination of tumor, tumor stroma, or tissue surrounding the tumor. According to the implementation plan, the biopsies include tumor tissue without normal tissue or necrotic areas.

[0073] According to some embodiments, the tumor is selected from melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmacytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colorectal cancer, kidney cancer, renal cell carcinoma, uterine cancer, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, testicular cancer, gastric cancer, esophageal cancer, multiple myeloma, liver cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL), or any combination thereof.

[0074] According to some embodiments, steps for adding culture medium to the culture vessel are provided. IL-2 addition can be provided in the following pre-REP days: 5+1, 9+1, and 12+1. According to some embodiments, the desired volume of IL-2 is aseptically extracted from a 100 mL bag

[151] using a custom-made sampling syringe

[180] . According to some embodiments, IL-2 is then added to G-Rex by welding the tube

[200] of syringe

[180] to the port of G-Rex

[121] via its port

[200] .

[0075] According to some embodiments, a sampling step is provided. Sampling can be provided at pre-REP days 9+1, 12+1, and REP days 11+1. Cells or culture medium are sampled using a custom-made syringe

[180] aseptically welded to G-Rex [121, 121] via the tube

[200] .

[0076] According to some embodiments, a pre-REP harvesting step is provided. Pre-REP culture harvesting comprises two steps: • first, depletion of the culture medium from G-Rex, and • then removal of tumor debris from single cells.

[0077] According to some embodiments, the culture medium is depleted by extracting the upper layer of the culture medium into a waste bag (e.g., using a GatheRex® instruction manual, page 8 / 13, 11 CN 121358541 A pump).

[0078] According to some embodiments, tumor debris is then separated from single cells using a filter bag

[130] , wherein a 40 pm screen filter separates the two sides of the filter bag.

[0079] According to some embodiments, the filter bag

[130] is welded to G-Rex via a tube

[200] , and the contents of G-Rex® are then transferred into the bag

[130] using a GatheRex® pump and then filtered through a screen, allowing only single cells to pass through while disallowing any tumor debris. The single cells are then collected in a 3L bag “cell master mix bag”

[110] .

[0080] According to some embodiments, a step of REP culture inoculation is provided. For REP culture inoculation, feeder cells and culture medium are added to a “cell master mix bag”

[110] containing single cells collected from the pre-REP process. The feeder cells are pre-packaged in a bag with PVC tubing and are added to the “cell master mix bag” by welding the bag

[110] and gravity. The culture medium packaged in a bag with PVC tubing (either received in a bag or pre-packaged in a suitable bag by the manufacturer's team) is added to the "cell master mix bag," which is done by welding two bags via tubing

[200] and measuring the precise volume using hooked weights. According to some embodiments, the REP culture is inoculated by welding the "cell master mix bag" to the port of each G-Rex500cs via tubing

[200] and transferring the precise volume of the mix using hooked weights.

[0081] According to some embodiments, another step of culture medium addition is provided. Culture medium addition can be provided at REP days 5 / 6, 7+1, and 11+1. Culture medium supplemented with IL-2 is added during the REP phase. The desired volume of IL-2 is then aseptically extracted from a 100 mL bag into a custom-made sampling syringe

[180] and subsequently added to the culture medium bag (received in a bag or pre-packaged in a suitable bag by the manufacturer's team). The culture medium is added to G-Rex®.

[0082] According to some embodiments, a cell harvesting step is provided. REP culture harvesting comprises two steps: • First, the culture medium is depleted, and • then, the cells are harvested.

[0083] According to some embodiments, the upper layer of the culture medium (cell-free) is removed from each G-Rex500cs

[122] into a waste bag using a GatheRex® pump, the waste bag being welded to a port of the G-Rex500cs via a tube

[200] . The remaining cell-containing culture medium is then collected into a collection bag or bottle using a GatheRex® pump, the collection bag or bottle being welded to another port of the G-Rex500cs via another tube

[200] .

[0084] According to some embodiments, a final preparation step is provided.A bag containing cells collected from all G-Rex500 containers is welded to a LOVO® sterile kit

[170] via a tube

[200] . The cells are washed, formulated, and packaged through a LOVO® system operating in a closed system.

[0085] According to some embodiments, any one or a combination of the embodiments mentioned above are configured to avoid or at least minimize the use of a laminar flow hood.

[0086] Example: TIL Manufacturing Process and Process Control Purpose: To demonstrate the ability to culture TILs in a closed system described in detail herein that meet all acceptance criteria for TILs intended to be used as pharmaceutical products.

[0087] Method: TILs are manufactured in a continuous process, beginning with a tumor biopsy of a patient and ending with a product configured for delivery to a patient via infusion for autologous therapy. All processing is performed within a closed system

[100] as disclosed in any of the embodiments mentioned above.

[0088] The treatment begins with a 14+2 day rapid amplification protocol pre-REP process, which involves isolating TILs from the patient's tumor and establishing a primary culture of 5-50 × 10⁶ TILs. Then, during the REP process, the TILs are further amplified for another 14+2 days to produce ≥10 × 10⁹ TILs for subsequent re-infusion back into the patient. The manufacturing process

[700] includes manufacturing steps [701-Instruction Manual 9 / 13 pages 12 CN 121358541 A 705], as described and illustrated in the following paragraphs.

[0089] Pre-REP Culture Inoculation (pre-REP Day 0)

[701] Tumor samples are excised in a hospital, collected into sterile tubes containing a static preservation solution of SPS-1® (UW solution) containing 50 μg / mL gentamicin, and transferred to a manufacturing facility at 2°C-8°C, where they are kept refrigerated until use. Within 48 hours of resection, tumor tissue processing began. The tumor tissue was first washed four (4) times, the first three washes being filtered through a Steriflip filter tube using phosphate-buffered saline (PBS) supplemented with 50 μg / ml gentamicin, and the last wash using 50 μg / ml gentamicin. The tumor sample was then dissected into fragments less than or equal to 8 mm3 using a sharp scalpel. Approximately 100–400 fragments were placed in 250 mL of PRIME-XV T cytochemical composition determination medium (CDM) supplemented with 3,000 IU / mL IL-2 in up to two units of G-RexlOOcs

[121] (closed system; 100–200 fragments per unit). The G-RexlOOcs flasks were then incubated in a humidified incubator at 37°C and 5% CO2.

[0090] IL-2 supplementation (pre-REP days 5±1, 9±1, 12±1)

[702] At pre-REP days 5+1, 9+1, and 12+1, the culture medium was supplemented with 3,000 IU / ml IL-2, removed from a sterile bag, and connected by welding a syringe

[180] to a G-Rex

[121] without opening the system (as shown in Figure 1).

[0091] Pre-REP culture harvest (pre-REP days 9±1, 12±1, or 14±2) and REP culture inoculation / REP day 0

[703] At pre-REP days 9+1 and 12+1, the likelihood of early harvest was assessed based on lactate concentration, cell number, viability, and identity. First, the lactate concentration in each G-Rex 100cs

[121] was measured. If the lactate concentration in one of the G-Rex100cs containers is equal to or greater than 2 mM, cell counting is performed on each G-Rex100cs. If the total cell count (two G-Rex100cs together) is equal to or greater than 35 × 10⁶, with at least 70% viability, cell identity testing using flow cytometry is performed. If the percentage of CD45+ cells in each G-Rex100cs is at least 70%, pre-REP culture is harvested within 24 hours. Cells are harvested at pre-REP day 14+2, regardless of the standard.

[0092] Pre-REP culture harvesting is performed using a closed system

[100] . First, the upper layer of cell-free culture medium (approximately 150 mL) is removed from the G-Rex100cs

[121] using a Gatherex pump. Then, the lower layer of culture medium containing cells and tumor debris is collected into a cell collection bottle via a standard infusion device, wherein a 200 μm screen filter separates the two sides of the standard infusion device. The contents of the G-Rex100cs were transferred and filtered through a sieve

[130] , allowing only cells without tumor debris to pass through. The harvesting process was repeated for the second G-Rex100cs, harvested into the same cell collection bottle to pool the TILs from the two G-Rex100cs. The pooled collection bottles were sampled and tested for cell count, viability, and identity. The acceptance criteria for REP were a cell count of at least 7 × 10⁶, viability of at least 70%, and CD45+ cells of at least 70%. At this point, the pre-REP TILs were directly seeded for rapid expansion (REP).

[0093] Cells were seeded in 625 mL of culture medium at a cell density of 4–10 × 10⁶ TILs / G-Rex500 using a closed system

[100] with one (1) to four (4) G-Rex500cs containers

[122] .The culture medium contained PRIME-XV T cell CDM supplemented with 30 ng / mL anti-CD3 antibody (OKT-3) and 3,000 IU / mL IL-2.

[0094] Culture medium addition (REP days 5±1, 7±1, 11±1)

[704] At REP day 5+1, 625 mL of PRIME XV CDM culture medium supplemented with 3,000 IU / mL IL-2 was added to each G-Rex500cs. At REP days 7+1 and 11+1, 1,875 mL of culture medium supplemented with 3,000 IU / mL IL-2 was added to each G-Rex500cs, for a total final volume of 5 L. All culture medium additions were performed in a closed system.

[0095] TIL Harvesting and Final Preparation (REP days 14±1)

[705] The top layer of cell-free culture medium (approximately 4.5 L) was removed from each G-Rex500cs using a Gatherex pump. The remaining cell-containing culture medium was then collected from all G-Rex500cs containers and pooled into a single collection bag. (Instruction manual 10 / 13 pages 13 CN 121358541 A) Next, the collection bag was welded to the LOVO aseptic kit. LOVO is an automated and fully enclosed cell processing device. Cells were washed and prepared via the LOVO system in Plasma-Lyte containing 4% (w / v) human serum albumin (HSA). The final product was packaged in single 500 mL bags at a volume of 100–500 mL and a cell concentration of 100–200 × 10⁶ cells / mL.

[0096] Results: TILs were prepared from tumors of three patients (designated ER03, ER05, and ER06) in a closed system in the R&D laboratory.

[0097] Table 1 shows the engineering run data for the three TIL batches. Table 1: Engineering Run Data:

[0098] Table 2 shows the test results for the three TIL batches produced in a closed system located in a standard R&D laboratory. It demonstrates the successful manufacture of sterile TIL products using a closed system. Safety tests included bacterial endotoxin, sterility, and mycoplasma tests, all of which confirmed that the cultures were uncontaminated.

[0099] Table 2: Release test instructions for TILs produced in the R&D laboratory, 11 / 13 pages, 14 CN 121358541 A

[0100] TILs were prepared from three qualified batches (referred to as 100-pp-QB201DP-IL001090, 100-PP-QB201DP-IL00052 and 100-PP-QB201DP-IL00058) in a closed system of a cleanroom in a GMP facility.

[0101] Table 3 shows the qualification data for the three TILs.

[0102] Table 3: Qualified Batch (QB) Data:

[0103] Safety tests included bacterial endotoxin, sterility, and mycoplasma tests, all of which confirmed that the cultures were uncontaminated.

[0104] Table 4 shows the test results for three TIL batches produced in a closed system in a clean room located in a GMP facility. It demonstrates the successful manufacture of sterile TIL products using a closed system. Safety tests included bacterial endotoxin, sterility, and mycoplasma tests, all of which confirmed that the cultures were uncontaminated.

[0105] Table 4: Release Tests for TILs Produced in a Clean Room of a GMP Facility:

[0106] Conclusion: The examples provided herein demonstrate the successful preparation of TILs from tumors collected from six patients, three of whom were prepared in an R&D laboratory (engineered operation) and three of whom were prepared in a clean room (qualified batch). The test results met all acceptance criteria for TILs as pharmaceutical products. Advantageously, the closed system described herein allows for the production of TILs with reduced risk of contamination and does not involve the use of non-aseptic processes or biosafety cabinets. Therefore, the systems and related methods described herein allow for the cultivation of TILs suitable for therapeutic use with less human intervention and at a reduced cost.

[0107] Different embodiments are disclosed herein. Features of certain embodiments may be combined with features of other embodiments; thus, certain embodiments may be combinations of features of multiple embodiments.

[0108] While certain features of the invention have been described and illustrated herein, many modifications, substitutions, alterations, and equivalents will now occur to those skilled in the art. Therefore, it will be understood that the appended claims are intended to cover all such modifications and alterations falling within the true spirit of the invention. Instruction manual, page 13 / 13, 16 CN 121358541 A, Figure 1; Instruction manual, Figure 1 / 8, page 17 CN 121358541 A, Figure 2, Figure 3, Figure 4A; Instruction manual, Figure 2 / 8, page 18 CN 121358541 A, Figure 4B; Instruction manual, Figure 3 / 8, page 19 CN 121358541 A, Figure 5; Instruction manual, Figure 4 / 8, page 20 CN 121358541 A, Figure 6A; Instruction manual, Figure 5 / 8, page 21 CN 121358541 A, Figure 6B; Instruction manual, Figure 6 / 8, page 22 CN 121358541 A, Figure 7; Instruction manual, Figure 7 / 8, page 23 CN 121358541 A, Figure 8; Instruction manual, Figure 8 / 8, page 24 CN 121358541 A.

Claims

1. A sterile bioprocessing system for cell culturing, the system comprising at least two closed sterile elements configured to hold fluids, wherein: each of the elements comprises at least one sterile tube configured for fluid communication; and all of the sterile tubes are made of the same material, suitable for welding to each other, to allow fluid communication between different sterile elements of the system; and thereby enable the system to be a closed sterile system.

2. The system of claim 1, wherein the sterile tubes are at least partially flexible.

3. The system of claim 1, wherein the material of the sterile tubes comprises polyvinyl chloride (PVC) or thermoplastic elastomer (TPE).

4. The system of claim 1, wherein each of the sterile tubes is initially provided with a sterile fluid connection to its respective element at its proximal end, and is sealed at its distal end.

5. The system of claim 1, wherein the sterile elements are selected from the group consisting of: rigid containers, flexible containers, at least partially flexible containers, and any combination thereof.

6. The system of claim 1, wherein the elements of the system comprise at least two of: at least one culture container; at least one container configured to hold tissue and / or cells; at least one bottle configured to hold tissue and / or cells; at least one syringe; at least one filter bag; at least one filtration device; at least one waste bag; at least one fluid bag; at least one manual or automatic pipette; at least one harvesting element; at least one washing element; and any combination thereof.

7. The system of claim 1, wherein the cells comprise at least one of: tumor infiltrating lymphocytes (TILs), T cells, CAR-T, engineered T cell receptor (TCR) cells, natural killer (NK) cells, CAR-NK, natural killer T (NKT) cells, and CAR-NKT cells, and any combination thereof.

8. The system of claim 1, wherein the closed sterile system is configured to avoid the use of laminar flow hoods and / or any open processes.

9. A method for closing a sterile bioprocessing system for cell culturing according to claim 1; the method comprising: sterile cutting of two sterile tubes of the system, providing each sterile tube with a cutting edge; and sterile welding of the cutting edges of the two sterile tubes to each other, the two sterile tubes being configured to allow sterile fluid communication between their respective elements.

10. The method of claim 9, wherein the step of cutting is provided simultaneously.

11. The method of claim 9, further comprising sterile cutting and sterile sealing of at least one sterile tube configured to be sterilely disconnected from its fluid communication.

12. The method of claim 9, further comprising sterile cutting and sterile welding of any of the communicating tubes of the system more than once, to allow sequential fluid communication between their respective elements and several different elements of the system, via their respective sterile tubes.

13. The method of claim 9, wherein the steps of aseptic cutting and aseptic welding are provided via an aseptic welder.

14. The method of claim 9, further comprising avoiding the use of a laminar flow hood and / or any open process.

15. A closed aseptic bottle configured for cell bioprocessing, the bottle comprising at least one aseptic tube configured for fluid communication; wherein the aseptic tube is made of polyvinyl chloride (PVC) or thermoplastic elastomer (TPE), suitable for welding to any other PVC tube or TPE tube, respectively.

16. A closed aseptic syringe configured for cell bioprocessing, the syringe comprising at least one aseptic tube configured for fluid communication; wherein the aseptic tube is made of polyvinyl chloride (PVC) or thermoplastic elastomer (TPE), suitable for welding to any other PVC tube or TPE tube, respectively.

17. A method for culturing cells, comprising inoculating cells in the aseptic bioprocessing system of claim 1.

18. The method of claim 17, wherein the cells comprise at least one of the following: tumor infiltrating lymphocytes (TILs), T cells, CAR-T, engineered T cell receptor (TCR) cells, natural killer (NK) cells, CAR-NK, natural killer T (NKT) cells, and CAR-NKT cells, and any combination thereof.

19. The method of claim 17, further comprising avoiding the use of a laminar flow hood and / or any open process.

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