Flexible facility configuration for therapeutic product manufacture

Abstract

Disclosed herein are flexible facilities for manufacturing at least one therapeutic product, wherein the flexible facilities include reconfigurable single use areas and stainless steel areas that may optionally be separated by partitions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Priority is claimed to U.S. Provisional Patent Application No. 63 / 356,361, filed on June 28, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to flexible manufacturing facilities that enable the production of therapeutic products, such as drug substances, cell therapies, etc., the manufacture of which may require different equipment (e.g., single-use systems and stainless steel equipment) in sequential or simultaneous campaigns. In some embodiments, the flexible manufacturing facility combines a reconfigurable single-use area with a conventional stainless steel area, wherein the reconfigurable single-use area and the stainless steel area are optionally separated by a divider. Background Art

[0004] Biologics are used worldwide in a variety of therapeutic areas, ranging from oncology to cardiology and beyond. However, the costs associated with manufacturing biologics are high due to their complex production methods, and the single-modality and / or single-product focus of typical biomanufacturing facilities. The manufacture of biologics typically utilizes multi-step processes that include cell culture scale-up, large-scale production cell culture, and purification of the desired biologic after cell harvest; these steps are often specific to a given modality or product. In recent years, the number of biologics manufactured has increased significantly due to the multitude of new modalities (e.g., monoclonal antibodies, Due to the availability of new biologics (e.g., genomics, genomics, cytotoxicity, and / or cytotoxicity), manufacturing complexity has increased. While improvements in all aspects of production have reduced the cost of commercial-scale manufacturing for many biologics, the upfront costs associated with building and maintaining biomanufacturing facilities (including upfront costs associated with changing or expanding the work-life of modalities and / or product types manufactured in a given facility) can still be prohibitive. Therefore, there is a need in the art for new, reconfigurable biomanufacturing facility configurations that enable multiple products and / or modalities to be manufactured at a single facility throughout its life at reduced cost and in shorter time to facilitate long-term supply planning.

[0005] Biological products are usually produced in an industry standard platform known as "fed-batch". The fed-batch process uses a partially open system, wherein during cell culture, one or more nutrient boluses are fed to the bioreactor. The fed-batch production process is often performed in large stainless steel bioreactors, and most of the costs associated with biopharmaceutical manufacturing can be attributed to the capital investment required for building and maintaining infrastructure, which is required for the production and purification of specific biological products. Demonstration, the construction of fed-batch facilities often requires a capital investment of approximately $1.5 billion and approximately 6 years of licensing. In addition, the conventional structure for manufacturing biological products (such as monoclonal antibodies and microbial products) does not allow efficient use of facility space, for example, due to the need for large stainless steel tanks and a large number of stainless steel hard pipes to support the fed-batch process. In addition, the layout of conventional biomanufacturing facilities (often including a large number of hard pipes and considerable equipment) may limit its ability to reconfigure or expand to manufacture new product lines at any time. This limitation is made more complicated by the diverse platforms used to manufacture biological products, which often have product specificity and lack interchangeability.

[0006] An ideal biomanufacturing facility would be inexpensive to build and able to be rapidly expanded and reconfigured to quickly and cost-effectively integrate new products and processes. In addition, it would be advantageous for such a manufacturing facility to be able to maintain and improve upon the high quality levels required by current good manufacturing practices (cGMP) for drug manufacturing (e.g., in compliance with 21 CFR Part 11).

[0007] Single-use production technology is becoming more and more popular in biomanufacturing facilities, and these single-use production technologies simplify equipment and facility design relative to the reusable system that needs to be cleaned and disinfected between each production batch. Single-use parts do not need to be cleaned after each batch production, and are cost-effective means for manufacturing biological products. However, the manufacturing requirements of some biological products have exceeded the current volume or other capacity that single-use technology can reach. Therefore, for some biological products with high quantity requirements, reusable large-volume stainless steel containers cannot be avoided. Such containers must be connected to other unit operations, culture medium and buffer supply, water supply and local cleaning and local steam systems by stainless steel pipes. As mentioned above, the manufacture and installation of these stainless steel containers and all utilities supporting them are expensive, and require a considerable lead time for design and manufacture.

[0008] Therefore, there is a need for a flexible manufacturing facility that allows users to efficiently deploy common resources between product lines and can be easily modified to accommodate new manufacturing processes or new manufacturing lines and reduce facility downtime. Summary of the invention

[0009] The present disclosure provides a flexible manufacturing facility for producing biological products, wherein the flexible manufacturing facility can be customized according to the specific needs of one or more biological products (e.g., one or more biological products of different modalities). This novel facility arrangement utilizes reconfigurable single-use areas and stainless steel areas that can be optionally isolated by dividers.

[0010] Disclosed herein is a flexible facility for manufacturing at least one therapeutic product, the flexible facility comprising:

[0011] A reconfigurable single-use area comprising at least one single-use bioreactor, wherein one or more of the at least one or more single-use bioreactors has a volume of at least 1000 L (e.g., at least 2000 L, greater than 2000 L); and

[0012] A stainless steel area, comprising at least one stainless steel bioreactor, wherein each stainless steel bioreactor in the stainless steel area has a volume greater than 5000 L,

[0013] Wherein, one or more of the at least one stainless steel bioreactor is fluidly connected to one or more of the at least one single-use bioreactor.

[0014] In some embodiments, one or more of the at least one single use bioreactor has a volume of 1000 L. In some embodiments, a plurality of the at least one single use bioreactor has a volume of 1000 L.

[0015] In some embodiments, one or more of the at least one single-use bioreactor has a volume of at least 2000 L. In some embodiments, one or more of the at least one single-use bioreactor has a volume of 2000 L. In some embodiments, a plurality of the at least one single-use bioreactor has a volume of 2000 L.

[0016] In some embodiments, one or more of the at least one single-use bioreactor has a volume of at least 3000 L. In some embodiments, one or more of the at least one single-use bioreactor has a volume of 3000 L. In some embodiments, a plurality of the at least one single-use bioreactor has a volume of 3000 L.

[0017] In some embodiments, one or more of the at least one single-use bioreactor has a volume of at least 5000 L. In some embodiments, one or more of the at least one single-use bioreactor has a volume of 5000 L. In some embodiments, a plurality of the at least one single-use bioreactor has a volume of 5000 L.

[0018] In some embodiments, one or more of the at least one single-use bioreactor has a volume of 1000 L, 2000 L, 3000 L, or 5000 L. In some embodiments, a plurality of the at least one single-use bioreactor has a volume independently selected from 1000 L, 2000 L, 3000 L, and 5000 L.

[0019] In some embodiments, one or more of the at least one single-use bioreactor has a volume of 200 L. In some embodiments, one or more of the at least one single-use bioreactor has a volume of 500 L.

[0020] Non-limiting examples of equipment that may be employed in a flexible facility include bioreactors, disc stack centrifuges, single-use centrifuges, tangential flow filtration (TFF) skids, depth filtration skids, in-line dilution skids, chromatography columns with associated control equipment, media tanks, harvest tanks, purification vessels, depth filter racks, water softening and / or dechlorination systems, clean steam generators, water for injection (WFI) storage tanks, WFI break tanks, WFI stills, cooling towers, switchboards, emergency generators, chillers, pumps, autoclaves, air handling units, process waste neutralization (e.g., fiberglass reinforced plastic (FRP)) equipment, biological waste collection and / or inactivation systems, clean-in-place systems, steam-in-place systems, parts washers, and / or other equipment.

[0021] By way of non-limiting example and not limitation, U.S. Publication Nos. 2013 / 0280797, 2012 / 0077429, 2011 / 0280797, and 2009 / 0305626 and U.S. Patent Nos. 8,298,054; 7,629,167; and 5,656,491 (which are incorporated herein by reference) describe example facility areas, equipment, and / or systems having features that may be suitable for use in the flexible facilities described herein.

[0022] In some embodiments, the stainless steel areas as a whole have a lower air classification than the reconfigurable single use areas as a whole. In some embodiments, the stainless steel areas as a whole have a lower building finish than the reconfigurable single use areas as a whole. In some embodiments, the stainless steel areas as a whole have a lower air classification and lower building finish than the reconfigurable single use areas as a whole.

[0023] In some embodiments, the stainless steel area is an unclassified manufacturing space. In some embodiments, at least a portion of the reconfigurable single-use area (e.g., a seed culture area; a purification area) is a classified manufacturing space. The integrity and environmental control within at least a portion of the reconfigurable single-use area facilitates the use of the unclassified manufacturing space.

[0024] In some embodiments, one or more of the at least one stainless steel bioreactor is supported on a ground surface.

[0025] In some embodiments, one or more of the at least one stainless steel bioreactor may be suspended from the structure itself.

[0026] In some embodiments, the stainless steel zone comprises a plurality of stainless steel bioreactors, each stainless steel bioreactor having a volume of at least 10,000 L.

[0027] In some embodiments, each stainless steel bioreactor in the stainless steel region has a volume between 5000 L and 20,000 L. In some embodiments, each stainless steel bioreactor in the stainless steel region has a volume between 5000 L and 15,000 L. In some embodiments, each stainless steel bioreactor in the stainless steel region has a volume between 5000 L and 10,000 L.

[0028] In some embodiments, the stainless steel area has no physical barrier separating it from the reconfigurable single use area.

[0029] In some embodiments, the reconfigurable single-use area and the stainless steel area are separated by a divider. In some embodiments, the divider is a wall. In some embodiments, the divider is a plastic sheet.

[0030] In some embodiments, the stainless steel area is completely enclosed. In some embodiments, the stainless steel area is completely enclosed by one or more walls and ceilings.

[0031] In some embodiments, the stainless steel area has separate air handling from the reconfigurable single use area.

[0032] In some embodiments, a portion of the at least one stainless steel bioreactor penetrates the wall and is fluidly connected to one or more of the at least one single-use bioreactor via tubing. In some embodiments, the portion is sufficient to facilitate connections between instruments and / or process units. In some embodiments, the portion is minimally sufficient to facilitate connections between instruments and / or process units.

[0033] In some embodiments, at least 1% (e.g., at least 2%, at least 3%, at least 4%, at least 5%; 1%, 2%, 3%, 4%, 5%) of the outer container surface area of ​​the at least one stainless steel bioreactor penetrates the wall and is fluidly connected to one or more of the at least one single-use bioreactor via tubing. In some embodiments, 3% to 7% (e.g., 3.5% to 6.5%, 4% to 6%, 4.5% to 5.5%; 3%, 4%, 5%, 6%, 7%) of the outer container surface area of ​​the at least one stainless steel bioreactor penetrates the wall and is fluidly connected to one or more of the at least one single-use bioreactor via tubing.

[0034] In some embodiments, the portion that penetrates the wall includes one or more components selected from the following: a probe strip to allow access to bioreactor instruments, a connection for perfusion, and a connection for a feeding solution (e.g., a nutrient feed; a solution containing an anti-foam agent).

[0035] In some embodiments, the fluid connector is configured to enable bypassing of stainless steel areas. In some embodiments, the fluid connector is removable. In some embodiments, the fluid connector can be moved between different bioreactors within a flexible facility.

[0036] In certain embodiments, the stainless steel area further includes a harvesting area. In certain embodiments, the harvesting area in the stainless steel area includes one or more centrifuges. In certain embodiments, the harvesting area in the stainless steel area includes one or more stainless steel centrifuges. In certain embodiments, the equipment in the stainless steel area is connected to tubing and / or one or more valves to allow the fluid connection required for the specific process being run to be completed. In certain embodiments, one or more valves are used to bypass a certain equipment that is not needed for the process being run. In certain embodiments, one or more valves are used to close fluid flow to allow replacement of equipment. For example, in certain embodiments, there is an inlet conduit connected to each stainless steel bioreactor from the seed culture section. In certain embodiments, there is an outlet conduit from each stainless steel bioreactor to the centrifuge to harvest.

[0037] In some embodiments, the reconfigurable single-use area may include one or more additional spaces independently selected from the group consisting of a cell culture unit, a pre-viral unit, a post-viral unit, a utility area, a warehouse, a media buffer facility, an office, a personnel unit, and a production unit. A production unit may be useful for manufacturing therapeutic products.

[0038] In some embodiments, the reconfigurable single-use areas may include reagent preparation areas, seed culture areas, and purification areas.

[0039] In some embodiments, the reconfigurable single-use area further includes equipment for performing downstream purification. In some embodiments, the equipment for performing downstream purification is single-use or stainless steel. In some embodiments, the equipment for performing downstream purification is single-use. In some embodiments, the equipment for performing downstream purification is stainless steel. In some embodiments, some of the equipment for performing downstream purification is stainless steel and some of the equipment for performing downstream purification is single-use.

[0040] In some embodiments, the stainless steel area further includes equipment for performing downstream purification. In some embodiments, the equipment for performing downstream purification is single use or stainless steel. In some embodiments, the equipment for performing downstream purification is single use. In some embodiments, the equipment for performing downstream purification is stainless steel.

[0041] In some embodiments, the reconfigurable single-use area further includes one or more centrifuges. In some embodiments, the one or more centrifuges are single-use and / or stainless steel equipment. In some embodiments, the one or more centrifuges are single-use equipment. In some embodiments, the one or more centrifuges are stainless steel equipment.

[0042] In some embodiments, the stainless steel area further comprises one or more centrifuges. In some embodiments, the one or more centrifuges are single use and / or stainless steel equipment. In some embodiments, the one or more centrifuges are single use equipment. In some embodiments, the one or more centrifuges are stainless steel equipment.

[0043] In some embodiments, the reconfigurable single-use area includes a reagent preparation area. In some embodiments, the reconfigurable single-use area includes walls surrounding the reagent preparation area. In some embodiments, the reagent preparation area has separate air handling. In some embodiments, the reconfigurable single-use area includes a seed cultivation area. In some embodiments, the reconfigurable single-use area includes walls surrounding the seed cultivation area. In some embodiments, the seed cultivation area has separate air handling. In some embodiments, the reconfigurable single-use area includes a purification area. In some embodiments, the reconfigurable single-use area includes walls surrounding the purification area. In some embodiments, the purification area has separate air handling.

[0044] In some embodiments, the reconfigurable single-use area includes a reagent preparation area, a seed culture area, and a purification area. In some embodiments, the reconfigurable single-use area includes a reagent preparation area, a seed culture area, and a purification area, together with one or more partitions, which at least partially isolate one or more of the reagent preparation area, the seed culture area, and the purification area. In some embodiments, the reconfigurable single-use area includes a reagent preparation area, a seed culture area, and a purification area, wherein the reagent preparation area, the seed culture area, and the purification area are not all served by the same air handling system.

[0045] In some embodiments, the stainless steel region is adjacent to the seed culture region, or adjacent to the purification region, or adjacent to both the seed culture region and the purification region.

[0046] In some embodiments, the reconfigurable single-use area includes one or more seed culture sections. In some embodiments, the reconfigurable single-use area includes one or more wave bioreactors, such as one or more wave bioreactors having a volume of at least 50 L (e.g., 50 L). In some embodiments, the reconfigurable single-use area includes one or more seed bioreactors, such as one or more seed bioreactors having a volume of at least 500 L (e.g., 500 L) or a volume of at least 2,000 L (e.g., 2,000 L).

[0047] In some embodiments, the reconfigurable single-use area further includes one or more purification skids. In some embodiments, each of the one or more purification skids includes one or more components independently selected from the following: a chromatography skid, a chromatography column, a virus inactivation system, a virus filtration system, an ultrafiltration diafiltration system, and a batch filtration system. In some embodiments, the reconfigurable single-use area further includes a chromatography skid. In some embodiments, the reconfigurable single-use area further includes a virus inactivation system. In some embodiments, the reconfigurable single-use area further includes a virus filtration system. In some embodiments, the reconfigurable single-use area further includes an ultrafiltration / diafiltration system. In some embodiments, the reconfigurable single-use area further includes a batch filtration system. In some embodiments, the reconfigurable single-use area further includes a virus inactivation system and a virus filtration system.

[0048] In some embodiments, one or more purification skids in a flexible facility have separate air handling.

[0049] In some embodiments, the flexible facility includes a plurality of connections that can be reconfigured to connect different equipment (eg, bioreactors, purification skids, etc.) in the flexible facility.

[0050] In some embodiments, utilities are provided in the reconfigurable single-use area by overhead, from one or more walls, or from one or more floor mounted utility distribution, or a combination of the above utility distribution options. In some embodiments, utilities are provided overhead in the reconfigurable single-use area. In some embodiments, utilities are readily available in the reconfigurable single-use area.

[0051] In some embodiments, multiple devices are within the reconfigurable single use area and are moveable around the reconfigurable single use area.

[0052] In some embodiments, the flexible facility further includes a quality assurance (QA) section, an administration / office section, plant utilities (eg, air handling, electrical, and plumbing), a quality control area, a laboratory area, and a receiving area.

[0053] In some embodiments, flexible facilities further include one or more additional spaces independently selected from the following: utility space, culture medium buffer facilities, personnel locker rooms and production units. In some embodiments, flexible facilities further include utility space. In some embodiments, flexible facilities further include culture medium buffer facilities. In some embodiments, flexible facilities further include personnel locker rooms. In some embodiments, flexible facilities further include production units. In some embodiments, flexible facilities further include utility space, culture medium buffer facilities, personnel locker rooms and production units. In some embodiments, the at least one additional space (e.g., utility space, culture medium buffer facilities, personnel locker rooms and / or production units) is located in a reconfigurable single-use area.

[0054] In some embodiments, the flexible facility includes at least one of: a fermentation unit, a pre-viral unit, a post-viral unit, utility space, a warehouse, a media buffer facility, an office, a personnel unit, and a production unit.

[0055] In some embodiments, any typical manufacturing and clean room equipment may be included in the flexible facility.

[0056] In some embodiments, the flexible facility is configured to produce one or more therapeutic products independently selected from the group consisting of: monoclonal antibodies, In some embodiments, the flexible facility is configured to produce monoclonal antibodies. In some embodiments, the flexible facility is configured to produce In some embodiments, the flexible facility is configured to produce RNA-based drug substances. In some embodiments, the flexible facility is configured to produce cell therapy products. In some embodiments, the flexible facility is configured to produce fermentation products.

[0057] In some embodiments, the stainless steel bioreactor(s) in the stainless steel area are dedicated to a single therapeutic product. In some embodiments, the entire flexible facility is dedicated to the production of a single therapeutic product. In some embodiments, the stainless steel bioreactor(s) in the stainless steel area contain only one cell line that produces a therapeutic product.

[0058] In some embodiments, a flexible facility is dedicated to producing more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, etc.) therapeutic products simultaneously (e.g., multiple products simultaneously in, for example, a seed culture section, a production bioreactor, and a purification skid). In some embodiments, a flexible facility is dedicated to producing two therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing three therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing four therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing five therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing six therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing seven therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing eight therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing nine therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing ten therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing eleven therapeutic products simultaneously. In some embodiments, a flexible facility is dedicated to producing twelve therapeutic products simultaneously.

[0059] In some embodiments, more than one (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, etc.) therapeutic product is produced in a flexible facility during a change in work period. For example, in some embodiments, after cell harvest and during purification, production of a second therapeutic product can be initiated in the seed culture section. In some embodiments, the stainless steel bioreactor(s) in the stainless steel area contain more than one cell line producing a therapeutic product.

[0060] In some embodiments, a flexible facility is dedicated to producing more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, etc.) therapeutic product during its lifetime. In some embodiments, a flexible facility is dedicated to producing up to 10 therapeutic products during its lifetime. In some embodiments, a flexible facility is dedicated to producing up to 15 therapeutic products during its lifetime. In some embodiments, a flexible facility is dedicated to producing up to 20 therapeutic products during its lifetime. In some embodiments, a flexible facility is dedicated to producing up to 25 therapeutic products during its lifetime. In some embodiments, a flexible facility is dedicated to producing up to 30 therapeutic products during its lifespan. In some embodiments, a flexible facility is dedicated to producing up to 35 therapeutic products during its lifespan. In some embodiments, a flexible facility is dedicated to producing up to 40 therapeutic products during its lifespan. In some embodiments, a flexible facility is dedicated to producing up to 45 therapeutic products during its lifespan. In some embodiments, a flexible facility is dedicated to producing up to 50 therapeutic products during its lifespan.

[0061] In some embodiments, flexible facilities are configured to support cGMP (current good manufacturing practice) processes. In some embodiments, flexible facilities are configured to support GMP processes. In some embodiments, flexible facilities are configured to support non-GMP processes. In some embodiments, flexible facilities support the production of GMP clinical products. In some embodiments, flexible facilities support the production of GMP commercial products. In some embodiments, flexible facilities support the production of non-GMP products. In some embodiments, flexible facilities support the production of GMP clinical products and GMP commercial products. In some embodiments, flexible facilities support the production of GMP clinical products and non-GMP products. In some embodiments, flexible facilities support the production of GMP commercial products and non-GMP products. In some embodiments, flexible facilities support the production of GMP clinical products, GMP commercial products, and non-GMP products. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Depicted are non-limiting examples of flexible manufacturing facilities in accordance with the present disclosure. Figure 1 is a top plan view of an example embodiment of a flexible facility. In some embodiments, Figure 1 The flexible facility can be constructed in a series of phases, such as phase one, phase two, and subsequent phases.

[0063] Figure 2A It is a depiction Figure 1 Schematic diagram of the various components of the flexible manufacturing facility.

[0064] Figure 2B It is a depiction Figure 1 and Figure 2A A schematic diagram of the components of this portion of at least one stainless steel bioreactor.

[0065] Figure 2C It is a depiction Figure 1 and Figure 2A Schematic diagram of at least one purification skid of one or more purification skids. DETAILED DESCRIPTION

[0066] The present disclosure relates to a new manufacturing facility configuration that integrates industry-leading technologies from next-generation biomanufacturing platforms based on single-use equipment to achieve enhanced processes in conventional fed-batch facilities based on reusable stainless steel equipment. The new facility configuration can be conducive to the simultaneous or sequential production of multiple biopharmaceuticals that are manufactured using a diversified manufacturing platform. This facility configuration also provides enhanced flexibility relative to conventional biomanufacturing facility layouts. For example, the facility can be configured or reconfigured to support any of the following manufacturing technologies: (a) fed-batch cell culture processing (e.g., up to a scale of 10,000L, at a scale greater than 10,000L) (e.g., using single-use and / or stainless steel equipment); (b) single-use cell culture processing (e.g., using only single-use technology) up to a scale of 2,000L; and (c) continuous upstream manufacturing up to a scale of 500L.

[0067] In addition to providing platform flexibility, the biomanufacturing facilities described herein are also expected to exhibit one or more of the following advantages when compared to conventional fed-batch facilities with similar production capacity: (a) reduced financial risk (e.g., less than 50% capital expenditure) due to lower space requirements, lower capacity equipment, less stainless steel pipes, and fewer automated stainless steel valves; (b) faster construction and earlier licensing due to improved space utilization; and (c) reduced carbon emissions and water consumption (e.g., up to 50% reduction) when sustainable building materials are employed, and reduced reliance on stainless steel equipment that requires regular cleaning and sterilization procedures.

[0068] Reference now Figures 1 to 2C, depicts a flexible manufacturing facility 10 for manufacturing at least one therapeutic product. The flexible facility 10 includes a reconfigurable single-use area 12 that includes at least one single-use bioreactor 14. In this embodiment, and as explained more below, one or more of the at least one single-use bioreactor 14 has a volume of at least 1000 L. In another example, the one or more of the at least one single-use bioreactor 14 has a volume of at least 2000 L and still falls within the scope of the present disclosure.

[0069] The flexible facility 10 also includes a stainless steel area 16 that includes at least one stainless steel bioreactor 18. Each stainless steel bioreactor 18 in the stainless steel area 16 has a volume greater than 5000 L. In addition, and as also explained more below, one or more of the at least one stainless steel bioreactor 18 can be fluidly connected to one or more of the at least one single-use bioreactor 14. In another example, and as Figure 1 As depicted in FIG. 1 , the stainless steel region 16 includes a plurality of stainless steel bioreactors 20 , each stainless steel bioreactor having a volume of at least 10,000 L.

[0070] In one example, the at least one stainless steel bioreactor 18 is a large stainless steel bioreactor and includes at least one valve assembly. The at least one stainless steel bioreactor 18 may include an on-site steam bioreactor with large-scale capacity, such as in one example and as described, configured to accommodate up to 10,000L or more fluid volume. The at least one valve assembly of the on-site steam-type large bioreactor enables the on-site steam-type large bioreactor to be fully sterilized before cell culture (e.g., perfusion cell culture) begins. Specifically, the at least one valve assembly may include a plurality of valves, and steam (e.g., clean steam CS) flows into a valve, enters other valves upward and reaches the port. Additionally, after flowing into the first valve, the steam also flows downward into another valve and flows through the steam trap to sterilize the bioreactor and produce a sterile environment sterilized by steam. In addition, as understood by those of ordinary skill in the art, during cleaning, process waste PW flows out of some valves and is injected into the sewer. After use, the on-site steam bioreactor is cleaned on-site, and the on-site steam sterilization process occurs again before any further use. The at least one valve assembly also includes a port for coupling to a sterile connector valve assembly (e.g., an autoclaved sterile connector valve assembly) or other device. The sterile connection valve assembly enables sterile connectivity of a single-use perfusion device or a reusable perfusion device to the at least one stainless steel bioreactor 18 (e.g., a steam-in-place large-scale bioreactor). In addition, the at least one valve assembly enables sterile connectivity of the single-use bioreactor 14 to the at least one stainless steel bioreactor 18.

[0071] In another example, the at least one stainless steel bioreactor 18 may include a side and a first valve assembly (e.g., an autoclaved valve assembly) coupled to the side of the at least one stainless steel bioreactor 18. The autoclaved valve assembly may include a plurality of valves, and steam (e.g., clean steam CS) flows into the first valve, reaching the other valves and ports upward. Additionally, for example, after flowing into the first valve, the steam flows downward through another valve and through the steam trap to sterilize the at least one stainless steel bioreactor 18 and produce an in-situ steam sterilization process before further use. In this example, there may be only at least one sterile connector coupled to the valve assembly (e.g., an autoclaved valve assembly). For example, the autoclaved valve assembly realizes connection to a factory-assembled and irradiated perfusion device or an autoclave-sterilized perfusion device via the at least one sterile connector of the autoclaved valve assembly, which may be a single-use perfusion device or a reusable perfusion device. In addition, a plurality of perfusion devices can be aseptically coupled to the at least one stainless steel bioreactor 18 without additional steam sterilization. Further, the autoclaved valve assembly also enables connectivity of the at least one single-use bioreactor 14 to the at least one stainless steel bioreactor 18. In yet another example, the at least one stainless steel bioreactor 18 can enable connection to an autoclaved perfusion device and in-place cleaning of the valve assembly without using a sterile connector. Instead, a hose assembly can be used as an alternative connector to connect the at least one stainless steel bioreactor 18 to the autoclaved perfusion device and / or the at least one single-use bioreactor 14.

[0072] like Figure 1 and Figure 2A As further depicted in FIG. 1 , in another example, the reconfigurable single-use area 12 and the stainless steel area 16 are separated by a divider 22. In one example, the divider is a wall. In another example, the stainless steel area 16 is completely enclosed and has separate air handling, as explained more below. Additionally, a portion 24 of the at least one stainless steel bioreactor 18 penetrates the dividing wall 22 and is fluidly connected to one or more of the at least one single-use bioreactor 14 via tubing 26. The fluid connections are configured to enable bypassing of the stainless steel area 16, also as explained more below.

[0073] In another example, the at least one stainless steel bioreactor 18 includes an adapter assembly (e.g., a single-use adapter assembly and / or a tee assembly) that is configured as a sterile connector and / or coupled to a sterile connector. The adapter assembly (e.g., a tee assembly) includes a connector that is directly coupled to a sterile connector that is ultimately coupled to a pair of tubes (e.g., tubing 26). For example, the sterile connector or each of the connectors of the tee assembly is configured to couple to a reusable or single-use perfusion device or Figure 1 and Figure 2A The at least one single-use bioreactor 14 of the reconfigurable single-use area 12. For example, this enables multiple perfusion devices or multiple single-use bioreactors 14 to be operatively coupled to the at least one stainless steel bioreactor 18.

[0074] In another example, the portion 24 of the at least one stainless steel bioreactor 18 that penetrates the dividing wall 22 includes one or more components, including but not limited to a probe belt 27a, a perfusion connection 27b for perfusion, and a feed solution tank connection 27c, such as Figure 2B As depicted in .

[0075] like Figure 1 and Figure 2A As depicted in FIG. 1 , and in one example, the stainless steel area 16 includes one or more centrifuges 30. Additionally, the reconfigurable single-use area 12 also includes one or more centrifuges 32. In another example, the reconfigurable single-use area 12 includes one or more seed culture sections 34. In yet another example, the reconfigurable single-use area 12 includes one or more purification skids 36. In one example, and as Figure 2C As described in the drawings, each of the one or more purification skids 36 includes one or more components independently selected from the following and / or including the following: a chromatography skid 37a, a chromatography column 37b, a virus inactivation system 37c, a virus filtration system 37d, an ultrafiltration diafiltration system 37e, a batch filtration system 37f, a conjugate tank system 37g, an oxidation tank system 37h and a reduction tank system 37i.

[0076] In some embodiments, the flexible facility 10 of the present disclosure exhibits or is configured for one or more of the following properties:

[0077] 1) Extensive deployment of reconfigurable spaces to accommodate cell culture processing (e.g., high titers at 10,000L scale (e.g., 1 to 15 g / L titers or greater)) and even higher titers at 2,000L scale (e.g., up to 30 g / L or greater). Fed-batch and perfusion cultures can be accommodated in one facility, including as part of a single therapeutic product manufacturing process.

[0078] 2) Includes a stainless steel bioreactor 18 (eg, a 10,000 L scale stainless steel production bioreactor) that uses a high binding capacity commercially available chromatography resin to maximize purification capacity.

[0079] 3) includes a stainless steel bioreactor 18 (e.g., a 10,000 L scale stainless steel production bioreactor) that matches a typical maximum split ratio from the 2,000 L (N-1) bioreactor to the production bioreactor (N bioreactor). Illustratively, for the (N-1) stage, a 5:1 split ratio enables 10,000 L production bioreactor processing and a 2,000 L seed reactor.

[0080] 4) The sterile connection between the stainless steel bioreactor 18 and the single-use bioreactor 14 facilitates enhanced fed-batch operation.

[0081] 5) The majority of the stainless steel bioreactor 18 is located in the stainless steel area 16 with lower air classification and lower building finish, thereby reducing construction costs and operating costs (eg, air purification costs).

[0082] 6) The downstream purification region is reconfigurable to accommodate a wide range of outputs from various upstream technologies, for example by utilizing a variety of easily movable production skids. The reconfigurable downstream purification region is configured to allow operation in either batch mode, which involves separate pool vessels for the various purification steps, or connected mode, which eliminates the pool vessels and employs direct connections between purification steps.

[0083] 7) The reconfigurable downstream purification region is positioned adjacent to the reconfigurable seed culture region within the reconfigurable single use region 12 .

[0084] In some embodiments, each of the reconfigurable single-use area 12 and the stainless steel area 16 can include a sub-area that is partially or completely isolated (e.g., by a partition 22, such as a wall or plastic sheet) from other portions of the reconfigurable single-use area 12 and the stainless steel area 16. Additionally, in some embodiments, the reconfigurable single-use area 12 can include one or more pieces of non-single-use equipment (e.g., a stainless steel centrifuge), as long as the piece of equipment is not a bioreactor. Similarly, in some embodiments, the stainless steel area 16 can include one or more pieces of equipment that are not made of stainless steel, as long as the piece of equipment is not a bioreactor.

[0085] The flexible facility 10 disclosed herein is useful for manufacturing at least one therapeutic product (e.g., one; more than one (e.g., two, three, four, etc.)) at a given time. Illustratively, the flexible facility 10 is adapted to manufacture a variety of different therapeutic products simultaneously or sequentially by deploying common resources where possible, while facilitating the interchangeability of process-specific equipment.

[0086] Non-limiting example features

[0087] Without limitation, some example embodiments / features of the present disclosure include:

[0088] 1. A flexible facility for manufacturing at least one therapeutic product, the flexible facility comprising:

[0089] A reconfigurable single-use area comprising at least one single-use bioreactor, wherein one or more of the at least one single-use bioreactor has a volume of at least 1000 L (e.g., 1000 L, 2000 L, 3000 L, 5000 L, at least 2000 L, at least 3000 L, at least 5000); and

[0090] A stainless steel area, comprising at least one stainless steel bioreactor, wherein each stainless steel bioreactor in the stainless steel area has a volume greater than 5000 L,

[0091] Wherein, one or more of the at least one stainless steel bioreactor is fluidly connected to one or more of the at least one single use bioreactor.

[0092] 2. A flexible facility according to feature 1, wherein one or more (e.g., one, two, three, four, five, six, seven, eight, nine or ten) of the at least one single-use bioreactor has a volume of 1000L, 2000L, 3000L or 5000L.

[0093] 3. A flexible facility according to feature 1 or 2, wherein the at least one single-use bioreactor has a volume of 1000L, 2000L, 3000L or 5000L.

[0094] 4. A flexible facility according to any one of features 1 to 3, wherein no single-use bioreactor in the reconfigurable single-use area has a volume greater than 2000 L.

[0095] 5. A flexible facility according to any one of features 1 to 4, wherein one or more (e.g., one, two, three, four, five, six, seven, eight, nine or ten) of the at least one single-use bioreactor has a volume of less than 2000 L (e.g., 1000 L, 500 L, 50 L).

[0096] 6. A flexible facility according to any one of features 1 to 5, wherein the reconfigurable single-use area includes one or more wave bioreactors (e.g., one or more wave bioreactors having a volume of at least 50 L (e.g., 50 L)) and / or one or more seed bioreactors (e.g., one or more seed bioreactors having a volume of at least 500 L (e.g., 500 L) or a volume of at least 2,000 L (e.g., 2,000 L)).

[0097] 7. A flexible facility according to any one of features 1 to 6, wherein the stainless steel area comprises a plurality of stainless steel bioreactors, each stainless steel bioreactor having a volume of at least 10,000 L.

[0098] 8. A flexible facility according to any one of features 1 to 7, wherein each stainless steel bioreactor in the stainless steel area has a volume between 5000L and 20,000L.

[0099] 9. A flexible facility according to any one of features 1 to 8, wherein each stainless steel bioreactor in the stainless steel area has a volume between 5000L and 15,000L.

[0100] 10. A flexible facility according to any of features 1 to 9, wherein each stainless steel bioreactor in the stainless steel area has a volume between 5000L and 10,000L.

[0101] 11. A flexible facility according to any one of features 1 to 10, wherein the stainless steel area comprises five 10,000 L bioreactors.

[0102] 12. A flexible facility according to any one of features 1 to 11, wherein one or more of the at least one stainless steel bioreactor is supported on a ground surface.

[0103] 13. A flexible facility according to any one of features 1 to 12, wherein one or more of the at least one stainless steel bioreactor is suspended from the structure itself.

[0104] 14. A flexible facility according to any one of features 1 to 13, wherein the reconfigurable single-use area includes a reagent preparation area, a seed culture area, and a purification area.

[0105] 15. A flexible facility according to any one of features 1 to 14, wherein the reconfigurable single-use area includes a reagent preparation area, a seed culture area and a purification area, together with one or more partitions that at least partially isolate one or more of the reagent preparation area, the seed culture area and the purification area.

[0106] 16. A flexible facility according to features 14 or 15, wherein the seed culture area includes one or more pieces of equipment independently selected from the following: tanks for storing cell culture media; tanks for storing acids, bases and buffer solutions; inoculation preparation equipment; bioreactors (e.g., suitable for culturing cells), such as rocker bioreactors, 500L bioreactors and 2000L single-use bioreactors; tanks suitable for holding cells; and tanks suitable for holding products produced by the cells.

[0107] 17. A flexible facility according to any one of features 14 to 16, wherein the equipment in the seed cultivation area is in a single use format.

[0108] 18. A flexible facility according to any one of features 1 to 17, wherein the stainless steel area is adjacent to the seed cultivation area.

[0109] 19. The flexible facility according to any one of features 1 to 17, wherein the stainless steel zone is adjacent to the purification zone.

[0110] 20. A flexible facility according to any one of features 1 to 17, wherein the stainless steel area is adjacent to both the seed culture area and the purification area.

[0111] 21. The flexible facility according to any one of features 1 to 20, wherein the stainless steel area further comprises a harvesting area.

[0112] 22. A flexible facility according to any one of features 1 to 21, wherein the stainless steel area is an unclassified manufacturing space.

[0113] 23. A flexible facility according to any one of features 1 to 22, wherein at least a portion of the reconfigurable single-use area (e.g., a seed culture area; a purification area) is a classified manufacturing space.

[0114] 24. A flexible facility according to any one of features 1 to 23, wherein the reconfigurable single use area and the stainless steel area are separated by a divider.

[0115] 25. A flexible facility according to feature 24, wherein the divider is a plastic sheet.

[0116] 26. A flexible facility according to feature 25, wherein the partition is a wall.

[0117] 27. A flexible facility according to feature 26, wherein the stainless steel area is completely enclosed and has separate air handling.

[0118] 28. A flexible facility according to feature 26 or 27, wherein a portion of the at least one stainless steel bioreactor penetrates the wall and is fluidly connected to one or more of the at least one single-use bioreactor by tubing.

[0119] 29. A flexible facility according to any one of features 26 to 28, wherein at least 5% of the outer container surface area of ​​the at least one stainless steel bioreactor penetrates the wall and is fluidly connected to at least one of the at least one single-use bioreactor by tubing.

[0120] 30. A flexible facility according to feature 28 or 29, wherein the portion penetrating the wall comprises one or more components independently selected from the following: a probe strip to allow access to bioreactor instruments, a connection for perfusion, and a connection for a feed solution.

[0121] 31. A flexible facility according to any one of features 1 to 30, wherein the fluid connection comprises a sterile connection module.

[0122] 32. A flexible facility according to any one of features 1 to 31, wherein a sterile connection module connects the one or more seed culture bioreactors in the reconfigurable single-use area to the one or more stainless steel bioreactors in the stainless steel area.

[0123] 33. A flexible installation according to any of features 1 to 32, wherein the fluid connection is configured to enable bypassing of the stainless steel area.

[0124] 34. A flexible installation according to any of features 1 to 33, wherein the fluid connection is configured to enable the stainless steel region to be bypassed through the use of one or more valves.

[0125] 35. A flexible facility according to any one of features 1 to 34, wherein the fluid connection is removable.

[0126] 36. A flexible facility according to any of features 1 to 35, wherein the fluid connection is movable between different bioreactors within the flexible facility.

[0127] 37. A flexible facility according to any one of features 1 to 36, wherein the flexible facility includes a plurality of connectors that can be reconfigured to connect different equipment in the flexible facility (e.g., bioreactors, purification skids, etc.).

[0128] 38. A flexible facility according to any one of features 1 to 37, wherein the reconfigurable single use area and / or stainless steel area further comprises one or more centrifuges.

[0129] 39. A flexible facility according to any one of features 1 to 38, wherein the reconfigurable single-use area includes one or more seed cultivation sections.

[0130] 40. A flexible facility according to any one of features 1 to 39, wherein the reconfigurable single-use area and / or the stainless steel area includes one or more purification skids (e.g., one or more purification skids containing only single-use equipment; one or more purification skids containing only stainless steel equipment; one or more purification skids containing single-use equipment and stainless steel equipment).

[0131] 41. A flexible facility according to feature 40, wherein each of the one or more purification skids comprises one or more components independently selected from the following: a chromatography skid, a chromatography column, a viral inactivation system, a viral filtration system, an ultrafiltration diafiltration system and a batch filtration system.

[0132] 42. A flexible facility according to any one of features 1 to 41, further comprising one or more additional spaces independently selected from the group consisting of utility spaces, media buffer facilities, personnel changing rooms, and production units.

[0133] 43. A flexible facility according to any one of features 1 to 42, wherein the flexible facility is configured for the production of one or more drug substances independently selected from the group consisting of: monoclonal antibodies, molecules, RNA-based drug substances and fermentation products.

[0134] 44. A flexible facility according to any one of features 1 to 43, wherein the flexible facility is configured to implement one or more processes independently selected from the following: a mammalian cell culture fed-batch process, a mammalian cell culture perfusion process, a continuous mammalian cell culture process, a microbial fermentation fed-batch process and a combination of any of the foregoing.

[0135] 45. A flexible facility according to any one of features 1 to 44, wherein the flexible facility can be constructed in a series of stages.

[0136] 46. ​​A flexible facility according to any one of features 1 to 45, wherein the flexible facility is expandable to add additional product lines.

[0137] 47. A flexible facility according to any of features 1 to 46, wherein the stainless steel bioreactor(s) in the stainless steel area contains only one cell line producing a therapeutic product.

[0138] 48. A flexible facility according to any one of features 1 to 46, wherein the stainless steel bioreactor(s) in the stainless steel area contain more than one cell line producing a therapeutic product.

[0139] 49. A flexible facility according to any one of features 1 to 48, wherein the single-use bioreactors in the reconfigurable single-use area contain only one cell line producing a therapeutic product.

[0140] 50. A flexible facility according to any one of features 1 to 48, wherein the single-use bioreactors in the reconfigurable single-use areas contain more than one cell line producing a therapeutic product.

[0141] 51. A flexible facility according to any one of features 47 to 50, wherein each therapeutic product producing cell line is independently selected from a prokaryotic cell line and a eukaryotic cell line.

[0142] 52. A flexible facility according to any one of features 47 to 50, wherein each therapeutic product producing cell line is independently selected from a prokaryotic cell line.

[0143] 53. A flexible facility according to any one of features 47 to 50, wherein each therapeutic product producing cell line is independently selected from a mammalian cell line.

[0144] 54. A flexible facility according to any one of features 47 to 50, wherein each cell line producing a therapeutic product is independently selected from a cell line capable of producing a viral therapeutic agent (e.g., an anti-cancer oncolytic virus, a viral vector for gene therapy and / or a viral immunotherapy).

[0145] 55. A flexible facility according to any one of features 47 to 50, wherein each therapeutic product producing cell line is independently selected from a CHO cell line.

[0146] definition:

[0147] As used herein, the terms "a" and "an" mean one or more unless expressly indicated otherwise. Additionally, "one or more" and "at least one" are used interchangeably herein. Furthermore, unless the context requires otherwise, singular terms include pluralities and plural terms include the singular.

[0148] As used herein, the term "bioreactor" means any container useful for the growth of cell cultures (e.g., mammalian cell cultures or bacterial cell cultures). "Bioreactor" encompasses the term "fermenter" herein (i.e., a container useful for the growth of bacterial cell cultures that typically contains a more stringent agitator and increased gas flow relative to a container for the growth of mammalian cell cultures). Non-limiting examples of bioreactors include agitated tanks, airlifts, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed beds, and / or spouted bed bioreactors. In some embodiments, an example bioreactor may perform one or more (e.g., one, two, three, all) of the following steps: feeding of nutrients and / or carbon sources, injection of a suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, stirring (e.g., agitation), and / or cleaning / disinfection. Unless otherwise indicated by the context, bioreactors can be suitable for batch, semi-fed batch, fed batch, perfusion and / or continuous fermentation processes. Any suitable bioreactor diameter can be used. Unless otherwise indicated by the context, in some embodiments, bioreactors can have a volume between 100mL and 50,000L. Unless otherwise indicated, bioreactors can be any size, as long as it is useful for cell culture; typically, the size of the bioreactor is suitable for the volume of the cell culture grown inside it. In a non-limiting embodiment, unless otherwise indicated by the context, bioreactors can be at least 1 liter (L), or can be 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or larger, or any volume between the two. The internal conditions of the bioreactor can be controlled during the culture period, including but not limited to pH and temperature. Those of ordinary skill in the art will be aware of suitable bioreactors for use in the flexible facilities disclosed herein, and will be able to select such suitable bioreactors based on relevant considerations.

[0149] As used herein, the term "cell culture" or "cultivation" refers to the growth and reproduction of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells and bacterial cells are known in the art. (See, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992)). Mammalian cells can be suspended cultured or attached to a solid substrate for cultivation. In certain embodiments, fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake bottles and / or agitated tank bioreactors (with or without microcarriers) can be used for cell culture. In certain embodiments, a bioreactor of 500L to 2000L is used for cell culture (for example, as a part of seed culture). In certain embodiments, a bioreactor of 1000L to 2000L is used for cell culture (for example, as a part of seed culture).

[0150] As used herein, the term "cell culture medium" (also referred to as "media / culture medium", "cell culture medium", "tissue culture medium", etc.) refers to any nutrient solution used to grow cells (e.g., bacterial cells or mammalian cells). Cell culture media typically provide one or more of the following components: an energy source (e.g., in the form of a carbohydrate, such as glucose); one or more essential amino acids (e.g., all essential amino acids; the twenty basic amino acids, plus cysteine); vitamins and / or other organic compounds that are typically required at low concentrations; lipids or free fatty acids; and trace elements, such as inorganic compounds or naturally occurring elements, that are typically required at very low concentrations (e.g., concentrations in the micromolar range). As used herein, cell culture media encompasses nutrient solutions that are typically employed and / or known for use with any cell culture process, including but not limited to batch, expanded batch, fed-batch, and / or perfusion or continuous culture of cells.

[0151] As used herein, "fed-batch culture" refers to a form of suspension culture, specifically a method of culturing cells, in which additional components are provided to the culture at one or more times after the culture process begins. The components provided typically include nutritional supplements that are depleted for the cells during the culture process. Additionally or alternatively, the additional components may include supplementary components (e.g., cell cycle inhibitory compounds). In some embodiments, the fed-batch cell culture medium formulation may be richer or more concentrated than the basal cell culture medium formulation. The fed-batch culture can be stopped at a certain point in time, and the cells and / or components in the culture medium can be harvested and optionally purified.

[0152] As used herein, "perfusion" cell culture medium refers to a cell culture medium that is typically used in cell cultures maintained by perfusion or continuous culture methods and is sufficiently complete to support the cell culture during the process. In some embodiments, the perfusion cell culture medium formulation may be richer or more concentrated than the basal cell culture medium formulation to accommodate the methods used to remove spent culture medium. In some embodiments, the perfusion cell culture medium may be used during both the growth and production phases.

[0153] As used herein, "production" cell culture medium refers to a cell culture medium that is typically used in cell culture during the transition when exponential growth ends and protein production takes over (i.e., the "transition" and / or "product" phase) and is sufficiently complete to maintain a desired cell density, viability, and / or product titer during this phase.

[0154] As used herein, a "therapeutic product" is a biological product used in a subject (e.g., a human) in connection with the prevention, diagnosis, treatment, or alleviation of a disease, illness, defect, or injury. Non-limiting examples of therapeutic products include monoclonal antibodies, molecules, RNA-based drug substances, cell therapeutics and fermentation products.

[0155] Seed cultivation area

[0156] The seed culture area in the flexible facility 10 (e.g., the area of ​​the single-use area 12 in which the one or more seed culture sections 34 are located) can accommodate equipment suitable for cell culture. Equipment for cell culture includes, but is not limited to: tanks for storing cell culture media; tanks for acid, base, and buffer solutions; inoculation preparation equipment; bioreactors (e.g., suitable for culturing cells), including swing-arm bioreactors, 500L bioreactors, and 2000L single-use bioreactors (e.g., single-use bioreactors 14); tanks suitable for containing cells or products produced by cells; centrifuges, such as centrifuge 32; pumps; and other equipment useful for product harvesting. In some embodiments, the equipment in the seed culture area is in a single-use format.

[0157] In some embodiments, the seed culture area demonstrates the flexibility of the manufacturing facility of the present disclosure. For example, in some configurations that also employ stainless steel bioreactors 18, the seed culture section can include bioreactor units up to and including the N-1 bioreactor. In some embodiments, the product of the N-1 bioreactor is transferred to the stainless steel N bioreactor. In this configuration, equipment for product harvesting is bypassed in favor of equipment in the stainless steel area.

[0158] In some configurations where the stainless steel sections are bypassed, the largest bioreactor unit is considered to be the N bioreactor (e.g., a 500L or 2000L N bioreactor), and product harvesting is performed using equipment in the seed culture area.

[0159] In certain embodiments, the seed culture area comprises one or more culture bottles (flask), culture bag and bioreactor unit suitable for culturing cells. In certain embodiments, the cell culture unit in the seed culture area gradually becomes larger in the seed culture section, to adapt to larger cell culture. In certain embodiments, the seed culture section may include a wave bioreactor and two seed bioreactors. In certain embodiments, the seed culture section may include a 50L wave bioreactor, a 500L single-use bioreactor and a 2000L single-use bioreactor, wherein the 2000L single-use bioreactor (such as, single-use bioreactor 14) is optionally configured to be used for perfusion culture (such as, ATF perfusion culture). In certain embodiments, there may also be a parallel group of gradually increasing bioreactors, wherein each parallel group may include cells expressing the same product, or may include cells expressing different products.

[0160] In certain embodiments, the seed culture area can comprise 1,2,3,4,5,6,7,8,9 or 10 bioreactors, or comprise its multiple bioreactors when manufacturing multiple products.In certain embodiments, each bioreactor in the seed culture area is suitable for being independently selected from the following one or more processing methods: batch, semi-fed batch, fed batch, perfusion and / or continuous process.In certain embodiments, the bioreactor is a stirred tank reactor.In certain embodiments, the bioreactor is an airlift reactor.In certain embodiments, the bioreactor can have a volume between about 100 milliliters and about 5,000 liters. Non-limiting example bioreactor volumes include 100 ml, 250 ml, 500 ml, 750 ml, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, and 5000 liters. Typically, the bioreactor units in the seed culture area are smaller than the bioreactor units in the stainless steel area. In certain configurations, each seed culture bioreactor is at least 2 times, at least 3 times, at least 4 times, or at least 5 times smaller than the stainless steel production bioreactor. In some embodiments, the seed culture section can accommodate four 2,000 liter containers for single-use technology operations.

[0161] In some embodiments, the seed culture area complies with good manufacturing process and biosafety standards. In some embodiments, the seed culture area complies with biosafety level 1 (BSL1), biosafety level 2 (BSL2), biosafety level 3 (BSL3) or biosafety level 4 (BSL4) standards.

[0162] In some embodiments, the seed culture area may include sub-compartments, wherein each sub-compartment may be used to perform different functions or aspects of supporting a cell culture production process. For example, in some embodiments, the seed culture area may include sub-compartments housing one or more bioreactors and sub-compartments housing equipment for product harvesting.

[0163] Single-use production areas

[0164] In some embodiments (where the stainless steel area 16 is bypassed), the reconfigurable single-use area 12 includes one or more production bioreactors. The flexible facility 10 is easily expandable and scalable, and different production bioreactors within the reconfigurable single-use area 12 can be used to produce completely different therapeutic products in the same flexible facility 10.

[0165] For example, in some embodiments, in a modular unit configured as a first bioreactor module, a user can manufacture one type of therapeutic product, such as a monoclonal antibody product derived from a mammalian cell line. In a second modular unit, a user can manufacture a completely different therapeutic product, such as a microbial product. In some embodiments, the flexible facility 10 of the present disclosure can use production bioreactors in one or both of the single-use production area 12 and the stainless steel area 16 to support multiple product lines simultaneously. In addition, in some embodiments, the flexible facility (e.g., flexible facility 10) of the present disclosure can be expanded to add additional product lines.

[0166] Stainless steel area

[0167] In some embodiments, the stainless steel section 16 houses equipment suitable for cell culture. Equipment suitable for cell culture includes, but is not limited to, bioreactors (e.g., suitable for culturing cells), tanks (e.g., suitable for containing cells, culture media, or products produced by cells), centrifuges, pumps, and other equipment useful for product harvesting, and cleaning systems (CIP). The CIP unit is typically a modular skid and has several tanks to hold cleaning solutions (e.g., caustic solutions and / or bleach), pumps, and sensors to deliver the cleaning solutions to the appropriate tanks to be cleaned.

[0168] In some embodiments, the stainless steel section 16 comprises one or more stainless steel bioreactor units suitable for culturing cells, such as (multiple) stainless steel bioreactors 18. The bioreactor units can perform one or more (e.g., one, two, three, four, all) of the following: feeding of nutrients and / or carbon sources; injection of suitable gases (e.g., oxygen); flow of cell culture medium; separation of gas and liquid phases; maintenance of growth temperature; maintenance of pH levels; agitation (e.g., stirring); and / or cleaning / sterilization. In some embodiments, the stainless steel section can comprise 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors. In some embodiments, each bioreactor in the stainless steel region 16 is suitable for one or more process methods independently selected from the following: batch, semi-fed batch, fed batch, perfusion, and / or continuous process. In some embodiments, at least one stainless steel bioreactor 18 is a stirred tank reactor. In some embodiments, at least one stainless steel bioreactor 18 is an airlift reactor. In some embodiments, at least one stainless steel bioreactor 18 can have a volume between 1000 liters and 50,000 liters. Non-limiting examples include the following volumes: 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, or 50,000 liters.

[0169] In some embodiments, approximately 95% of the stainless steel bioreactor(s) reside in an unclassified space within the flexible facility 10, with a small portion (approximately 5%) of the stainless steel bioreactor(s) 18 being exposed to the classified reconfigurable single-use area 12 of the flexible facility 10. In some embodiments, the exposed portion(s) of the stainless steel bioreactor(s) 18 may include a tank probe strip 27a to allow access to tank instrumentation, connections 27b for perfusion, and any other connections 27c for feeding and adding solutions to the tank, including connections for pH control, nutrient feeds (culture media), or other additions (i.e., anti-foam addition).

[0170] In some embodiments, the exposed portion(s) 24 of the stainless steel bioreactor(s) 18 are exposed directly to the classified space, such as by means of a probe strip 27a, or through one or more connections to one or more lines in the classified space which may enter the tank through a tube at another tank location (e.g., with media addition).

[0171] In some embodiments, the stainless steel area 16 is designed to accommodate several bioreactors 18. In some embodiments, the stainless steel area 16 can accommodate five 10,000 liter vessels for mammalian cell lines.

[0172] In some embodiments, the stainless steel area 16 includes five or more 10,000 liter containers and harvesting equipment. In some embodiments, the stainless steel area 16 includes five 10,000 liter containers and harvesting equipment. In some embodiments, the bioreactor in the stainless steel area is configured to manufacture a monoclonal antibody product derived from a mammalian cell line.

[0173] In some embodiments, one or more stainless steel bioreactors 18 in the stainless steel area 16 can be ground-based reactors. In some embodiments, one or more stainless steel bioreactors 18 can be suspended from the structure itself.

[0174] In some embodiments, the stainless steel region 16 complies with good manufacturing process and biosafety standards. In some embodiments, the stainless steel region 16 complies with biosafety level 1 (BSL1), biosafety level 2 (BSL2), biosafety level 3 (BSL3) or biosafety level 4 (BSL4) standards.

[0175] In some embodiments, the stainless steel area 16 may include sub-compartments, each of which may be used to perform a different function or aspect of supporting a cell culture production process. For example, in some embodiments, the stainless steel area 16 may include sub-compartments for housing one or more bioreactors, sub-compartments for housing equipment for product harvesting, sub-compartments for inoculation, and sub-compartments for cleaning and decontamination equipment and operators handling such equipment.

[0176] In some embodiments, different production bioreactors within stainless steel area 16 can be used to produce completely different therapeutic products in the same flexible facility, such as flexible facility 10. For example, in some embodiments, in a modular unit configured as a first bioreactor module, a user can produce one type of therapeutic product, such as a monoclonal antibody product derived from a mammalian cell line. In a second modular unit, a user can produce a completely different therapeutic product, such as a microbial product.

[0177] Downstream processing area

[0178] A standard downstream processing (DSP) unit is typically an assembly of chromatography, mixing, and filtration equipment. In a non-limiting embodiment of the present disclosure, the filtration equipment in the downstream processing area may include ultrafiltration, microfiltration, viral filtration, and disinfection equipment, including, for example, a pre-viral separation subunit and a post-viral separation subunit. Viral reduction may occur throughout a typical mammalian cell-derived protein purification process; for example, in some embodiments, viral reduction may occur through (multiple) viral reduction chromatography steps, viral inactivation in the case of mixed containers, and / or viral filtration - all within a mammalian process. The post-viral separation subunit houses equipment and utilities suitable for any of the following: ultrafiltration (tangential filtration); normal filtration; chromatography; formulation; titration; mixing; concentration; buffer exchange; and bulk drug substance container filling and freezing.

[0179] A non-limiting example purification zone may include: a tank with an agitator, to which an inlet conduit of the crude product from the separation module is connected; an outlet conduit provided with a volumetric pump, the outlet conduit being connected to an inlet conduit in a first chromatography column run by a computerized unit, the conduit also being connected to the computerized unit; an outlet conduit of the chromatography column, the outlet conduit being connected to an inlet conduit in a tank provided with an agitator, the outlet conduit of the tank provided with a volumetric pump being connected to an inlet conduit in a second chromatography column run by a computerized unit, the conduit also being connected to the computerized unit; an outlet conduit of the chromatography column, which is connected to an inlet conduit in a tank provided with an agitator, the outlet conduit of which is in turn connected in a discontinuous manner to an inlet of a vacuum freeze dryer provided with an outlet.

[0180] Classification level

[0181] Different areas within the flexible facility 10 (e.g., the reconfigurable single-use area 12 and the stainless steel area 16, and portions thereof) may have different classification levels based on the classification criteria. For example, in some embodiments, different areas may have different classification levels based on the classification criteria set by a regulatory agency in the jurisdiction in which the flexible facility 10 is regulated. In some embodiments, different areas may have different classification levels based on the classification criteria set by a regulatory agency in the jurisdiction in which the flexible facility 10 is located. In some embodiments, different areas may have different classification levels based on the classification criteria set by a regulatory agency in the jurisdiction in which the therapeutic products produced in the flexible facility 10 are sold.

[0182] In some embodiments, different regions may have different classification levels based on the classification standards set by the U.S. Food and Drug Administration or the classification standards set by EudraLex (EU Medicinal Products Regulation, Volume 4, EU Good Manufacturing Practice Guide for Human and Veterinary Medicinal Products (supplemented by Annex 1, EU Sterile Medicinal Products Manufacturing)).

[0183] Control System

[0184] In some embodiments, any of the analytical techniques known in the art can be used to monitor and evaluate samples from cell cultures. In some embodiments, various parameters can be monitored over the duration of the culture, including but not limited to the quality and properties of the recombinant protein and culture medium. Samples can be obtained and intermittently monitored at a desired frequency, including continuous monitoring, real-time monitoring, or near real-time monitoring.

[0185] In some embodiments, seed culture equipment, stainless steel bioreactor and purification equipment can each include a sensor for monitoring reaction parameters and / or product quality parameters. The monitored parameters can include but are not limited to conductivity, temperature, pH, oxygen and CO2. The sensor can be any type of invasive sensor known in the art for monitoring these parameters, wherein the sensor is in contact with the process fluid. In addition, in some embodiments, the sensor can be a non-invasive photochemical sensor, such as those described in, for example, U.S. Patent Nos. 6,673,532 and 7,041,493 and U.S. Patent Application Publication No. 20110065084. In addition, spectrometers known in the art can be used in seed culture equipment, production bioreactors and / or purification equipment to monitor product flow and / or the input of each device. The parameters measured by such spectrometers can include but are not limited to absorbance, multi-angle light scattering, fluorescence, Raman scattering, circular dichroism and infrared spectral characteristics.

[0186] In some embodiments, the equipment and processes in each zone are computer controlled and monitored via a computer system and corresponding software, which also allows the system to be automated. The equipment located in each zone may include data collection sensors and controls that can communicate with a central controller via a hardwired connection or a wireless connection.

[0187] In some embodiments, each area may additionally include an electronic control panel that may also be used to monitor, collect, and control the equipment and processes occurring therein. The central controller may also include a data storage device for storing data, and application programs and / or operating software for the central controller and system operations. The software may include a graphical user interface (GUI) that may be displayed on the workstation. The GUI may display a number of different screens for setting up, monitoring, and controlling the equipment and the overall manufacturing process.

[0188] Thus, in some embodiments, such software may be used to control a program within a zone, and optionally may be customized via a central computer and / or electronic control board. A program is a strategy for implementing a particular process within a zone (i.e., a material produced or produced by a single execution of a batch process). In some embodiments, a process may be a series of chemical, physical, or biological activities used to transform, transport, or store materials or energy.

[0189] In some embodiments, the sterile connection module connects one or more seed culture bioreactors in the reconfigurable single use area to one or more stainless steel bioreactors in the stainless steel area.

[0190] In some embodiments, a sterile connection module connects the harvesting unit to one or more downstream purification units.

[0191] Cell lines

[0192] In some embodiments, each bioreactor in the flexible facility 10 of the present disclosure is suitable for culturing prokaryotic cells or eukaryotic cells. In some embodiments, at least one bioreactor in the flexible facility 10 is suitable for culturing prokaryotic cells. In some embodiments, at least one bioreactor in the flexible facility 10 is suitable for culturing eukaryotic (e.g., mammalian) cells. In some embodiments, at least one bioreactor in the flexible facility 10 is suitable for culturing cells capable of producing viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy, and / or viral immunotherapy). In some embodiments, at least one bioreactor in the flexible facility 10 is suitable for culturing prokaryotic cells capable of producing viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy, and / or viral immunotherapy). In some embodiments, at least one bioreactor in the flexible facility 10 is suitable for culturing eukaryotic (e.g., mammalian) cells capable of producing viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy, and / or viral immunotherapy).

[0193] Examples of suitable cells for culturing in the bioreactors of the flexible facility 10 of the present disclosure include, but are not limited to, bacterial cells (e.g., E. coli, Pichia pastoris), yeast cells (e.g., Saccharomyces cerevisiae, Trichoderma reesei), plant cells, insect cells (e.g., Sf9), Chinese hamster ovary cells (CHO, and any genetically modified or derived CHO cell lines), mouse cells (e.g., mouse embryonic fibroblasts, cells derived from mouse cancer models), human cells (e.g., cells from any tissue or organ, cells from cancer or other diseased cell lines, stem cells), hybridoma cells or other genetically modified or hybrid cells. Suitable host cells for use in the bioreactors contained in the flexible facility of the present disclosure include those that can be purchased from, for example, culture collections (e.g., DSMZ (German Microbiological Collection, Brunswick, Germany) or American Type Culture Collection (ATCC)).

[0194] In certain embodiments, cell is bacterium or prokaryotic cell.In certain embodiments, prokaryotic cell is selected from gram-positive cell, such as: bacillus (such as, bacillus subtilis (such as, bacillus subtilis 3NA and bacillus subtilis 168), bacillus amyloliquefaciens, bacillus licheniformis, bacillus natto or bacillus megaterium), streptomyces, streptococcus, staphylococcus or lactobacillus cell.In certain embodiments, prokaryotic cell is selected from gram-negative cell, such as Salmonella species or Escherichia coli (such as, TGI, TG2, W3110, DH1, DHB4, DH5a, HMS174, HMS174 (DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue and Origami cell) and the cell (such as, BL-21 or BL21 (DE3)) derived from Escherichia coli B-strain.Prokaryotic cell or bacterial cell can be obtained from commercial supplier. Bacillus cells can be obtained, for example, from the Bacillus Genetic Collection, Biosciences 556, 484 West 12th Avenue, Columbus, OH 43210-1214.

[0195] In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell. The mammalian cell can be, for example, a human or rodent or bovine cell line or cell strain. Examples of such cells, cell lines or cell strains include, but are not limited to, for example, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, FIT 1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BF1K (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK-293, VERO, PER.C6, HeLa, EB1, EB2, EB3, oncolytic or hybridoma cell lines. In some embodiments, the mammalian cell is a CHO cell line. In some embodiments, the mammalian cell is a CHO cell. In some embodiments, the mammalian cell is selected from the group consisting of CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS cells, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN cells, and CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. Additionally, CHO FUT8 knockout cells are, for example, CHOK1SV (Lonza Co., Ltd.). In some embodiments, the eukaryotic cell may also be an avian cell, cell line or cell strain, such as cells, EB14, EB24, EB26, EB66 or EBv13.

[0196] In some embodiments, any mammalian cell line can be used in a bioreactor included in the flexible facility 10 of the present disclosure. As non-limiting examples, a wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, VA) and commercial suppliers. Non-limiting examples of cell lines commonly used in industry include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture (Graham et al., 1977, J. Gen Virol. Journal of General Virology 36:59)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. Reproduction Biology 23:243-251); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC 5 cells or FS4 cells; mammalian myeloma cells, as well as several other cell lines and Chinese hamster ovary (CHO) cells.

[0197] CHO cells, including CHOK1 cells (ATCC CCL61), are widely used to produce complex recombinant proteins. In some embodiments, mutant cell lines deficient in dihydrofolate reductase (DHFR) (Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), DXB11 and DG-44 are desirable CHO host cell lines because the highly efficient DHFR selectable and amplifiable gene expression system allows high-level recombinant protein expression in these cell lines (Kaufman RJ, 1990, Meth Enzymol 185:537-566). Also included are glutamine synthetase (GS)-knockout CHOK1SV cell lines using methionine sulfoximine (MSX) selection based on glutamine synthetase (GS). Other CHO host cells suitable for use in the flexible facility bioreactor of the present disclosure include, but are not limited to, the following (ECACC accession numbers are in parentheses): CHO (85050302); CHO (protein-free) (00102307); CHO-K1 (85051005); CHO-K1 / SF (93061607); CHO / dhFr- (94060607); CHO / dhFr-AC-free (05011002); and RR-CHOKI (92052129).

[0198] Large-scale production of proteins for commercial applications can be performed in suspension culture. Thus, the mammalian host cells used to generate the recombinant mammalian cells described herein can, but need not, be adapted to grow in suspension culture. Various host cells suitable for growing in suspension culture are known, including mouse myeloma NS0 cells and CLIO cells from the CFIO-S, DG44, and DXB11 cell lines. Other suitable cell lines include, but are not limited to, mouse myeloma SP2 / 0 cells, baby hamster kidney BF1K-21 cells, human PER. Cells, human embryonic kidney F1EK-293 cells, and cell lines derived from or engineered from any of the cell lines disclosed herein.

[0199] In some embodiments, the eukaryotic cell is selected from the following: a lower eukaryotic cell, such as a yeast cell (e.g., a Pichia spp. (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angus)), a Saccharomyces spp. (e.g., Saccharomyces pastoris, Saccharomyces pseudopasteur, or Saccharomyces filipinosus), a cell of the Saccharomyces spp. (e.g., Saccharomyces cerevisiae, Kluyveromyces, Saccharomyces vitis), a cell of the Kluyveromyces spp. (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), a cell of the genus Candida (e.g., Candida utilis, Candida theobrominus, Candida boidinii), a cell of the genus Geotrichum (e.g., Geotrichum fermentum), Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces pombe. In some embodiments, the eukaryotic cell is selected from a Pichia pastoris strain. Non-limiting examples of Pichia pastoris strains include X33, GS115, KM71, KM71H, and CBS7435.

[0200] In some embodiments, the eukaryotic cell is selected from a fungal cell (e.g., a cell of Aspergillus (e.g., Aspergillus niger, Aspergillus fumigatus, Aspergillus erinaceus, Aspergillus nidulans), Acremonium (e.g., Acremonium thermophilum), Chaetomium (e.g., Chaetomium thermophilum), Chrysosporium (e.g., Chrysosporium thermophilum), Cordyceps (e.g., Cordyceps militaris), Coryneformis, Ctenophora, Fusarium (e.g., Fusarium oxysporum), Aspergillus niger (e.g., Aspergillus nidulans), grisea), Hypocrea (e.g., Hypocrea rubra), Rice blast fungus (e.g., Magnaporthe oryzae), Myceliophthora (e.g., Myceliophthora thermophila), Erythrocrista (e.g., Erythrocrista rhamnosus), Neurospora (e.g., Neurospora crassa), Penicillium, Sporothrix (e.g., Thermospora thermophila), Thielavia (e.g., Thielavia terrestris, Thielavia heterocystis), Trichoderma (e.g., Trichoderma reesei), or Verticillium dahliae (e.g., Verticillium dahliae)).

[0201] In some embodiments, the eukaryotic cell is selected from insect cells (e.g., Sf9, Mimic TM Sf9, Sf21, HighFive TM The invention relates to cells of the invention to cells of the invention comprising: a plant cell (e.g., a cell from a monocotyledonous plant (e.g., corn, rice, wheat or foxtail grass) or a cell from a dicotyledonous plant (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella or Arabidopsis thaliana) and a plant cell (e.g., a cell from a monocotyledonous plant (e.g., corn, rice, wheat or foxtail grass) or a cell from a dicotyledonous plant (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens or Arabidopsis thaliana)).

[0202] In some embodiments, cells express or produce products, such as recombinant therapeutic products. As described in more detail below, examples of therapeutic products produced by cells include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimics (specifically bound to antigens but structurally unrelated to antibodies, such as DARPins, affibodies, adnectins or IgNARs), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapies (e.g., anticancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cell therapies (e.g., pluripotent stem cells, mesenchymal stem cells and adult stem cells), vaccines or lipid-coated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA) or DNA (e.g., plasmid DNA), antibiotics or amino acids. In some embodiments, flexible facilities according to the present disclosure can be used to produce biosimilars. In some embodiments, flexible facilities according to the present disclosure can be used to produce therapeutic products, which have a certain range of complexity and can include post-translational modifications (e.g., glycosylation).

[0203] In some embodiments, flexible facilities according to the present disclosure allow production of: eukaryotic cells, such as mammalian cells or lower eukaryotic cells (e.g., yeast cells or filamentous fungal cells); or prokaryotic cells, such as Gram-positive or Gram-negative cells; and / or products of eukaryotic or prokaryotic cells, such as proteins, peptides, antibiotics, amino acids, nucleic acids (e.g., DNA or RNA), which are synthesized by eukaryotic cells in a large-scale manner.

[0204] Cultivation methods

[0205] Various methods that may be used in commercial processes for producing recombinant proteins by mammalian cell culture include, but are not limited to: batch culture; fed-batch culture; and perfusion culture.

[0206] Batch culture is a discontinuous process in which cells are grown in a fixed volume of culture medium for a short period of time and then harvested completely. Cultures grown using batch methods experience an increase in cell density until a maximum cell density is reached, followed by a decrease in viable cell density as medium components are consumed and levels of metabolic byproducts (e.g., lactate and ammonia) accumulate. Harvesting typically occurs when a maximum cell density is reached (e.g., 5 x 10 6Cells / mL or higher, depending on media formulation, cell line, etc.). Batch processing is the simplest culture method; however, feasible cell density is limited by nutrient availability, and once cells are at maximum density, the culture declines and yields decrease. There is no ability to extend the production phase in batch cultures because the accumulation of waste products and depletion of nutrients quickly leads to culture decline, typically in about 3 to 7 days.

[0207] Fed-batch cultures improve upon batch processes by providing bolus or continuous media feeds to replenish those media components that have been consumed. Because fed-batch cultures receive additional nutrients throughout the run, they have the potential to achieve higher cell densities (>10 to 30 x 10 6 Cells / mL, depending on culture medium formulation, cell line, etc.) and the potential for increasing product titer. Different from batch process, biphasic culture can be produced and maintained by manipulating feed strategy and culture medium formulation to distinguish between the cell proliferation phase (growth phase) and the suspension or slow cell growth phase (production phase) that realize the desired cell density. Thus, compared with batch culture, fed-batch culture has the potential for obtaining higher product titer. Typically, a batch process is used during the growth phase, and a fed-batch process is used during the production phase, but the feeding strategy of fed-batch can be used throughout the entire process. However, different from batch process, bioreactor volume is the limiting factor that limits the amount of feed. And, like batch process, the accumulation of metabolic byproducts will cause the culture to decline, which limits the duration of the production phase, often about 10 to 21 days. Fed-batch culture is discontinuous, and harvest typically occurs when metabolic byproduct levels or culture vigor reach a predetermined level. When compared to batch cultures (where no feeding occurs), fed-batch cultures can produce larger amounts of recombinant protein. (See, e.g., U.S. Pat. No. 5,672,502.)

[0208] Perfusion methods provide potential improvements to batch and fed-batch methods by adding fresh medium during the culture period and removing spent medium at the same time. Perfusion culture is a culture in which the cell culture receives fresh perfusion feed medium while removing spent medium at the same time. Typical perfusion culture begins with a batch culture start-up that lasts for one or two days, followed by continuous, stepwise and / or intermittent addition of fresh feed medium to the culture and simultaneous removal of spent medium, wherein cells and additional high molecular weight compounds, such as proteins (based on the molecular weight cut-off of the filter) are retained throughout the growth and production phases of the culture. Various methods (e.g., precipitation, centrifugation or filtration) can be used to remove spent medium while maintaining cell density. A non-limiting example of a filtration method is alternating tangential flow filtration. Alternating tangential flow is maintained by pumping the culture medium through a hollow fiber filter module. See, for example, U.S. Patent No. 6,544,424; Furey, 2002, Gen. Eng. News. 22 (7): 62-63.

[0209] Perfusion can be continuous, stepwise, intermittent, or any combination of any of these or all. The perfusion rate can be less than one working volume to many working volumes per day. The cells are retained in the culture, and the spent medium that is removed is substantially free of cells or has significantly fewer cells than the culture. Recombinant proteins expressed by the cell culture can also be retained in the culture.

[0210] Typical large-scale commercial cell culture strategies strive to achieve high cell densities, 60–90(+) x 10 6 cells / mL, with almost one-third to more than half of the reactor volume being biomass. With perfusion culture, >1x10 8 The perfusion process has the potential to produce a culture that can be maintained for a longer period of time than batch or batch culture methods. However, increased preparation, use, storage and disposal of the culture medium are necessary to support long-term perfusion culture, especially for cultures with high cell density, which also require even more nutrients. All this increases production costs compared to batch and fed-batch methods. In addition, higher cell densities may cause problems during production, such as problems maintaining dissolved oxygen levels and increased aeration, including supplying more oxygen and removing more carbon dioxide, which may lead to greater foaming and the need to change the defoaming strategy; as well as problems during harvesting and downstream processing, where the effort required to remove excess cell material can result in product loss, thereby negating the benefits of increased titer due to increased cell mass.

[0211] An alternative large-scale cell culture strategy suitable for use in the flexible facility 10 of the present disclosure combines fed-batch feeding during the growth phase with continuous perfusion during the production phase. In some embodiments, fed-batch culture with bolus feeding is used to maintain cell culture during the growth phase. Perfusion feeding can then be used during the production phase. In some embodiments, perfusion begins when the cells have reached the production phase.

[0212] Therapeutic products

[0213] Various therapeutic products can be produced in flexible facilities according to the present disclosure. Polypeptides and proteins of interest (which represent a subset of therapeutic products) may have scientific or commercial interests, including, for example, protein-based treatments. Proteins of interest include, but are not limited to, secretory proteins, non-secretory proteins, intracellular proteins, or membrane-bound proteins. Polypeptides and proteins of interest can be produced by recombinant animal cell lines using cell culture methods, and can be referred to as "recombinant proteins". The expressed (multiple) proteins can be produced intracellularly or secreted into a culture medium, from which the protein can be recovered and / or collected. The term "isolated protein" or "isolated recombinant protein" refers to a polypeptide or protein of interest that is purified from proteins or polypeptides or other contaminants that interfere with its therapeutic, diagnostic, preventive, research, or other uses. Proteins of interest include, but are not limited to, proteins that exert therapeutic effects by binding to targets, such as targets among those listed below, including targets derived therefrom, targets associated therewith, and modifications thereof.

[0214] Proteins of interest may include, but are not limited to, "antigen binding proteins". "Antigen binding protein" refers to a protein or polypeptide that includes an antigen binding region or antigen binding portion that has affinity for another molecule (antigen) to which it binds. Antigen binding proteins include, but are not limited to, antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including, for example, single-chain variable fragments (scFv), double-chain (bivalent) scFv and IgGscFv (see, for example, Orcutt et al., 2010, Protein Eng Des Sel 23:221-228)), hybrid IgG (see, for example, Liu et al., 2015, Journal of Biol. Chem. 290:7535-7562), mutant proteins and (Xencor, Inc., Monrovia, California). Also included are bispecific T cell engagers of Bispecific T cell engagers (e.g., HLE BiTE, HeteroIg BITE, etc.), chimeric antigen receptors (CAR, CAR T), and T cell receptors (TCR) with extended (e.g., half-life extension).

[0215] In some embodiments, the protein of interest may include a colony stimulating factor, such as granulocyte colony stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to (filgrastim) and (Pegfilgrastim). Also included are erythropoiesis-stimulating agents (ESAs), such as (Epoetin α), (darbepoetin alfa), (Epoetine δ), (methoxy polyethylene glycol-epoetin beta), MRK-2578, INS-22, (Epoetin ζ), (Epoetin β), (Epoetin ζ), (Epoetin α), Epoetin α Hexal, (Epoetin α), (Epoetine θ), (Epoetine θ), (Epoetin theta), Epoetin alpha, Epoetin beta, Epoetin zeta, Epoetin theta and Epoetin delta, Epoetin omega, Epoetin iota, tissue plasminogen activator and GLP-1 receptor agonist, and variants or analogs thereof and biosimilars thereof of any of the foregoing.

[0216] In some embodiments, the protein of interest binds to one or more of the following, alone or in any combination: CD proteins, including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174; HER receptor family proteins, including, for example, HER2, HER3, HER4, and EGF receptor, EGFRvIII, cell adhesion molecules (e.g., LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCA M and αv / β3 integrins); growth factors, including, but not limited to, for example, vascular endothelial growth factor ("VEGF"); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian inhibitory substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factor (e.g., NGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor (including, for example, aFGF and bFGF), epidermal growth factor (EGF), Cripto, transforming growth factor (TGF) (especially including TGF-α and TGF-β (including TG F-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5)), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and osteoinductive factors, insulin and insulin-related proteins (including but not limited to insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding protein); blood proteins and coagulation-related proteins, especially factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators (such as urokinase and tissue plasminogen activator ("t-PA")), bombazine, thrombin, thrombopoietin and thrombopoietin receptors, colony stimulating factors (CSFs) (including, inter alia, the following: M-CSF, GM-CSF and G-CSF), other blood and serum proteins (including, but not limited to, albumin, IgE and blood group antigens), receptors and receptor-associated proteins (including, for example, flk2 / flt3 receptors, obesity (OB) receptors, growth hormone receptors and T cell receptors); neurotrophic factors, including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6);Relaxin A chain, relaxin B chain and relaxin pro-like protein, interferons (including, for example, interferon α, interferon β and interferon γ), interleukins (IL) (e.g., IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 or IL-17 receptor, IL-1RAP); viral antigens, including but not limited to AIDS envelope virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic peptide, lung surface Active agents, tumor necrosis factor-α and tumor necrosis factor-β, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (normal T cell expressed and secreted factor regulated by activation), mouse gonadotropin-related peptide, DNA enzyme, FR-α, inhibin and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay accelerating factor (DAF), AIDS envelope, transporter, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen binding protein, growth hormone, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2 , NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv [PE38] conjugate, Legionella pneumophila (lly), IFNγ, interferon gamma inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / Kexin 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1)), transforming growth factor β (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and biologically active fragments or variants of any of the foregoing. ;

[0217] In some embodiments, the protein of interest comprises abciximab, adalimumab, adelimumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosozumab, blinatumomab, brentuximab, brodalumab, mocantuzumab, canakinumab, cetuximab, certolizumab, canatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, etanercept, evolocumab, galiximab, ganitazumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab muma, lerdizumab, ruximab, lxdkizumab, mapatumumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, oremumab, ofatumumab, omalizumab, oprelleukin, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab, rituximab, rituximab, romiplostim, lomosozumab, sargramostim, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, visilizumab, volocizumab, zalumab and zalutumumab, and biosimilars of any of the foregoing.

[0218] In some embodiments, the protein of interest may also include genetically engineered receptors, such as chimeric antigen receptors (CAR or CAR-T) and T cell receptors (TCR) and other proteins including antigen binding molecules that interact with the targeted antigen. By incorporating antigen binding molecules that interact with the targeted antigen, CAR can be engineered to bind to antigens (e.g., cell surface antigens). CAR typically connects antigen binding domains (e.g., scFv) in series with one or more costimulatory ("signaling") domains and one or more activation domains.

[0219] All documents or parts of documents cited in this application, including but not limited to patents, patent applications, articles, books and monographs, are hereby expressly incorporated by reference. Unless the context clearly indicates otherwise, the content described in the embodiments of the present disclosure can be combined with one or more other embodiments of the present disclosure.

[0220] The disclosed subject matter is not intended to be limited in scope by the specific embodiments described herein, which are instead intended as non-limiting illustrations of various aspects of the present disclosure. Functionally equivalent methods and components are within the scope of the present disclosure. In fact, various modifications of the disclosed subject matter, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the disclosed subject matter.

[0221] The descriptions of various embodiments and / or examples of the disclosed subject matter are presented for the purpose of illustration, but are not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terms used herein are selected to best explain the principles of the embodiments, practical applications, or technical improvements over technologies existing in the marketplace, and / or to enable others of ordinary skill in the art to understand the disclosed subject matter.

Claims

1. A flexible facility for manufacturing at least one therapeutic product, the flexible facility comprising: a reconfigurable single-use area comprising at least one single-use bioreactor, wherein one or more of the at least one single-use bioreactor has a volume of at least 1000 L; and A stainless steel area, comprising at least one stainless steel bioreactor, wherein each stainless steel bioreactor in the stainless steel area has a volume greater than 5000 L, Wherein, one or more of the at least one stainless steel bioreactor is fluidly connected to one or more of the at least one single-use bioreactor.

2. The flexible facility according to claim 1, wherein: One or more of the at least one single-use bioreactor has a volume of at least 2000L.

3. A flexible facility according to claim 1 or claim 2, wherein: The stainless steel area includes a plurality of stainless steel bioreactors, each stainless steel bioreactor having a volume of at least 10,000 L.

4. A flexible facility according to any one of claims 1 to 3, wherein: The reconfigurable single use area and the stainless steel area are separated by a divider.

5. The flexible facility of claim 4, wherein: The divider is a wall.

6. The flexible facility of claim 5, wherein: This stainless steel area is completely enclosed and has separate air handling.

7. The flexible facility of claim 5, wherein: A portion of the at least one stainless steel bioreactor penetrates the wall and is fluidly connected to one or more of the at least one single-use bioreactor via tubing.

8. The flexible facility of claim 7, wherein: The portion penetrating the wall includes one or more components independently selected from the group consisting of: a probe strip, a connection for perfusion, and a feed solution tank connection.

9. A flexible facility according to any one of claims 1 to 8, wherein: The fluid connection is configured to enable bypassing of the stainless steel region.

10. A flexible facility according to any one of claims 1 to 9, wherein: The reconfigurable single use area and / or the stainless steel area further comprises one or more centrifuges.

11. A flexible facility according to any one of claims 1 to 10, wherein: The reconfigurable single-use area includes one or more seed cultivation sections.

12. A flexible facility according to any one of claims 1 to 11, wherein: The reconfigurable single-use area further includes one or more purification skids.

13. The flexible facility of claim 12, wherein: Each of the one or more purification skids comprises one or more components independently selected from the group consisting of a chromatography skid, a chromatography column, a virus inactivation system, a virus filtration system, an ultrafiltration diafiltration system, and a batch filtration system.

14. The flexible facility according to any one of claims 1 to 13, further comprising one or more additional spaces independently selected from the group consisting of utility spaces, media buffer facilities, personnel changing rooms, and production units.

15. A flexible facility according to any one of claims 1 to 14, wherein: The flexible facility is configured to produce one or more therapeutic products independently selected from the group consisting of: monoclonal antibodies, molecules, RNA-based drug substances, cell therapeutics and fermentation products.

16. A flexible facility according to any one of claims 1 to 15, wherein: The flexible facility is configured to implement one or more processes independently selected from the group consisting of a mammalian cell culture fed-batch process, a mammalian cell culture perfusion process, a continuous mammalian cell culture process, a microbial fermentation fed-batch process, and a combination of any of the foregoing.

Images