Fixed bed bioreactor with reduced bypass and related methods

Abstract

A bioreactor system is provided that includes a cell culture vessel having an interior reservoir defined by an interior wall. A fixed-bed cell culture matrix having a plurality of layers of substrate is contained within the interior reservoir and has a bed height and a bed width in a direction perpendicular to the direction of the bed height. The system further includes a compression sleeve surrounding at least a portion of the fixed-bed cell culture matrix. The compression sleeve exerts a compressive force in a direction of the bed width. The compressive force is sufficient to compress the bed width to a compressed bed width that is less than the reservoir width, and the compression sleeve is configured to release the compressive force so that the bed width increases to an expanded bed width.

Description

Attorney Docket No.: SP24-308FIXED BED BIOREACTOR WITH REDUCED BYPASS AND RELATED METHODSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 724,653 filed on November 25, 2024, the content of which is relied upon and incorporated herein by reference in its entirety their entireties.FIELD OF DISCLOSURE

[0002] This disclosure general relates to cell culture substrates and perfusion-based, fixed-bed bioreactors incorporating such substrates, as well as systems and methods for culturing cells. In particular, the present disclosure relates to fixed bed cell culture substrates and bioreactors with such substrates with reduced bypass around the fixed bed under perfusion cell culture conditions.BACKGROUND

[0003] In the bioprocessing industry, large-scale cultivation of cells is performed for purposes of the production of hormones, enzymes, antibodies, vaccines, and cell therapies. Cell and gene therapy markets are growing rapidly, with promising treatments moving into clinical trials and quickly toward commercialization. However, one cell therapy dose can require billions of cells or trillions of viruses. As such, being able to provide a large quantity of cell products in a short amount of time is critical for clinical success.

[0004] A significant portion of the cells used in bioprocessing are anchorage dependent, meaning the cells need a surface to adhere to for growth and functioning. Traditionally, the culturing of adherent cells is performed on two-dimensional (2D) cell-adherent surfaces incorporated in one of a number of vessel formats, such as T-flasks, petri dishes, cell factories, cell stack vessels, roller bottles, and HYPERStack® vessels. These approaches can have significant drawbacks, including the difficulty in achieving cellular density high enough to make it feasible for large scale production of therapies or cells.Attorney Docket No.: SP24-308

[0005] Alternative methods have been suggested to increase volumetric density of cultured cells. These include microcarrier cultures performed in stir tanks. In this approach, cells that are attached to the surface of microcarriers are subject to constant shear stress, resulting in a significant impact on proliferation and culture performance. Another example of a high-density cell culture system is a hollow fiber bioreactor, in which cells may form large three-dimensional aggregates as they proliferate in the interspatial fiber space. However, the cells growth and performance are significantly inhibited by the lack nutrients. To mitigate this problem, these bioreactors are made small and are not suitable for large scale manufacturing

[0006] Another example of a high-density culture system for anchorage dependent cells is a packed-bed bioreactor system. In this this type of bioreactor, a cell substrate is used to provide a surface for the attachment of adherent cells. Medium is perfused along the surface or through the semi-porous substrate to provide nutrients and oxygen needed for the cell growth. For example, packed bed bioreactor systems that contain a packed bed of support or matrix systems to entrap the cells have been previously disclosed U.S. Patent Nos. 4,833,083; 5,501,971; and 5,510,262. Packed bed matrices usually are made of porous particles as substrates or non-woven microfibers of polymer. Such bioreactors can function as recirculation flow-through bioreactors. One of the significant issues with such bioreactors is the non-uniformity of cell distribution inside the packed bed. The packed bed often functions as depth filter with cells predominantly trapped at the inlet regions, resulting in a gradient of cell distribution during the inoculation step. In addition, due to random fiber packaging, flow resistance and cell trapping efficiency of cross sections of the packed bed are not uniform. For example, medium flows fast though the regions with low cell packing density and flows slowly through the regions where resistance is higher due to higher number of entrapped cells. This creates a channeling effect where nutrients and oxygen are delivered more efficiently to regions with lower volumetric cells densities and regions with higher cell densities are being maintained in suboptimal culture conditions.

[0007] Another significant drawback of packed bed systems disclosed in a prior art is the inability to efficiently harvest intact viable cells at the end of culture process. Harvesting of cells is important if the end product is cells, or if the bioreactor is being used as part of a “seed train,” where a cell population is grown in one vessel and then transferred to another vessel for furtherAttorney Docket No.: SP24-308 population growth. U.S. Patent No. 9,273,278 discloses a bioreactor design to improve the efficiency of cell recovery from the packed bed during cells harvesting step. It is based on loosening the packed bed matrix and agitation or stirring of packed bed particles to allow porous matrices to collide and thus detach the cells. However, this approach is laborious and may cause significant cells damage, thus reducing overall cell viability.

[0008] Certain examples of packed-bed bioreactors currently on the market use small strips of cell substrate material consisting of randomly oriented fibers in a non-woven arrangement. These strips are randomly packed into a vessel to create a packed bed. However, the non-uniform packing of the substrate strips creates visible channels within the packed bed, leading to preferential and non-uniform media flow and nutrient distribution through the packed bed. Studies of such commercially available bioreactors have noted a “systemic inhomogeneous distribution of cells, with their number increasing from top to bottom of fixed bed,” as well as a “nutrient gradient... leading to restricted cell growth and production,” all of which lead to the “unequal distribution of cells [that] may impair transfection efficiency.” (Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells. BiotechnoL J. 2016, 11, 290-297). Studies have noted that agitation of the packed bed may improve dispersion, but would have other drawbacks (i.e., “necessary agitation for better dispersion during inoculation and transfection would induce increased shear stress, in turn leading to reduced cell viability.” Id.). Another study noted that the uneven distribution of cells makes monitoring of the cell population using biomass sensors difficult (“... if the cells are unevenly distributed, the biomass signal from the cells on the top carriers may not show the general view of the entire bioreactor.” Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale.Human Gene Therapy, Vol. 26, No. 8, 2015).

[0009] In addition, because of the random arrangement of fibers in the substrate strips and the variation in packing of strips between one packed bed and another of a given packed bed reactor system, it can be difficult for users to predict cell culture performance, since the substrate varies between cultures. Furthermore, the packed substrate of many existing bioreactors makesAttorney Docket No.: SP24-308 efficiently harvesting cells very difficult or impossible, as it is believed that cells are entrapped by the packed bed.

[0010] Existing fixed bed or packed bed bioreactor systems are designed around the concept of a loosely -packed bed, which has been suggested to improve fluid flow. This loose packing results in a low-occupancy bed region (i.e., open spaces are left in the reactor volume due to the reactor not being filled with a substrate to maximum occupancy, let alone having the substrate being compressed into a tightly packed bed).

[0011] While manufacturing of viral vectors for early-phase clinical trials is possible with existing platforms, there is a need for a platform that can produce high-quality product in greater numbers in order to reach late-stage commercial manufacturing scale.

[0012] There is a need for cell culture substrates and / or matrices, bioreactors, systems, and methods that enable culturing of cells in a high-density format, with uniform cell distribution, and easily attainable and increased harvesting yields. In particular, there is a need for cell culture substates for fixed bed bioreactors that have improved fluid flow characteristics with reducing fluid flow channeling and reduced fluid flow bypass around the cell culture substrate.SUMMARY

[0013] According to embodiments of this disclosure, a bioreactor system for culturing cells is provided. The system comprises a cell culture vessel comprising at least one interior reservoir defined by an interior wall; and a cell culture matrix disposed in the reservoir, the cell culture matrix comprising a plurality of substrates configured for adhering cells thereto, the plurality of substrates being in a stacked arrangement, wherein the cell culture matrix comprises a matrix width extending across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted.

[0014] According to embodiments of this disclosure, a bioreactor system for culturing cells is provided that has a cell culture vessel with at least one interior reservoir defined by an interior wall. The system also includes a cell culture matrix disposed in the reservoir. The cell culture matrix includes a multi-layered substrate configured for adhering cells thereto. The multi-layered substrate includes at least one substrate sheet rolled about a central cylinder axis in a cylindricalAttorney Docket No.: SP24-308 rolled arrangement. The cell culture matrix extends across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted.

[0015] In aspects of embodiments, the cell culture matrix includes a plurality of substrate sheets rolled together about a central cylinder axis in a cylindrical rolled arrangement. The plurality of substrate sheets can be stacked and then rolled, such that there are alternating substrate sheets in the roll, or the plurality of substrate sheets can be rolled separately, with one or more sheets making an inner core of the cylindrical rolled arrangement and one or more sheets making an outer section of the cylindrical rolled arrangement. In aspects of embodiments, the cell culture matrix includes a plurality of rolled substrate sheets arranged as a plurality of cylindrical substrate sections in a stacked arrangement.

[0016] In aspects of embodiments, the plurality of substrates is under compression in a compression direction that is substantially parallel to a stacking direction in which the plurality of substrates is stacked and substantially perpendicular to the reservoir width. The cell culture matrix comprises a height measured in the stacking direction, and the height is compressed by at least 10% compared to the plurality of substrates when under no compression. The height is compressed by at least 20% compared to the plurality of substrates when under no compression. The system can further comprise an O-ring disposed at a top or a bottom of the cell culture matrix in the stacking direction, the O-ring being disposed along a circumference of the interior wall. The O-ring comprises an elastomeric material.

[0017] In aspects of embodiments, one or more of the substrates of the plurality of substates have a peripheral edge with an edge thickness that is greater than a thickness of an interior of the one or more substrates. The peripheral edge is compressible in the edge thickness direction. Two or more consecutive layers of the plurality of substrates are joined at peripheral edges of the layers forming a jointed layer. The plurality of substrates can comprise a plurality of jointed layers. The peripheral edges can be jointed by at least one of an adhesive material, a molded material, and a melted edge of one or more of the peripheral edges. The jointed layer can include layers of differing properties. The differing properties can include at least one of a physical dimension, a material composition, a stiffness, and a porosity. The plurality of substates can beAttorney Docket No.: SP24-308 comprises of fibers, with the physical dimension comprising a fiber diameter. Also, the physical dimension can comprises a pore diameter.

[0018] In aspects of embodiments, each of the plurality of substrates comprising a first major surface separated from a second major surface by a thickness, the plurality of substrates being stacked such that the first major surface of one of the plurality of substrates faces a second major surface of another of the plurality of substrates. The system can further include an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir. The cell culture matrix is disposed between the inlet and the outlet. The cell culture vessel is configured to flow fluid into the interior reservoir via the inlet, through the cell culture matrix, and out of the interior reservoir via the outlet in substantially a linear path between the inlet and the outlet. The cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix.

[0019] In aspects of embodiments, the cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix, such that the uniform flow satisfies the following expressions:^ E(0)d0 = P,AO = 02 - 0i, and A0 <N, where P is greater than or equal to at least one of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.95, and where A is less than or equal to at least one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0

[0020] In aspects of embodiments, the uniform flow is maintained through a bed height of the fixed bed, the bed height being greater than or equal to 10 cm; 20 cm; 30 cm; 40 cm; 50 cm; 60 cm; 70 cm; 80 cm; 90 cm; or 100 cm. In aspects, each of the plurality of substrates comprises a physical structure and a porosity that is substantially regular and uniform. In aspects, the plurality of substates comprises at least one of a molded polymer lattice, a 3D-printed lattice, and a woven substrate. In aspects, the cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix, such that E(0) is less than or equal to 0.2 or less than or equal to 0.1, when 0 is less than or equal to 0.8, 0.75, 0.7, 0.6, or 0.5.Attorney Docket No.: SP24-308BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A shows a perspective view of a three-dimensional model of a cell culture substrate, according to one or more embodiments of this disclosure.

[0022] Figure IB is a two-dimensional plan view of the substrate of Figure 1A.

[0023] Figure 1C is a cross-section along line A-A of the substrate in Figure IB.

[0024] Figure 2 showis a perspective of a cell culture system with a fixed bed of vertically stacked substrate layers, according to embodiments.

[0025] Figure 3 is a plan view of a cell culture system with a fixed bed with a cylindrical rolled configuration, according to embodiments.

[0026] Figure 4A shows a plan view of a modeled multi-layer woven mesh cell culture substrate in a tightly packed arrangement, according to one or more embodiments of this disclosure.

[0027] Figure 4B shows a side cross-section view of the multi-layer woven mesh cell culture substrate of Figure 4A, according to one or more embodiments of this disclosure.

[0028] Figure 5A shows a plan view of a modeled multi-layer woven mesh cell culture substrate in a loosely packed arrangement, according to one or more embodiments of this disclosure.

[0029] Figure 5B shows a side cross-section view of the multi-layer woven mesh cell culture substrate of Figure 5 A, according to one or more embodiments of this disclosure.

[0030] Figure 6 A shows the modeled empty space in the dotted-line volume shown in Figures5 A and 5B.

[0031] Figure 6B shows the modeled empty space in the dotted-line volume shown in Figures 5 A and 5B.

[0032]

[0033] Figure 7 is a schematic drawing of an experimental setup for measuring residence time distribution of different cell culture substrate samples.

[0034] Figure 8 A is a photograph of a stack of substrate disks, according to embodiments of this disclosure, at 100% occupancy for an experimental bioreactor,Attorney Docket No.: SP24-308

[0035] Figure 8B is a photograph of a stack of substrate disks, according to embodiments of this disclosure, at 95% occupancy for an experimental bioreactor.

[0036] Figure 9 A is a photograph of a bioreactor with a cell culture matrix of stacked layers of substate packed at 95% occupancy with compressed packing, according to one or more embodiments.

[0037] Figure 9B is a photograph of a bioreactor with a cell culture matrix of stacked layers of substate packed at 95% occupancy with loose packing, according to one or more embodiments.

[0038] Figure 10A is a graph of the residence time distribution data from three different packing densities in compress packing configurations, according to one or more embodiments.

[0039] Figure 10B is a graph of the residence time distribution data from three different packing densities in loose packing configurations, according to one or more embodiments.

[0040] Figure 11 A is a graph of the residence time distribution data from a fixed bed at 102.5% occupancy at different flow rates, according to one or more embodiments.

[0041] Figure 1 IB is a graph of the residence time distribution data from a fixed bed at 95% occupancy with compressed packing at different flow rates, according to one or more embodiments.

[0042] Figure 11C is a graph of the residence time distribution data from a fixed bed at 95% occupancy with loose packing at different flow rates, according to one or more embodiments.

[0043] Figure 12A is a graph of the residence time distribution data from fixed beds in various occupancy percentages with compressed packing and the presence of bubbles, according to one or more embodiments.

[0044] Figure 12B is a graph of the residence time distribution data from fixed beds in various occupancy percentages with loose packing and the presence of bubbles, according to one or more embodiments.

[0045] Figure 13A is a photograph of an example mesh substrate of a first size, according to embodiments.

[0046] Figure 13B is a photograph of an example mesh substrate of a second size that is a higher- density mesh than that of the first size in Figure 13 A, according to embodiments.Attorney Docket No.: SP24-308

[0047] Figure 13C is a photograph of an example mesh substrate of a third size that is a higher- density mesh than that of the second size in Figure 13B, according to embodiments.

[0048] Figure 13D is a photograph of an example mesh substrate of a fourth size that is a higher-density mesh than that of the third size in Figure 13C, according to embodiments.

[0049] Figure 14 is a model of the effect on fluid flow of a gap between the fixed bed and the vessel wall, according to embodiments.

[0050] Figure 15 is a graph of the effect of gap size between the fixed bed and the vessel wall on fluid bypass, according to embodiments.

[0051] Figure 16 is a photograph of crystal violet-stained mesh desks from a bioreactor showing the effect of edge bypass, according to embodiments.

[0052] Figure 17 is a side view of a wrapped fixed bed and bioreactor, according to embodiments.

[0053] Figure 18A is a side view illustration of an unrolled substrate sheet with a film on a portion of the sheet to reduce bypass when the substrate sheet is rolled into a rolled fixed bed, according to embodiments.

[0054] Figure 18B is a plan view of a bioreactor with a fixed bed made from the substrate sheet of Figure 18 A, according to embodiments.

[0055] Figure 19A is a side view of a fixed bed substrate and bioreactor vessel with edge sealing prior to inserting the fixed bed, according to embodiments.

[0056] Figure 19B is a side view of the fixed bed substrate and bioreactor vessel of Figure 19A after insertion of the fixed bed, according to embodiments.

[0057] Figure 20 is a side view of a fixed bed and bioreactor vessel with edge sealing, according to embodiments.

[0058] Figure 21 A is a side view of rolled fixed bed and a bioreactor vessel with an expandable sealant between the fixed bed and the vessel wall, according to embodiments.

[0059] Figure 21 AB is a side view of the system of Figure 21 A after expansion of the sealant to reduce edge bypass, according to embodiments.Attorney Docket No.: SP24-308

[0060] Figure 22 is a side view of a fixed bed made from stacked substrate layers with side tabs of the substrate layers extending out from the fixed bed to seal the edge from bypass, according to embodiments.

[0061] Figure 23 is a side view of a fixed bed and vessel, where the vessel is made from a shrinkable material, according to embodiments.DETAILED DESCRIPTION

[0062] Various embodiments of the disclosure will be described in detail with reference to drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments of the claimed invention.

[0063] Embodiments of this disclosure include a cell culture substrate, as well as cell culture or bioreactor systems incorporating such a substrate, and methods of culturing cells using such a substrate and bioreactor systems. The substrate and bioreactor systems incorporating the same exhibit improved flow characteristics through the substrate. For example, more uniform flow is achieved through the substrate, and non-uniform flow resulting from channeling or turbulent flow is reduced or eliminated. Flow “dead zones” in the substrate or fixed bed of the bioreactor are greatly reduced or eliminated compared to existing solutions. The result is a substrate or fixed bed that allows for uniform perfusion throughout the substrate or fixed bed, which promotes cell health during cell culture and an efficient cell culture process in terms of not only the culturing of cells, but also cell seeding, and harvesting of cells or cell by-products.

[0064] In conventional large-scale cell culture bioreactors, different types of fixed bed or packed bed bioreactors have been used. Usually these packed beds contain porous matrices to retain adherent or suspension cells, and to support growth and proliferation. Packed-bed matrices provide high surface area to volume ratios, so cell density can be higher than in the other systems. However, the packed bed often functions as a depth filter, where cells are physically trapped in the entangled fibers of the matrix. Thus, because of linear flow of the cell inoculum through the packed bed, cells are subject to heterogeneous distribution inside the packed bed,Attorney Docket No.: SP24-308 leading to variations in cell density through the depth or width of the packed bed. For example, cell density may be higher at the inlet region of a bioreactor and significantly lower nearer to the outlet of the bioreactor. This non-uniform distribution of the cells inside of the packed bed significantly hinders scalability and predictability of such bioreactors in bioprocess manufacturing, and can even lead to reduced efficiency in terms of growth of cells or viral vector production per unit surface area or volume of the packed bed.

[0065] Another problem encountered in packed bed bioreactors disclosed in prior art is the channeling effect. Due to random nature of packed nonwoven fibers, the local fiber density at any given cross section of the packed bed is not uniform. Liquid media flows quickly in the regions with low fiber density (high bed permeability) and much slower in the regions of high fiber density (lower bed permeability). The resulting non-uniform media perfusion across the packed bed creates the channeling effect, which manifests itself as significant nutrient and metabolite gradients that negatively impact overall cell culture and bioreactor performance. Cells located in the regions of low media perfusion will starve and very often die from the lack of nutrients or metabolite poisoning. The flow can be so non-uniform that there are effectively flow “dead zones” in the packed bed, where perfusion does not occur and the delivery of any nutrients to cells in those areas is limited to diffusion mechanics in the media.

[0066] Cell harvesting is yet another problem encountered when bioreactors packed with nonwoven fibrous scaffolds are used. Due to packed-bed functions as depth filter, cells that are released at the end of cell culture process are entrapped inside the packed bed, and cell recovery is very low. This significantly limits utilization of such bioreactors in bioprocesses where live cells are the products or where live cells need to be harvested for further processing to capture cell by-products. Thus, the non-uniformity leads to areas with different exposure to flow and shear, effectively reducing the usable cell culture area, causing non-uniform culture, and interfering with transfection efficiency and cell release.

[0067] In addition, while existing solutions suggest that loosely packed beds are preferable, the current disclosure will illustrate the converse: densely packed beds, in accordance with embodiments of the present disclosure, can actually improve performance of a fixed bed for cell culture. For example, embodiments of this disclosure show superior flow uniformity at highAttorney Docket No.: SP24-308 packed densities of substrate material in the bioreactor. Embodiments also show a reduction in the presence of bubbles in the fixed bed and the bioreactor system, in general. Nonetheless, even if bubbles are present in the system, the uniform fluid flow characteristics described herein can be maintained. Bubbles are commonly observed during cell cultures and can easily be trapped in fixed bed bioreactors, including the substrates. Once trapped, they disturb flow direction and reduce flow uniformity, therefore interfering with cell seeding and nutrient supply. For cell culture in a fixed bed reactor, higher flow rates are consistently used to maintain oxygen and nutrient supply to support high density cell growth. High flow rates can potentially lift mesh disks in the packed bed if the disks are not restricted. This can change local permeability and disturb flow direction. By using high compression and high occupancy, the disks are restricted in place and the negative impacts from bubbles and high flow rates can be mitigated. The loose packing or shifting of the packed bed can also mean inconsistent packing of packed beds in conventional systems, which can result in variations in permeability and channeling. However, when the bed is compacted according to embodiments of this disclosure, these negative flow characteristics can be avoided.

[0068] The above advantages of embodiments of this disclosure are quite unexpected in light of the preference for loosely-packed beds in the marketplace. These improvements also exist in embodiments regardless of fluid flow rate (including, e.g., channeling) or size of the fixed bed or bioreactor. Thus, the advantages are present across a range of bioreactor scales. A further advantage includes reducing or eliminating shifting of the fixed bed or a change in compaction of the fixed bed due to being subjected to various flow rates or due to mechanical forces during handling and / or shipping. In addition, the higher compaction of the fixed bed results in more surface area for cells.

[0069] As used herein, “occupancy” is defined as percentage of substrate packed into the bed volume of the reactor, relative to 100% occupancy. To determine 100% occupancy, the space height of the reactor bed space is divided by the thickness of each substrate layer to obtain the number of substrate layers for 100% occupancy. Occupancies of less than 100% (also referred to as loose packing) is obtained by removing the requisite percentage of mesh layers. For example, removing 5% of the layers from the 100% occupancy condition results in 95% occupancy. It isAttorney Docket No.: SP24-308 also possible to achieve greater than 100% occupancy. For example, due to some elasticity in the material of a substrate, the bed could be compressed to some extent and thus create additional space for most substate layers. If additional layers are added to that space above the 100% occupancy number of layers, then the resulting occupancy would be greater than 100%. For example, if 2.5% extra layers are added, the occupancy would be 102.5%.

[0070] To address these and other problems of existing cell culture solutions, embodiments of the present disclosure provide cell growth substrates, matrices of such substrates, and / or packed- bed systems using such substrates that enable efficient and high-yield cell culturing for anchorage-dependent cells and production of cell products (e.g., proteins, antibodies, viral particles). Embodiments include a porous cell-culture matrix made from an ordered and regular array of porous substrate material that enables uniform cell seeding and media / nutrient perfusion, as well as efficient cell harvesting. Embodiments also enable scalable cell-culture solutions with substrates and bioreactors capable of seeding and growing cells and / or harvesting cell products from a process development scale to a full production size scale, without sacrificing the uniform performance of the embodiments. For example, in some embodiments, a bioreactor can be easily scaled from process development scale to product scale with comparable viral genome per unit surface area of substrate (VG / cm2) across the production scale. The harvestability and scalability of the embodiments herein enable their use in efficient seed trains for growing cell populations at multiple scales on the same cell substrate. In addition, the embodiments herein provide a cell culture matrix having a high surface area that, in combination with the other features described, enables a high yield cell culture solution. In some embodiments, for example, the cell culture substrate and / or bioreactors discussed herein can produce 1016to 1018viral genomes (VG) per batch.

[0071] In one embodiment, a matrix is provided with a structurally defined surface area for adherent cells to attach and proliferate that has good mechanical strength and forms a highly uniform multiplicity of interconnected fluidic networks when assembled in a packed bed or other bioreactor. In particular embodiments, a mechanically stable, non-degradable woven mesh can be used as the substrate to support adherent cell production. The cell culture matrix disclosed herein supports attachment and proliferation of anchorage dependent cells in a high volumetricAttorney Docket No.: SP24-308 density format. Uniform cell seeding of such a matrix is achievable, as well as efficient harvesting of cells or other products of the bioreactor. In addition, the embodiments of this disclosure support cell culturing to provide uniform cell distribution during the inoculation step and achieve a confluent monolayer or multilayer of adherent cells on the disclosed matrix, and can avoid formation of large and / or uncontrollable 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. Thus, the matrix eliminates diffusional limitations during operation of the bioreactor. In addition, the matrix enables easy and efficient cell harvest from the bioreactor. The structurally defined matrix of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of the bioreactor.

[0072] According to some embodiments, a method of cell culturing is also provided using bioreactors with the matrix for bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0073] In contrast to existing cell culture substrates used in cell culture bioreactors (i.e., nonwoven substrates of randomly ordered fibers), embodiments of this disclosure include a cell culture substrate having a defined and ordered structure. The defined and order structure allows for consistent and predictable cell culture results. In addition, the substrate has an open porous structure that prevents cell entrapment and enables uniform flow through the packed bed. This construction enables improved cell seeding, nutrient delivery, cell growth, and cell harvesting. According to one or more particular embodiments, the matrix is formed with a substrate material having a thin, sheet-like construction having first and second sides separated by a relatively small thickness, such that the thickness of the sheet is small relative to the width and / or length of the first and second sides of the substrate. In addition, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is of a size and geometry that allows cells to adhere to the surface of the substrate material as if it were approximately a two-dimensional (2D) surface, while also allowing adequate fluid flow around the substrate material and through the openings. In some embodiments, the substrate is a polymer-based material, and can be formed as a molded polymer sheet; a polymer sheet with openings punched through the thickness; a number of filaments that are fused into a mesh-likeAttorney Docket No.: SP24-308 layer; a 3D-printed substrate; or a plurality of filaments that are woven into a mesh layer. The physical structure of the matrix has a high surface-to-volume ratio for culturing anchorage dependent cells. According to various embodiments, the matrix can be arranged or packed in a bioreactor in certain ways discussed here for uniform cell seeding and growth, uniform media perfusion, and efficient cell harvest.

[0074] Embodiments of this disclosure can achieve viral vector platforms of a practical size that can produce viral genomes on the scale of greater than about 1014viral genomes per batch, greater than about 1015viral genomes per batch, greater than about 1016viral genomes per batch, greater than about 1017viral genomes per batch, or up to or greater than about g 1016viral genomes per batch. In some embodiments, productions is about 1015to about 1018or more viral genomes per batch. For example, in some embodiments, the viral genome yield can be about 1015to about 1016viral genomes or batch, or about 1016to about 1019viral genomes per batch, or about 1016-l 018viral genomes per batch, or about 1017to about 1019viral genomes per batch, or about 1018to about 1019viral genomes per batch, or about 1018or more viral genomes per batch.

[0075] In addition, the embodiments disclosed herein enable not only cell attachment and growth to a cell culture substrate, but also the viable harvest of cultured cells. The inability to harvest viable cells is a significant drawback in current platforms, and it leads to difficulty in building and sustaining a sufficient number of cells for production capacity. According to an aspect of embodiments of this disclosure, it is possible to harvest viable cells from the cell culture substrate, including between 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, of the cells that are harvested, at least 80% are viable, at least 85% are viable, at least 90% are viable, at least 91% are viable, at least 92% are viable, at least 93% are viable, at least 94% are viable, at least 95% are viable, at least 96% are viable, at least 97% are viable, at least 98% are viable, or at least 99% are viable. Cells may be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0076] Figures 1 A and IB show a three-dimensional (3D) perspective view and a two- dimensional (2D) plan view, respectively, of a cell culture substrate 100, according to an example of one or more embodiments of this disclosure. The cell culture substrate 100 is a woven mesh layer made of a first plurality of fibers 102 running in a first direction and a secondAttorney Docket No.: SP24-308 plurality of fibers 104 running in a second direction. The woven fibers of the substrate 100 form a plurality of openings 106, which can be defined by one or more widths or diameters (e.g., Di, D2). The size and shape of the openings can vary based on the type of weave (e.g., number, shape and size of filaments; angle between intersecting filaments, etc.). A woven mesh may be characterized as, on a macro-scale, a two-dimensional sheet or layer. However, a close inspection of a woven mesh reveals a three-dimensional structure due to the rising and falling of intersecting fibers of the mesh. Thus, as shown in Figure 1C, a thickness T of the woven mesh 100 may be thicker than the thickness of a single fiber (e.g., ti). As used herein, the thickness T is the maximum thickness between a first side 108 and a second side 110 of the woven mesh. Without wishing to be bound by theory, it is believed that the three-dimensional structure of the substrate 100 is advantageous as it provides a large surface area for culturing adherent cells, and the structural rigidity of the mesh can provide a consistent and predictable cell culture matrix structure that enables uniform fluid flow.

[0077] In Figure IB, the openings 106 have a diameter Di, defined as a distance between opposite fibers 102, and a diameter D2, defined as a distance between opposite fibers 104. Di and D2 can be equal or unequal, depending on the weave geometry. Where Di and D2 are unequal, the larger can be referred to as the major diameter, and the smaller as the minor diameter. In some embodiments, the diameter of an opening may refer to the widest part of the opening. Unless otherwise specified, the opening diameter, as used herein, will refer to a distance between parallel fibers on opposite sides of an opening.

[0078] A given fiber of the plurality of fibers 102 has a thickness ti, and a given fiber of the plurality of fibers 104 has a thickness t2. In the case of fibers of round cross-section, as shown in Figure 1 A, or other three-dimensional cross-sections, the thicknesses ti and t2 are the maximum diameters or thicknesses of the fiber cross-section. According to some embodiments, the plurality of fibers 102 all have the same thickness ti, and the plurality of fiber 104 all have the same thickness t2. In addition, ti and t2 may be equal. However, in one or more embodiments, ti and t2 are not equal such as when the plurality of fibers 102 are different from the plurality of fiber 104. In addition, each of the plurality of fibers 102 and plurality of fibers 104 may contain fibers of two or more different thicknesses (e.g., tia, tib, etc., and t2a, t2b, etc.). According toAttorney Docket No.: SP24-308 embodiments, the thicknesses ti and t2 are large relative to the size of the cells cultured thereon, so that the fibers provide an approximation of a flat surface from the perspective of the cell, which can enable better cell attachment and growth as compared to some other solutions in which the fiber size is small (e.g., on the scale of the cell diameter). Due to three-dimensional nature of woven mesh, as shown in Figures 1A-1C, the 2D surface area of the fibers available for cell attachment and proliferation exceeds the surface area for attachment on an equivalent planar 2D surface.

[0079] In one or more embodiments, a fiber may have a diameter in a range of about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm; or about 150 pm to about 300 pm. On a microscale level, due to the scale of the fiber compared to the cells (e.g., the fiber diameters being larger than the cells), the surface of monofilament fiber is presented as an approximation of a 2D surface for adherent cells to attach and proliferate. Fibers can be woven into a mesh with openings ranging from about 100 pm x 100 pm to about 1000 pm x 1000 pm. In some embodiments, the opening may have a diameter o about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; or about 200 pm to about 300 pm. These ranges of the filament diameters and opening diameters are examples of some embodiments, but are not intended to limit the possible feature sizes of the mesh according to all embodiments. The combination of fiber diameter and opening diameter is chosen to provide efficient and uniform fluid flow through the substrate when, for example, the cell culture matrix is comprises a number of adjacent mesh layers (e.g., a stack of individual layers or a rolled mesh layer).

[0080] Factors such as the fiber diameter, opening diameter, and weave type / pattern will determine the surface area available for cell attachment and growth. In addition, when the cell culture matrix includes a stack, roll, or other arrangement of overlapping substrate, the packing density of the cell culture matrix will impact the surface area of the packed bed matrix. Packing density can vary with the packing thickness of the substrate material (e.g., the space needed for a layer of the substrate). For example, if a stack of cell culture matrix has a certain height, each layer of the stack can be said to have a packing thickness determined by dividing the total heightAttorney Docket No.: SP24-308 of the stack by the number of layers in the stack. The packing thickness will vary based on fiber diameter and weave, but can also vary based the alignment of adjacent layers in the stack. For instance, due to the three-dimensional nature of a woven layer, there is a certain amount of interlocking or overlapping that adjacent layers can accommodate based on their alignment with one another. In a first alignment, the adjacent layers can be tightly nestled together, but in a second alignment, the adjacent layers can have zero overlap, such as when the lower-most point of the upper layer is in direct contact with the upper-most point of the lower layer. It may be desirable for certain applications to provide a cell culture matrix with a lower density packing of layers (e.g., when higher permeability is a priority) or a higher density of packing (e.g., when maximizing substrate surface area is a priority). According to one or more embodiments, the packing thickness can be from about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm.

[0081] The above structural factors can determine the surface area of a cell culture matrix, whether of a single layer of cell culture substrate or of a cell culture matrix having multiple layers of substrate). For example, in a particular embodiment, a single layer of woven mesh substrate having a circular shape and diameter of 6 cm can have an effective surface area of about 68 cm2. The “effective surface area,” as used herein, is the total surface area of fibers in a portion of substrate material that is available for cell attachment and growth. Unless stated otherwise, references to “surface area” refer to this effective surface area. According to one or more embodiments, a single woven mesh substrate layer with a diameter of 6 cm may have an effective surface area of about 50 cm2to about 90 cm2; about 53 cm2to about 81 cm2; about 68 cm2; about 75 cm2; or about 81 cm2. These ranges of effective surface area are provided for example only, and some embodiments may have different effective surface areas. The cell culture matrix can also be characterized in terms of porosity, as discussed in the Examples herein.

[0082] The substrate mesh can be fabricated from monofilament or multifilament fibers of polymeric materials compatible in cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene,Attorney Docket No.: SP24-308 polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide. Mesh substrates may have a different patterns or weaves, including, for example knitted, warp-knitted, or woven (e.g., plain weave, twilled weave, dutch weave, five needle weave).

[0083] The surface chemistry of the mesh filaments may need to be modified to provide desired cell adhesion properties. Such modifications can be made through the chemical treatment of the polymer material of the mesh or by grafting cell adhesion molecules to the filament surface. Alternatively, meshes can be coated with thin layer of biocompatible hydrogels that demonstrate cell adherence properties, including, for example, collagen or Matrigel®.Alternatively, surfaces of filament fibers of the mesh can be rendered with cell adhesive properties through the treatment processes with various types of plasmas, process gases, and / or chemicals known in the industry. In one or more embodiments, however, the mesh is capable of providing an efficient cell growth surface without surface treatment.

[0084] Although from afar the mesh used in embodiments for the substrate appears to be a flat sheet, close inspection reveals the three-dimensional quality of the meshes due to the undulating fibers of the weave, which provides increased surface area for cell attachment and proliferation compared to a planar 2D surface of comparable size. This increased surface area aids in the scalable performance achieved by embodiments of this disclosure. For process development and process validation studies, small-scale bioreactors are often required to save on reagent cost and increase experimental throughput. Embodiments of this disclosure are applicable to such small- scale studies, but can be scaled-up to industrial or production scale, as well. For example, if 100 layers of a mesh material having a fiber diameter of about 158 pm and openings of about 244 pm by 244 pm is formed in circular disks having a diameter of 2.2 cm that are packed into a cylindrical fixed bed (e.g., inside a cell culture vessel with a 2.2 cm internal diameter), the total surface area available for cells to attach and proliferate is equal to about 935 cm2. To scale such bioreactor ten times, one could use a similar setup of a cylindrical packed bed with 7 cm internal diameter and 100 layers of the same mesh. In such a case, the total surface area would be equal 9,350 cm2. In some embodiments, the available surface area is about 99,000 cm2 / L or more.Because of the plug-type perfusion flow in a packed bed, the same flow rate expressed inAttorney Docket No.: SP24-308 ml / min / cm2of cross-sectioned packed bed surface area can be used in smaller-scale and larger- scale versions of the bioreactor. A larger surface area allows for higher seeding density and higher cell growth density. According to one or more embodiments, the cell culture substrate described herein has demonstrated cell seeding densities of up to 22,000 cells / cm2or more. For reference, the Corning HyperFlask® has a seeding density on the order of 20,000 cells / cm2on a two-dimensional surface. As used herein, “plug-type perfusion flow” or “plug flow” refers to laminar flow through the bioreactor having a fixed bed according to embodiments herein, where the flow through any cross-section of the fixed bed perpendicular to the flow direction proceeds at the same rate across the cross section.

[0085] Another advantage of the higher surface areas and high cell seeding or growing densities is that the cost of the embodiments disclosed herein can be the same or less than competing solution. Specifically, the cost per cellular product (e.g., per cell or per viral genome) can be equal to or less than other packed bed bioreactors.

[0086] By using a structurally defined culture matrix of sufficient rigidity, high-flow- resistance uniformity across the matrix or packed bed is achieved. According to various embodiments, the matrix can be deployed in monolayer or multilayer formats. This flexibility eliminates diffusional limitations and provides uniform delivery of nutrients and oxygen to cells attached to the matrix. In addition, the open matrix lacks any cell entrapment regions in the packed bed configuration, allowing for complete cell harvest with high viability at the end of culturing. The matrix also delivers packaging uniformity for the packed bed, and enables direct scalability from process development units to large-scale industrial bioprocessing unit. The ability to directly harvest cells from the packed bed eliminates the need of resuspending a matrix in a stirred or mechanically shaken vessel, which would add complexity and can inflict harmful shear stresses on the cells. Further, the high packing density of the cell culture matrix yields high bioprocess productivity in volumes manageable at the industrial scale.

[0087] The geometry of the mesh substrate layers is designed to allow efficient and uniform flow through one or multiple substrate layers. In addition, the structure of the matrix can accommodate fluid flow through the matrix in multiple orientations. For example, the direction of bulk fluid flow can be perpendicular to the major side surfaces of the first and secondAttorney Docket No.: SP24-308 substrate layers. However, the matrix can also be oriented with respect to the flow such that the sides of the substrate layers are parallel to the bulk flow direction. In addition to fluid flow being perpendicular or parallel to the first and second sides of the mesh layers, the matrix can be arranged with multiple pieces of substrate at intermediate angles, or even in random arrangements with respect to fluid flow. This flexibility in orientation is enabled by the essentially isotropic flow behavior of the woven substrate. In contrast, substrates for adherent cells in existing bioreactors do not exhibit this behavior and instead their packed beds tend to create preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the matrix of the current disclosure allows for its use in various applications and bioreactor or container designs while enabling better and more uniform permeability throughout the bioreactor vessel.

[0088] As discussed herein, the cell culture substrate can be used within a bioreactor vessel, according to one or more embodiments. For example, the substrate can be used in a packed bed bioreactor configuration, or in other configurations within a three-dimensional culture chamber. However, embodiments are not limited to a three-dimensional culture space, and it is contemplated that the substrate can be used in what may be considered a two-dimensional culture surface configuration, where the one or more layers of the substrate lay flat, such as within a flat- bottomed culture dish, to provide a culture substrate for cells. Due to contamination concerns, the vessel can be a single-use vessel that can be disposed of after use.

[0089] A cell culture system is provided, according to one or more embodiments, in which the cell culture matrix is used within a culture chamber of a bioreactor vessel. Figure 2 shows an example of a cell culture system 300 that includes a bioreactor vessel 302 having a cell culture chamber 304 in the interior of the bioreactor vessel 302. Within the cell culture chamber 304 is a cell culture matrix 306 forming a fixed-bed cell culture substrate that is made from a stack of substrate layers 308. The substrate layers 308 are stacked with the first or second side of a substrate layer facing a first or second side of an adjacent substrate layer. The bioreactor vessel 300 has an inlet 310 at one end for the input of media, cells, and / or nutrients into the culture chamber 304, and an outlet 312 at the opposite end for removing media, cells, or cell products from the culture chamber 304. By allowing stacking of substrate layers in this way, the systemAttorney Docket No.: SP24-308 can be easily scaled up without negative impacts on cell attachment and proliferation, due to the defined structure and efficient fluid flow through the stacked substrates. While the vessel 300 may generally be described as having an inlet 310 and an outlet 312, some embodiments may use one or both of the inlet 310 and outlet 312 for flowing media, cells, or other contents both into and out of the culture chamber 304. For example, inlet 310 may be used for flowing media or cells into the culture chamber 304 during cell seeding, perfusion, or culturing phases, but may also be used for removing one or more of media, cells, or cell products through the inlet 310 in a harvesting phase. Thus, the terms “inlet” and “outlet” are not intended to restrict the function of those openings.

[0090] However, embodiments of this disclosure include other arrangements for the fixed-bed substrate shown in Figure 2, including, for example: one or more rolled sheets (the center longitudinal axis of the roll being parallel to the direction between the inlet 310 and outlet 312 of the bioreactor vessel 300); a three-dimensional monolithic substrate matrix; sheets of substrate material stacked such that their major faces are parallel to a direction between the inlet 310 and the outlet 312; etc. In Figure 2, the bulk flow direction is in a direction from the inlet 310 to the outlet 312, and, in this example, the first and second major sides of the substrate layers 308 are perpendicular to the bulk flow direction.

[0091] Figure 3 shows an example of embodiments of a bioreactor 400 that uses a fixed bed made of at least one rolled substrate 402 made from at least one substrate sheet 404. The repeated layers of the substrate sheet 404 in the rolled substrate 402 can be considered to form a multi-layered fixed bed substrate. In embodiments, there can be more than one substrate sheet 404 rolled together in the fixed bed. The rolled substrate 402 has a central cylinder axis, which, in embodiments, coincides with a central longitudinal axis of the interior reservoir and / or bioreactor vessel. In embodiments, the guide rode 406 is provided at the center of the rolled substrate 402, with the rolled substrate disposed between the guide rod 406 and the interior wall 408 of the cell culture vessel. The rolled substrate can be attached to or unattached to the guide rod 406.

[0092] In one or more embodiments, flow resistance and volumetric density of the packed bed can be controlled by interleaving substrate layers of different geometries. In particular, mesh sizeAttorney Docket No.: SP24-308 and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the fluid flow resistance in packed bed format. By interlaying meshes of different sizes and geometries, flow resistance can be controlled or varied in one or more specific portions of the bioreactor. This will enable better uniformity of liquid perfusion in the packed bed. Various combinations of meshes of different sizes are possible to obtain different profiles of volumetric density of cells growth surface and flow resistance. For example, a packed bed column with zones of varying volumetric cells densities (e.g., a series of zones creating a pattern of low / high / low / high, etc. densities) can be assembled by interleaving meshes of different sizes.

[0093] The cell culture matrix can be arranged in multiple configurations within the culture chamber depending on the desired system. For example, in one or more embodiments, the system includes one or more layers of the substrate with a width extending across the width of a defined cell culture space in the culture chamber. Multiple layers of the substrate may be stacked in this way to a predetermined height. As discussed above, the substrate layers may be arranged such that the first and second sides of one or more layers are perpendicular to a bulk flow direction of culture media through the defined culture space within the culture chamber, or the first and second sides of one or more layers may be parallel to the bulk flow direction. In one or more embodiments, the cell culture matrix includes one or more substrate layers at a first orientation with respect to the bulk flow, and one or more other layers at a second orientation that is different from the first orientation. For example, various layers may have first and second sides that are parallel or perpendicular to the bulk flow direction, or at some angle in between.

[0094] In one or more embodiments, the cell culture system includes a plurality of discrete pieces of the cell culture substrate in a packed bed configuration, where the length and or width of the pieces of substrate are small relative to the culture chamber. As used herein, the pieces of substrate are considered to have a length and / or width that is small relative to the culture chamber when the length and / or width of the piece of substrate is about 50% or less of the length and / or width of the culture space. Thus, the cell culture system may include a plurality of pieces of substrate packed into the culture space in a desired arrangement. The arrangement of substrate pieces may be random or semi-random, or may have a predetermined order or alignment, such asAttorney Docket No.: SP24-308 the pieces being oriented in a substantially similar orientation (e.g., horizontal, vertical, or at an angle between 0° and 90° relative to the bulk flow direction).

[0095] The “defined culture space,” as used herein, refers to a space within the culture chamber occupied by the cell culture matrix and in which cell seeding and / or culturing is to occur. The defined culture space can fill approximately the entirety of the culture chamber, or may occupy a portion of the space within the culture chamber. As used herein, the “bulk flow direction” is defined as a direction of bulk mass flow of fluid or culture media through or over the cell culture matrix during the culturing of cells, and / or during the inflow or outflow of culture media to the culture chamber.

[0096] In one or more embodiments, the cell culture matrix is secured within the culture chamber by a fixing mechanism. The fixing mechanism may secure a portion of the cell culture matrix to a wall of the culture chamber that surrounds the matrix, or to a chamber wall at one end of the culture chamber. In some embodiments, the fixing mechanism adheres a portion of the cell culture matrix to a member running through the culture chamber, such as member running parallel to the longitudinal axis of the culture chamber, or to a member running perpendicular to the longitudinal axis. In embodiments, the member can be a guide rod. However, in one or more other embodiments, the cell culture matrix may be contained within the culture chamber without being fixedly attached to the wall of the chamber or bioreactor vessel. For example, the matrix may be contained by the boundaries of the culture chamber or other structural members within the chamber such that the matrix is held within a predetermined area of the bioreactor vessel without the matrix being fixedly secured to those boundaries or structural members.

[0097] The bioreactor vessel optionally includes one or more outlets capable of being attached to inlet and / or outlet means. Through the one or more outlets, liquid, media, or cells can be supplied to or removed from the chamber. A single port in the vessel may act as both the inlet and outlet, or multiple ports may be provided for dedicated inlets and outlets. In embodiments, inlet and outlet are disposed on the top and / or bottom of the bioreactor vessel. For example, the bioreactor can have an inlet on the bottom of the bioreactor vessel and an outlet on the top of the bioreactor vessel, such that fluid is designed to flow into the interior cavity where the fixed bed is located via the inlet, then up through the fixed bed, and out of the bioreactor via the outlet.Attorney Docket No.: SP24-308

[0098] The packed bed cell culture matrix of one or more embodiments can consist of the woven cell culture mesh substrate without any other form of cell culture substrate disposed in or interspersed with the cell culture matrix. That is, the woven cell culture mesh substrate of embodiments of this disclosure are effective cell culture substrates without requiring the type of irregular, non-woven substrates used in existing solution. This enables cell culture systems of simplified design and construction, while providing a high-density cell culture substrate with the other advantages discussed herein related to flow uniformity, harvestability, etc.

[0099] As discussed herein, the cell culture substrates and bioreactor systems provided offer numerous advantages. For example, the embodiments of this disclosure can support the production of any of a number of viral vectors, such as AAV (all serotypes) and lentivirus, and can be applied toward in vivo and ex vivo gene therapy applications. The uniform cell seeding and distribution maximizes viral vector yield per vessel, and the designs enable harvesting of viable cells, which can be useful for seed trains consisting of multiple expansion periods using the same platform. In addition, the embodiments herein are scalable from process development scale to production scale, which ultimately saves development time and cost. The methods and systems disclosed herein also allow for automation and control of the cell culture process to maximize vector yield and improve reproducibility. Finally, the number of vessels needed to reach production-level scales of viral vectors (e.g., 1016to 1018AAV VG per batch) can be greatly reduced compared to other cell culture solutions.

[0100] As discussed herein, the embodiments of this disclosure provide cell culture substrates, bioreactor systems, and methods of culturing cells or cell by-products that are scalable and can be used to provide a cell seed train to gradually increase a cell population. One problem in existing cell culture solutions is the inability for a given bioreactor system technology to be part of a seed train. Instead, cell populations are usually scaled up on various cell culture substrates. This can negatively impact the cell population, as it is believed that cells become acclimated to certain surfaces and being transferred to a different type of surface can lead to inefficiencies. Thus, it would be desirable to minimize such transitions between cell culture substrates or technologies. By using the same cell culture substrate across the seed train, as enabled by embodiments of this disclosure, efficiency of scaling up a cell population is increased. ForAttomey Docket No.: SP24-308 example, the seed train can begin with a vial of starter cells which are seeded into a first vessel (such as a T175 flask from Corning Incorporated), then into a second vessel (such as a HyperFlask® from Corning Incorporated), then into a process-development scale bioreactor system according to embodiments of this disclosure (e.g., with effective surface area of substrate of about 20,000 cm2), and then into a larger bioreactor pilot system according to embodiments of this disclosure (e.g., with effective surface area of substrate of about 300,000 cm2). At the end of this seed train, the cells can be seeded into a production-scale bioreactor vessel according to embodiments of this disclosure, with a surface area of about 5,000,000 cm2, for example.Harvest and purification steps can then be performed when the cell culture is complete. Harvest can be accomplished via in situ cell lysis with a detergent (such as, for example, Triton X-100), or via mechanical lysis; and further downstream processing can be performed, as needed. This description of a seed train is to illustrate an example process for scaling a cell culture population, and embodiments of this disclosure are not limited to this example.

[0101] The benefits of using the same cell culture substrate within the seed train (e.g., from process development level to pilot level, or even to production level) include efficiencies gained from the cells being accustomed to the same surface during the seed train and production stages; a reduced number of manual, open manipulations during seed train phases; more efficient use of the packed bed due to uniform cell distribution and fluid flow, as described herein; and the flexibility of using mechanical or chemical lysis during viral vector harvest.Examples

[0102] To demonstrate the efficacy of the cell culture matrix, cell culture systems, and related methods of this disclosure, studies were conducted on the seeding and culturing of cells, according to the following examples.

[0103] Example 1

[0104] To further examine the flow uniformity or permeability of the substrates of this disclosure, modeling was used to understand the porosity of the three-dimensional cell culture matrix. Sheets of woven PET mesh substrate, as examples of substate material according to embodiments of this disclosure, were modeled in a tight-packed configuration and a loose-Attorney Docket No.: SP24-308 packed configuration, which represent upper and lower boundaries of the packing density of a substrate stack for the particular mesh sheet that was modeling. In particular, Figure 4A shows a plan view of the tight-packed configuration, and Figure 4B shows a cross-section side view of the same stack. Figures 5A and 5B show plan and cross-section views, respectively, of the loose- packed configuration. For each modeled configuration, a sample cell 600, 602 was defined that encloses the same volume of mesh material to analyze the porosity per unit volume of the sample cell 600, 602. The modeled volume of open space within each cell is shown in Figures 7A (for tight-packed stack) and 7B (for loose-packed stack). The porosity in terms of percentage of open space was about 40.8% for the loose-packed cell, and 61.4% for the tight-packed cell. Because the modeled stacks in Figures 4A5B represent the tightest- and loosest-packed configurations for the given mesh material, the porosities of 40.8% and 61.4% are the upper and lower bounds of porosity for this particular mesh material. Depending on the alignment and real- world packing density when using this mesh material, the porosity may fall in between these extremes.However, embodiments of this disclosure are not limited to this porosity range, as variations in the mesh dimensions and arrangement of the substrate within the cell culture vessel can lead to a different range of porosities.

[0105] In addition to the modeled porosity range, porosity was measured using real packed beds of PET woven mesh substrate. The measurements were made using one hundred disks, each with a diameter of 22.4 mm, stacked with random alignment. The total weight of the 100-disk stack was 5.65±0.2 g. Volume of the PET material of the stack was calculated, assuming a PET density of 1.38 g / cm3, using the following formula:VPET = (total weight of stack) / (density of PET) Equation 2Thus, the PET volume VPET of 5.65 g of PET (for 100 disks of 22.4 mm diameter) was calculated to be 4.1 ml. The total volume Vtotai of the stack, including the PET volume VPET and the volume of the open space within the stack, was then calculated using the following formula:Vtotai = 7t * (0.5 x disk diameter) x (stacked bed height) Equation 3The 100-disk stack had a stack height of 25±1 mm. Thus, with a disk diameter of 22.4 mm, Vtotai was found to be 9.85 ml. Accordingly, porosity of the stacked bed can be calculated using the following:Attorney Docket No.: SP24-308Porosity = (Vtotai - VPET) / Vtotai Equation 4Using Equation 4 and the above values, the porosity was calculated to be 58.4%, which is within the range predicted by the model. The values discussed above are only an example of embodiments of this disclosure. It is contemplated that substrates of higher or lower porosity can be used in accordance with embodiments discussed herein.

[0106]

[0107] Example 2

[0108] In order to understand the flow performance of a bioreactor and fixed bed, including the presence of fluid bypass within or around the fixed bed, it is useful to have qualitative and / or quantitative measures of the fluid flow through a fixed bed and vessel. Residence time distribution (RTD) is a useful tool to study the flow in a vessel. Its theory, measurement, and analysis can be found in the textbook: Levenspiel, O. Chemical Reaction Engineering. 3ed. 1999. Wiley. New York. Figure 7 shows the schematic drawing of the setup to measure RTD. In this example, the bioreactor vessel containing the fixed bed is cylindrical with diameter of 29 mm and the total fixed bed volume is 36 ml. The fixed bed consisted of 3.6 g of non-woven mesh or, according to embodiments of this disclosure, 200 layers of woven mesh. 1 :2000 diluted McCormick Green Food Color was used as a tracer for the RTD measurement. A UV-vis with a Flowcell was used to monitor the change of tracer concentration. A flow rate of 22.5 m 1 / ml was used for all experiment. The chamber was first filled with water. After switched to green dye, the change of the optical density (OD) was recorded. The results in terms of normalized dye concentration versus time.

[0109] The following equations were used to calculate mean residence time t (Equation (6)) and variance c (Equation (7)): Equation (6)Equation (7) where F is normalized concentration in a step tracer response. Table 1 summarizes the calculated mean residence time and variance from the measurement, according to example experimentsAttorney Docket No.: SP24-308 conducted using the setup of Figure 7. The woven mesh shows shorter mean residence time, which was likely caused by the lower porosity and decreased dead zones. In a fixed bed of open woven mesh, the porosity was about 60% while the porosity of the non-woven mesh has higher porosity which was about 93%. The much higher normalized variance detected in packed bed of non-woven mesh suggests that the flow in non-woven mesh was less uniform or ideal.Table 1. Means residence time and variance of non-woven and open woven mesh from measurement.

[0110] Using the same setup as described above, the RTDs of other commercially available fixed or packed bed substrate materials were measured. The packing density was calculated based on the vendor recommendation and to avoid loose packing. The measured changes of dyes were used to calculate their normalized variance as described in previous section. The calculated variances are listed in Table 2.Table 2. Normalized variance of different commercially available packed bed substrates vs. a woven mesh substrate of the present disclosure.

[0111] From the above permeability and residence time experiments, it is shown that the type of non-woven, irregular cell culture substrate used in some bioreactors has lower permeability than substrates according to embodiments of the present disclosure. These non-woven substrates also have different permeability or flow rates depending on the direction of flow relative to the non-woven substrate, whereas the substrates of the present disclosure exhibit essentially isotropic flow behavior. Due to the non-uniform flow and lower residence time of the non-woven substrates, nutrients and transfection reagents can take longer to reach to the cells on the substrate surface or the other side of a substrate layer, as compared to the uniform, woven meshAttorney Docket No.: SP24-308 substrate of the present disclosure. Adding to this is the higher permeability of the randomly packed non-woven substrate, which suggest a strong channeling effect and thus non-uniform delivery of cells or nutrients.

[0112] Example 3

[0113] To further quantify the flow uniformity in substrates according to embodiments of this disclosure, the compaction or occupancy of the fixed bed was controlled in experiments using a setup as described above. Specifically, to compare and quantify the flow uniformity through different fixed beds, the RTD was measured. In each experiment, while maintaining flow through the bioreactor fixed bed, a small volume of a green dye solution was injected at the inlet of the bioreactor and the change of dye concentration over time was measured at the outlet at the outlet. The dye was injected over a direction of within 2 seconds, which is considered as a pulse input. The change in dye concentration at the outlet was recorded using a UV-vis spectrometer. Different flow rates were used during the measurement depending on the experimental designs.

[0114] The reactor used in these experiments had an internal diameter of 63 mm and 201.7 mm bed space height to hold substrate material as a fixed bed or packed bed. Substrates included open woven mesh disks, according to embodiments of this disclosure. To pack the reactor, each disk was aligned relative to a disk according to a 45° rotation about a z-axis, where the z-axis is parallel to the longitudinal axis of the bioreactor bed space and is perpendicular to the first and second major surfaces of the disk. Thus, the orientation of the fibers in each woven disk were rotated 45° relative to the fibers in the neighboring disk. This rotation prevented interlocking of the fibers between disks and provided more consistent permeability. To control the packing density or occupancy, we used the space height of the reactor divided by the thickness of each mesh disk to obtain the number of disks for 100% occupancy. In this situation, the mesh disks filled all the way to the top of reactor space. Figure 8A shows this stack of disks at 100% occupancy. By removing 5% of the disk count from the 100% occupancy condition, 95% occupancy was obtained, as shown in Figure 8B.

[0115] Figure 9A is a photograph of a bioreactor with a cell culture matrix of stacked layers of substate packed at 95% occupancy with compressed packing, according to one or more embodiments, when fluid is present in the bioreactor, as it is during a cell culture. Figure 9B is aAttorney Docket No.: SP24-308 photograph of a bioreactor with a cell culture matrix of stacked layers of substate packed at 95% occupancy with loose packing, according to one or more embodiments, when fluid is present in the bioreactor.

[0116] At lower occupancy, either the disks were compressed down, in which case empty space was created above the packed bed (this is referred to as compressed packing), or the extra space was distributed across the packed bed without clear space on the top (this is referred to as loose packing). Even at 100% occupancy, due to some elasticity of the mesh layer, the bed can still be compressed to some extent and result in a little space left above the fixed bed. This is called compressed packing at 100% occupancy, whereas without the applied compression there would be loose packing. In compressed configuration, about 2.5% extra disks were added to create 102.5% occupancy which means that the mesh disks were always maintained at highly compressed condition.

[0117] The experiments showed that compressed packing, including different occupancies from 95% to 102.5%, surprisingly provided good and comparable RTD profiles, demonstrating flow uniformity, as shown in Figure 10A. However, loose packing generated comparably worse results, which indicated more bypass at 100 ml / min, as shown in Figure 10B, as represented by a larger earlier peak of the E(0) curve. With increased flow rate, such as 300ml / min, the bypass was increased further, and more significant for lower occupancies and loose packing conditions, as demonstrated in Figure 10B and Figures 11 A-C. This means that, when a higher flow rate is needed during higher density cell culture, the flow uniformity will suffer more.

[0118] Figures 11 A, 1 IB, and 11C show the E(0) curves using substate disks according to embodiments of this disclosure under different packing conditions. In Figure 11 A, the bed is packed at 102.5% occupancy. In Figure 1 IB, the bed is packed at 95% occupancy with compressed packing. In Figure 11C, the bed is paced at 95% occupancy with loose packing. As shown, the loose packing condition in Figure 11C results in an early peak (indicating channeling), a wider base of the E(0) curve, and more variability between flow rates, with a higher flow rate creating worse flow. In comparison, compressed packing, at both 102.5% andAttorney Docket No.: SP24-30895% occupancy, creates more uniform flow conditions that do not vary significantly with flow rate.

[0119] It should be noted that the E(0) curve is normalized and dimensionless, and thus bioreactors of different sizes can be easily compared on the same curve. According to embodiments of this disclosure, the substate and fixed beds show remarkably consistent E(0) curves regardless of the size of the reactor. For example, a fixed bed having a substrate surface area of 5 m2and one having a surface area of 100 m2have similar overlapping E(0) curves. These sizes are given for example, only, as it is anticipated that fixed beds of any size according to embodiments herein will have similar flow characteristics.

[0120] As would be understood by a person of ordinary skill in the art, an “early” peak means that the peak of the E(0) comes earlier 0 = 1 (and, in the above experiment, earlier than those of the compressed packing). A distribution of the RTD curve closer to 0 = 1 indicates more uniform flow or flow closer to a true “plug flow” condition. For example, a single, sharp peak in the E(0) curve indicates that all (or nearly all) dye particles injected spend about the same amount of time in the reactor, which indicates uniform flow that is close to idealized “plug flow”. However, in some substrates or beds, channeling can occur meaning that the permeability of the bed will be variable and the particles of dye used in the RTD experiment will spend variable amounts of time in the reactor. This can be indicated by an “early” peak on the E(0) curve, indicating dye that reached the bioreactor outlet early, and / or by a wider E(0) curve, indicating a longer length of time to clear the dye from the reactor.

[0121] Without wishing to be bound by theory, it is believed that with loose packing, the substrate disks may not be able to keep flat in the fixed bed due to less restriction from neighboring disks. Lifted or curved disks can create more gaps between the disks and vessel wall, and lead to more bypass. At higher flow rate, the flow can further lift the disks and make greater gaps and more bypass flow.

[0122] Example 4

[0123] To simulate the effect of bubbles, the setup described above for Example 3 was used, but 8 ml of air was injected at the inlet before corresponding RTD measurements. The obtained RTD profiles were compared with the same reactor before bubble introduction. As shown inAttorney Docket No.: SP24-308Figures 12A and 12B, at lower occupancies, the RTD profile, or E(0) curve, has a wider spread, suggesting poor uniformity compared to those at higher occupancies. This was similar for both compressed packing (Figure 12A) and loose packing (Figure 12B).

[0124] Without wishing to be bound by theory, it is believed that, at high occupancy such as 100% or 102.5%, there is not much extra space in the packed bed for bubbles and most of the bubbles were pushed out of the bed by perfusion flow through the reactor, which made the impact much less. While at lower occupancies, bubbles were easily trapped in the extra spaces in the bed and then disturbed the perfusion flow through the bed. Since bubbles are difficult to avoid during cell culture, using high occupancy can help minimize the negative impact of bubbles, and maintain uniform flow to support better cell growth.

[0125] High Density Substrates and Bypass

[0126] According to embodiments of this disclosure, fixed-bed bioreactors are provided that increase the cell numbers per unit volume able to be cultured in a bioreactor by increasing the available surface area of the cell culture substrate. In embodiments, this increase in surface area is achieved by so-called “high-density” substrates. As used herein, “high density” refers to using substrates that enable more available surface area per unit volume of the bioreactor. In embodiments, high-density substrates can include substrates, such as woven substrates, with pores or openings that have a diameter less than 250 pm, less than 200 pm, less than 150 pm, less than 100 pm, or less than 50 pm. By having more available surface area per unit volume in the bioreactor, it can be possible to seed and / or grown more cells in the same physical space of the bioreactor. Accordingly, increased cell density can enable increased bioreactor capacity and also lead to lower costs through efficiency. For example, high-density mesh can be achieved by decreasing the diameter of the fibers and and / or the diameter of the openings between fibers in the substrate.

[0127] However, as the substrate density increases (i.e., the opening size decreases), the resistance to fluid flow can also increase (i.e., lower permeability). During bioreactor assembly, it is important that alternative flow paths (e.g., gaps or bypass flow paths) around the fixed-bed substrate core are on a similar size scale or smaller. For example, in mesh substrates with openings of 250 pm, gaps of 250 pm result in significant fluid bypass. Thus, as the mesh densityAttorney Docket No.: SP24-308 is increased, the pore size also decreases (decreasing permeability), and bypass gaps that larger than the pore size will be favored flow paths and a non-trivial amount of the cell culture media can bypass the fixed-bed substrate core of the bioreactor. In addition, it is possible that sheets of woven substrates according to embodiments of this disclosure can become, with finer fibers and narrower openings, thinner sheets and more pliable. As a result, such high-density substrates can deform more easily under the conditions of fluid perfusion through the bioreactor. These effects can lead to negative consequences, such as higher variability, lower media efficiency, and lower maximum cell density. In general, significant bypass limits the maximum cell density in the reactor. Accordingly, embodiments of this disclosure include fixed-bed bioreactors and methods of assembling fixed-bed bioreactors that minimize gaps for fluid bypass to achieve maximum cell density and uniformity.

[0128] Embodiments of this disclosure include various fixed-bed bioreactor designs and methods of assembling fixed-bed bioreactors that seek to close and / or minimize bypass gaps, particularly such gaps around the edge or periphery of the fixed bed, between the fixed bed and the wall of the interior reservoir containing the fixed bed in the bioreactor. Aspects of embodiments include leveraging mechanical interference and compressibility of the substrate to seal bypass gaps; these being bioreactor designs and methods that are practical to implement from a manufacturing and cost perspective. Aspects of embodiments include using various types of sealing to seal the edge of the fixed bed or seal the space between the fixed bed and the interior wall of the vessel. Aspects of embodiments include fixed beds with stacked substrate layers where some or all of the layers have tabs extending outside the diameter needed for the fixed bed, such tabs be used to seal the space between the fixed bed and the interior wall of the vessel. Aspects of embodiments also include surrounding the fixed bed with a shrink-to-fit material (e.g., thermal shrink tubing) to conform to the width of the fixed bed. Advantages of the aforementioned embodiments of bioreactors and bioreactor assembly methods is the enablement of using higher density mesh in a bioreactor and increasing cell density.

[0129] Table 3 shows some examples of wove mesh materials used for cell culture substrates, according to embodiments of this disclosure. It should be understood that these are examples only, and are not intended to be limiting on substrate materials that can be used in embodimentsAttorney Docket No.: SP24-308 contemplated herein. As shown in Table 3, with decreasing pore size and fiber size, the overall thickness of the mesh also decreases. When thinner mesh substrate is used, the accessible surface area per disk / layer (leading to cell density) may be similar, but, because each layer is thinner, more layers of that mesh can be assembled into the reactor leading to more surface area for the entire fixed bed.

[0130] Figures 13A, 13B, 13C, and 13D show photographs of mesh substrates Samples A, B, D, and E, respectively, from Table 3. In the context of this disclosure, the 250 pm opening size of Sample A can be considered a “standard” mesh size, while the smaller-opening meshes are “high density” substrates. In this context, the standard mesh of Sample A can be can be considered to have a standard or normalized density of “IX” and SampleE, for example, can be considered to have a density of “4X” (i.e., four times the density of the standard mesh of Sample A).

[0131] Figure 14 shows the result of computational fluid dynamics (CFD) modeling of a fixed-bed bioreactor using a mesh substrate in the fixed bed and illustrates flow fields when a gap is present at the periphery of the fixed bed. The gap is an area between the core of the reactor and the inner sidewall of the reactor shell. If this gap is significant (relative to pore size), a significant quantity of the media can bypass the reactor, reducing oxygen and nutrient flow to cells in the fixed bed. While bypass gaps may primarily be discussed as being at the periphery of the fixed bed between the outer edge of the fixed bed and the inner wall of the interior reservoir,Attorney Docket No.: SP24-308 it is possible for there to be bypass gaps in other areas. For example, if a guide rod is used in the bioreactor where the guide rod passes through one or more openings in the substrate material, a gap can form between the guide rod and the fixed bed. Accordingly, embodiments discussed herein also contemplate systems and methods to address these other bypass gaps, even if not explicitly stated or even if only the peripheral bypass gap is expressly mentioned.

[0132] Figure 15 shows additional results from the CFD modeling of the bioreactor using various types of mesh substrate and gap sizes. This model illustrates that, when the high-density substrate is used, a larger percentage of the media could bypass the core of the fixed bed for a given gap size. This effect is independent of media flow rate. Thus, according to embodiments, to maintain the same low bypass level with higher-density substrates, the gap must proportionally be reduced. The modeling in Figure 15 is based on a bioreactor with a fixed bed height of 38 mm and a perfusion rate of 0.5 L / min.

[0133] Figure 16 is a photograph of fifteen disks taken from a fixed-bed bioreactor using a plurality of stacked disks for the fixed bed and illustrates the actual bypass that has occurred. Crystal violet staining was used to qualitatively compare cell density across the mesh. More intense color indicates higher cell density. Uniform cell density resulting from uniform flow is observed through mesh disks closer to the inlet (bottom row of disks in Figure 16). Through the center of the reactor, the density variation becomes apparent and at the top of the reactor near the outlet, cells are primary dense at the edge of the fixed bed (e.g., the edge of the disks show a darker stain). The cells at the edge survive due to media preferentially flowing through the bypass gap, while the cell density in the center of the disks is much lower due to lack of sufficient media flow through the center of the fixed bed. The substrate used in Figure 16 is that of Sample E from Table 3. While Figures 14-16 illustrate the problem of bypass flow, particular for higher-density substrates, attention will now be turned to embodiments of this disclosure to address and overcome these challenges.

[0134] Figure 17 shows an example according to embodiments that using a type of compression sleeve 702 around the fixed bed 700 that has a width that is slightly oversized relative to the width of an interior reservoir of a cell culture vessel 704. The compression sleeve 702 is used to temporarily compress C the width of the fixed bed 700 prior to inserting it into theAttorney Docket No.: SP24-308 cell culture vessel 704. The compression allows the fixed bed 700 to be easily inserted in a direction D into an opening in the vessel 704. After insertion, the fixed bed 700 can expand to close the gap between the edge of the fixed bed 700 and the interior wall of the vessel 704. This expansion results in the closing of any bypass gap between the fixed bed 700 and the interior wall of the vessel 704. The compression sleeve 702 can be a type of shrinking wrap or layer surrounding at least part of the fixed bed 700. For example, the compression sleeve 702 can be a vacuum sleeve or can shrink due to the presence of some kind of external stimuli (e.g., temperature, radiation, chemical or physical reaction, etc.). In some examples, the compression sleeve 702 may cover a top and / or bottom of the fixed bed, in which case the compression sleeve 702 may need to be at least partially opened, perhaps after insertion of the fixed bed 700 into the vessel 704. For example, openings 706, 708 can be formed by cutting or otherwise puncturing or opening the compression sleeve 702. In some embodiments, at least part of the compression sleeve 702 may remain in bioreactor after insertion of the fixed bed 700, so the compression sleeve can be formed from a biocompatible material that will not adversely impact the cell culture. Figure 17 depicts a rolled fixed bed, but this embodiments can be used with other styles of fixed bed, including stacked layers of substrate.

[0135] Figure 18A and Figure 18B show a substrate material 720 used for a rolled fixed bed. Applied to a portion of the substrate material 720 that will form the outer-most layer of the rolled fixed bed is a sealing material 722 that can seal the bypass gap between the rolled fixed bed 724 and the interior wall of the vessel 726 when the fixed bed 724 is inserted into the vessel 726. In one example, the sealing material 722 is a double-sided pressure-sensitive adhesive film to seal the outer-most layer of the substrate in the fixed bed. The sealing material 722 increases the flow resistance on the outside part of the fixed bed. In addition, the sealing material 722 can help stabilize the rolled fixed bed and hold it together prior to insertion into the vessel 726. In embodiments, the sealing material 722 can be a reactive material that expands under certain conditions, preferably after insertion into the vessel. For example, the sealing material can swell due to the presence of some kind of external stimuli (e.g., temperature, radiation, chemical or physical reaction, etc.). The coefficient of thermal expansion (CTE) of the sealing material 722 and the vessel 726 can be chosen to close the gap, according to aspects of embodiments. ForAttorney Docket No.: SP24-308 example, based on material CTE and a temperature differential of, e.g., 100°C between the fixed bed and the vessel, a 300 pm, for example, dimensional change is possible to close the gap. These embodiments are compatible with various styles and configurations of fixed beds, including rolled and stacked fixed beds.

[0136] Figures 19A and 19B show additional aspects of embodiments that use interference fit to close the bypass gap between the fixed bed 740 and vessel 742. Specifically, internal features 744 can be supplied on the inner wall of the interior reservoir of the vessel 742 to block fluid flow through the gap between the fixed bed 740 and the vessel 742. In embodiments, the internal features 744 are part of the injection mold design of the vessel 742. The internal features 744 can be shaped (e.g., smooth bumps) to ease insertion of the fixed bed while slightly compressing the substrate. In other examples, the internal features 744 can be formed as rings or as internal threads. Rotation of the fixed bed during insertion could also reduce friction to ease insertion.

[0137] Figure 20 shows a fixed-bed bioreactor having a fixed bed 760 and vessel 762. A sealant coating 764 is applied to the outside of the fixed bed 760 to fill any gap between the fixed bed 760 and the vessel 762. The sealant coating 764 can be a film, adhesive, polymer, or paint applied to the exterior of the fixed bed 760 prior to insertion into the vessel 762. The final dimensions can be matched to that of the bioreactor vessel 762 inner diameter. Flexibility of the sealant coating 764 aids in sealing by physical blocking of the gap. Alternatively, the bioreactor fixed bed 760 and sealant coating 764 can be intentionally undersized for ease of assembly. To seal the gap, a gasket 766 may be required on at least one end of the reactor core, especially if the fixed bed 760 is undersized relative to the inner wall of the vessel 762. In that case, sealing is achieved by preventing any lateral flow of fluid to outside of the fixed bed 760 core. Common materials that are cell compatible and could be used include silicones, polyurethanes, polyethylene, and polyesters.

[0138] According to embodiments, as shown in Figures 21A and 21B, physical sealing with a film can be accomplished by use of a material (e.g., a foam, polymer film, adhesive, etc.) that is irreversibly reactive to media, irradiation, and / or heat. Upon assembly of the reactor, the typical radiation treatment for sterility, an intentional heat treatment or exposure to media can cause the film to expand into the gap and seal. The material can be applied to the reactor core or the innerAttorney Docket No.: SP24-308 reactor shell wall. With reference to Figure 21 A, the sealing film 780 is applied to the inside wall 782 of the vessel 784. Prior to expansion, the sealing film 780 may not fill the gap 786 between the fixed bed 788 and the inside wall 782. However, as shown in Figure 21B, the sealing film 780 can expand to close the gap.

[0139] Figure 22 shows a schematic of a fixed bed cell culture substrate designed to reduce fluid bypass around the periphery of the fixed bed. The fixed bed 800 is made of a plurality of stacked substrate layers. For clarity and simplicity, only two substrate layers 802, 804 are shown. The two substrate layers 802, 804 have tabs 803, 805 that extend beyond the edge 801 of the fixed bed. When inserted into the bioreactor vessel (not shown), these tabs 803, 805 can be deflected and pressed by the interior wall of the vessel so that the tabs 803, 805 are approximately vertical, as shown in Figure 22. Thus, a gap between the edge 801 of the fixed bed 800 and the vessel wall can be filled by the tab 803, 805 material. The fixed bed 800 may have one, two, or more than two substrate layers with tabs. In some embodiments, all the layers in the fixed bed have tabs. In embodiments where not all of the layers have tabs, the layers with tabs (e.g., layers 802 and 804) may be separated by a first height hi. Additionally, the top of a deflected tab 805 may be separated a tabbed layer above it by a distance 112. In embodiments, 112 may be zero such that the tops of the tabs of a lower layer extend to the next layer up that has tabs. In embodiments, hi may be less than a height hi of the tabs, such that the tabs of a lower layer overlap with the tabs of the next layer up that has tabs. In embodiments, the tabs of subsequent layers may be offset from one another such that the tabs of one layer are center on the valleys between the tabs of the next layer. According to aspects of these embodiments, the gap between the fixed bed and the vessel wall can be effectively sealed to prevent bypass.

[0140] Figure 23 shows an embodiment where the fixed bed 820 is placed within a vessel 822 that shrinks in a direction S to close the gap between it and the fixed bed 820. Ends caps 824, 826 having inlets and / or outlets (and optionally with distributor plates 825 and 827) can be provided within the vessel 822 so that the shrinking of the vessel also, at least in part, acts to hold the endcaps 824, 826 in place. The vessel 822 can be a type of heat-shrink tubing, for example. The nature of the heat shrink wrap eliminates gaps between the walls and reactor core while heating / shrinking.Attorney Docket No.: SP24-308

[0141] Embodiments of this disclosure includes aspects of embodiments of the substrate materials, fixed bed configurations, and bioreactors disclosed in U.S. Patent Application Nos. 16 / 781,685; and 16 / 781,723; and U.S. Provisional Patent Application No. 63 / 227,693, the contents of which are hereby incorporated herein in their entireties.

[0142] Illustrative Implementations

[0143] The following is a description of various aspects of implementations of the disclosed subject matter. Each aspect may include one or more of the various features, characteristics, or advantages of the disclosed subject matter. The implementations are intended to illustrate a few aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible implementations.

[0144] Aspect 1 pertains to a bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width; a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed- bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height; and a compression sleeve surrounding at least a portion of the fixed-bed cell culture matrix, the compression sleeve exerting a compressive force in a direction of the bed width of the fixed-bed cell culture matrix, wherein the compressive force is sufficient to compress the bed width to a compressed bed width that is less than the reservoir width, and wherein the compression sleeve is configured to release the compressive force so that the bed width increases to an expanded bed width.

[0145] Aspect 2 pertains to the bioreactor system of Aspect 1, wherein the cell culture vessel further comprises an opening with an opening width greater than or equal to the compressed bed width.Attorney Docket No.: SP24-308

[0146] Aspect 3 pertains to the bioreactor system of Aspect 1 or Aspect 2, wherein the compression sleeve is configured to exert the compressive force by shrinking in response to an external stimulus.

[0147] Aspect 4 pertains to the bioreactor system of Aspect 3, wherein the external stimulus is at least one of vacuum, heat, and radiation.

[0148] Aspect 5 pertains to a method of assembling a fixed-bed bioreactor, the method comprising: providing a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed- bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height; providing a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width; surrounding at least a portion of the fixed-bed cell culture matrix with a compression sleeve; compressing the compression sleeve so that it decreases the bed width to a compressed bed width; after compressing the compression sleeve, inserting the fixed-bed cell culture matrix with the compression sleeve in compression into the interior reservoir.

[0149] Aspect 6 pertains to the method of Aspect 5, further comprising, after inserting the fixed-bed cell culture matrix, releasing the tension in the compression sleeve so that the bed width increases from the compressed bed width to the reservoir width.

[0150] Aspect 7 pertains to a bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width; a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed- bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height; and an adhesive around a circumference of the fixed-bed cell culture matrix, wherein the adhesive is configured to adhere an exterior of the fixed-bed cell culture matrix to the interior wall.Attorney Docket No.: SP24-308

[0151] Aspect 8 pertains to the bioreactor system of Aspect 7, wherein the adhesive extends across at least a portion the bed height.

[0152] Aspect 9 pertains to the bioreactor system of Aspect 8, wherein the adhesive extends across an entirety of the bed height.

[0153] Aspect 10 pertains to the bioreactor system of any one of Aspects 7-9, wherein the adhesive is a pressure-sensitive adhesive film.

[0154] Aspect 11 pertains to a bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width, the interior wall comprising a plurality of protrusions extending inward into the interior reservoir; and a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed- bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height, wherein the plurality of protrusions are sized to at least partially compress the fixed-bed cell culture matrix in a direction of the bed width.

[0155] Aspect 12 pertains to the bioreactor system of Aspect 11, wherein the plurality of protrusions are sized to fill a gap between the fixed-bed cell culture matrix and the interior wall.

[0156] Aspect 13 pertains to a bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width; a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed- bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height; and a sealant disposed between the fixed-bed cell culture vessel and the interior wall, wherein the sealant comprises an expandable material that expands to fill a gap between the fixed-bed cell culture matrix and the interior wall.

[0157] Aspect 14 pertains to the bioreactor system of Aspect 13, wherein the sealant is disposed on an exterior side of the fixed-bed cell culture matrix.Attorney Docket No.: SP24-308

[0158] Aspect 15 pertains to the bioreactor system of Aspect 13, wherein the sealant is disposed on the interior wall.

[0159] Aspect 16 pertains to the bioreactor system of any one of Aspects 13-15, wherein the sealant comprises at least one of a silicone, a polyurethane, a polyethylene, and a polyester.

[0160] Aspect 17 pertains to the bioreactor system of any one of Aspects 13-16, wherein the sealant comprises film, adhesive, polymer, or coating.

[0161] Aspect 18 pertains to the bioreactor system of any one of Aspects 13-17, wherein the sealant is configured to expand or contract in response to a stimulus.

[0162] Aspect 19 pertains to the bioreactor system of Aspect 18, wherein the stimulus comprises at least one of a change in temperature, a presence of fluid, a chemical reaction, and an exposure to radiation.

[0163] Aspect 20 pertains to the bioreactor system of any one of Aspects 1-4 and 7-19, wherein the fixed-bed cell culture substrate comprises a plurality of layers of substrate material in a stacked arrangement.

[0164] Aspect 21 pertains to the bioreactor system of any one of Aspects 1-4 and 7-19, wherein the fixed-bed cell culture substrate comprises a rolled substrate material.

[0165] Aspect 22 pertains to the bioreactor system of any one of Aspects 1-4 and 7-21, further comprising an O-ring disposed at a top or a bottom of the fixed-bed cell culture matrix, the Ciring being disposed along a circumference of the interior wall.

[0166] Aspect 23 pertains to the bioreactor system of Aspect 22, wherein the O-ring comprises an elastomeric material.Definitions

[0167] “Wholly synthetic” or “fully synthetic” refers to a cell culture article, such as a microcarrier or surface of a culture vessel, that is composed entirely of synthetic source materials and is devoid of any animal derived or animal sourced materials. The disclosed wholly synthetic cell culture article eliminates the risk of xenogeneic contamination.

[0168] ‘ ‘Include,” “includes,” or like terms means encompassing but not limited to, that is, inclusive and not exclusive.Attorney Docket No.: SP24-308

[0169] ‘ ‘Users” refers to those who use the systems, methods, articles, or kits disclosed herein, and include those who are culturing cells for harvesting of cells or cell products, or those who are using cells or cell products cultured and / or harvested according to embodiments herein.

[0170] ‘ ‘About” modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, viscosities, and like values, and ranges thereof, or a dimension of a component, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example: through typical measuring and handling procedures used for preparing materials, compositions, composites, concentrates, component parts, articles of manufacture, or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term “about” also encompasses amounts that differ due to aging of a composition or formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture.

[0171] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0172] The indefinite article “a” or “an” and its corresponding definite article “the” as used herein means at least one, or one or more, unless specified otherwise.

[0173] Abbreviations, which are well known to one of ordinary skill in the art, may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” for gram(s), “mL” for milliliters, and “rt” for room temperature, “nm” for nanometers, and like abbreviations).

[0174] Specific and preferred values disclosed for components, ingredients, additives, dimensions, conditions, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The systems, kits, and methods of the disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values described herein, including explicit or implicit intermediate values and ranges.Attorney Docket No.: SP24-308

[0175] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that any particular order be inferred.

[0176] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the disclosed embodiments. Since modifications, combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the embodiments may occur to persons skilled in the art, the disclosed embodiments should be construed to include everything within the scope of the appended claims and their equivalents.

Claims

Attorney Docket No.: SP24-308What is claimed:

1. A bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width; a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed-bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height; and a compression sleeve surrounding at least a portion of the fixed-bed cell culture matrix, the compression sleeve exerting a compressive force in a direction of the bed width of the fixed- bed cell culture matrix, wherein the compressive force is sufficient to compress the bed width to a compressed bed width that is less than the reservoir width, and wherein the compression sleeve is configured to release the compressive force so that the bed width increases to an expanded bed width.

2. The bioreactor system of claim 1, wherein the cell culture vessel further comprises an opening with an opening width greater than or equal to the compressed bed width.

3. The bioreactor system of claim 1 or claim 2, wherein the compression sleeve is configured to exert the compressive force by shrinking in response to an external stimulus.

4. The bioreactor of claim 3, wherein the external stimulus is at least one of vacuum, heat, and radiation.

5. A method of assembling a fixed-bed bioreactor, the method comprising: providing a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell cultureAttorney Docket No.: SP24-308 matrix further comprising a bed height in direction of a longitudinal axis of the fixed-bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height; providing a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width; surrounding at least a portion of the fixed-bed cell culture matrix with a compression sleeve; compressing the compression sleeve so that it decreases the bed width to a compressed bed width; after compressing the compression sleeve, inserting the fixed-bed cell culture matrix with the compression sleeve in compression into the interior reservoir.

6. The method of claim 5, further comprising, after inserting the fixed-bed cell culture matrix, releasing the tension in the compression sleeve so that the bed width increases from the compressed bed width to the reservoir width.

7. A bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width; a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed-bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height; and an adhesive around a circumference of the fixed-bed cell culture matrix, wherein the adhesive is configured to adhere an exterior of the fixed-bed cell culture matrix to the interior wall.

8. The bioreactor system of claim 7, wherein the adhesive extends across at least a portion the bed height.Attorney Docket No.: SP24-3089. The bioreactor system of claim 8, wherein the adhesive extends across an entirety of the bed height.

10. The bioreactor system of any one of claims 7-9, wherein the adhesive is a pressuresensitive adhesive film.

11. A bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width, the interior wall comprising a plurality of protrusions extending inward into the interior reservoir; and a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed-bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height, wherein the plurality of protrusions are sized to at least partially compress the fixed-bed cell culture matrix in a direction of the bed width.

12. The bioreactor system of claim 11, wherein the plurality of protrusions are sized to fill a gap between the fixed-bed cell culture matrix and the interior wall.

13. A bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising at least one interior reservoir defined by an interior wall and having a reservoir width; a fixed-bed cell culture matrix comprising a plurality of layers of substrate, the substrate comprising a surface configured for adhering cells thereto, the fixed-bed cell culture matrix further comprising a bed height in direction of a longitudinal axis of the fixed-bed cell culture matrix and a bed width in a direction perpendicular to the direction of the bed height; and a sealant disposed between the fixed-bed cell culture vessel and the interior wall,Attorney Docket No.: SP24-308 wherein the sealant comprises an expandable material that expands to fill a gap between the fixed-bed cell culture matrix and the interior wall.

14. The bioreactor system of claim 13, wherein the sealant is disposed on an exterior side of the fixed-bed cell culture matrix.

15. The bioreactor system of claim 13, wherein the sealant is disposed on the interior wall.

16. The bioreactor system of any one of claims 13-15, wherein the sealant comprises at least one of a silicone, a polyurethane, a polyethylene, and a polyester.

17. The bioreactor system of any one of claims 13-16, wherein the sealant comprises film, adhesive, polymer, or coating.

18. The bioreactor system of any one of claims 13-17, wherein the sealant is configured to expand or contract in response to a stimulus.

19. The bioreactor of claim 18, wherein the stimulus comprises at least one of a change in temperature, a presence of fluid, a chemical reaction, and an exposure to radiation.

20. The bioreactor of any one of claims 1-4 and 7-19, wherein the fixed-bed cell culture substrate comprises a plurality of layers of substrate material in a stacked arrangement.

21. The bioreactor of any one of claims 1-4 and 7-19, wherein the fixed-bed cell culture substrate comprises a rolled substrate material.

22. The bioreactor of any of claims 1-4 and 7-21, further comprising an O-ring disposed at a top or a bottom of the fixed-bed cell culture matrix, the O-ring being disposed along a circumference of the interior wall.Attorney Docket No.: SP24-30823. The bioreactor system of claim 22, wherein the O-ring comprises an elastomeric material.

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