Filtering apparatus and methods for purifying biological processes and cell populations

Abstract

The present invention provides a method for purifying cell populations and a filter device suitable for use in bioreactors. [Solution] A filter device for withdrawing fluid culture medium from a bioreactor during cell culture growth in the bioreactor is disclosed. A method for culturing cells in a bioreactor is also disclosed. The filter device includes a hollow tubular member attached to a filter member. The filter member has a pore size and volume that allows for the withdrawal of fluid culture medium from the bioreactor at a relatively high flow rate. Without withdrawing biological cells from the bioreactor and without damaging or harming the cells, the filter device of this disclosure enables many process improvements.

Description

Background Art

[0001] A bioreactor is a device that can perform biological reactions or processes on a laboratory or industrial scale and is widely used in the biopharmaceutical industry. A bioreactor can be used for batch applications, and the biological material supplied to the bioreactor remains in the bioreactor until the reaction time is over. Alternatively, a bioreactor can be used for perfusion applications, and the fluid medium contained in the bioreactor is periodically or continuously removed and resupplied to the bioreactor to replenish the nutrients contained in the fluid medium and to enable the removal of damaging by-products produced during the process.

[0002] For example, bioreactors are used to produce biological agents that are biological drugs produced from living organisms. Bioreactors are also used in immunotherapy, a type of treatment that strengthens a patient's immune system to fight cancer, infections, and other diseases. The immunotherapy process can include, for example, the production of T cells and / or natural killer (NK) cells. During T cell therapy, for example, T cells are removed from a patient's blood. The T cells are then sent to a bioreactor and grown or cultured. Additionally, the T cells can be altered to have specific proteins called receptors. The receptors on the T cells are designed to recognize and target unwanted cells in the body, such as cancer cells. The modified T cells are cultured in a bioreactor to achieve a certain cell density and then supplied to the patient's body to fight cancer or other diseases. T cell therapy is typically referred to as chimeric antigen receptor (CAR) T cell therapy. The use of T cells in CAR therapy has recently skyrocketed due to great success in the fight against blood diseases.

[0003] Similarly, NK cells can be cultured and grown in a bioreactor for injection into a patient's body. NK cells are a type of cytotoxic lymphocyte that can search for and destroy infected cells in the body. NK cells can exhibit a very rapid immune response. As a result, the use of NK cells in anticancer therapy has grown considerably with interest and popularity. However, only a limited number of NK cells are present in the blood of mammals, and NK cells need to be grown to a relatively high cell density in a bioreactor.

[0004] During the proliferation of T cells, NK cells, or other mammalian cells for the production of biological agents, the regulation of key metabolites in the bioreactor's fluid medium can directly affect the quality of the produced product. For example, nutrient levels, lactate concentration, dissolved oxygen, pH, etc., should be carefully controlled and monitored during cell growth and survival.

[0005] In addition to maintaining cells in a carefully controlled environment, culturing cells for human use also requires a somewhat complex process from seeding to patient use. For example, when producing CAR T cells, the cells are typically first activated and then subjected to gene editing. Once edited, the cells proliferate to a certain cell density. Proliferation can exceed 15 days, for example, more than 20 days. After proliferation, the cells are purified by removing undesirable biological byproducts and unusable cells. During this process, the cells also need to be washed and removed from the growth medium. This process may also require multiple cycles. Finally, the cells are combined with buffers and either administered to the patient or placed in containers for freezing.

[0006] The therapeutic cells are concentrated and transferred from one solution to another during the process, in multiple stages. This can occur and may require a considerable amount of time and manual labor. However, washing and separating the therapeutic cells can be an important part of the overall effectiveness of cell therapy and can prevent side effects in patients.

[0007] As can be understood, the processes or methods described above not only require a considerable amount of time but may also require a significant amount of human handling. However, time is of the essence when it comes to treating cancer patients. Therefore, there is a need for streamlined processes for producing therapeutic cells and biological agents.

[0008] Furthermore, many past methods are not scalable. Therefore, many of the above processes were carried out in small bioreactors, such as those with a volume of less than 2 liters. Thus, there is also a need for processes and methods for culturing therapeutic cells and biological agents that are scalable to produce larger quantities of products in the same amount of time.

[0009] There is also a need for processes and methods for culturing cells and / or for manufacturing biological products that can be automated to reduce the use of manual labor. For example, there is a need for closed systems for culturing and purifying cells such as T cells and NK cells. [Overview of the project]

[0010] In general, this disclosure relates to filter devices capable of removing fluid culture medium from a bioreactor without damaging the cells contained within the bioreactor or reactor. More specifically, this disclosure relates to filter devices specifically designed to remove fluid medium from a bioreactor at relatively high flow rates. As will be described in more detail below, the filter devices are particularly well adapted to remove fluid without removing or harming the cells contained within the bioreactor. This disclosure also relates to a method for promoting cell growth in a bioreactor system, using a filter device to remove fluid medium for cell replenishment and further growth, and / or purification.

[0011] In one embodiment, for example, the present disclosure relates to a method for purifying a cell population. The method comprises growing a biological cell population in a fluid medium. The biological cell population comprises biological cells in an unsupported state, meaning that the cells are not bound to any adjacent surface. The biological cell population contained in a bioreactor contains at least 1 × 10⁶ cells. 6 The fluid medium has a cell density of cells / mL. According to this disclosure, the fluid medium is withdrawn from the bioreactor and filtered. More specifically, the fluid medium is filtered through a filtering device including a filter member. The filter member has a pore size that prevents biological cells from being withdrawn from the bioreactor when the fluid medium is withdrawn. The method further includes the step of adding a buffer medium to the biological cell population to replace the withdrawn fluid medium. Through this process, the biological cell population is washed with the buffer medium, while the method is also well suited to removing biological byproducts that may be present with the biological cell population. For example, the filter member may have a pore size that allows the passage of biological byproducts in the fluid medium that are withdrawn without removing or harming the biological cells. Biological byproducts may include, for example, proteins, serum, and mixtures thereof. For example, after the fluid medium has been withdrawn from the bioreactor, the biological cell population may contain biological byproducts (or any of the individual byproducts described above) in amounts of less than about 0.1% by weight.

[0012] During the above method, more than 40% of the volume of the fluid medium, for example more than 50%, for example more than 60%, for example more than 70%, for example more than 80%, can be withdrawn and replaced at least partially with buffer medium. The biological cell population and the fluid medium are, for example, in one embodiment The bioreactor may have a volume of approximately 1 L to approximately 10 L, or in another embodiment, approximately 5 L to approximately 75 L. The fluid medium can be withdrawn from the bioreactor at a flow rate such that at least 50% of the volume of fluid medium in the bioreactor is withdrawn in a period of less than approximately 1 hour. The filter device of this disclosure can operate at relatively high flow rates, for example, without obstruction formed on the outer surface of the filter device.

[0013] As described above, the above method can wash biological cell populations and remove biological byproducts. This method can be repeated multiple times to purify the cells. For example, this method can be repeated approximately 2 to 5 times.

[0014] As described above, after purifying the biological cells, the biological cell population and buffer medium can be placed in a flexible bag container for cryogenic storage. In one embodiment, the cryogenic buffer medium can be combined with the biological cell population.

[0015] All different types of biological cells can be purified according to the method described above. For example, the biological cells may include any suitable mammalian cells. The method of this disclosure is particularly well suited for the proliferation and purification of T cells and NK cells.

[0016] In some applications, a biological cell population may contain different cell types, such as a first cell and a second cell. One of the cell types may be particularly suitable for administration to a patient, while other cell types may have other uses or be discarded. The method of this disclosure provides an efficient way to separate a first cell type from a second cell type for further purification of the biological cell population.

[0017] For example, a biological cell population containing at least two different cell types may be arranged in contact with one or more microcarriers within a bioreactor. The microcarriers may be added to a fluid culture medium containing the biological cell population. The microcarriers may be designed so that first cells adhere to and bind to the surface of the microcarriers, while second cells do not. According to this disclosure, the fluid culture medium within the bioreactor can be removed and filtered through a second filter device. The second filter device may have pore sizes that allow the passage of second cells but inhibit the passage of microcarriers for separating first cells from second cells.

[0018] The second cells, once separated from the first cells, may then be subjected to further purification and washing steps as described above, and then either placed in use or stored for future use.

[0019] The first cells can also be isolated and used as desired. For example, in one embodiment, the microcarriers added to the bioreactor may be soluble. For example, the microcarriers may be soluble in a fluid medium or may dissolve when in contact with a solvent added to the fluid medium. Once the microcarriers are soluble, the first cells remain unsupported in the bioreactor. The first cells can then be purified, washed according to the method described above, and used as desired.

[0020] As described above, the method of the present disclosure is carried out using a filter device. In one embodiment, the filter device may include a hollow tubular member for filtering fluid from a bioreactor.

[0021] The hollow tubular member may be long enough to be inserted into the bioreactor. For example, the hollow tubular member may be long enough to extend toward the bottom of the bioreactor. A hollow tubular member may extend through a port on the top or side of the bioreactor. The hollow tubular member has a first end defining a first opening and an opposing second end defining a second opening. The second opening is for insertion into and withdrawal of the fluid medium within the bioreactor. The second opening of the hollow tubular member may have a cross-sectional area designed to allow a desired volumetric flow rate to be drawn from the bioreactor.

[0022] According to this disclosure, the filter device further includes a filter member located at a second end of a hollow tubular member. The filter member can completely enclose and seal the second opening. The filter member defines an inner surface and an outer surface. The filter member comprises a porous material. According to this disclosure, the porous material has an absolute pore size of about 1 micron to about 9 microns, for example, about 1 micron to about 6 microns. The above pore size has been found to allow the fluid culture medium to be withdrawn from the bioreactor without removing biological cells. In one embodiment, the filter member comprises a porous mesh. In another embodiment, the filter member comprises a nonwoven mesh formed from sintered metal fibers. For example, the sintered metal fibers may include stainless steel. Alternatively, the filter member (and the hollow tubular member) may be made of a polymer material. For example, the filter member may be made of a polymer mesh or a nonwoven fabric. The polymer material may include, for example, polyamide or polyolefin.

[0023] The filter components of a filter device may have a surface area sufficient to allow for relatively high volumetric fluid flow rates. For example, the surface area may be approximately 0.5 in. 2 It can be larger. The outer surface area is, for example, about 3 in. 2 Larger than that, for example, about 4 inches 2 Larger than that, for example, about 6 inches 2 Larger than that, and generally about 50 inches. 2 It may be less than [a certain length]. The length of the filter member may depend on various factors. In one embodiment, the filter member may have a length along the axial direction of a hollow tubular member of about 2 inches to about 8 inches.

[0024] The hollow tubular member and the second opening may generally have a diameter greater than about 2 mm, for example, greater than about 4 mm, for example, greater than about 8 mm, for example, greater than about 10 mm, for example, greater than about 12 mm, for example, greater than about 14 mm, for example, greater than about 16 mm, for example, greater than about 18 mm, for example, greater than about 20 mm. The diameter of the hollow tubular member is generally less than about 50 mm, for example, less than about 30 mm, for example, less than about 20 mm, for example, less than about 14 mm.

[0025] The ratio of the cross-sectional area of ​​the second opening to the surface area of ​​the filter member is generally about 1:5 to about 1:200, for example, about 1:15 to about 1:100.

[0026] In one embodiment, the inner surface of the filter member may have a different absolute pore diameter than the outer surface of the filter member. For example, the inner surface of the filter member may have a larger pore diameter than the outer surface. The pore diameter of the outer surface may be, for example, about 1 micron to about 9 microns, while the absolute pore diameter of the inner surface of the filter member may be about 5 microns to about 20 microns.

[0027] As described above, in one embodiment, a second type of filter device can be used that allows the passage of a first type of cell while preventing the passage of a second type of cell that may bind to a microcarrier. In this application, the filter device may be as described above, but may have a larger pore size. For example, the filter member of the filter device may have an absolute pore size of more than about 60 microns, for example more than about 70 microns, for example more than about 80 microns, for example more than about 90 microns, and generally less than about 150 microns, for example less than about 130 microns, for example less than about 120 microns, for example less than about 110 microns.

[0028] Furthermore, in one aspect, the disclosure generally relates to filtering fluids from a bioreactor. A filter device including a hollow tubular member is targeted. The hollow tubular member can have a length sufficient to be inserted into a bioreactor. For example, the hollow tubular member can have a length sufficient to extend towards the bottom of the bioreactor. The hollow tubular member can extend through a port in the upper or side surface of the bioreactor. The hollow tubular member has a first end defining a first opening and an opposing second end defining a second opening. The second opening is for inserting into and withdrawing the fluid medium within the bioreactor. The second opening of the hollow tubular member can have a cross-sectional area designed to be able to withdraw a desired volumetric flow rate from the bioreactor.

[0029] The filter member of the filter device can have a surface area sufficient to allow a relatively high volumetric fluid flow rate. For example, the surface area can be greater than about 0.5 in 2 It may be larger. The outer surface area, for example, is larger than about 3 in 2 more, for example, larger than about 4 in 2 more, for example, larger than about 6 in 2 more, and generally can be less than about 50 in 2 The length of the filter member may depend on various factors. In one aspect, the filter member can have a length along the axial direction of the hollow tubular member of about 2 inches to about 8 inches.

[0030] In one embodiment, the hollow tubular member is straight from a first end to a second end. In an alternative embodiment, the hollow tubular structure may have a shape such that the second end does not interfere with an impeller that can be rotated in the bioreactor. For example, in one embodiment, the hollow tubular member may include a first straight section, a second straight section, and an angled section positioned between the first and second straight sections. The angled section may extend from the first straight section at an angle of about 25° to about 45°. Similarly, the angled section may extend from the second straight section at an angle of about 25° to about 45°. In one embodiment, the first and second straight sections are parallel to a vertical axis extending through the bioreactor.

[0031] In one embodiment, the hollow tubular member may also include an angled member located at a second end. The hollow tubular member may include a straight member that transitions into the angled member. The angled member may be at an angle to the straight section of about 50° to about 90°. For example, in one embodiment, the angled member is at a right angle at the end of the hollow tubular member. In this regard, when the filter device is extended into a bioreactor, the angled member may be positioned toward the bottom of the bioreactor and may be substantially parallel to the bottom surface of the bioreactor. For example, in one embodiment, the angled member may be designed to position the filter member below an impeller contained within the bioreactor.

[0032] In one embodiment, the hollow tubular member and the filter member can be completely sealed for sterile closure connection to the bioreactor port. A flexible plastic bellows can seal the hollow tubular member and the filter member. The sterile connection port can be attached to one end of the bellows. The bioreactor port may have a matching sterile connector. Once the matching sterile connectors of the bioreactor and the bellows are connected, the bellows may collapse, allowing the filter member and the hollow tubular member to be inserted into the bioreactor port.

[0033] In one embodiment, the filter device may include a filter member in the side wall or bottom wall of the bioreactor. The filter member may be a mesh patch on the side wall or bottom wall of the bioreactor. A flexible cone can connect the mesh patch to a hollow tubular member for fluid output from the bioreactor.

[0034] This disclosure also relates to a method for culturing cell proliferation. This method is for T cells or N cells. The method includes seeding biological cells, such as K cells, into a bioreactor. The bioreactor contains a fluid medium for cell proliferation. The fluid medium is perfused by inserting a filter device into the bioreactor as described above. The filter components of the filter device may have pore sizes that prevent biological cells from being withdrawn from the bioreactor when the fluid medium is withdrawn. The method further includes the step of replenishing the fluid medium in the bioreactor to promote cell viability.

[0035] In one embodiment, for example, the filter device may be designed to remove the fluid medium at a rate of more than approximately 0.5 L per day, for example more than approximately 1 L per day, for example more than approximately 2 L per day, for example more than approximately 5 L per day, for example more than approximately 10 L per day, for example more than approximately 15 L per day, for example more than approximately 25 L per day.

[0036] According to this disclosure, the filter device further includes a filter member located at a second end of a hollow tubular member. The filter member can completely enclose and seal the second opening. The filter member defines an inner surface and an outer surface. The filter member comprises a porous material. According to this disclosure, the porous material has an absolute pore size of about 1 micron to about 9 microns, for example, about 1 micron to about 6 microns. In one embodiment, the filter member comprises a porous mesh. In another embodiment, the filter member comprises a nonwoven mesh formed from sintered metal fibers. For example, the sintered metal fibers may include stainless steel. Alternatively, the filter member (and the hollow tubular member) may be made of a polymer material. For example, the filter member may be made of a polymer mesh or a nonwoven fabric. The polymer material may include, for example, polyamide or polyolefin.

[0037] This disclosure also relates to a method for culturing cells. This method includes seeding biological cells into a bioreactor. The bioreactor includes a stirred-tank bioreactor. The bioreactor contains a fluid medium for cell growth that is stirred during cell growth. The biological cells are present in the bioreactor in an unsupported state. According to this disclosure, the fluid medium is perfused from the bioreactor through a filter device that comes into contact with the fluid medium in the bioreactor. The filter device includes a filter member having a pore size that prevents the biological cells from being drawn out of the bioreactor when the fluid medium is drawn out. The fluid medium in the bioreactor is replenished when the fluid medium in the bioreactor is drawn out of the bioreactor in order to promote cell viability.

[0038] The biological cells contained within the bioreactor may include any suitable cells, such as any suitable mammalian cells. In certain embodiments, the biological cells are T cells or NK cells. The initial cell density after seeding is generally about 0.5 × 10⁻⁶. 6 Less than cells / mL, e.g., approximately 0.4 × 10⁻⁶ 6 Less than cells / mL, e.g., approximately 0.3 × 10⁻⁶ 6The cell density is less than 0.1 × 10⁻⁶ cells / mL. The initial cell density is generally about 0.1 × 10⁻⁶. 4 Exceeding cells / mL.

[0039] In one embodiment, perfusion is initiated after the biological cells in the bioreactor have reached a desired cell density. For example, perfusion is initiated when the biological cells have reached approximately 1 × 10⁻⁶ 6 Cells / mL greater than, for example, about 1.5 × 10⁻⁶ 6 cells / mL greater than, for example, about 1.7 × 10⁻⁶ 6 Perfusion may be initiated after a cell density exceeding cells / mL is reached. For example, perfusion may be initiated at least 4 days, or for example, at least 7 days, after the bioreactor has been seeded with biological cells.

[0040] Biological cells can proliferate rapidly and dramatically during the process. For example, biological cells in a bioreactor can grow to at least 1 × 10⁶ 7 cells / mL, e.g., at least about 1.2 × 10⁶ 7 Cell density can reach cells / mL. Cells can be harvested from the bioreactor after approximately 14 days, for example, after approximately 13 days.

[0041] During the process, various parameter levels can be controlled due to the mode in which the bioreactor operates. For example, the glucose level in the fluid medium can be maintained above 4 g / L, for example, above about 5 g / L. On the other hand, the lactate level can be maintained below about 1.5 g / L, for example, below about 1.3 g / L, throughout the entire process. In one embodiment, the dissolved oxygen in the fluid medium during the process decreases rapidly to 0 or near 0. The reduction of dissolved oxygen is thought to increase the desired phenotype. For example, the process can be manipulated to have a proportional increase in a particularly desired phenotype. This phenotype is, for example, T scm It may contain cells.

[0042] During perfusion, the fluid culture medium is withdrawn from the bioreactor using a filter device having a filter element with an absolute pore size of approximately 1 to 9 microns, for example, approximately 1 to 6 microns. The filter element is generally about 0.5 in2 For example, about 2 in 2 Over, and generally about 10 inches 2 It may have a surface area of ​​less than . During perfusion, at least about 30%, e.g., at least about 40%, e.g., at least about 50%, e.g., at least about 60%, e.g., at least about 70% of the volume of the fluid medium in the bioreactor is perfused and replaced every 24 hours.

[0043] Other features and aspects of this disclosure are described in more detail below. [Brief explanation of the drawing]

[0044] The complete and possible disclosure of this disclosure is more specifically provided in the remainder of this specification, including references to the accompanying drawings.

[0045] [Figure 1] This is a cross-sectional view of one embodiment of the bioreactor system according to the present disclosure. [Figure 2] This is a side view of one embodiment of a filter device manufactured in accordance with this disclosure. [Figure 3] This is a side view of another embodiment of a filter device fabricated in accordance with this disclosure. [Figure 4A] This is a perspective view of one embodiment of a filter member attached to a filter device according to the present disclosure. [Figure 4B] Figure 4A is a side view of the filter component shown. [Figure 4C] This is another side view of the filter component shown in Figure 4A. [Figure 5A] This is a side view of another embodiment of the filter device according to the present disclosure. [Figure 5B] Figure 5A is a partial side view of the filter device shown. [Figure 6] This is a perspective view of another embodiment of the bioreactor system according to the present disclosure. [Figure 7A] This is a perspective view of one embodiment of a bioreactor system having a sterile closed connection between the bioreactor and the filter device. [Figure 7B] This is a perspective view of one embodiment of a bioreactor system having a sterile closed connection between the bioreactor and the filter device. [Figure 7C] This is a perspective view of one embodiment of a bioreactor system having a sterile closed connection between the bioreactor and the filter device. [Figure 8] Figure 8A is a perspective view of another embodiment of the bioreactor system according to this disclosure. Figure 8B is a side view of a filter device that may be used in conjunction with the bioreactor system shown in Figure 8A. [Figure 9] This is a side view of another embodiment of the bioreactor system according to the present disclosure. [Figure 10] This is a side view of another embodiment of a filter device that may be used in accordance with this disclosure. [Figure 11] The following is a graphic representation of the results obtained in the examples described below. [Figure 12] The following is a graphic representation of the results obtained in the examples described below. [Figure 13] The following is a graphic representation of the results obtained in the examples described below. [Figure 14] The following is a graphic representation of the results obtained in the examples described below. [Figure 15] The following is a graphic representation of the results obtained in the examples described below. [Figure 16] The following is a graphic representation of the results obtained in the examples described below. [Figure 17] The following is a graphic representation of the results obtained in the examples described below. [Figure 18] This is a cross-sectional view of a bioreactor illustrating the method according to this disclosure for separating different types of cells. [Figure 19-1] Figures 19A and 19B are representations of results obtained before and after the use of magnetic beads as microcarriers in accordance with this disclosure. [Figure 19-2]Figures 19C, 19D, and 19E are representations of results obtained before and after the present disclosure using magnetic beads as microcarriers as shown in Figures 19A and 19B. [Figure 20A] This is a representation of the results obtained in accordance with this disclosure using magnetic beads as microcarriers in the presence of a magnet. [Figure 20B] This is a representation of the results obtained in accordance with this disclosure using magnetic beads as microcarriers in the presence of a magnet. [Figure 20C] This is a representation of the results obtained in accordance with this disclosure using magnetic beads as microcarriers in the presence of a magnet. [Figure 20D] Figures 20A-20C represent the results obtained according to this disclosure using magnetic beads as microcarriers 24 hours after the removal of the magnets. [Figure 20E] Figures 20A-20C represent the results obtained according to this disclosure using magnetic beads as microcarriers 24 hours after the removal of the magnets. [Figure 20F] Figures 20A-20C represent the results obtained according to this disclosure using magnetic beads as microcarriers 24 hours after the removal of the magnets. [Figure 21A] This disclosure shows a cross-sectional view of a bioreactor having a skimmer filter. [Figure 21B] This disclosure shows a cross-sectional view of a bioreactor having a skimmer filter.

[0046] The repeated use of reference numerals in this specification and drawings is intended to represent identical or similar features or elements of the present invention. [Modes for carrying out the invention]

[0047] Those skilled in the art will understand that this discussion is for illustrative purposes only and is not intended to limit any broader aspects of the present disclosure.

[0048] Generally, this disclosure relates to methods and systems for culturing and growing cells and / or cell products in a bioreactor. The bioreactor contains a population of biological cells in a fluid medium, such as a fluid culture medium. The biological cells are cultured under conditions and in a suitable culture medium suitable for promoting cell reproduction and growth until a desired amount of cells can be harvested from the bioreactor.

[0049] According to this disclosure, the bioreactor is designed to operate in perfusion mode during the cell culture process. Specifically, at selected times, the fluid medium contained within the bioreactor is continuously or at least periodically removed and replenished. In the past, there have been problems in removing the fluid medium from the bioreactor without significantly harming or damaging the cells contained within it. Therefore, in the past, bioreactors were typically operated under static conditions or in batch mode in oscillating bioreactors. These systems have been found to be extremely inefficient in the proliferation of cell populations. Conventional systems are also not scalable and therefore only operate in small bioreactor volumes.

[0050] This disclosure relates to an improved bioreactor system including a stirred tank bioreactor combined with a filter device. According to this disclosure, the filter device is a biological filter. The fluid medium can be rapidly removed from the bioreactor without removing the cell population, without damaging the cells, and / or without contamination-related problems. The agitated tank bioreactor combined with the filtration device of this disclosure can offer many advantages and benefits. For example, through the system and process of this disclosure, cell cultures, particularly T cell cultures and NK cell cultures, can grow more rapidly and efficiently compared to previous bioreactor systems. For example, the process of this disclosure can reach cell densities, and especially viable cell densities, that were not possible with previous equipment and protocols. Furthermore, the process and system of this disclosure also allows for careful control of parameters and metabolites within the bioreactor, which further promotes cell growth and cell health and viability. For example, the ability to maintain cells in an unsupported state (i.e., unsupported on microcarriers) within the agitated tank reactor and the ability to operate in perfusion mode allows for careful control of nutrient levels, including lactate levels and glucose levels, as well as ammonia levels, in addition to controlling pH, dissolved oxygen, and other parameters. Particularly advantageous is that all of the above parameters can be carefully controlled and monitored in a closed system that eliminates manual handling of the cells or the fluid medium in which the cells are maintained.

[0051] The processes and systems of this disclosure have also been found to offer a variety of other advantages and benefits compared to systems used in the past. For example, the processes and systems of this disclosure are fully scalable, and similar results can be achieved not only with small bioreactors but also with much larger bioreactors. For example, the bioreactors incorporated into the process may have volumes exceeding 1 liter, e.g., exceeding 3 liters, e.g., exceeding 5 liters, e.g., exceeding 10 liters, e.g., exceeding 15 liters, e.g., exceeding 20 liters, e.g., exceeding 30 liters, e.g., exceeding 40 liters, and even exceeding 50 liters. Being fully scalable, the allogeneic cell therapy process can be run to produce much larger quantities of product for administration to many patients by taking only a fraction of the time required by previous processes.

[0052] Initiating cell proliferation is also much simpler and more automated with respect to the use of this system and process. For example, this system and process eliminates the 2D activation / seed train required in previous bioreactor systems such as rocker-type bioreactors. Instead, biological cell populations cultured in this system can be activated / proliferated directly.

[0053] However, in one embodiment, the biological cells may still be subjected to 2D seed train activation and / or proliferation before use in the system or process according to this disclosure. In one embodiment, the biological cells may be thawed in a 2D seed train flask. The biological cells are approximately 0.1 × 10⁶ 6 cells / cm 2 ~Approx. 1.5×10 6 cells / cm 2 Seed density, for example, approximately 0.25 × 10 6 cells / cm 2 ~Approx. 1.25×10 6 cells / cm 2 Seed density, for example, about 0.5 × 10 6 cells / cm2 ~Approx. 1×10 6 cells / cm 2 Seeds may be sown at the following seed densities, or at any range or value between them.

[0054] As used herein, nutrient medium or matrix means any fluid, compound, molecule, or substance that can increase the mass of biological products, such as those on which organisms live and grow, or which can otherwise be used to supplement biomass. For example, a nutrient feed may contain gases such as oxygen or carbon dioxide used for respiration or any type of metabolism. Other nutrient mediums may contain carbohydrate sources. Carbohydrate sources include complex sugars and simple sugars, such as glucose, maltose, fructose, galactose, and mixtures thereof. Nutrient mediums may also contain amino acids. Amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, celery The amino acids may include threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid, their single stereoisomers, and racemic mixtures thereof. The term “amino acid” may also refer to known nonstandard amino acids, such as 4-hydroxyproline, ε-N,N,N-trimethyllysine, 3-methylhistidine, 5-hydroxylysine, O-phosphoserine, γ-carboxyglutamic acid, γ-N-acetyllysine, ω-N-methylarginine, N-acetylserine, N,N,N-trimethylalanine, N-formylmethionine, γ-aminobutyric acid, histamine, dopamine, thyroxine, citrulline, ornithine, β-cyanoalanine, homocysteine, azasin, and S-adenosylmethionine. In some embodiments, the amino acids are glutamates, glutamine, lysine, tyrosine, or valine.

[0055] Nutrient media may also contain one or more vitamins. Vitamins that may be contained in nutrient media include B vitamins such as B12. Other vitamins include vitamin A, vitamin E, riboflavin, thiamine, biotin, and mixtures thereof. Nutrient media may also contain one or more fatty acids and one or more lipids. For example, nutrient medium feed may contain cholesterol, steroids, and mixtures thereof. Nutrient media may also supply proteins and peptides to the bioreactor. Proteins and peptides include, for example, albumin, transferrin, fibronectin, fetwin, and mixtures thereof. Growth media in this disclosure may also contain growth factors and growth inhibitors, trace elements, inorganic salts, hydrolysates, and mixtures thereof. Trace elements that may be contained in growth media include trace metals. Examples of trace metals include cobalt and nickel. For example, in one embodiment, and for example only in one embodiment, the nutrient medium / matrix may be X-Vivo® Matrix Medium sold by Lonza.

[0056] Nevertheless, the thawed biological cells are seeded for growth in a 2D seed train flask containing nutrient medium and maintained in the 2D seed train flask until the biological cells reach a cell density of at least 2 million cells / mL for T cells. For example, in one embodiment, the biological cells may be maintained in the 2D seed train flask for about 3 to 9 days, e.g., about 4 to 8 days, e.g., about 5 to 7 days, to achieve a desired cell density.

[0057] After the desired cell density is achieved, the biological cells contained in the 2D seed train can be harvested using a permeable solution. Regardless of the selected permeable solution, the harvested biological cells can be used to seed the processes or systems described herein.

[0058] Nevertheless, as discussed, in the embodiment, 2D seed train activation and / or proliferation is not used; instead, cells are activated and / or proliferated using the system and / or process.

[0059] When proliferating T cell or NK cell populations, the processes and systems of this disclosure can also facilitate many downstream processes after the desired cell density has been reached. For example, the filtering apparatus of this disclosure can greatly improve the efficiency of washing cells, separating by-products from cells, purifying cells, and / or transferring cells to cryogenic storage containers. Therefore, the systems and processes of this disclosure are particularly well suited to the proliferation of cell therapy populations such as T cell populations and NK cell populations. Using these systems and processes, it is possible to facilitate not only the proliferation of autologous cell therapy but also allogeneic cell therapy. With respect to autologous cell therapy, the systems and processes of this disclosure can dramatically reduce the amount of time required to reach the desired viable cell density.

[0060] When producing allogeneic CAR T cells or NK cells, the processes and systems of this disclosure can immediately provide off-the-shelf cell therapy to many patients while significantly reducing costs. The processes and systems are also particularly efficient and appropriately suited for removing undesirable biological byproducts such as proteins, serum, and T cell receptors from the final cell culture, and for significantly improving the purity of the final product, which not only provides immediate better patient care but also allows for the storage of the product at higher cell densities. Similar advantages and benefits are also achieved when producing virus-specific T cells and CAR NK cells.

[0061] Referring to Figure 1, one embodiment of a bioreactor system according to the present disclosure is shown. The bioreactor system comprises a bioreactor 10. The bioreactor 10 comprises a hollow container or vessel having a bioreactor volume 12 for receiving a cell culture suspended in a fluid growth medium. According to the present disclosure, the biological cells contained in the biological 10 may be suspended in the fluid growth medium in an unsupported state, meaning that the cells are not attached to any adjacent surface such as microcarriers. The ability to process cells in an unsupported state is thought to increase the growth rate of the cell culture and allow the system to be scalable. As shown in Figure 1, the bioreactor system may further include a rotatable shaft 14 coupled to a stirrer such as an impeller 16.

[0062] The bioreactor 10 can be made of various different materials. In one embodiment, for example, the bioreactor 10 may be made of a metal such as stainless steel. Metal bioreactors are typically designed to be reusable.

[0063] Alternatively, the bioreactor 10 may include a single-use bioreactor made of a flexible polymer film. This film or shape-conforming material may be liquid-impermeable and may have an internally hydrophilic surface. In one embodiment, the bioreactor 10 may consist of a flexible polymer film designed to be inserted into a rigid structure such as a metal container to exhibit a desired shape. Polymers that can be used to make up the flexible polymer film include polyolefin polymers such as polypropylene and polyethylene. Alternatively, the flexible polymer film may be made of polyamide. In another embodiment, the flexible polymer film may be formed from multiple layers of different polymer materials. In one embodiment, the flexible polymer film may be gamma-irradiated.

[0064] Since the process is scalable, the bioreactor 10 can have any suitable volume. For example, the volume of the bioreactor 10 may range from 100 mL to approximately 10,000 L or more. For example, the volume 12 of the bioreactor 10 may be greater than approximately 0.5 L, for example, greater than approximately 1 L, for example, greater than approximately 2 L, for example, greater than approximately 3 L, for example, greater than approximately 4 L, for example, greater than approximately 5 L, for example, greater than approximately 6 L, for example, greater than approximately 7 L, for example, greater than approximately 8 L, for example, greater than approximately 10 L, for example, greater than approximately 12 L, for example, greater than approximately 15 L, for example, greater than approximately 20 L, for example, greater than approximately 25 L, for example, greater than approximately 30 L, for example, greater than approximately 35 L, for example, greater than approximately 40 L, for example, greater than approximately 45 L, and so on. The volume of the bioreactor 10 may generally be less than about 20,000 L, for example less than about 15,000 L, for example less than about 10,000 L, for example less than about 5,000 L, for example less than about 1,000 L, for example less than about 800 L, for example less than about 600 L, for example less than about 400 L, for example less than about 200 L, for example less than about 100 L, for example less than about 50 L, for example less than about 40 L, for example less than about 30 L, for example less than about 20 L, for example less than about 10 L. In one embodiment, for example the volume of the bioreactor may be about 1 L to about 5 L. In another embodiment the volume of the bioreactor may be about 25 L to about 75 L. In yet another embodiment the volume of the bioreactor may be about 1,000 L to about 5,000 L.

[0065] In addition to the impeller 16, the bioreactor 10 may include various additional instruments such as baffles, dispersers, gas supplies, and ports to enable the culture and propagation of biological cells. Furthermore, the bioreactor system may include various probes for measuring and monitoring pressure, foaming, pH, dissolved oxygen, dissolved carbon dioxide, and the like.

[0066] In one embodiment, the bioreactor 10 includes a top section defining a plurality of ports. The ports can allow supply and feed lines to the bioreactor 12 for adding and removing fluids and other materials. In addition, the bioreactor system may be arranged in association with a load cell for measuring the mass of the culture in the bioreactor 10.

[0067] In alternative embodiments, multiple ports may be located at different positions on the bioreactor 10. For example, in one embodiment, ports may be located on the side walls of the bioreactor, as shown in Figures 6-8. In another embodiment, ports may be located at the bottom of the bioreactor, as shown in Figure 9. For example, a bioreactor made of a flexible polymer film may include ports located at the bottom of the container.

[0068] As shown in Figure 1, the bioreactor 10 may include a rotatable shaft 14 attached to at least one impeller 16. The rotatable shaft 14 may be coupled to a motor to rotate the shaft 14 and the impeller 16. The impeller 16 may be made of any suitable material, such as metal or a biocompatible polymer. Examples of impellers suitable for use in a bioreactor system include hydrofoil impellers, high-solidity pitch-blade impellers, high-solidity hydrofoil impellers, Rushton impellers, pitch-blade impellers, and gentle marine-blade impellers. In addition, the rotatable shaft 14 may be coupled to a single impeller 16 as shown in Figure 1, or to two or more impellers. If two or more impellers are included, the impellers may be spaced apart along the rotating shaft 14. In one embodiment, the impeller 16 is rotated at an amount sufficient to maintain the biological cells contained in the bioreactor 10 suspended in a fluid medium without damaging the biological cells.

[0069] The amount of energy imparted to the fluid medium by one or more agitators can affect cell viability and cell proliferation. In one embodiment, it has been found that optimal conditions in the bioreactor are maintained when the propeller 16 is rotated at a speed of about 35 rpm or more, e.g., about 40 rpm or more, e.g., about 50 rpm or more, e.g., about 60 rpm or more, e.g., about 70 rpm or more, and generally less than about 200 rpm, e.g. less than about 120 rpm, e.g. less than about 100 rpm. The tip speed of the impeller may be greater than about 0.07 m / s, e.g., greater than about 0.09 m / s, e.g., greater than about 0.1 m / s, e.g., greater than about 0.12 m / s, and generally less than 0.4 m / s, e.g. less than about 0.3 m / s. Regarding the power input to a 1L agitated tank bioreactor, this is equivalent to a power input greater than 0.0012 watts and generally less than 0.0143 watts, for example, less than approximately 0.006 watts.

[0070] In one embodiment, the bioreactor system may also include a controller which may include one or more programmable devices or microprocessors. The controller can be used to maintain optimal conditions within the bioreactor 10 for promoting cell proliferation. The controller may be located, for example, in a communication and control heat circulator, load cell, or control pump, and may receive information from various sensors and probes. For example, the controller may control and / or monitor pH, dissolved oxygen tension, dissolved carbon dioxide, temperature, stirring conditions, alkalinity conditions, fluid growth medium conditions, pressure, foam level, etc. For example, based on pH readings, the controller may be configured to adjust the pH level by adding the required amount of acid or alkali. The controller may also use a carbon dioxide gas supply to lower the pH. Similarly, the controller may receive temperature information and control the fluid supplied to the water jacket surrounding the bioreactor to raise or lower the temperature.

[0071] In one embodiment, various parameters contained within a bioreactor may be monitored using Raman spectroscopy. A Raman spectrometer can, for example, measure the biomass concentration and / or various other parameters contained within the bioreactor. This information can then be supplied to a controller, which can automatically adjust the feed and withdrawal rates from the bioreactor to maintain various parameters within controlled limits. For example, the use of Raman spectroscopy in monitoring cell cultures is described in U.S. Patent Publication 2019 / 0137338, incorporated herein by reference.

[0072] According to this disclosure, the bioreactor 10 can also communicate with one or more filter devices 20, as shown in Figure 1. The filter devices 20 may extend through a port in the upper part of the bioreactor 10. As shown, the filter devices 20 may extend within the bioreactor 10 without interfering with the impeller 16 and may be positioned adjacent to the bottom of the bioreactor. The filter devices 20 are for continuously or periodically withdrawing liquid culture medium from the bioreactor without removing the biological cells contained within the bioreactor 10. The filter devices 20 of this disclosure can, for example, withdraw fluid at a relatively high flow rate without removing or damaging cells.

[0073] Referring to Figures 2, 4A, 4B, and 4C, one embodiment of a filter device 20 that may be used in accordance with this disclosure is shown. Referring to Figure 2, the filter device 20 includes a hollow tubular member 22. The hollow tubular member 22 may include a first end 24 defining a first opening and a second end 26 defining an opposing second opening. The hollow tubular member 22 may be made of any suitable material that is biologically compatible with the cell culture. For example, the hollow tubular member 22 may be made of a metal such as stainless steel.

[0074] In alternative embodiments, the hollow tubular member may be made of a polymer. In one embodiment, for example, the filter device 20 may be designed to be discarded after a single use. In this embodiment, the hollow tubular member 22 may be made of a polymer material. For example, the hollow tubular member may be made of polyolefin such as polypropylene or polyethylene. Alternatively, the hollow tubular member 22 may be made of polyamide. Otherwise, the hollow tubular member 22 may be made of a plastic material that can be irradiated with gamma rays.

[0075] The hollow tubular member 22 may be flexible or rigid. The hollow tubular member 22, the first opening, and the second opening may generally have diameters sized for a particular application and the amount of fluid to be drawn from the bioreactor 10. For example, the diameter of the hollow tubular member 22 may generally be greater than about 2 mm, e.g., greater than about 4 mm, e.g., greater than about 6 mm, e.g., greater than about 8 mm, e.g., greater than about 10 mm. The diameter of the hollow tubular member 22 may generally be less than about 60 mm, e.g., less than about 40 mm, e.g., less than about 20 mm, e.g., less than about 15 mm, e.g., less than about 11 mm, e.g., less than about 10 mm, e.g., less than about 8 mm.

[0076] The first end 24 of the hollow tubular member 22 may include a tube connection for connecting the hollow tubular member 22 to a plastic tube. The tube connection may be any of various weldable tube types. The outer diameter of the tube connection portion of the first end 24 is approximately The outer diameter may be sized for specific applications and the amount of fluid that needs to be withdrawn from the bioreactor. For example, the outer diameter of the tube connection may generally be about 3 mm or more, e.g., about 6 mm or more, e.g., about 13 mm or more, e.g., about 19 mm or more, e.g., about 26 mm. The outer diameter of the tube connection is generally about 26 mm or less.

[0077] The hollow tubular member 22 may consist of a single material or of multiple members connected together. The hollow tubular member 22 may be straight from a first end 24 to a second end 26. Alternatively, the hollow tubular member 22 may include an angled member 28, as shown in Figure 2. In the embodiment illustrated in Figure 2, the angled member 28 extends from the bioreactor 10 to direct the fluid flow from the bioreactor in a desired direction. As shown in the figure, the angled member 28 is generally perpendicular to the straight section 30 of the hollow tubular member 22. However, the angled member 28 may be at any preferred angle with respect to the straight or vertical section 30 of the hollow tubular member 22.

[0078] When used to extract fluid from the bioreactor 10, the filter device must be long enough so that the second end 26 of the hollow tubular member 22 is adjacent to the bottom surface of the bioreactor 10. In this regard, the straight section 30 of the filter device 20 is generally longer than the length (or depth) of the bioreactor 10. For example, the length of the straight section 30 may be more than about 110% of the length of the bioreactor 10, for example more than about 120%, for example more than about 150%. Generally, the straight section 30 is less than about 500% of the length of the bioreactor 10, for example less than 300%, for example less than about 200%.

[0079] According to this disclosure, the filter device 20 further includes a filter member 32 positioned at the second end 26 of the hollow tubular member 22. The filter member 32 is shown in detail in Figures 4A, 4B, and 4C. According to this disclosure, the filter member 32 has a pore size and surface area that allows a relatively high flow rate of the fluid culture medium through the filter device 20 while inhibiting the flow of biological cells through the filter member 32. For example, in one embodiment, the filter member 32 may be made of a porous mesh such as a stainless steel nonwoven, knitted, or woven material. In one embodiment, the filter member 32 may be made of a nonwoven mesh formed from sintered metal fibers such as stainless steel fibers. Alternatively, the filter member 32 may be made of a polymer material. For example, in one embodiment, the filter member 32 may be made of a polyamide screen mesh such as a nonwoven material. For example, polymer meshes may be more flexible and less susceptible to damage than filter elements made of metal. The filter device 20, which has a polymer hollow tubular member 22 and a filter member 32, may further include a polymer shell (not shown) surrounding the filter member 32.

[0080] The pore size of the filter member 32 generally depends on the size of the cells contained in the bioreactor. For example, the pore size of the filter member 32 is sufficient to allow the flow of the fluid medium without allowing the biological cells to be drawn out along with the fluid medium. The pore size may be uniform or non-uniform on the filter member 32. In one embodiment, the average pore size of the filter member 32 is less than about 12 microns, e.g., less than about 10 microns, e.g., less than 9 microns, e.g., less than 8 microns, e.g., less than 7 microns, e.g., less than 6 microns, e.g., less than 5 microns, e.g., less than 4 microns. The average pore size of the filter member 32 is generally greater than about 1 micron, e.g., greater than about 2 microns, e.g., greater than 3 microns, e.g., greater than 4 microns. Of course, while the pore size has been described as the average pore size so far, it should be understood that the above ranges of pore size may also refer to the nominal pore size or absolute pore size. For example, and as is well known in the art, a nominal pore size of 5 microns represents approximately 99% of particles with pore sizes greater than 5 microns. It will capture them. Conversely, an absolute pore size of 5 microns would indicate that all pores are 5 microns or smaller in size so that all, or substantially all, particles larger than 5 microns are captured by the filter. Therefore, in one embodiment, the pore size of the filter member 32 according to this disclosure has an absolute pore size within the above range.

[0081] The filter member 32 includes an outer surface that is in direct contact with the fluid culture medium contained in the bioreactor, and an opposing inner surface. The pore diameter of the outer surface of the filter member 32 may differ from the pore diameter of the inner surface of the filter member 32. In one embodiment, for example, the pore diameter on the inner surface may be larger than the pore diameter on the outer surface. For example, the absolute pore diameter on the outer surface may be about 1 micron to about 9 microns, while the absolute pore diameter on the inner surface of the filter member 32 may be about 4 microns to about 20 microns, for example, about 6 microns to about 15 microns. For example, the absolute pore diameter on the inner surface may be at least about 20% larger than the absolute pore diameter on the outer surface, for example, at least about 40% larger, for example, at least about 60% larger, for example, at least 80% larger. In this way, the pores passing through the filter member 32 may have a funnel shape that can allow for a larger flow rate through the filter member and prevent deposits and blockages.

[0082] As shown in Figures 4A, 4B, and 4C, the filter member 32 is attached to the second end 26 of the hollow tubular member 22. For example, in the illustrated embodiment, the filter member 32 completely encloses and seals the opening located at the second end 26 of the hollow tubular member 22. The filter member 32 can be attached to the hollow tubular member 22 using any preferred method or technique. For example, the filter member 32 can be welded to the hollow tubular member 22, bonded to the hollow tubular member 22, or mechanically attached to the hollow tubular member. In one particular embodiment, for example, the filter member 32 can be resin-welded to the hollow tubular member 22.

[0083] As shown in Figures 4A, 4B, and 4C, the filter member 32 has a length designed to optimize its surface area and sealed volume to ensure that the filter member 32 can maintain a desired flow rate. The size of the sealed volume 34 may depend on the flow requirements of the system and may be proportional to the cross-sectional area of ​​the opening at the second end 26. For example, the sealed volume 34 may be large enough to allow sufficient fluid flow through the filter member into the hollow tubular member 22, which may be desired for a particular application. The sealed volume 34 may increase the surface area of ​​the filter member 32, for example, and thus provide a larger area for the fluid to enter the filter member, allowing for a larger flow rate through the hollow tubular member 22.

[0084] For example, in one embodiment, the ratio of the cross-sectional area of ​​the opening at the second end 26 to the surface area of ​​the filter member 32 may be greater than about 1:5, for example, greater than about 1:10, for example, greater than about 1:15, for example, greater than about 1:20, for example, greater than about 1:25, for example, greater than about 1:30, for example, greater than about 1:35, for example, greater than about 1:40. The ratio between the cross-sectional area of ​​the opening at the second end 26 and the surface area 34 of the filter member 32 may generally be less than about 1:1000, for example, less than about 1:500, for example, less than 1:200, for example, less than about 1:150, for example, less than about 1:100, for example, less than about 1:80. For example, if the second opening of the second end 26 has a diameter of about 2 mm to about 20 mm, the filter member 32 may generally have a length L of more than about 20 mm, for example more than about 50 mm, for example more than about 100 mm, for example more than about 500 mm, and generally less than about 1000 mm, for example less than about 500 mm, for example less than about 200 mm.

[0085] The outer surface area of ​​the filter member 32 is generally about 0.5 in. 2 It is larger than that. For example, the surface area of ​​the filter member 32 is approximately 1 in. 2 For example, about 1.5 inches 2 For example, about 2 inches 2 For example, about 2.5 inches 2 For example, about 3 inches2 For example, about 3.5 inches 2 For example, about 4 inches 2 For example, about 5 inches 2 It can be more than that. Surface area is generally, Approximately 100 inches 2 Less than, for example, 10 inches 2 It is less than.

[0086] In the embodiment shown in Figure 4A, the filter member 32 has an elongated shape ending at an inclined end 38. However, it should be understood that the filter member 32 may have any preferred shape. For example, the shape of the filter member 32 may depend on a shape that maximizes the surface area while being conveniently configurable in the bioreactor 10. However, it should be understood that the length of the filter member 32 can be much greater than the above-mentioned desired length. For example, an almost entire tubular member may consist of the filter member 32.

[0087] For example, alternative shapes of the filter member are shown in Figures 5A and 5B. Referring to Figures 5A and 5B, the filter device 220 is shown to include a hollow tubular member 222 connected to the filter member 232. In this embodiment, the filter member 232 has a non-porous end 233. The closed end 233 can protect the filter member 232 from damage when it is inserted into or removed from the bioreactor.

[0088] According to this disclosure, the cross-sectional area of ​​the hollow tubular member 22, the sealed volume 34 of the filter member 32, and the pore diameter of the filter member 32 are all selected to optimize the flow rate. In particular, the filter device 20 of this disclosure is designed to allow a relatively high flow rate from the bioreactor 10. In one embodiment, for example, the flow rate through the filter device 20 may depend on the volume of the bioreactor 10. For example, the filter device 20 may be designed to extract more than approximately 40% of the bioreactor's volume per day (24 hours), for example, more than 50% of the bioreactor's volume, for example, more than 60% of the bioreactor's volume, for example, more than 70% of the bioreactor's volume, for example, more than 80% of the bioreactor's volume, for example, more than 100% of the bioreactor's volume, for example, more than 110% of the bioreactor's volume, for example, more than 120% of the bioreactor's volume, for example, more than 130% of the bioreactor's volume, for example, more than 140% of the bioreactor's volume, for example, more than 150% of the bioreactor's volume. Generally, the flow rate through the filter device 20 is generally less than approximately 500% of the bioreactor's volume per day, for example, less than approximately 200% of the bioreactor's volume per day.

[0089] In one particular example, the filter device 20 is designed to draw out more than approximately 0.5 L of fluid per day, for example more than approximately 1 L of fluid per day, for example more than approximately 2 L of fluid per day, for example more than approximately 5 L of fluid per day, for example more than approximately 10 L of fluid per day, for example more than approximately 20 L of fluid per day, for example more than approximately 30 L of fluid per day, for example more than approximately 40 L of fluid per day, and generally less than approximately 100 L of fluid per day from the bioreactor 10. For example, the flow rate through the filter device 20 may be more than approximately 10 mL / min, for example more than approximately 15 mL / min, for example more than approximately 20 mL / min, for example more than approximately 30 mL / min, for example more than approximately 40 mL / min, for example more than approximately 50 mL / min, for example more than approximately 100 mL / min, for example more than approximately 200 mL / min, and generally less than approximately 2 liters / min, for example less than approximately 1 liter / min.

[0090] An embodiment of the filter device 20, as shown in Figure 2, includes a straight or vertical section 30 intended to be inserted into the bioreactor 10. When inserted into the bioreactor 10, the straight or vertical section 30 remains substantially parallel to the vertical axis and / or rotatable shaft 14 of the bioreactor. Thus, the straight or vertical section 30 has a length that is at least the same as the length or depth of the bioreactor 10. However, in one embodiment, the straight or vertical section 30 may interfere with the impeller 16 contained within the bioreactor 10. Therefore, in other embodiments, the shape of the filter device 20 may be modified to provide a better fit within the bioreactor.

[0091] Referring to Figure 3, for example, another embodiment of the filter device 120 is shown. The filter device 120 includes a hollow tubular member 122 having a first end 124 and a second opposing end 126. Attached to the second end 126 is a filter member 132 manufactured according to this disclosure. The hollow tubular member 122 further includes an angled member 128 positioned at the first end 124.

[0092] In the embodiment illustrated in Figure 3, the filter device 120 includes a first straight section 140, a second straight section 142, and an angled section 144. The angled section 144 is positioned between the first straight section 140 and the second straight section 142. As shown in Figure 1, the angled section 144 may be included in a hollow tubular member 22 to prevent interference between the impeller 16 contained within the bioreactor 10 and the filter device 120. In particular, the angled section 144 positions the second end 126 of the hollow tubular member 122 adjacent to the wall of the bioreactor 10. In one embodiment, the angled section 144 may be at a certain angle with the first straight section 140, such as about 10° to about 80°, for example, about 25° to about 45°. For example, the angle between the angle section 144 and the first straight section 140 may generally be greater than about 20°, e.g., greater than about 30°, e.g., greater than about 40°, and generally less than about 60°, e.g., less than about 50°. Similarly, the angle between the angle section 144 and the second straight section 142 may be between about 10° and about 80°, e.g., between about 25° and about 45°.

[0093] The lengths of the straight sections 140 and 142 and the angled section 144 may also vary depending on the shape of the bioreactor 10 and various other factors. In one embodiment, for example, the angled section 144 may be more than about 5% of the total length of the first straight section 140, the second straight section 142, and the angled section 144, for example, more than about 10%, for example more than about 15%, for example more than about 20%, and generally less than about 50%, for example less than about 40%, for example less than about 30%, for example less than 20%.

[0094] Referring to Figure 5A, yet another embodiment of a filter device 220 fabricated according to this disclosure is shown. The filter device 220 includes a hollow tubular member 222 having a first end 224 and a second opposing end 226. A filter member 232 is attached to the second end 226 of the hollow tubular member 222. The hollow tubular member 222 includes a first straight section 250, a second straight section 242, and an angled section 244 positioned between the first straight section 240 and the second straight section 242. The filter device 220 further includes a first angled member 228 positioned at the first end 224 of the hollow tubular member 222.

[0095] In the embodiment shown in Figure 5A, the filter device 220 further includes a second angle member 250 positioned at the second end 226 of the hollow tubular member 222. The second angle member 250 is for positioning the filter member 232 adjacent to the bottom of the bioreactor 10. For example, the second angle member 250 may be angled with the first straight section 240 at a rate of generally more than about 40°, e.g., more than about 50°, e.g., more than about 60°, e.g., more than about 70°, e.g., more than 80°, and generally less than about 120°, e.g., less than about 100°. For example, as shown in Figure 5A, in one embodiment, the second angle member 250 is perpendicular to the first straight section 240 of the hollow tubular member 222. In this way, the filter device 220 may be positioned in the bioreactor to avoid interference with the impeller. On the other hand, the second angle member 250 allows the filter member 232 to extend along the bottom of the bioreactor toward the center of the bioreactor or toward the walls of the bioreactor, depending on the specific application. Therefore, the second angle member 250, The filter member 232 may have a length suitable for positioning it in a desired location. For example, in one embodiment, the length of the second angle member 250 may generally be greater than about 20 mm, e.g., greater than about 30 mm, e.g., greater than about 40 mm, e.g., greater than about 50 mm, e.g., greater than about 60 mm, e.g., greater than about 70 mm, e.g., greater than about 80 mm, e.g., greater than about 90 mm, e.g., greater than about 100 mm, and generally less than about 500 mm, e.g., less than about 300 mm, e.g., less than about 200 mm, e.g., less than about 180 mm, e.g., less than about 160 mm, e.g., less than about 140 mm. However, the length of the second angle member 250 may depend on the size and volume of the bioreactor 10. Therefore, this length may be greater or smaller than the dimensions provided above.

[0096] Referring to Figure 6, yet another embodiment of a bioreactor system fabricated according to this disclosure is shown. The bioreactor system includes a bioreactor 310 having a port 318 located on the side wall of the bioreactor. The bioreactor system further includes a filter device 320 having a hollow tubular member 322 and a filter member 332. The filter device 320 may be inserted into the port 318. The filter member 332 is similar to that shown in more detail in Figures 4A, 4B, and 4C. The filter device 320 may minimize the amount of space occupied within the bioreactor, which may, in some embodiments, allow the filter member 332 to include a longer mesh with a larger surface area. By allowing access of the filter device 320 to the bioreactor 310 at the bottom side wall, the total amount of material penetrating the bioreactor 310 is reduced, as shown in Figure 6, compared to an embodiment of the filter device being inserted through a port at the top of the bioreactor, as shown in Figure 1, for example.

[0097] Referring now to Figures 7A-7C, additional embodiments of a bioreactor system fabricated according to this disclosure are shown. The bioreactor system includes a bioreactor 410 having a port 418 located on the lower wall of the bioreactor. The embodiments in Figures 7A-7C further include a filter device 420 having a hollow tubular member 422 and a filter member 432.

[0098] In the embodiments shown in Figures 7A-7C, the filter device 420 further includes a foldable bellows structure 440 for a completely closed sterile entry. The bellows 420 may be made of plastic. The hollow tubular structure 422 and the filter member 432 are completely enclosed within the bellows 440. The bellows 440 forms a sealed environment that can be sterilized to accommodate the hollow tubular structure 422 and the filter member 432. The filter device 420 further includes a rigid tunnel 446 within the bellows 440 that leads to a sterile connection port 442. The sterile connection port 442 may be any commercially available sterile connection port compatible with the bioreactor 410. For example, the sterile connection port may be a Kleenpak® sterile connector from Pall Biotech, an Opta® sterile connector from Sartorius, a ReadyMate single-use connector from GE Healthcare Life Sciences, or other commercially available sterile connectors. The bioreactor 410 includes a sterile connector 444 that fits into a port 418 on the bioreactor wall.

[0099] As shown in Figure 7B, the sterile connectors 442 and 444 of the filter device 420 and the bioreactor 410 are first connected to each other. A seal is formed between the sterile connectors 442 and 444. Then, as shown in Figure 7C, an opening is formed between the sterile connectors 442 and 444. The bellows 440 can then be folded, the hollow tubular member 422 can be pushed into the bioreactor 410, and the filter member 432 can extend into the bioreactor 410. The bellows 440 collapses when the hollow tubular member 422 and the filter member 432 are pushed into the bioreactor.

[0100] Referring to Figures 8A and 8B, yet another embodiment of a bioreactor system fabricated according to this disclosure is shown. The bioreactor system includes a bioreactor 510 having a conical filter device 520. The filter member 532 of the filter device 520 is formed as a mesh patch on the wall 511 of the bioreactor 510. The mesh patch may be located on the side wall 511 of the bioreactor 510, as shown in Figure 8B. The filter device 520 has a sealed volume 534 formed by a cone 536 that connects the filter member 532 to an outlet hollow tubular member 522. In some embodiments, the cone 536 may be flexible.

[0101] Referring to Figure 9, an additional embodiment of a bioreactor system fabricated according to this disclosure is shown. The bioreactor system includes a bioreactor 610 having a conical filter device 620 which can function as a filter drain for the bioreactor 610. The filter member 632 of the filter device 620 is formed as a mesh patch on the bottom wall of the bioreactor 610. The filter device 620 has a sealed volume 634 formed by a cone 636 that connects the filter member 632 to an outlet hollow tubular member 622. In some embodiments, the cone 636 may be flexible. The design of the embodiment shown in Figure 9 allows for the draining of a maximum amount of liquid from the bioreactor.

[0102] Referring to Figure 10, another embodiment of a filter device 720 manufactured according to the present disclosure is shown. In this embodiment, the filter device 720 includes a hollow tubular member 722 attached to a filter member 732 manufactured as described above according to the present disclosure. However, in this embodiment, the filter device 720 includes an inner hollow tube 740 including a flow-allowing section 742. During operation, the outer tubular member 722 is mounted on a motor and rotates, as indicated by the arrows. Thus, the outer tubular member 722 and the filter member 732 rotate while the inner tubular member 740 remains stationary. Rotating the outer tubular member 722 can prevent contamination and blockage.

[0103] The filter devices of this disclosure are generally resistant to contamination and other blockages, but various methods and techniques can also be used to prevent or disrupt flow blockages. For example, in one embodiment, the filter device may be operated periodically in a backflush mode. For example, when a fluid medium is withdrawn from a bioreactor, the flow of the fluid medium may be reversed at periodic intervals. For example, in one embodiment, the flow through the filter device may be reversed at periodic time intervals of about 30 minutes to about 4 hours, for example, at periodic time intervals of about 45 minutes to about 90 minutes. At a given time interval, the flow through the filter device may be reversed for a short amount of time, for example, less than about 10 minutes, for example less than 8 minutes, for example less than 5 minutes, and generally more than about 2 seconds. In this way, forward flow may occur for more than approximately 80%, for example, more than 85%, for example, more than 90%, for example, more than 95%, while reverse flow may occur for less than approximately 15% of the time the filter device is operating, for example, less than 10%, for example, less than 5%. By periodically returning the fluid through the device in the reverse direction, any material or debris accumulated on the outer surface of the filter member can be removed.

[0104] The filtration apparatus of this disclosure, in particular, in combination with a stirred tank bioreactor, can be used in a number of different processes to improve the viability of biological cell populations and / or to purify or otherwise harvest biological cells. In one application, for example, the bioreactor may first be filled with a fluid medium, such as a culture medium containing a food source for the biological cells. Before adding the biological cells to the bioreactor, various parameter monitoring devices associated with the bioreactor can be calibrated. For example, the bioreactor may be calibrated with probes for measuring dissolved oxygen, pH, temperature, carbon dioxide, and / or oxygen. It can be arranged in association with a b. Furthermore, the bioreactor can be arranged in fluid communication with an air source, a nitrogen gas source, an oxygen gas source, and / or a carbon dioxide gas source. The bioreactor may include an impeller that can be used to agitate or mix the fluid culture medium. For example, the impeller can rotate at a speed of about 40 rpm to about 100 rpm, for example, about 85 rpm to about 93 rpm.

[0105] Once the pH / dissolved oxygen and / or temperature probes are calibrated and the pH, dissolved oxygen, and temperature are stabilized within the bioreactor, biological cells can be seeded into the bioreactor. According to this disclosure, any suitable biological cells, such as mammalian cells, can be added to the bioreactor. For example, the processes and systems of this disclosure are particularly suitable for receiving therapeutic cells, including T cells and NK cells.

[0106] The source of biological cells for seeding a bioreactor can vary. In one embodiment, the biological cells may be obtained from cryogenic bags that need to be thawed and diluted before seeding. When seeding T cells, the proportion of PBMCs (which are T cells) can be determined empirically and used to estimate the subsequent yield.

[0107] The cell density of biological cells during seeding can vary depending on the cell type, the type of bioreactor, and various other factors. Generally, the initial cell density in a bioreactor when growing a cell population is approximately 4 × 10⁻⁶. 6 Less than cells / mL, for example, about 2 × 10⁻⁶ 6 Less than cells / mL, e.g., approximately 1.5 × 10⁻⁶ 6 Less than cells / mL, e.g., about 1 × 10⁶ 6 Less than cells / mL, e.g., approximately 0.7 × 10⁻⁶ 6 Less than cells / mL, e.g., about 0.5 × 10⁻⁶ 6 Less than cells / mL, e.g., approximately 0.3 × 10⁻⁶ 6 Less than cells / mL, and generally about 1 × 10⁻⁶ 3 cells / mL greater than, e.g., 1 × 10⁻⁶ 4 cells / mL greater than, e.g., 1 × 10⁻⁶ 5 The cell / mL is greater than [value missing].

[0108] During sowing, the stirring speed inside the bioreactor can be reduced. For example, the impeller can rotate at a speed of less than about 79 rpm, for example, less than about 65 rpm, and generally more than about 40 rpm.

[0109] The seeded cells are contained in an unsupported bioreactor suspended in a liquid medium, such as a growth medium containing a food source like glucose. In certain embodiments, the seeded cells need to be activated for cell proliferation to occur. For example, T cell activation occurs after activation of the TCR complex and co-stimulation of CD28 by CD80 or CD86. The stimulation can be either antigen-dependent or antigen-independent. Antigen-dependent activation, for example, proliferates only antigen-specific T cells, while antigen-independent activation proliferates all T cells in a biological cell population.

[0110] In one embodiment, for unsupported T cells, activation is initiated by adding a bioreactor-soluble tetrameric antibody complex bound to CD3 and CD28 cell surface ligands. The addition of the antibody results in crosslinking of the CD3 and CD28 cell surface ligands, thereby providing the primary and co-signals necessary for T cell activation. For example, one commercially available activator is marketed under the name IMMUNOCULT Human CD3 / CD28 T Cell Activator. The activator can be added to the bioreactor using any preferred method, such as the use of a sterile syringe.

[0111] After the cells are seeded and activated, cell proliferation is promoted within the bioreactor. In one embodiment, the bioreactor operates in batch mode for a predetermined period, e.g., more than about 1 day, e.g., more than 2 days, e.g., more than 3 days, e.g., more than 4 days, e.g., more than 5 days, and generally less than 9 days. After a specific period, e.g., perfusion mode is activated using the filter device of this disclosure. Perfusion mode is activated, e.g., on day 5, day 6, day 7, or day 8 It can be initiated on day 10. In one embodiment, the perfusion mode is activated when a certain cell density is reached. For example, the perfusion mode is activated when the cell density is 1 × 10⁻⁶ 6 Cells / mL greater than, for example, about 1.5 × 10⁻⁶ 6 cells / mL greater than, for example, about 1.8 × 10⁻⁶ 6 Cells / mL greater than, for example, about 2 × 10⁻⁶ 6 The cell density is greater than 5 cells / mL, and generally about 5 × 10⁻⁶. 6 It may be initiated after the cell / mL is less than 1 / mL.

[0112] During batch mode, the stirring speed may be increased to accommodate a higher volume of media within the bioreactor. For example, the impeller may rotate at a speed of over approximately 80 rpm, e.g., over approximately 85 rpm, e.g., over approximately 95 rpm, and generally less than approximately 105 rpm.

[0113] During perfusion mode, the fluid medium is removed along with the bioreactor as new medium is added to replenish the medium being withdrawn. During perfusion mode, more than 30% of the fluid volume in the bioreactor, e.g., more than 40% of the fluid volume in the bioreactor, e.g., more than 45% of the fluid volume in the bioreactor, e.g., more than 45% of the fluid volume in the bioreactor, e.g., more than 100% of the total medium volume in the bioreactor per day, e.g., less than 75%, e.g., less than 60%.

[0114] In one embodiment, the mass or weight of the bioreactor may be monitored periodically or continuously to ensure that the volume of the fluid medium contained in the bioreactor does not change by more than about 10%, for example, more than about 5%, during the cell proliferation process.

[0115] The proliferation of biological cells by the process described above has been found to not only dramatically improve the quality of the product but also to enable the control of various parameters and metabolites in the fluid medium in a fully automated manner that allows for proliferation. For example, remarkably high cell densities can be achieved in a short period of time, less than about 15 days, e.g., less than about 14 days, e.g., less than about 13 days, e.g., less than about 12 days, according to the process of this disclosure. 7 Cells / mL greater than, for example, about 1.2 × 10⁻⁶ 7 Cells / mL greater than, for example, about 1.5 × 10⁻⁶ 7 Cells / mL greater than, for example, about 2 × 10⁻⁶ 7 A cell density greater than cells / mL can be achieved. In addition, cell viability can be greater than approximately 90%, for example, greater than approximately 92%, for example, greater than approximately 95%, for example, greater than approximately 96%.

[0116] During this process, the glucose level in the fluid medium within the bioreactor can be controlled to a desired level. For example, the glucose level can be maintained above approximately 4 g / L, e.g., above approximately 4.5 g / L, e.g., above approximately 5 g / L, and generally below approximately 8 g / L. On the other hand, the lactate level can be maintained at a relatively low level. The lactate level during the process may be below approximately 1.5 g / L, e.g., below approximately 1.3 g / L, e.g., below approximately 1 g / L. The ammonia level may also remain relatively low during the process. The ammonia level may be below approximately 3 mmol / L.

[0117] After a certain period or after reaching a certain cell density, the biological cell population in the bioreactor can be harvested, washed, purified, or subjected to various other processes. In one embodiment, the biological cell population may be divided into smaller portions, optionally concentrated, and placed in cryogenic bags for cryogenic storage. For example, the cryogenic bags may first be frozen and then moved to a liquid nitrogen atmosphere for long-term storage.

[0118] In addition to washing biological cell populations, in some applications, the biological cell population may comprise multiple cell types. For example, the biological cell population may comprise a first type of cell that may be desired and a second type of cell that may not be desired. According to this disclosure, different cell types can be easily and efficiently separated from one another.

[0119] For example, when producing CAR T cells, such as allogeneic CAR T cells, the resulting biological cell population may contain TCR+ cells. In one application, TCR+ cells need to be isolated from CAR T cells. For instance, TCR+ cells can have adverse effects on patients and may lead to graft-versus-host disease. Similarly, when producing NK cells, the resulting biological cell population may also contain CD3+ T cells, which need to be isolated and extracted from the NK cells. CD3+ T cells can cause undesirable side effects in patients, such as graft-versus-host disease. In addition, in some applications, the resulting proliferated biological cell population may also contain various cell subsets. For example, a T cell population may contain CD4+ and CD8+ cells, while an NK cell population may contain CD16+ cells. In some applications, it may also be desirable to isolate the different cell subsets identified above before administering the desired cell type to the patient.

[0120] Therefore, according to this disclosure, after a biological cell population has grown to a desired cell density as described above, in some embodiments it may be desirable to separate different cell types that may be contained within the cell population. One method for separating different cell types in a biological cell population according to this disclosure is shown in Figure 18. As shown in Figure 18, the bioreactor 10 contains a biological cell population at a desired cell density. The biological cell population contains different types of cells. In Figure 18, for example, the biological cell population contains a first cell type 70 and a second cell type 72. The cell population is contained in a fluid medium within the bioreactor 10.

[0121] To separate the first cells 70 from the second cells 72, a microcarrier 74 is added to the bioreactor 10. The microcarrier 74 consists of a material that adheres to and binds the first cells 70 to the surface of the microcarrier. However, the second cells 72 do not bind to or bind to the microcarrier 74. For example, in some embodiments, as shown in Figure 19, the microcarrier may be a magnetic bead, such as a paramagnetic bead, which may include one or more surface coatings or functionalizations. Furthermore, it should be understood that separating the first cells from the second cells may include positive selection or negative selection, as understood in the art.

[0122] For example, a microcarrier 74 containing magnetic bead microcarriers may contain antibody-conjugated beads. The antibodies present on the beads bind to one of the cell types before they bind to other cell types. The microcarriers 74 may have any suitable particle size, generally greater than about 0.5 microns, e.g., greater than about 1 micron, e.g., greater than about 2 microns, e.g., greater than about 3 microns, e.g., greater than about 4 microns, e.g., greater than about 5 microns, e.g., greater than about 6 microns, e.g., greater than about 7 microns, e.g., greater than about 8 microns, e.g., greater than about 15 microns, e.g., greater than about 50 microns, e.g., greater than about 60 microns, e.g., greater than about 70 microns, e.g., greater than about 100 microns, and generally less than about 500 microns, e.g., less than about 200 microns, e.g., less than about 175 microns, e.g., less than about 150 microns, e.g., less than about 125 microns, e.g., less than about 100 microns, e.g., less than about 75 microns, e.g., less than about 50 microns, e.g., less than about 25 microns, e.g., less than about 15 microns, or any range or value in between.

[0123] As shown in Figure 18, a filter device 820 can be inserted into the bioreactor 10 to separate the first cells 70 from the second cells 72 after the microcarriers 74 have been added. The filter device 820 is similar to the filter device described above with respect to Figures 2-10. As shown, for example, the filter device 820 includes a hollow tubular member 822 that is in fluid communication with a filter member 832. However, in this embodiment, the filter member 832 has a larger pore size that allows the second cells 72 to pass through while preventing the flow of microcarriers 74 through the filter member 832.

[0124] For example, the filter device 820 may be similar to the filter device described and disclosed in U.S. Patent Publication 2019 / 0136173, which is incorporated herein by reference. For example, the filter member 832 may generally have an absolute pore size of more than about 5 microns, e.g., more than about 10 microns, e.g., more than about 20 microns, e.g., more than about 60 microns, e.g., more than about 70 microns, e.g., more than about 80 microns, e.g., more than 90 microns. The absolute pore size of the filter member 832 is generally less than about 150 microns, e.g., less than about 130 microns, e.g., less than about 120 microns, e.g., less than 110 microns. The filter device 820 can rapidly and efficiently separate the first cells 70 from the second cells 72.

[0125] Additionally or alternatively, in embodiments where the microcarriers are magnetic beads, magnets including, but not limited to, neodymium magnets can be used to further separate cells within the bioreactor. For example, in one embodiment, magnets may be used to separate cells by introducing the magnets into the bioreactor chamber, for example, through a hollow tubular member 822, or additionally or alternatively, in the form of a patch 836 of magnets 834 (for example, shown by one or more of the 836 in Figure 18), attached to the outer wall of the bioreactor formed from a material that does not interfere with the magnetic field of the magnets (for example, referred to herein as non-reactive).

[0126] In one such embodiment, the bioreactor may be formed from a rigid or flexible nonreactive plastic, and the magnet 834 is attached to the outer portion of one or more portions of the side wall 838 of the bioreactor 10. In particular, the disclosure has found that when the magnet is applied to one or more portions of the side wall 838 in the form of a patch 836, a more uniform magnetic field extending along the length of the magnet can be generated, instead of being focused near the tip / distal end. Thus, greater separation can be obtained using magnets of the same size. Furthermore, although shown in rectangular form, it should be understood that the patch may have any shape or cross-section, such as circular, cylindrical, cruciform (or X-shaped), square, or triangular.

[0127] It should be understood that any pattern can be formed on the outer wall of the bioreactor using magnets 834, but in one embodiment, each patch 836 may be large enough to separate the desired size of sample. For example, in one embodiment, the magnet, or magnetic patch, has a relatively small surface area (e.g., the area in contact with the outer surface of the bioreactor), e.g., about 0.4 in 2 ~approximately 1.5in 2 For example, about 0.5 in 2 ~Approximately 1.4 inches 2 For example, about 0.6 in 2 ~about 1.25in 2 For example, about 0.7 in 2 ~about 1.2in 2 For example, approximately 0.8 in 2 ~approximately 1.1in 2 It has, for example, in one embodiment, about 1 in 2 Using approximately 5 x 10 9 One or more magnetic beads, for example, approximately 5.5 × 10 9 More than one magnetic bead, for example, about 6 x 10 9 More than one magnetic bead, for example, about 7 x 10 9 More than one magnetic bead, for example, about 8 x 10 9 More than one magnetic bead, for example, about 9 x 10 9 More than 10 magnetic beads, for example, about 10 x 10 9One or more magnetic beads, for example, at least about 12 × 10 9 They can be separated as individual magnetic beads. Therefore, the size of the magnetic patch can be increased or decreased based on the number of microcarriers to be separated. The above size can be used in relation to a magnet 836 in the form of a patch or to be placed in a tubular member 822, but in one embodiment, the above magnet size can be used in relation to an externally mounted patch 836.

[0128] Nevertheless, as shown in Figures 19 and 20, the present disclosure relates to a method in which a larger proportion of the first cells are seeded, for example, about 80% or more of the targeted first cells, for example, about 85% or more, for example, about 87.5% or more, for example, about 90% or more, for example, about 92.5% or more, for example, about 95% or more, for example, about 97% or more It has been found that separation can occur from 0.5% or more, for example, approximately 99% or more, or any range or value in between. For example, referring to Figures 19A-C, less than 0.1% of CD4+ cells remained in the purified second cell population removed from the bioreactor after purification using magnetic beads (e.g., the CD4+ cells remained bound to microcarriers held in place by one or more magnets 834).

[0129] Furthermore, as shown in Figures 19C-E, after magnetic separation, a very small percentage of beads remain, e.g., less than 10%, less than 5%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, or any range or value in between. This is further illustrated in Figures 20A-E. For example, Figures 20A-C show a sample containing magnetic bead microcarriers in the presence of a magnet, where larger dark circles represent magnetic beads and smaller bright circles represent T cells. As shown in Figures 20A-C, very few magnetic beads remain in the sample because the magnetic beads are largely captured by the magnet, allowing unbound cells to be removed from the bioreactor. Furthermore, Figures 20D-E show the same sample 24 hours after removal of the magnet, with magnetic beads redispersed in the sample. Therefore, it is clear that magnets placed on one or more sides of the bioreactor can effectively separate microcarriers from the seeding material, and that the magnetic beads can be re-released for washing or removal after the separation and filtration of the second cell.

[0130] In a further embodiment, the microcarriers 74 can be recirculated using additional or alternative filters as shown in Figures 21A and B. For example, as shown in Figure 21A, the microcarriers 74 and any cells bound to them can be recirculated through an outlet 910 located on the bottom surface 906 of an external filter 900, which may be in parallel with a suitable bioreactor and any other components discussed herein, thereby recirculating the microcarriers 74 and any suspended cells to a suitable bioreactor. In such an embodiment, the recirculated material is encouraged to move away from the skimming filter 914 due to the density of the microcarriers 74, allowing the prepared medium 912 to pass through the skimming filter 914 without damage by the microcarriers 74 or any suspended cells, thus allowing the prepared medium 912 to be filtered through the skimming filter 914, reducing the amount of deposits that form on the skimming filter 914. Such a skimming filter 914 can further prevent damage to the microcarriers that is sometimes experienced during other filtration methods.

[0131] Furthermore, referring to Figure 21B, in one embodiment, the skimming filter 914 may also include a baffle 916. In one embodiment, the baffle 916 may be positioned near the inlet 918 to allow the introduction of new or recirculated media from a suitable bioreactor. Nevertheless, the baffle 916, which may be solid or microporous, deflects the microcarriers 908 or suspended cells toward the outlet 910 (returning the microcarriers and any suspended cells to a suitable bioreactor), while allowing the conditioned medium 912 to pass through the skimming filter 914. In one embodiment, the baffle 916 may be microporous, but it should be understood that the micropores have an average diameter smaller than the average diameter of the microcarriers.

[0132] After filtration, the bioreactor 10 contains the first cells 70 attached to the microcarrier 74. Meanwhile, the second cells 72 may be transferred to a new bioreactor. Each cell population (first cells and second cells) can then be further purified and washed as necessary. If either the first or second cells are not desired, either cell population may be discarded.

[0133] The above process, for example, efficiently and easily separates TCR+ cells from CAR T cells. This can be done (for example, by selectively binding to CD3+, CD4+, or by isolating CD3+ T cells from NK cells).

[0134] In one application, it may be desirable to separate the first cells 70 from the microcarrier 74 after the second cells 72 have been separated from the first cells. The first cells 70 can be separated from the microcarrier 74 using different methods and techniques. For example, in one embodiment, a separation agent that separates cells from the microcarrier can be added to the bioreactor 10. After separation, the first cells 70 can be removed from the bioreactor 10 using a filter device 820 and separated from the microcarrier 74.

[0135] In an alternative embodiment, the microcarrier 74 may be designed to be soluble in a fluid medium contained within the bioreactor 10. For example, the microcarrier 74 may be soluble in the fluid medium, or a solvent may be added to the fluid medium to dissolve the microcarrier 74. Once the microcarrier is dissolved, the first cells 70 remain unsupported in the bioreactor 10 and may be further washed and purified.

[0136] Once a desired biological cell population is optionally isolated and separated from other cells, the cell population may be washed and purified according to the methods of this disclosure using a filter device, as shown and described with respect to Figures 1-10. In one embodiment, for example, a fluid medium containing the biological cell population is withdrawn from a bioreactor using a filter device of this disclosure, such as filter device 20. The filter device can withdraw at least about 30%, e.g., at least about 40%, e.g., at least about 50%, e.g., at least about 60%, e.g., at least about 70%, e.g., at least about 80% of the volume of the fluid medium in the bioreactor. A buffer medium may then be added to the bioreactor to replace the withdrawn fluid medium in order to wash and purify the cells.

[0137] During the washing process described above, the biological cell population is purified by removing and separating the fluid medium from the cells and any biological byproducts or other contaminants that may be present in the fluid medium. For example, during cell proliferation, biological cells may produce byproducts such as proteins. In addition, serum may also be present in the fluid medium. Through the process of this disclosure, such biological byproducts such as proteins and serum can be removed and separated from the cells. The washing process may be carried out multiple times (i.e., multiple cycles) to reach a desired level of purity. For example, cells can be washed so that the resulting cell population contains biological byproducts such as proteins and serum in amounts of less than 0.1% by weight. For example, a cell population may be washed by the method described above for more than one cycle, e.g., more than two cycles, e.g., more than three cycles, and generally less than about ten cycles, e.g., less than eight cycles, e.g., less than six cycles, e.g., less than five cycles.

[0138] Particularly advantageous, the biological cell population can be washed in accordance with this disclosure in an automated manner that does not require centrifugation or a considerable amount of manual labor. Furthermore, the washing can be carried out very efficiently and quickly. For example, the fluid medium can be withdrawn from the bioreactor at a flow rate such that at least about 50% of the volume of the fluid medium, for example, at least about 60% of the volume of the fluid medium, for example, at least about 70% of the volume of the fluid medium can be withdrawn from the bioreactor in less than about 4 hours, for example, less than about 3 hours, for example, less than about 2 hours, for example, less than 1 hour.

[0139] Furthermore, this method is fully scalable. For example, the cell population washed according to this disclosure may be contained in a smaller bioreactor having a volume of about 1 to about 10 L, or it may occur in a larger bioreactor having a volume of about 5 L to about 75 L.

[0140] After washing the cell population to a desired level of purity, the cell population is placed in a buffer medium. The resulting product may be transferred to a flexible bag container for cryogenic storage. In one embodiment, the cryogenic buffer medium may also be added to the cell population before freezing. The cryogenic buffer medium may be, for example, CRYOSTOR 10, sold by Biolife Solutions. The cryogenic buffer medium may be serum-free and may contain an alkyl sulfoxide such as dimethyl sulfoxide. Dimethyl sulfoxide may be present in the cryogenic buffer medium in an amount greater than about 5% by weight and less than about 25% by weight.

[0141] Cell populations for storage can have a relatively high level of purity. Because the process is automated, a high level of purity can be maintained batch by batch without fluctuation. Therefore, due to the high level of purity, higher cell densities can be frozen and stored and later delivered to patients. For example, the cell density in a flexible storage bag can be over 9,000,000 cells per mL, over 10,000,000 cells per mL, or over 12,000,000 cells per mL.

[0142] Through the process of this disclosure, the cell density, including T cell populations and NK cell populations, can proliferate by more than 200%. Once the cell population proliferates, the cells can be separated and / or purified from undesirable cells using the method described above. In addition, the process according to this disclosure can produce cell populations with a greater desired amount of phenotype. Ultimately, a cell population with a purity of more than 90% can be achieved. For example, a cell population with a purity of more than 90% of CD4 T cells and / or CD8 T cells can be achieved. Furthermore, this process can be carried out with a total cell loss of less than 10% and a cell viability of more than 90%, e.g., more than 95%. The cell concentration can be increased by up to 3 times compared to previous processes, thereby reducing the required volume of filter medium. In addition, the final cell product can contain residual serum / protein levels of less than 0.1% by weight. Finally, the post-cold viability of polar cells can be more than 90% due to more than 30-fold amplification after reactivation.

[0143] This disclosure can be better understood by referring to the following examples. [Examples]

[0144] Example 1 The following examples were performed to test the ability of a filtration device fabricated in accordance with this disclosure to extract liquid culture medium from a stirred tank bioreactor without extracting or harming any biological cells contained in the bioreactor.

[0145] The filter apparatus used in the following experiment is similar to the design shown in 4A. The filter element is approximately 1.5 inches. 2 It has a length of 0.65 in, and the outer surface of the filter member is 0.65 in 2 It had a surface area of ​​[specified area]. The filter element was made of sintered stainless steel fibers and had an absolute pore size of 3-4 microns. The tubular member attached to the filter element had an inner diameter of 6.35 mm.

[0146] T cells, 3-6.5 × 10 6Cells were seeded at a density of cells / mL in a 1-liter bioreactor. The bioreactor was a stirred-tank bioreactor. The cell culture was maintained in the bioreactor for 1 day, during which time the fluid medium was withdrawn from the bioreactor using the aforementioned filter device.

[0147] The filtration system was operated at three different flow rates: a first flow rate of 5–10 mL / min, an intermediate flow rate of 10–15 mL / min, and a high flow rate of 20–25 mL / min. Samples of the fluid medium withdrawn from the bioreactor were collected and analyzed. At all three flow rates, cell loss in the bioreactor was less than 2%.

[0148] In another experiment, the filter was operated with a medium-low flow rate (5 mL per minute = 25 mL drawn every hour for 5 minutes) using an intermittent perfusion regimen (on for 5 minutes every hour). The initial seeding density was 0.5 × 10⁶, accompanied by T cell activation and post-seed proliferation. 6 The cell / mL was [number] cells. Perfusion was initiated 5 days after T cell seeding and activation (day 5 of culture). On day 6 of culture (e.g., 24 hours after initiating perfusion), we successfully perfused 522 mL of fluid medium from the bioreactor. On day 12, we successfully perfused 3,650 mL of fluid medium from the bioreactor. After 12 days of service, some clogging was noticed. Cell proliferation was observed during perfusion mode.

[0149] Example 2 In this embodiment, a stirred-tank bioreactor operating in batch mode was compared to a stirred-tank bioreactor operating in accordance with the disclosure using the filter device described in Example 1.

[0150] For both bioreactors, CD3+ T cells were isolated from PBMCs and seeded into a 1-liter stirred-tank bioreactor. The set pH value was less than 7.2. The set dissolved oxygen value was greater than 50%. On day 0, an IMMUNOCULT CD3 / CD28 activator was added. The growth medium added to the bioreactor was XVIVO15, 5% human serum, and 25 IU IL-2. The initial medium volume was 400 mL, and the initial cell density was 220×10 6 cells / mL.

[0151] Each bioreactor was operated for 18 days. 800 mL of fresh liquid growth medium was added to each bioreactor on day 3. No further changes were made to the batch-mode stirred-tank bioreactor.

[0152] For the bioreactor system fabricated according to the present disclosure, perfusion was started on day 5. During perfusion, approximately half of the volume of the fluid medium in the bioreactor was withdrawn and replaced. An intermittent perfusion regimen was used, and 25 mL of the fluid medium was withdrawn every hour over a fixed 5-minute period. Perfusion was continued for 9 days and terminated on day 14.

[0153] The results are shown in FIGS. 11 to 14. For example, FIG. 11 shows the viable cell density and percentage viability of both systems. As shown, the bioreactor operated according to the present disclosure shows a dramatic improvement, especially after day 12.

[0154] FIGS. 12, 13, and 14 show the dissolved oxygen levels, glucose levels, lactate, ammonia levels, and IL-2 concentrations over the course of the experiment. As shown in FIG. 13, in particular, the glucose and lactate levels were better controlled using the bioreactor of the present disclosure.

[0155] Example 3 In this example, for Example 2, T cells were grown in a stirred tank bioreactor using the filter device of this disclosure, following the same process as described above. However, in this example, the perfusion was performed when the T cell population reached 2 x 10⁶ 6 Perfusion was initiated after the cell density exceeded m / L, which occurred on day 8. The results are shown in Figures 15–18. As shown in Figure 15, cell proliferation increased dramatically after perfusion was initiated. Cell viability was also maintained above 96% throughout the entire process.

[0156] As shown in Figure 16, dissolved oxygen rapidly decreased to zero between day 10 and day 13. Normally, such a decrease in dissolved oxygen is expected to have a negative impact on cell proliferation. Conversely, cell proliferation increased rapidly during the same period.

[0157] As shown in Figure 17, glucose, lactate, and ammonia levels were controlled and maintained at optimal levels throughout the process.

[0158] The T cell phenotype was also tested from day 13 onward and compared with the batch agitated tank reactor process. The following results were obtained. [Table 1]

[0159] As shown above, the preferred phenotype is T scm There was an unexpected and dramatic increase in the cells. It is unknown, but T scm The increase in cell volume may be due to, or may occur in conjunction with, lower dissolved oxygen levels.

[0160] The devices, facilities, and methods described herein are suitable for use with and in culture any desired cell line, including prokaryotic and / or eukaryotic cell lines. Furthermore, in embodiments, the devices, facilities, and methods are suitable for culturing suspension cells or anchorage-dependent (adherent) cells and are suitable for production operations comprising the production of pharmaceutical and biopharmaceutical products such as polypeptide products, nucleic acid products (e.g., DNA or RNA), or cells and / or viruses (e.g., those used in cell therapy and / or viral therapy).

[0161] In embodiments, cells express or produce products such as recombinant therapeutic or diagnostic products. Examples of products produced by cells, as will be described in more detail below, include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimes (polypeptide molecules that specifically bind to an antigen but are not structurally related to antibodies, e.g., DARPin, aphidobodies, adonectin, or IgNAR), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutics (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cell therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA), or DNA (e.g., plasmid DNA), antibiotics, or amino acids. In embodiments, devices, facilities, and methods may be used to produce biosimilars.

[0162] As mentioned, in embodiments, the devices, facilities, and methods enable the production of eukaryotic cells, e.g., mammalian cells or lower eukaryotic cells, e.g., yeast cells or filamentous fungal cells, or prokaryotic cells, e.g., Gram-positive or Gram-negative cells, and / or eukaryotic or prokaryotic cell products, e.g., proteins, peptides, antibiotics, amino acids, nucleic acids (such as DNA or RNA), which are synthesized on a large scale by eukaryotic cells. Unless otherwise stated herein, the devices, facilities, and methods include, but are not limited to, bench-scale, pilot-scale, and full-production-scale capacities, and may include any desired volume or production capacity.

[0163] Furthermore, unless otherwise stated herein, devices, equipment, and methods may include, but are not limited to, any suitable reactors, including, agitated tanks, airlifts, fibers, microfibers, hollow fibers, ceramic matrices, fluidized beds, fixed beds, and / or jet-bed bioreactors. As used herein, “reactor” may include a fermenter or fermentation unit, or any other reaction vessel, and the term “reactor” is used synonymously with “fermenter.” For example, in some embodiments, an exemplary bioreactor unit may perform one or more or all of the following: supplying nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), fermentation or inlet and outlet flows of cell culture media, separation of gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or washing / sterilization. Exemplary reactor units, such as fermentation units, may include multiple reactors within the unit. For example, a unit may have one, two, three, four, five, ten, fifteen, twenty, twenty-five, thirty, thirty, thirty-five, forty, forty-five, fifty, sixty, seventy, eighty, ninety, or one hundred or more bioreactors within each unit, and / or a facility may include multiple units, each having one or more reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-feed batch, feed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter may be used. In embodiments, the bioreactor may have a volume of about 100 mL to about 50,000 L.Non-restrictive examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, and 550 liters. Volumes include 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Furthermore, preferred reactors may be multi-use, single-use, disposable, or non-disposable and may be formed from any preferred material including stainless steel (e.g., 316L or any other preferred stainless steel) and metal alloys such as Inconel, plastic, and / or glass.

[0164] In embodiments, unless otherwise specified herein, the devices, facilities, and methods described herein may also include any preferred unit operations and / or equipment not specifically mentioned, such as operations and / or apparatus for the separation, purification, and isolation of such products. Conventional stick-built equipment, modules Any suitable equipment and environment can be used, such as modular, mobile, and temporary structures, or any other suitable construction, equipment, and / or layout. For example, in some embodiments, a modular cleanroom can be used. Furthermore, unless otherwise stated, the devices, systems, and methods described herein may be housed and / or operated in a single location or facility, or in separate or multiple locations and / or facilities.

[0165] As non-limiting examples, U.S. Patent Publications 2013 / 0280797, 2012 / 0077429, 2011 / 0280797, 2009 / 0305626, and U.S. Patents 8,298,054, 7,629,167, and 5,656,491, which are incorporated herein by reference in their entirety, describe exemplary equipment, apparatus, and / or systems that may be preferred.

[0166] In embodiments, the cells are eukaryotic cells, such as mammalian cells. Mammalian cells may be, for example, human, rodent, or bovine cell lines or cell lines. Examples of such cells, cell lines, or cell lines include, for example, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, for example, COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EB1, EB2, EB3, oncolytic or hybridoma cell lines. Preferably, the mammalian cells are CHO cell lines. In one embodiment, the cells are CHO cells. In one embodiment, the cells are CHO-K1 cells, CHO-K1SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN, or CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. CHO FUT8 knockout cells are, for example, Potelligent® CHOK1 SV (Lonza Biologics, Inc.). Eukaryotic cells may also be, for example, avian cells, cell lines, or cell lines such as EBx® cells, EB14, EB24, EB26, EB66, or EBvl3.

[0167] In one embodiment, eukaryotic cells are stem cells. Stem cells may be pluripotent stem cells, including, for example, embryonic stem cells (ESCs), adult stem cells, inducible pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs).

[0168] In one embodiment, the cells are any differentiated form of the cells described herein. In one embodiment, the cells are cells derived from any primary cells in a culture.

[0169] In embodiments, the cells are hepatocytes such as human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, the cells may be adherent metabolically qualified human hepatocytes, adherent induced qualified human hepatocytes, adherent Qualyst Transporter Certified® human hepatocytes, suspension qualified human hepatocytes (including 10-donor and 20-donor pooled hepatocytes), human hepatic Kupffer cells, human hepatic stellate cells, canine hepatocytes (including single and pooled Beagle hepatocytes), mouse hepatocytes (including CD-1 and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including Cynomolgus or Rhesus monkey hepatocytes), feline hepatocytes (including domesticated Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand white hepatocytes). Exemplary hepatocytes are commercially available from Triangle Research Labs, LLC, 6 Davis Drive Research Triangle Park, North Carolina, USA 27709. It is.

[0170] In one embodiment, eukaryotic cells include, for example, yeast cells (e.g., Pichia genus (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta)), Komagataella genus (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii), Saccharomyces genus (e.g., Saccharomyces cerevisae, cerevisiae, Saccharomyces kluyveri, Saccharomyces uvarum), Kluyveromyces genus (e.g., Kluyveromyces lactis, Kluyveromyces marxianus), Candida genus (e.g., Candida utilis, Candida cacaoi, Candida boidinii), Geotrichum genus (e.g., Geotrichum fermentans), Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces These are lower eukaryotic cells such as pombe. Pichia yeasts are preferred as seed cells. Examples of Pichia yeast strains include X33, GS115, KM71, KM71H, and CBS7435.

[0171] In one embodiment, eukaryotic cells are fungal cells (e.g., Aspergillus (e.g., A. niger, A. fumigatus, A. orzyae, A. nidula), Acremonium (e.g., A. thermophilum), Chaetomium (e.g., C. thermophilum), Chrysosporium (e.g., C. thermophile), Cordyceps (e.g., C. militaris), Corynascus, Ctenomyces, Fusarium (e.g., F. oxysporum), Glomerella (e.g., G. graminicola), Hypo These include crea (e.g., H. jecorina), Magnaporthe (e.g., M. orzyae), Myceliophthora (e.g., M. thermophile), Nectria (e.g., N. heamatococca), Neurospora (e.g., N. crassa), Penicillium, Sporotrichum (e.g., S. thermophile), Thielavia (e.g., T. terrestris, T. heterothallica), Trichoderma (e.g., T. reesei), or Verticillium (e.g., V. dahlia).

[0172] In one embodiment, eukaryotic cells are insect cells (e.g., Sf9, Mimic® Sf9, Sf21, High Five® (BT1-TN-5B1-4), or BT1-Ea88 cells), algal cells (e.g., Amphora, Basilariophyseae, Dunaliella, Chlorella, Chlamydomonas, Cyanofyta (cyanobacteria), Nanochloropsis, Spirulina, or Ochromonas), or plant cells (e.g., cells derived from monocots (e.g., maize, rice, wheat, or setaria), or cells derived from dicots (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Fiscomitrella paten, or Arabidopsis).

[0173] In one embodiment, the cells are bacteria or prokaryotic cells.

[0174] In the embodiment, the prokaryotic cell is a Gram-positive cell, such as a bacilli, Streptomyces, Streptococcus, Staphylococcus, or Lactobacillus. Other bacilli that can be used include, for example, B. subtilis, B. amyloliquefaciens, B. licheniformis, B. natto, and This is B. megaterium. In embodiments, the cells are B. subtilis, such as B. subtilis 3NA and B. subtilis 168. The bacilli are available, for example, from the Bacillus Genetic Stock Center, Biological Sciences 556, 484 West 12th Avenue, Columbus OH 43210-1214.

[0175] In one embodiment, the prokaryotic cells are Gram-negative cells, such as Salmonella spp. or Escherichia coli, e.g., TG1, TG2, W3110, DH1, DHB4, DH5a, HMS174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue and Origami, as well as those derived from E. coli B strains such as BL-21 or BL21(DE3), all of which are commercially available.

[0176] Suitable host cells include, for example, DSMZ (Deutsche Sammlung von They are commercially available from culture stock preservation institutions such as Mikroorganismen and Zellkulturen GmbH (Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0177] In embodiments, cultured cells are used to produce proteins, such as antibodies, such as monoclonal antibodies, and / or recombinant proteins, for therapeutic use. In embodiments, cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in embodiments, molecules having molecular weights ranging from about 4,000 daltons to over 140,000 daltons can be produced. In embodiments, these molecules may have a range of complexity and may include post-translational modifications, including glycosylation.

[0178] In the embodiment, the protein is, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alpha, daptomycin, YH-16, coliogonadotropin alpha, filgrastim, cetrorelix, interleukin-2, aldesleukin, teseleurine, denileukin difutitox, interferon alpha-n3 (injection), interferon alpha-nl, DL-8234, interferon, Suntory (gamma-1a), Interferon gamma, thymosin alfa 1, tasonelmin, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, levif, eptothermin alfa, teriparatide (osteoporosis), injectable calcitonin (bone disease), calcitonin (nasal cavity, osteoporosis), etanercept, hemoglobin glutamer 250 (bovine), drolecogin alfa, collagenase, carperitide, recombinant human epidermal growth factor (topical gel, wound healing), DWP401, darbepoetin alfa Epoetin omega, epoetin beta, epoetin alpha, decildin, repiridine, vivaliridine, nonacog alpha, mononin, eptacog alpha (activated), recombinant factor VIII + VWF, recombinant, recombinant factor VIII, factor VIII (recombinant), alfmato, octocog alpha, factor VIII, parifermin, indikinase, tenecteplase, alteplase, pamiteplase, leteplase, nateplase, monteplase, follitropin alpha, rFSH, hpFSH, mikafa Ngin, pegfilgrastim, lenograstim, naltograstim, selmorelin, glucagon, exenatide, plumrintide, iniglucerase, galsulfase, leukotropin, morglamostim, triptorelin acetate, histrelin (subcutaneous implant, Hydron), deslorerin, histrelin, nafarelin, leuprolide sustained-release depot (ATRIGEL), leuprolide implant (DUROS), goserelin, eutropin, KP-102 program, somatropin, mecasermin (growth disorder), enru Favirtide, Org-33408, insulin glargine, insulin glulisine, insulin (inhalation), insulin lispro, insulin deternyl, insulin (buccal, RapidMist), mecasermin linfabate, anakinra, cermoleukin, 99 mTc-apcitide injection, Myelopid, Betaseron, Glatyramer acetate, Gepon, Salglamostim, Oprelbequin, Human leukocyte-derived alpha interferon, Bilive, Insulin (recombinant), Recombinant human insulin, Insulin aspart, Mechasenin, Loferon-A, Interferon-Alpha 2, Alphaferon, Interferon Alphacon-1, Interferon Alpha, Avonex recombinant human luteinizing hormone, Dolnase Alpha, Trafermin, Diconotide, Taltirelin, Dibotermin Alpha, Atosiban, Becaprelmin, Eptifavatide, Zemyra, CTC-111, Shanvac-B, HPV vaccine (quadrivalent), Octreotide, Lanreotide, Ancestim, Agalsidase Beta, agalsidase alfa, laronidase, prezatide copper acetate (topical gel), rasburicase, ranibizumab, Actimune, PEG-intron, trichomin, recombinant house dust mite allergy desensitization injection, recombinant human parathyroid hormone (PTH) 1-84 (sc, osteoporosis), epoetin delta, transgenic antithrombin III, granditropin, vitorase, recombinant insulin, interferon alfa (oral tablets), GEM-21S, vapreotide, idursulfase, omnapatril rat, recombinant serum albumin, certolizumab pegol, glucarpidase, human recombinant C1 esterase inhibitor (angioedema), lanoteplase, recombinant human growth hormone, enfuvirtide (needleless injection, Biojector2000), VGV-1, Interferon (Alpha), Lucinactant, Abiptadil (Inhalation, Lung Disease), Icatibant, Ekalanchid, Omiganan, Aurograb, Pexiganan Acetate, ADI-PEG-20, LDI-200, Degarelix, Syntredelinbesdotox, Favld, MDX-1379, ISAtx-247, Liraglutide, Teriparatide (Osteoporosis), Tifacogin, AA4500, T4N5 Liposome Lotion, Kat Maxomab, DWP413, ART-123, Chrysaline, Desmoteplase, Amethyplase, Corifolitropin alfa, TH-9507, Teduglutide, Diamide, DWP-412, Growth hormone (sustained-release injection), Recombinant G-CSF, Insulin (inhalation, AIR), Insulin (inhalation, Technosphere), Insulin (inhalation, AERx), RGN-303, DiaPep277, Interferon beta (Hepatitis C virus infection (HC) V)) Interferon alpha-n3 (oral), belatacept, transdermal insulin patch, AMG-531, MBP-8298, Xerecept, Opevacan, AIDSVAX, GV-1001, LymphoScan, lampirase, lipoxisan, lusulbutide, MP52 (beta-tricalcium phosphate carrier, bone regeneration), melanoma vaccine, cyplucel-T, CTP-37, Insesia, vitespen, human thrombin (freezing, surgical bleeding) ), thrombin, TransMID, alfimeplase, precase, terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinant FGF-I (injectable, vascular disease), BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin (inhalation, cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman-Burk inhibitor concentrate, XMP-629, 99 mTc-Hynic-Annexin V, kahalalideF, CTCE-9908, teverelix (sustained release), Ozarelix, Lornidepsin, BAY-504798, Interleukin 4, PRX-321, Pepscan, Ivocatekin, Lalactoferrin, TRU-015, IL-21, ATN-161, Silengitide, Albuferon, Bifasix, IRX-2, Omega Interferon, PCK-3145, CAP-232, Pasire Otide, huN901-DMI, ovarian cancer immunotherapy vaccine, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16, multiepitope peptide melanoma vaccine (MART-1, gp100, tyrosinase), nemifitide, rAAT (inhalation), rAAT (dermatology), CGRP (inhalation, asthma), pegsnercept, thymosin beta-4, plutinate Depsin, GTP-200, Lamoplanin, GRASPA, OBI-1, AC-100, Salmon Calcitonin (oral, Erigen), Calcitonin (oral, osteoporosis), Examorelin, Capromorelin, Caldeva, Verafermin, 131I-TM-601, KK-220, T-10, Uralitide, Deperestat, Hematide, Chrysaline (topical), rNAPc2, Recombinant Factor V111 (PEGylated Liposome), bFGF, PEGylated Recombinant Staphylokinase Variant, V-10153, Sonolysis Prolase, NeuroVax, CZEN-002, Islet Cell Regeneration Therapy, rGLP-1, BIM-51077, LY-548806, Exenatide (sustained-release, Medisorb), AVE-0010, G A-GCB, Avorelin, ACM-9604, Linaclotide Acetate, CETi-1, Hemospan, VAL (injectable), Rapid-acting insulin (injectable, Viadel), Intranasal insulin, Insulin (inhaled), Insulin (oral, Erigen), Recombinant methionyl human leptin, Pitraquinra subcutaneous injection (eczema), Pitraquinra (inhaled dry powder, asthma), Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 (autoimmune disease / inflammation), Talactoferrin (topical), rEV-131 (eye), rEV-131 (respiratory disease), Oral recombinant human insulin (diabetes), RPI-78M, Oprelbequin (oral), CYT-99007 CTLA4-Ig, DTY-001, Valategrast, Interferon Alpha-n3 (topical), IRX-3, RDP-58, Tauferon, Bile salt-stimulated lipase, Merispase, Araline phosphatase, EP-2104R, Melanotan-II, Bremelanotide, ATL-104, Recombinant human microplasmin, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, Dynorphin A, SI-6603, LABGHRH, AER-002, BGC-728, Malaria vaccine (Virosome, PeviPRO), ALTU-135, Parvovirus B19 vaccine, Influenza vaccine (Recombinant neuraminidase), Malaria / HBV vaccine, Anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine, Tat toxoid, YSPSL, CHS-13340, PTH(1-34) liposome cream (Novaso (Vitamin C), Ostavorin-C, PTH analog (topical, psoriasis), MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85A vaccine (tuberculosis), FARA04, BA-210, recombinant FIV vaccine, AG-702, OxSODrol, rBetV1, Der-p1 / Der-p2 / Der-p7 allergen-targeted vaccine (house dust mite allergy), PR1 peptide antigen (leukemia), variant ras vaccine, HPV-16E7 lipopeptide vaccine, labyrinthin vaccine (adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentris, Norelin, CytoFab, P-9808, VT-111, Icrocaptide, Terbermine (dermatology, diabetic foot ulcers), Rupintrivir, Reticulose, rGRF, HA, Alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160 Angiotensin therapy vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828, ErbB2-specific immunotoxin (anti-cancer), DT3SSIL-3, TST-10088, PRO-1762, Combotox, cholecystokinin-B / gastrin receptor binding peptide, 111In-hEGF, AE-37, trastuzumab-DM1, antagonist G, IL-12 (recombinant), PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (local), L-19-based radioimmunotherapy (cancer), Re-188-P-2045, AMG-386, DC / 1540 / KLH vaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccine (peptide), NA17.A2 peptide, melanoma vaccine (pulsed antigen therapy) ), prostate cancer vaccine, CBP-501, recombinant human lactoferrin (dry eye), FX-06, AP-214, WAP-8294A (injectable), ACP-HIP, SUN-11031, peptide YY[3-36] (obesity, intranasal cavity), FGLL, Atasicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 (intranasal cavity, osteoporosis), F-18-CCR1, AT-1100 (celiac disease / diabetes). ), JPD-003, PTH(7-34) liposome cream (Novasome), duramycin (ophthalmology, dry eye), CAB-2, CTCE-0214, glycopegylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, factor XIII, aminocandin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, Teverelix (immediate release), EP-51216, hGH (controlled release, Biosphere), OGP-I, Scifvirtide, TV4710, ALG-889, Org-41259, rhCC10, F-991, Timopentin (lung disease), r(m)CRP, hepatic selective insulin, Subarin, L19-IL-2 fusion protein Elaphin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist (thrombocytopenic disorder), AL-108, AL-208, nerve growth factor antagonist (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide (eligen), GEM-OS1, AC-162352, PR These include X-302, LFn-p24 fusion vaccine (Serapore), EP-1043, pediatric pneumonia vaccine, malaria vaccine, group B meningococcal vaccine, neonatal group B streptococcal vaccine, anthrax vaccine, HCV vaccine (gpE1+gpE2+MF-59), otitis media treatment, HCV vaccine (core antigen + ISCMATRIX), hPTH(1-34) (percutaneous, ViaDerm), 768974, SYN-101, PGN-0052, abiscumin, BIM-23190, tuberculosis vaccine, multiepitope tyrosinase peptide, cancer vaccine, Encastem, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular targeted TNF (solid tumor), desmopressin (buccal sustained-release), Onercept, and TP-9201.

[0179] In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADE®), rituximab (RITUXAN® / MAB THERA®), etanercept (ENBREL®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), peglirgrastim (NEULASTA®), or any other suitable polypeptide, including biosimilars and biobetters.

[0180] Other suitable polypeptides are listed below and in Table 1 of US2016 / 0097074: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0181] In the embodiments, the polypeptide is a hormone, blood coagulation / coagulation factor, cytokine / growth factor, antibody molecule, fusion protein, protein vaccine, or peptide, as shown in Table 2. [Table 3-1] [Table 3-2] [Table 3-3]

[0182] In this embodiment, the protein is a multispecific protein, such as a bispecific antibody as shown in Table 3. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0183] These and other modifications and variations of the present invention can be implemented by those skilled in the art without departing from the spirit and scope of the invention as specifically described in the appended claims. In addition, it should be understood that the various embodiments may be interchangeable, either whole or in part. Furthermore, those skilled in the art will understand that the foregoing descriptions are illustrative only and are not intended to limit the invention to what is further described in such appended claims.

Claims

1. A method for purifying a cell population, Growing a population of biological cells in a fluid culture medium, wherein the population of biological cells includes unsupported biological cells, and the population of biological cells is contained within a bioreactor, and at least 1 × 10 ６ Having a cell density of cells / mL, and proliferating, The process of removing the fluid culture medium from the bioreactor and filtering it, wherein the fluid culture medium is filtered through a filter device including a filter member, and the filter member has a pore size that prevents the biological cells from being withdrawn from the bioreactor when the fluid culture medium is withdrawn. A method comprising adding a buffer medium to the biological cell population.

2. The method according to claim 1, wherein the fluid medium contains biological byproducts, the filter member has a pore size that allows the biological byproducts to pass through together with the fluid medium being withdrawn, preferably the biological byproducts include proteins, serum, and mixtures thereof, and preferably the biological cell population contains less than 0.1% by weight of the biological byproducts after the fluid medium has been withdrawn from the bioreactor.

3. The method according to any one of the prior claims, wherein more than about 50% of the volume of the fluid medium is withdrawn and at least partially replaced with the buffer medium, preferably the fluid medium is withdrawn from the bioreactor at a flow rate such that at least 50% of the volume of the fluid medium in the bioreactor is withdrawn in a period of less than one hour.

4. The method according to any one of the prior claims, wherein the biological cell population and fluid culture medium have a volume of about 1 L to about 10 L, preferably, the biological cell population and fluid culture medium have a volume of about 5 L to about 75 L.

5. The method according to any one of the prior claims, wherein the method is repeated for approximately two to five cycles.

6. The method according to any one of the prior claims, wherein the biological cells include T cells or NK cells.

7. The method according to any one of the prior claims, further comprising the step of distributing the biological cell population and buffer medium into a flexible bag container for cryogenic storage, preferably the biological cells and buffer medium are also combined with cryogenic buffer medium, and more preferably the cryogenic buffer medium contains an alkyl sulfoxide.

8. The method according to any one of the prior claims, wherein the filter member of the filter device has an absolute pore size of about 1 micron to 9 microns, preferably about 1 micron to 6 microns.

9. The filter device includes a hollow tubular member having a first end defining a first opening and an opposing second end defining a second opening, wherein the filter member is located at the second end of the hollow tubular member, the filter member completely surrounds and seals the second opening, the filter member defines an inner surface and an outer surface, preferably the outer surface of the filter member has a surface area, the surface area of ​​which is approximately 2 in. ２ Larger, approximately 50 inches ２ The method according to any one of the prior claims, which is smaller.

10. The biological cell population comprises at least two different cell types, including first cells and second cells, and the method involves arranging one or more microcarriers in the fluid culture medium containing the biological cell population, such that the first cells bind to the one or more microcarriers, but the second cells do not. The method according to any one of the prior claims, further comprising removing the fluid medium from the bioreactor and filtering it, wherein the fluid medium is filtered through a second filter device including a filter member, the filter member having a pore size that allows the passage of the second cells but inhibits the passage of one or more microcarriers to separate the first cells from the second cells, and preferably, at least a portion of the one or more microcarriers is magnetic.

11. The method according to claim 10, wherein at least one of the first cells or the second cells comprises a T cell or an NK cell, preferably an unsupported T cell, and more preferably the unsupported T cell is activated by adding an antibody that provides a primary signal and a co-signal for T cell activation, such as a soluble tetrameric antibody complex, to the bioreactor.

12. The method according to any one of the prior claims, wherein the filter device operates periodically in backflush mode or is configured for continuous perfusion.

13. A filter device suitable for use in bioreactors, A hollow tubular member for filtering fluid from a bioreactor, wherein the hollow tubular member has a first end defining a first opening and an opposing second end defining a second opening, A filter member located at the second end of the hollow tubular member, wherein the filter member completely surrounds and seals the second opening, defines an inner surface and an outer surface, and comprises a porous material, the porous material having an absolute pore size of about 1 micron to about 9 microns, A filter device for replenishing the fluid culture medium in the bioreactor in order to promote cell viability.

14. The filter member includes a porous mesh, The porous material has an absolute pore size of about 1 micron to about 6 microns, The filter member includes a nonwoven mesh formed from sintered metal fibers, preferably the sintered metal fibers include stainless steel, or The filter device according to claim 13, wherein the distal end of the filter member is a closed, non-porous end.

15. The filter device according to claim 13 or 14, wherein the filter member has a length along the axial direction of the hollow tubular member, and the length of the filter member is 1 inch or more and about 12 inches or less, preferably 2 inches or more and about 8 inches or less.

16. The outer surface of the filter member has a surface area, and the surface area is approximately 0.5 in. ２ Preferably, the surface area is approximately 2 in ２ It is super and approximately 10 inches ２ A filter device according to any one of claims 13 to 15, wherein the filter is less than [amount missing].

17. The filter device according to any one of claims 13 to 16, wherein the ratio of the cross-sectional area of ​​the second opening to the surface area of ​​the filter member is about 1:5 to about 1:200, for example, about 1:15 to about 1:

100.

18. The hollow tubular member is The filter member is made of stainless steel or a thermoplastic polymer, preferably comprising a polymer mesh or nonwoven fabric, and more preferably comprising a polyamide or polyolefin. A filter device according to any one of claims 13 to 17, having a diameter of approximately 0.2 inches to approximately 0.7 inches.

19. The filter device according to any one of claims 13 to 18, wherein the inner surface of the filter member has an absolute pore diameter, the outer surface of the filter member has an absolute pore diameter, the absolute pore diameter of the inner surface is larger than the absolute pore diameter of the outer surface, the absolute pore diameter of the outer surface is about 1 micron to about 9 microns, and preferably the absolute pore diameter of the inner surface of the filter member is about 5 microns to about 20 microns.

20. The filter device according to any one of claims 13 to 19, wherein the hollow tubular member includes a first straight section, a second straight section, and an angled section disposed between the first straight section and the second straight section, the angled section positions the second opening and the filter member within the bioreactor without contact with a rotating impeller.

21. The filter device according to claim 1, wherein the hollow tubular member includes an angled member located adjacent to the second end, the hollow tubular member includes a straight section transitioning to the angled member, and the angled member is at an angle of about 50° to about 90° with respect to the straight section.

22. The filter device according to claim 1, wherein the hollow tubular member and the filter member are movably sealed within a collapsible bellows, and the collapsible bellows includes a sterile connection port on one end for connecting to a corresponding sterile connection port of the bioreactor.

23. The filter device according to claim 1, wherein the filter member includes a mesh patch on the side wall or bottom wall of the bioreactor, and the filter member further includes a cone connecting the mesh patch to the hollow tubular member.

24. A method for culturing cells for proliferation, Seeding biological cells into a bioreactor, wherein the bioreactor contains a fluid medium for cell growth, The filter device according to any one of claims 13 to 23 is inserted into the bioreactor to perfuse the fluid culture medium contained in the bioreactor, A method comprising replenishing the fluid medium in the bioreactor in order to promote cell proliferation.

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