Perfusion bioreactor and related methods of use

Abstract

To provide a method of controlling a bioreactor useful for controlling a cell culture process for protein production.SOLUTION: A method of controlling a bioreactor system includes: providing a cell culture to a bioreactor such that conditions in the bioreactor enable the cell culture to produce a protein of interest (POI); measuring process parameters of the culture in the bioreactor by Raman; removing cell-free spent media from the cell culture using a first output conduit at a first specified rate; removing cells from the cell culture using a second output conduit at a second specified rate; and introducing one or both of fresh media or nutrients into the cell culture using an input conduit at a third specified rate.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATION(S) This patent application claims the benefit under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62 / 572,918, filed October 16, 2017, which is incorporated herein by reference in its entirety.

[0002] The present disclosure is directed to perfusion bioreactors and related methods of use. [Background technology]

[0003] Bioreactors can be used to maintain cell cultures for the production of biological products, such as proteins. In fed-batch bioreactors, one or more nutrients are fed into the bioreactor during cultivation, and the biological product remains in the bioreactor until the end of the batch. Perfusion bioreactors became popular in the late 1990s in response to some of the performance challenges associated with fed-batch reactors. However, state-of-the-art perfusion bioreactors suffer from a limited number of available control strategies, lack of data, and high cost.

[0004] For example, control solutions for perfusion reactors attempt to calibrate the volumetric flow rates of input and output feed pumps while accounting for pump drift and process variations. However, due to inherent differences (e.g., manufacturing variations) between any two given pumps and an inability to achieve tight control, a failed production run can result in the bioreactor being overfilled or empty. Existing control solutions also lack the ability to measure other parameters, such as ammonia, glucose, and protein quality attributes. Embodiments of the present disclosure address one or more limitations and shortcomings of existing perfusion bioreactors. Summary of the Invention

[0005] Embodiments of the present disclosure relate to methods of controlling a bioreactor and bioreactor systems useful, inter alia, for controlling cell culture processes for protein production. Each of the embodiments disclosed herein may include one or more of the features described in connection with any of the other embodiments.

[0006] The present disclosure relates to a method of controlling a bioreactor system, including feeding a cell culture to a bioreactor, measuring one or more process parameters of the cell culture in the bioreactor with a Raman probe, removing cell-free spent medium from the cell culture at a first specified rate using a first outlet conduit, removing cells from the cell culture at a second specified rate using a second outlet conduit, introducing one or both of fresh medium or nutrients to the cell culture at a third specified rate using an input conduit, and altering one or more of the first specified rate, the second specified rate, or the third specified rate based on the measurements of the Raman probe.

[0007] One embodiment of the present disclosure includes a method for producing a cell culture in a bioreactor, the method comprising: feeding a cell culture into a bioreactor, wherein conditions in the bioreactor enable the cell culture to produce a protein of interest (POI); measuring a process parameter (PP) of the culture in the bioreactor by Raman, the process parameter being selected from the group consisting of nutrient concentration, viable cell concentration, and protein attribute; measuring the PP; measuring the weight of the bioreactor including the cell culture contents; removing cell-free spent medium from the cell culture at a first specified rate using a first discharge conduit; and removing cell-free spent medium from the cell culture at a second specified rate using a second discharge conduit. and introducing one or both of fresh medium and nutrients to the cell culture at a third specified rate using an input conduit, wherein the input and output conduits are adjusted based on Raman probe measurements and weight measurements of the bioreactor to (i) maintain one or more of the process parameters within predetermined ranges, (ii) maintain the weight of the bioreactor containing the cell culture within predetermined ranges, and (iii) maintain the third specified rate of the input conduit and the first and second specified rates of each of the output conduits within their respective predetermined ranges.

[0008] In some embodiments, Raman measurements of one or more process parameters of the culture in the bioreactor are performed at regular intervals, e.g., at least hourly. In other embodiments, the method is configured to maintain the cell culture at an average viable cell concentration of at least about 30 million cells per mL for at least about 30 days at steady state. In one embodiment, the bioreactor has a volume of at least 2 L, at least 3 L, at least 10 L, at least 35 L, or at least 50 L, or more, and the method is configured to maintain the weight of the bioreactor containing the cell culture within 0.1 percent of the initial weight of the bioreactor containing the cell culture. For example, the bioreactor has a volume of at least about 10 L, and the method is configured to maintain the weight of the bioreactor and cell culture within a weight range determined based on the initial weight of the bioreactor and cell culture contents, e.g., within a range of about 20±2 g. In some embodiments, the bioreactor is controlled to remove cell-free medium, remove cells, and / or introduce fresh medium and / or nutrients when process parameters deviate from setpoint values ​​within their respective desired ranges, thereby adjusting the bioreactor to reduce the deviation. At least two bioreactor volumes of spent medium per day are removed through a first discharge conduit. Up to three bioreactor volumes of spent medium per day are removed through a first discharge conduit. The process parameters include cell culture temperature and cell culture pH, where the temperature is maintained at about 30-40°C, about 32-38°C, or about 34-38°C, and the pH is maintained at about 6.50-7.50, about 6.60-7.40, about 6.70-7.40, about 6.80-7.30, about 6.90-7.20, about 7.00-7.20, or about 7.20-7.20. The pH is maintained at about 7.10, about 6.50, about 6.55, about 6.60, about 6.65, about 6.70, about 6.75, about 6.80, about 6.85, about 6.90, about 6.95, about 7.00, about 7.05, about 7.10, about 7.15, about 7.20, about 7.25, about 7.30, about 7.35, about 7.40, about 7.45, or about 7.50.The process parameters include a cell-specific productivity, and the method is configured to maintain the cells in the cell culture at a cell-specific productivity of at least about 15-60 pg / cell / day, about 15-25 pg / cell / day, at least about 17-23 pg / cell / day, or at least about 19-21 pg / cell / day for at least 25-37 days. The process parameters include a glucose concentration, and the method is configured to maintain the glucose concentration at about 5 mM to about 85 mM, or about 0.5 g / L to about 15.5 g / L, about 1 g / L to about 15.5 g / L, about 0.5 g / L to about 8 g / L, about 2 g / L to about 6 g / L, or about 3 g / L to about 5 g / L. The process parameters include lactate concentration, and the method is configured to maintain a lactate concentration of less than about 60 mM, or less than about 6 g / L, less than about 5 g / L, less than about 4 g / L, less than about 3 g / L, less than about 2 g / L, or less than about 1 g / L. The process parameters include ammonia concentration, and the method is configured to maintain an ammonia concentration of less than about 15 mM, less than about 12 mM, less than about 10 mM, less than about 9 mM, less than about 8 mM, less than about 7 mM, or less than about 6 mM. The removal of cell-free spent medium, the removal of cells, and the introduction of one or both of fresh medium and nutrients are each controlled by a respective pump. The bioreactor includes a filter configured to retain cells and allow liquid to pass through.

[0009] In another embodiment, the present disclosure is directed to a method of controlling a bioreactor system, comprising: supplying a cell culture to a bioreactor; measuring one or more process parameters (PP) of the cell culture in the bioreactor with a Raman probe; and adjusting one or more inputs or outputs of the bioreactor based on the measurements from the Raman probe.

[0010] A method according to the present disclosure is shown as including the steps of: providing a cell culture to a bioreactor (302), where conditions in the bioreactor enable the cell culture to produce a protein of interest (POI); measuring a process parameter of the culture in the bioreactor by Raman (304), where the process parameter is selected from the group consisting of at least nutrient concentration, viable cell concentration, and protein attribute; measuring a predetermined weight of the bioreactor containing the cell culture (306); removing cell-free spent medium from the cell culture at a first specified rate using a first discharge conduit (308); and removing cell-free spent medium from the cell culture at a first specified rate using a second discharge conduit (309). removing cells from the cell culture at a second specified rate (310); introducing one or both of fresh medium and nutrients to the cell culture at a third specified rate using the input conduit; and adjusting the input and output conduits based on the Raman probe measurements and the weight measurements of the bioreactor (312) to (i) maintain one or more of the process parameters within predetermined ranges, (ii) maintain the weight of the bioreactor containing the cell culture within predetermined ranges, and (iii) maintain the third specified rate of the input conduit and the first and second specified rates of each of the output conduits within their respective predetermined ranges.

[0011] In yet another aspect, the present disclosure is directed to a bioreactor culture system comprising: a tank having an input conduit and at least one output conduit; at least one pump; a filter coupled to the tank; a Raman probe coupled to the tank; and a controller coupled to the at least one pump and the Raman probe, wherein the controller is configured to control the at least one pump based on an input from the Raman probe.

[0012] The at least one exhaust conduit includes a first exhaust conduit for connecting to a second pump configured to control removal of fluid from the tank and a second exhaust conduit for connecting to a third pump configured to control removal of cells from the tank. The filter is configured to retain cells in the tank and allow liquid to pass through the filter. The Raman probe is disposed in the tank. The controller is coupled to the first pump, the second pump, and the third pump. The bioreactor includes a scale configured to measure a weight of the tank containing the cell culture therein, and the controller is configured to receive weight data from the scale. The controller is configured to compare the weight of the tank with a weight setpoint and adjust one or more of the outputs of the first pump, the second pump, and the third pump based on the comparison. The controller is configured to receive spectral data from the Raman probe, determine a parameter of the cell culture based on the received spectral data, compare the determined parameter with a parameter setpoint, and adjust one or more of the outputs of the first pump, the second pump, or the third pump based on the comparison. Adjusting the output of one or more of the first pump, the second pump, and the third pump reduces the deviation between the determined parameter and the parameter setpoint, or the deviation between the received weight and the weight setpoint. The method is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per mL for 30 days at steady state. The tank has a volume of at least 10 L, and the method is configured to maintain a weight variation of the tank containing the cell culture within a 20 g range. The tank has a volume of at least 10 L, and the method is configured to maintain a weight variation of the bioreactor containing the cell culture within 0.1 percent of the initial weight of the tank containing the cell culture.The controller is configured to determine a plurality of parameters of the bioreactor culture based on the received spectral data, compare each of the plurality of parameters to a respective set point for the plurality of parameters, and adjust an output of one or more of the first pump, the second pump, and the third pump based on the comparison to reduce a deviation between the determined parameters and the respective set point. The plurality of parameters include temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity. The filter is configured to retain the cells and allow a liquid to pass through. The bioreactor includes a scale, and the tank and filter are mounted on the scale. The bioreactor includes a scale, and the tank is mounted on the scale. The bioreactor includes a scale, and the tank is in physical contact with the scale.

[0013] A bioreactor culture system comprising: a tank having an input conduit and at least one output conduit; at least one pump; a filter in contact with the tank; a Raman probe coupled to the tank; a scale in contact with the tank; and a controller coupled to the at least one pump, the scale, and the Raman probe. In some embodiments, the filter and the tank are in contact with the scale. In other embodiments, the filter comprises a mesh material. In some embodiments, the filter comprises a mesh having a pore size in the range of 0.2 μm to 30 μm.

[0014] In yet another embodiment, the present disclosure is directed to a bioreactor culture system comprising: a tank having an input conduit and at least one output conduit; at least one pump; a filter coupled to the tank; a scale in contact with the tank; a Raman probe coupled to the tank; and a controller coupled to the at least one pump, the scale, and the Raman probe, wherein the controller is configured to control the at least one pump based on input from the Raman probe and input from the scale.

[0015] In another embodiment, a bioreactor culture system is disclosed that includes a tank having an input conduit for connection to a first pump configured to control fluid delivery to the tank, a first output conduit for connection to a second pump configured to control fluid removal from the tank, and a third output conduit for connection to a third pump configured to control cell removal from the tank, a filter coupled to the tank, the filter configured to retain cells in the tank and allow liquid to pass through the filter, a scale configured to measure a weight of the tank including the cell culture in the tank, and a Raman probe disposed within the tank. This embodiment includes a controller coupled to the first pump, the second pump, the third pump, the scale, and the Raman probe, the controller configured to receive weight data from the scale, compare the weight of the tank to a weight setpoint, receive spectral data from the Raman probe, determine a parameter of the cell culture based on the received spectral data, compare the determined parameter to the parameter setpoint, and adjust one or more of the throughput of the first pump, the second pump, and the third pump based on the comparison.

[0016] Adjusting the throughput of one or more of the first pump, the second pump, and the third pump reduces a deviation between the determined parameter and a parameter set point or a deviation between the received weight and a weight set point. The controller is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per mL for 30 days at steady state. The tank has a volume of at least 3 L, and the controller is configured to maintain a variation in the weight of the tank containing the cell culture within a 20 g range. The tank has a volume of at least 3 L, and the controller is configured to maintain a variation in the weight of the bioreactor containing the cell culture within 0.1 percent of an initial weight of the tank containing the cell culture. The controller is configured to determine a plurality of parameters of the bioreactor culture based on the received spectral data, compare each of the plurality of parameters to a respective set point for the plurality of parameters, and adjust the throughput of one or more of the first pump, the second pump, and the third pump based on the comparison to reduce a deviation between the determined parameter and the respective set point. The plurality of parameters include temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity. The bioreactor culture system includes a filter configured to retain the cells and allow liquid to pass through. The tank and filter are mounted on a scale. The tank is mounted on a scale.

[0017] In certain embodiments, a bioreactor culture system according to the present disclosure is exemplified as including the following elements: a tank (10) having an input conduit for connection to a first pump (30) configured to control fluid delivery to the tank, a first output conduit for connection to a second pump (40) configured to control fluid removal from the tank, and a third output conduit for connection to a third pump (50) configured to control cell removal from the tank, a filter (100) coupled to, connected to, or otherwise in fluid communication with the tank, the filter configured to retain cells in the tank and allow liquid to pass through the filter, a scale (110) configured to measure the weight of the tank including the cell culture therein, a Raman probe (18) disposed within the tank, and a controller (200) coupled to the first pump (30), the second pump (40), the third pump (50), the scale (110), and the Raman probe (18).

[0018] In the bioreactor culture system, the controller (200) is configured to receive weight data from the scale (110), compare the weight of the tank (10) to a weight setpoint, receive spectral data from the Raman probe (18), determine a parameter of the cell culture based on the received spectral data, compare the determined parameter to the parameter setpoint, and adjust one or more of the throughputs of the first pump, the second pump, and the third pump based on the comparison.

[0019] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various examples and, together with the description, serve to explain the principles of the disclosed examples and embodiments.

[0020] Aspects of the present disclosure may be implemented in conjunction with the embodiments illustrated in the accompanying drawings. These drawings illustrate different aspects of the present disclosure, and where appropriate, reference numerals that designate similar structures, components, materials, and / or elements in different views are similarly labeled. It is understood that various combinations of structures, components, and / or elements other than those specifically shown are contemplated and are within the scope of the present disclosure.

[0021] Moreover, many embodiments are described and illustrated herein. The present disclosure is not limited to any single aspect or embodiment thereof, or to any combination and / or permutation of such aspects and / or embodiments. Moreover, each aspect and / or embodiment of the present disclosure may be used alone or in combination with one or more of the other aspects and / or embodiments of the present disclosure. For simplicity, specific permutations and combinations are not otherwise described and / or illustrated herein. In particular, any embodiment or implementation described herein as "exemplary" should not be construed as preferred or advantageous over, for example, other embodiments or implementations, but rather is intended to reflect or illustrate that the embodiment(s) are "examples" of the embodiment(s). [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram of a bioreactor system according to one example of the present disclosure. [Figure 2] FIG. 2 is a schematic diagram of an exemplary controller of the bioreactor system of FIG. 1 and its respective inputs and outputs. [Figure 3] 1 is a flowchart of an exemplary method according to the present disclosure. [Figure 4] 1 is a graph comparing the measured viable cell concentration in the perfusion bioreactor at day 37 of the batch with the measured viable cell concentration in the fed-batch bioreactor at day 6 of the batch. [Figure 5]FIG. 5 is a graph showing normalized cell-specific productivity over time between the perfusion bioreactor and the fed-batch bioreactor described with reference to FIG. 4. [Figure 6] FIG. 10 is a graph showing viable cell concentration over time for a perfusion bioreactor where viable cell concentration or glucose was not controlled. [Figure 7] FIG. 7 is a graph showing glucose concentration over time in the perfusion bioreactor described in FIG. 6. [Figure 8] FIG. 7 is a graph showing cell viability over time in the perfusion bioreactor described in FIG. 6. [Figure 9] 1 is a graph showing viable cell concentration over time for a perfusion bioreactor with viable cell concentration control. [Figure 10] FIG. 10 is a graph showing steady-state cell viability in the perfusion bioreactor described in FIG. [Figure 11] FIG. 10 is a graph showing normalized protein production (titer) over time in the perfusion bioreactor described in FIG. [Figure 12] FIG. 10 is a graph showing glucose concentration over time in the perfusion bioreactor described in FIG. [Figure 13] 1 is a graph comparing viable cell concentrations from a perfusion bioreactor and a fed-batch bioreactor. [Figure 14] 14 is a graph comparing normalized protein production (titer) achieved in the bioreactors described in FIG. 13. [Figure 15] 1 is a graph showing viable cell concentration over time in a perfusion bioreactor using a Raman probe to control viable cell concentration. [Figure 16] FIG. 16 is a graph showing cell viability over time in the perfusion bioreactor described in FIG. 15. [Figure 17] FIG. 16 is a graph showing normalized protein production (titer) over time in the perfusion bioreactor described in FIG. 15. [Figure 18] FIG. 16 is a graph showing glucose concentration over time in the perfusion bioreactor described in FIG. [Figure 19] 1 is a graph showing viable cell concentration over time in a perfusion bioreactor using a Raman probe to control viable cell concentration. [Figure 20] FIG. 20 is a graph showing normalized protein production (titer) over time in the perfusion bioreactor described in FIG. 19. [Figure 21] 1 is a graph showing viable cell concentration over time in a perfusion bioreactor using a Raman probe to control viable cell concentration. DETAILED DESCRIPTION OF THE INVENTION

[0023] Again, many embodiments are described and illustrated herein. The present disclosure is not limited to any single aspect or embodiment thereof, or to any combination and / or permutation of such aspects and / or embodiments. Each aspect and / or embodiment thereof of the present disclosure may be used alone or in combination with one or more of the other aspects and / or embodiments of the present disclosure. For simplicity, many of these combinations and permutations are not separately described herein.

[0024] In particular, for purposes of brevity and clarity of explanation, certain aspects of the figures are intended to illustrate the general structure and / or construction methods of various embodiments. Descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring other features. Elements in the figures are not necessarily drawn to scale. The dimensions of some features may be exaggerated relative to other elements to enhance understanding of the example embodiments. For example, those skilled in the art will understand that cross-sectional views are not drawn to scale and should not be taken to represent proportional relationships between the various components. Cross-sectional views are provided to help explain the various components of the illustrated assemblies and to show their relative placement with respect to one another.

[0025] Examples of the present disclosure will now be described in detail with reference to the accompanying drawings which illustrate the present disclosure. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. In the following description, relative terms such as "about," "substantially," and "approximately" are used to indicate that the stated numerical values ​​may vary by ±10%. Furthermore, in the claims, values, limits, and / or ranges refer to ±10% of the value, limit, and / or range.

[0026] The term "conduit" refers to a flow path, tubing, connection, passageway, etc. through which a fluid may travel. By way of example, a conduit may include bioprene thermoplastic tubing manufactured by Watson-Marlow.

[0027] "Batch culture" or "batch mode" refers to a unit (e.g., a culture vessel) that is filled with cells and an initial working volume of cell culture medium that is not replaced. In such batch culture, all components for cell culture are supplied to the culture vessel at the beginning of the culture process. The culture can proceed until nutrients are exhausted or waste products reach toxic levels, causing apoptosis.

[0028] The phrases "fed-batch cell culture" or "fed-batch culture" refer to batch culture in which animal cells and medium are initially fed to the culture vessel, and additional culture nutrients are provided to the culture during cultivation, either continuously or as discrete bolus additions, with or without periodic harvesting of cells and / or product prior to the end of the culture. Fed-batch culture includes "semi-continuous fed-batch culture," in which the entire culture (which may include cells and medium) is periodically removed and replaced with fresh medium. Fed-batch culture is distinguished from simple "batch culture" by the addition (or removal) of components to the vessel during cultivation. Fed-batch culture can be further distinguished from perfusion culture insofar as the medium is not replaced during the fed-batch process, whereas in perfusion culture, all or a portion of the cells are retained in the culture, e.g., by using a filter or cell retention device, and medium is supplied continuously or intermittently while growth-inhibitory by-products are constantly or periodically removed from the culture vessel. In a fed-batch process, which differs from a perfusion process, the culture is continued until a maximum or otherwise determined working volume and / or protein production is determined to have been reached, after which the fed-batch culture product is harvested.

[0029] Perfusion culture as a method for producing a protein of interest is also contemplated for use in the methods of the present disclosure. Perfusion cell culture methods for the production of proteins or antibodies of interest are known to those of skill in the art.

[0030] The term "cell" includes any cell suitable for expression of a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells. Eukaryotic cells include, but are not limited to, yeast and all mammalian cells (human and non-human), as well as cell fusions such as hybridomas or quadromas. In certain embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In other embodiments, the cell is selected from the following cells: Examples of cells that can be used include CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, lymphocytes, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epithelial), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cells contain one or more viral genes (e.g., retinal cells expressing viral genes (e.g., PER.C6® cells)). In some embodiments, the cells are CHO cells. In other embodiments, the cells are CHO K1 cells.

[0031] A "cell line" refers to one or more cells derived from a particular lineage through serial passage or subculture of cells. The term "cell" is used interchangeably with "cell population."

[0032] Given the current state-of-the-art supply strategy, CHO cells are expected to reach 10 x 10 cells per week, which is the standard industrial value for CHO cell fed-batch cultures. 6Cell numbers exceeding 1000 cells / mL and titers of human IgG exceeding 2 g / L (harvested after approximately two weeks) have been achieved. See Kim, BJ, et al., Biotechnol Bioeng. 2012 January;109(1):137-45. Antibody production exceeding 10 g / L has been reported from CHO cells, which are well established as an important industrial mammalian cell line. See Omasa et al., Current Pharmaceutical Biotechnology, 2010,11:233-240.

[0033] The terms "cell culture medium" and "culture medium" generally refer to a nutrient solution used to grow mammalian cells that provides nutrients necessary to promote cell growth, such as a carbohydrate energy source, essential amino acids, trace elements, and vitamins. Cell culture medium may contain extracts, such as serum or peptone (hydrolysate), that provide raw materials to support cell growth. Instead of animal-derived extracts, media may contain yeast-derived extracts or soy extracts. Chemically defined media refer to cell culture media in which all chemical components are known. Chemically defined media do not contain any animal-derived components, such as serum-derived peptones or animal-derived peptones. Media may also be protein-free. "Fresh medium" refers to medium that has not yet been introduced into a cell culture and / or utilized by cells in the cell culture. Fresh medium generally contains high levels of nutrients and may contain little to no waste products. "Spent medium" can refer to medium that has been used by cells in a cell culture and can generally contain lower levels of nutrients (because these nutrients can be utilized by the cells in the cell culture) and higher levels of waste products than are present in fresh medium.

[0034] In a perfusion bioreactor, medium can be continuously removed from a cell culture and replaced with fresh medium. The constant addition of fresh medium while removing waste products provides cells in the cell culture with the nutrients they need to achieve high cell concentrations. Unlike the constantly changing conditions experienced during batch and fed-batch cultures, perfusion provides a means to achieve and maintain a steady-state culture. Typically, approximately one culture volume is exchanged per day, and the cell concentration achieved with perfusion is typically two to ten times higher than that achieved at the peak of a batch or fed-batch culture. Nutrient exchange and / or removal of apoptotic cells allows cell viability to be maintained at steady state for extended periods. During steady-state production, the quality attributes of a protein (or other target compound) produced early in the batch can be substantially identical to those of a protein (or other target compound) produced later in the batch. Proteins can be evaluated based on various post-translational modifications, such as glycoforms, charge heterogeneity, aggregation, and various measures of purity. Achieving substantially identical protein quality in fed-batch reactors is not feasible because cell culture conditions within such reactors are constantly changing.

[0035] The culture conditions in the bioreactor enable the cell culture to produce the protein of interest (POI) to provide a consistent protein material. In some culture conditions of the cell culture, one or more process parameters can be at least selected from the group consisting of nutrient concentrations such as glucose concentration, glutamate concentration, and glutamine concentration; ammonia concentration; lactate concentration; total cell density; viable cell density; and protein attributes.

[0036] The bioreactor method allows for control of the flow of various constituents, such as media (e.g., nutrients), proteins, and cells, into and out of the bioreactor. The bioreactor method includes removing cell-free spent media from the cell culture at a first specified flow rate using a first outlet conduit. The method includes removing cells from the cell culture at a second specified flow rate using a second outlet conduit. The method includes introducing fresh media or nutrients, or both, into the cell culture at a third specified flow rate using an input conduit. One or more of the first specified flow rate, the second specified flow rate, and the third specified flow rate are adjusted based on Raman probe measurements of the bioreactor. One or more of the first specified flow rate, the second specified flow rate, and the third specified flow rate are adjusted based on the Raman probe measurements of the bioreactor to maintain one or more process parameters within a predetermined range. The first designated flow rate, the second designated flow rate, and the third designated flow rate are adjusted based on Raman probe measurements of the bioreactor to maintain the third designated flow rate of the input conduit and the first designated flow rate and the second designated flow rate of each of the output conduits within their respective predetermined ranges.

[0037] The removal of cell-free spent medium, the removal of cells, and the introduction of fresh medium and / or nutrients are each controlled by a respective pump. The bioreactor includes a filter configured to retain cells and allow liquid to pass through.

[0038] The disclosed methods and systems include a method for controlling the weight of a bioreactor and its contents, among other reasons, to employ a stable production process. The method includes measuring the weight of a bioreactor containing cell culture contents. In a further embodiment, the method employs controlling the weight of the bioreactor in combination with controlling the flow rates described above in connection with the conduits. The method includes measuring the weight of the bioreactor containing the cell culture contents, and adjusting one or more of a first designated flow rate, a second designated flow rate, and a third designated flow rate based on the measured weight. The first designated flow rate, the second designated flow rate, and the third designated flow rate are adjusted based on the measured weight to maintain the third designated flow rate of the input conduit and the first designated flow rate and the second designated flow rate of each of the output conduits within respective predetermined ranges. The first designated flow rate, the second designated flow rate, and / or the third designated flow rate are adjusted to maintain the weight of the cell culture and the bioreactor within the predetermined ranges. Measurement of a process parameter (PP) of the cell culture in the bioreactor with a Raman probe is performed at least hourly. The method is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per mL for at least about 30 days at steady state. The bioreactor has a volume of at least 10 L, and the method is configured to maintain a weight variation of the bioreactor and cell culture within a 20 g range. The bioreactor has a volume of at least 10 L, and the method is configured to maintain a weight variation of the bioreactor containing the cell culture within 0.1 percent of the initial weight of the bioreactor containing the cell culture. If the process parameter deviates from a setpoint value within a respective desired range, one or more of the removal of cell-free medium, the removal of cells, and the introduction of one or both of fresh medium and nutrients are adjusted to reduce the deviation. For example, at least two bioreactor volumes of spent medium per day are removed through the first discharge conduit, or up to three bioreactor volumes of spent medium per day are removed through the first discharge conduit.

[0039] The one or more process parameters also include a temperature of the cell culture and a pH of the cell culture, where the temperature is maintained at 35-36°C and the pH is maintained at 6.85-7.15. In other embodiments, the pH is maintained at about 6.50 to about 7.50, about 6.60 to about 7.40, about 6.70 to about 7.40, about 6.80 to about 7.30, about 6.90 to about 7.20, about 7.00 to about 7.10, about 6.50, about 6.55, about 6.60, about 6.65, about 6.70, about 6.75, about 6.80, about 6.85, about 6.90, about 6.95, about 7.00, about 7.05, about 7.10, about 7.15, about 7.20, about 7.25, about 7.30, about 7.35, about 7.40, about 7.45, or about 7.50.

[0040] The one or more process parameters include a cell-specific productivity, and the method is configured to maintain the cells in the cell culture at a cell-specific productivity of at least 15-25 pg / cell / day for at least 25-37 days.

[0041] The one or more process parameters include a glucose concentration, and the method is configured to maintain the glucose concentration between about 5 mM and about 85 mM, or between about 1 g / L and about 15.5 g / L.

[0042] The one or more process parameters include lactate concentration, and the method is configured to maintain a lactate concentration of less than about 60 mM, or less than about 6 g / L.

[0043] The one or more process parameters include an ammonia concentration, and the method is configured to maintain an ammonia concentration of less than about 15 mM.

[0044] The term "steady state" refers to maintaining a nutrient concentration, process parameter, or quality attribute of a cell culture at an unchanging, constant, or stable level. A unchanging, constant, or stable level is understood to refer to a predetermined set point or a level within a predetermined set range. The set point, and therefore the steady state level, may be shifted by an operator during cell culture production. The set point or steady state level may also include a set range or threshold of values.

[0045] The term "predetermined" refers to a quantity or set value, the value of which is established or calculated either manually by a user or according to one or more algorithms by a controller.

[0046] Throughout the manufacturing process for a particular therapeutic protein product, product or protein quality attributes can be identified that require control based on their potential impact on quality, particularly clinical impact. The relevant protein quality attributes may affect purity, safety, and / or efficacy. Quality attributes refer to physical, chemical, biological, or microbiological characteristics or properties of the manufactured formulation that should be within appropriate limits, ranges, or distributions that ensure the desired product (protein) quality. For example, International See the Council for Harmonization (ICH) Q8(R2) Pharmaceutical Development (ICH, August 2009). Protein product quality attributes may include, but are not limited to, high molecular weight species, aggregates, charge isomers, appearance, color, pH, potency, post-translational modifications (glycan content and distribution), conductivity, isoelectric point, charge heterogeneity, disulfide bond scrambling, free cysteines, and host cell proteins, and may be considered attributes that significantly impact product quality. Specific process parameters are controlled within appropriate limits, ranges, or distributions during production culture for operational reliability and consistency during the manufacturing process. Process parameters may include initial cell density, initial cell viability, final cell viability, total protein (titer), viable cell count (VCC), nutrient concentrations (glucose, phosphate, amino acids, etc.), ammonia, pH, lactate, etc. Formulations that are sensitive to specific process parameters during the manufacturing process require appropriate control because they may cause changes in the protein attribute above or below the threshold for that specific attribute. As such, process parameters may include those whose variations may affect the quality attributes above by more than the thresholds defined, and therefore need to be monitored or controlled to ensure that the process produces material of the desired quality.

[0047] The terms "cell-specific productivity," "cell-specific rate," and the like refer to a product expression rate in a specific, e.g., per cell, or in units of measure of cell mass or volume. Cell-specific productivity is measured, for example, in grams of protein produced per cell per day.

[0048] Bioreactor system 1 may include bioreactor tank 10, feed reservoir 28, feed pump 30, discharge pump 40, and harvest pump 50. Bioreactor system 1 may also include ATF pump 70, discharge tank 80, and harvest tank 90. ​​Pumps 30, 40, 50, and 70 may be operatively coupled to controller 200, although in some examples, ATF pump 70 may be coupled to and controlled by a separate controller 102.

[0049] The bioreactor tank 10 can be a vat, barrel, vessel, flask, or other suitable container sized for numerous operational scales. For example, the volume of the bioreactor tank 10 can be from about 1 L to about 20,000 L, from about 5 L to about 10,000 L, from about 10 L to about 1,000 L, from about 20 L to about 100 L, about 50 L, at least about 1 L, at least about 10 L, at least about 50 L, at least about 100 L, at least about 200 L, at least about 500 L, at least about 1,000 L, at least about 10,000 L, less than about 20,000 L, less than about 10,000 L, less than about 1,000 L, less than about 500 L, less than about 200 L, or less than about 100 L. In other embodiments, the bioreactor tank 10 has a volume of at least 2 L, at least 3 L, at least 10 L, at least 35 L, or at least 50 L, or more. The bioreactor tank 10 can be made from metal (e.g., steel or stainless steel), metal alloy, glass, and / or polymer (e.g., a disposable, single-use bioreactor).

[0050] Pumps 30, 40, and 50 may include any suitable pump, such as, for example, a peristaltic pump, a diaphragm pump, a piston pump, an electric pump, etc. In one example, pumps 30, 40, and 50 may be substantially identical to one another. In another example, one or more of pumps 30, 40, and 50 may be different from the other(s). In yet another example, pump 70 may be similar to any one of pumps 30, 40, and 50. Feed reservoir 28 may contain any suitable source of nutrient feed for bioreactor tank 10, and the nutrient feed may be directed to bioreactor tank 10 by feed pump 30 via suitable conduits. The nutrient feed (culture medium) may include a carbon source (e.g., glucose), water, salts, an amino acid source, and / or other nutrients.

[0051] A lid 12 may cover the top of the bioreactor tank 10, and various components and instruments may extend through the lid 12 into the interior of the bioreactor tank 10. For example, the aerator 14, the agitator 16, the Raman probe 18, the conduit 20, and the conduit 22 may extend through the lid 12. However, it is contemplated that any or all of the aerator 14, the agitator 16, the Raman probe 18, the conduit 20, and the conduit 22 may be operatively coupled to the bioreactor tank 10 in any other suitable manner, such as, for example, through the side of the bioreactor tank 10.

[0052] Aerator 14 may be a sparger configured to supply oxygen and / or other gases to the cell culture in bioreactor tank 10. Aerator 14 may be coupled to a source of oxygen or other gases and can direct the gas to the cell culture such that the gas bubbles through the cell culture, thereby aerating the cell culture. In some examples, a microsparger may be used in combination with a perforated tube sparger.

[0053] The agitator 16 may be any suitable agitator configured to mix the cell culture within the bioreactor tank 10. The agitator 16 may be top-driven or bottom-driven, with mechanical and / or magnetic mechanisms. A bottom-driven agitator may be desirable in some cases, as it may free up space within the lid 12 for instrumentation such as temperature, pH, dissolved oxygen, foam, carbon dioxide, and other sensors, as well as inlet and outlet ports for acid, alkali, foam, and fresh medium inlet. The agitator 16 may include a radial agitator, an axial agitator, a Rushton impeller, a pitched blade impeller, a Marine blade impeller, and the like.

[0054] For example, Raman probe 18 may be a fiber optic Raman probe, e.g., in a stainless steel enclosure, with a transparent, e.g., sapphire or glass, window. Raman probe 18 may be configured to enable Raman sampling of cell culture 2. Raman probe 18 may be configured to illuminate cell culture 2 with monochromatic light (e.g., a laser at 785 nm or another suitable wavelength) and detect scattered light from cell culture 2.

[0055] Raman spectroscopy is a form of vibrational spectroscopy that provides information about molecular vibrations, which can be used by inserting a Raman probe in situ for sample identification and quantification. In some embodiments, process variables are monitored using in situ Raman spectroscopy. In situ Raman analysis is a method of analyzing a sample in its original location without the need to extract a portion of the sample for analysis in a Raman spectrometer. In situ Raman analysis is advantageous in that the Raman spectrometer is non-invasive, which reduces the risk of contamination and is non-destructive, without affecting cell culture viability or protein quality. In situ Raman analysis can provide real-time assessment of one or more process variables in a cell culture. Manufacturers of Raman probes include, but are not limited to, tech5usa, Anton Paar, InPhotonics, Kaiser Optical Systems, Inc., and FiberTech Optica.

[0056] The bioreactor tank 10 may be coupled to a filter system 100 having a hollow fiber filter therein. The hollow filter membrane (e.g., polysulfone) may comprise one or more tubular membranes having an inner diameter of about 0.3 mm to about 6.0 mm, about 0.5 mm to about 3.0 mm, about 0.5 mm to about 2.0 mm, greater than about 0.3 mm, greater than about 0.5 mm, less than about 6.0 mm, less than about 3.0 mm, or less than about 2.0 mm. The mesh material in the membrane may be selected so that the pore size of the mesh approximates the diameter of the cells from the cell culture 2, effectively ensuring a high cell retention rate while allowing cell debris and spent medium to pass through the filter. In one example, the pore size of the mesh is about 0.2 μm to about 30 μm, although other suitable ranges and values ​​are also contemplated. The protein of interest or other biological material may be perfused or retained based on the pore size of the filter (e.g., 0.2 μm or 50 kD).

[0057] Fluid from the bioreactor tank 10 can be delivered to the filter system 100 via the conduit 20 and the pump 70. The pump 70 may be reversible, allowing fluid to flow back and forth from the filter system 100 to the bioreactor tank 10. The filter system 100 may operate under alternating tangential flow. In one example, alternating tangential flow may refer to one flow reciprocating in the same direction (e.g., tangential) as the membrane surface of the hollow fibers, and another flow in a direction substantially perpendicular to the filter surface. Alternating tangential flow can be achieved using one pump (e.g., pump 70) to circulate the cell culture across the filter module containing the hollow fibers, and another pump (e.g., pump 50) to remove the low-cell-density liquid prior to filter separation. Alternating tangential flow can be useful to prevent fouling and shear problems typical of other cell retention mechanisms.

[0058] Alternatively, other filtration mechanisms (such as membrane filtration mechanisms) may be utilized, such as, for example, ultrafiltration, microfiltration, and tangential flow filtration.

[0059] The discharge pump 40 may be configured to remove cells from the bioreactor tank 10 via the conduit 22. The conduit 22 may be a dip tube selected to avoid cell clumping and clogging (e.g., which may occur if the conduit 22 is too narrow for the viscosity of the culture 2). The conduit 22 may include a thermoplastic elastomer tubing (e.g., bioprene). The discharge pump 40 may be controlled, for example, by the processor 200. Cell discharge via the discharge pump 40 can remove cells from the cell culture 2 in the bioreactor tank 10. The cell discharge rate (controlled by the discharge pump 40 and the controller 200) may be determined based on the growth rate of the cells in the cell culture 2. To maintain a stable cell density in the cell culture 2, it may be desirable for the discharge rate and the cell growth rate to be approximately or substantially equal to each other. In some examples, if a significant amount of the cell culture 2 is removed from the cell discharge along with a valuable product, the discharge can be collected and processed to recover the product.

[0060] The bioreactor tank 10 may be placed on a scale 110 configured to measure the weight of the bioreactor tank 10 and the cell culture 2. The scale 110 may be coupled to the controller 200 and may continuously transmit the weight of the bioreactor tank 10 and the cell culture 2 to the controller 200. In some examples, at least a portion of the filter system 100, including, for example, the filter housing and hollow membrane filter therein, may also be placed on the scale 110. The scale 110 may be any suitable scale or load cell configured to measure the weight of a component resting on the scale.

[0061] 1 and 2, the controller 200 may be configured to receive data from the Raman probe 18, the scale 110, and other sensors, and may be configured to control the flow rate of fluid through one or more of the supply pump 30, the discharge pump 40, and the harvest pump 50 based on that data.

[0062] The controller 200 may be configured to receive raw spectral data from the Raman probe 18 and determine process parameters such as, for example, glucose concentration, glutamine concentration, glutamate concentration, ammonia concentration, lactate concentration, total cell density, titer, and viable cell density. The controller 200 may use these determined process parameters to establish a feedback loop that regulates one or more of the fluid flows through the feed pump 30, discharge pump 40, and harvest pump 50. That is, the controller 200 may be configured to determine a glucose concentration (e.g., about 5 mM to about 85 mM, or about 0.5 g / L to about 15.5 g / L, about 1 g / L to about 15.5 g / L, about 0.5 g / L to about 8 g / L, about 2 g / L to about 6 g / L, or about 3 g / L to about 5 g / L), a glutamine concentration (e.g., less than about 8 mM, less than about 7 mM, less than about 6 mM, less than about 5 mM, or less than about 4 mM), a glutamate concentration (e.g., less than about 5 mM, less than about 4 mM, less than about 3 mM, less than about 2 mM, or less than about 1 mM), an ammonia concentration (e.g., less than about 15 mM, less than about 12 mM, less than about 10 mM, less than about 9 mM, less than about 8 mM, less than about 7 mM, or less than about 6 mM), a milk protein ...15 mM, Set points for one or more of acid salt concentration (e.g., less than about 6 g / L, less than about 5 g / L, less than about 4 g / L, less than about 3 g / L, less than about 2 g / L, or less than about 1 g / L), total cell density (e.g., greater than about 30 MM, greater than about 35 MM, greater than about 40 MM, greater than about 45 MM, greater than about 50 MM, greater than about 55 MM, greater than about 60 MM, or greater than about 65 MM), and viable cell density (e.g., at least 30 million cells per mL, at least 35 million cells per mL, at least 50 million cells per mL, or at least 75 million cells per mL) can be established, and the determined values ​​(based on the Raman spectrum from Raman probe 18) can be compared to the respective set points for those values.

[0063] The controller 200 can utilize a negative feedback loop to correct for any difference between the setpoint value (or set range of values) and the determined value. For example, if the determined glucose concentration is greater than the setpoint glucose concentration, the controller 200 can, for example, decrease the output of the feed pump 30, decrease the output of the output pump 40, and / or increase the output of the harvest pump 50 to facilitate a decrease in the glucose concentration, or the controller 200 can decrease the output of the feed pump 30 and decrease the output of the harvest pump 50 to facilitate a decrease in the glutamine concentration. For example, if the determined glutamine concentration is greater than the setpoint glutamine concentration, the controller 200 can, for example, decrease the output of the feed pump 30, decrease the output of the output pump 40, and / or increase the output of the harvest pump 50 to facilitate a decrease in the glutamine concentration, or the controller 200 can decrease the output of the feed pump 30 and decrease the output of the harvest pump 50 to facilitate a decrease in the glutamine concentration. For example, if the determined glutamate concentration is greater than the setpoint glutamate concentration, controller 200 may, for example, decrease the discharge of feed pump 30, decrease the discharge of discharge pump 40, and / or increase the discharge of harvest pump 50 to facilitate a decrease in the glutamate concentration, or controller 200 may decrease the discharge of feed pump 30 and decrease the discharge of harvest pump 50. For example, if the determined ammonia concentration is greater than the setpoint ammonia concentration, controller 200 may, for example, decrease the discharge of feed pump 30, increase the discharge of discharge pump 40, and / or decrease the discharge of harvest pump 50 to facilitate a decrease in the ammonia concentration, or controller 200 may increase the discharge of feed pump 30 and increase the discharge of harvest pump 50. For example, if the determined lactate concentration is greater than the set point lactate concentration, the controller 200 may, for example, increase the discharge of the supply pump 30, decrease the discharge of the discharge pump 40, and / or increase the discharge of the harvest pump 50 to facilitate a decrease in the lactate concentration, or the controller 200 may decrease the discharge of the supply pump 30 and decrease the discharge of the harvest pump 50.For example, if the determined total cell density is greater than the set point total cell density, the controller 200 may, for example, decrease the discharge of the feed pump 30, increase the discharge of the discharge pump 40, and / or decrease the discharge of the harvest pump 50 to facilitate a decrease in the total cell density, or the controller 200 may decrease the discharge of the feed pump 30 and decrease the discharge of the harvest pump 50. For example, if the determined viable cell density is greater than the set point viable cell density, the controller 200 may, for example, decrease the discharge of the feed pump 30, increase the discharge of the discharge pump 40, and / or decrease the discharge of the harvest pump 50 to facilitate a decrease in the viable cell density, or the controller 200 may decrease the discharge of the feed pump 30 and decrease the discharge of the harvest pump 50.

[0064] For example, if the determined glucose concentration is less than the set point glutamine concentration, the controller 200 may, for example, increase the output of the feed pump 30, increase the output of the output pump 40, and / or decrease the output of the harvest pump 50 to facilitate an increase in the glucose concentration, or the controller 200 may increase the output of the feed pump 30 and increase the output of the harvest pump 50. For example, if the determined glutamine concentration is less than the set point glutamine concentration, the controller 200 may, for example, increase the output of the feed pump 30, increase the output of the output pump 40, and / or decrease the output of the harvest pump 50 to facilitate an increase in the glutamine concentration. For example, if the determined glutamate concentration is less than the setpoint glutamate concentration, the controller 200 may, for example, increase the discharge of the feed pump 30, increase the discharge of the discharge pump 40, and / or decrease the discharge of the harvest pump 50 to facilitate an increase in the glutamate concentration, or the controller 200 may increase the discharge of the feed pump 30 and increase the discharge of the harvest pump 50. For example, if the determined lactate concentration is less than the setpoint lactate concentration, the controller 200 may, for example, increase the discharge of the feed pump 30, increase the discharge of the discharge pump 40, and / or decrease the discharge of the harvest pump 50 to facilitate an increase in the lactate concentration. For example, if the determined total cell density is less than the set point total cell density, the controller 200 may, for example, increase the discharge of the feed pump 30, decrease the discharge of the discharge pump 40, and / or increase the discharge of the harvest pump 50 to facilitate an increase in the total cell density, or the controller 200 may increase the discharge of the feed pump 30 and increase the discharge of the harvest pump 50.For example, if the determined viable cell density is less than the set point viable cell density, the controller 200 may, for example, increase the discharge of the supply pump 30, decrease the discharge of the discharge pump 40, and / or increase the discharge of the harvest pump 50 to facilitate an increase in the viable cell density, or the controller 200 may increase the discharge of the supply pump 30 and increase the discharge of the harvest pump 50.

[0065] However, the overall perfusion through the system is maintained at a given set point (the perfusion rate does not vary based on the concentration in the reactor). Similarly, the controller 200 may control the bioreactor weight (and the weight of cell culture 2) using a negative feedback loop.

[0066] It should be noted that additions or subtractions of various nutrients input to the reactor can be combined with corresponding changes to other inputs to ensure that the total mass and / or volume of material input to the reactor remains the same. That is, since the perfusion rate is maintained constant, an increase in one nutrient, e.g., glucose solution, glutamine, glutamate, etc., can be accompanied by a corresponding decrease in the mass or volume of the initial nutrient feed stream.

[0067] In one embodiment, the system includes at least two feedback loops: one for weight control and one for control of a process parameter (e.g., VCC, glucose, glutamine, glutamate, ammonia, lactate, etc.). In one example, the various input and output pumps are not controlled by competing loops. For example, the perfusion rate may be set (e.g., 20 L / day), after which the Raman probe 18 measures one or more culture values, and the controller 200 evaluates steps to take based on the measurements from the Raman probe 18. If the controller 200 determines, for example, that the VCC is too high, the controller 200 can initiate cell removal via the output pump 40 while simultaneously reducing the flow rate of the harvest pump 50 so that the total volume through the system remains constant. Additional steps for the controller 200 to take if other process parameters (e.g., glucose, glutamine, glutamate, ammonia, lactate, and total cell density) are sensed to be too high or too low are described above.

[0068] A second feed pump may be added to add glucose, lactose, glutamine, glutamate, etc. In an alternative embodiment, or in addition, release can be adjusted in response to increased ammonia by removing cells.

[0069] The controller 200 may be located in a headless computer system (e.g., a system without a monitor, keyboard, and mouse). Thus, the controller 200 may be installed in a server that is controlled via a network connection or some other connection, such as a serial connection. The controller 200 may be cloned onto one or more redundant servers in case one or more of the servers fails.

[0070] The controller 200 may be configured to apply Kalman filtering, for example, linear quadratic estimation (LQE), to the Raman spectral data from the Raman probe 18. Kalman filtering may involve applying an algorithm that uses a series of measurements over time to the spectral data to generate estimates of unknown variables that tend to be more accurate than those based on a single measurement alone. The determined process parameters may therefore be based on the filtered model. It is also contemplated that the controller 200 may use other types of filtering to process the spectral data from the Raman probe 18.

[0071] The controller 200 may include or otherwise be coupled to a PI (Process Information) historian. The PI historian may be an application with a time series database that can record data from a process control system. The PI historian may enable a user to record, analyze, and monitor real-time information. For example, the controller 200 may store weight values ​​from the scale 110, spectral data from the Raman probe 18, and pump speeds for the feed pump 30, discharge pump 40, and harvest pump 50 in the PI historian.

[0072] 3 illustrates a method 300 according to the present disclosure. One or more steps of method 300 may be performed out of order, simultaneously with other steps, or omitted entirely. Method 300 may begin at step 302, in which bioreactor system 1 is assembled, cell culture 2 is provided into bioreactor tank 10, and cell culture 2 is inoculated with a cell line. Method 300 may then proceed to step 304, in which process parameters of cell culture 2 are measured within the bioreactor by Raman probe 18 and / or by additional or other sensors. The process parameters may include any of the aforementioned parameters determined from Raman spectral data acquired by Raman probe 18. Method 300 may proceed to step 306, in which the weight of bioreactor tank 10 (with cell culture 2 therein) is measured by scale 110 and provided to processor 200.

[0073] Method 300 may proceed from step 306 to step 308, where cell-free spent medium from cell culture 2 is removed at a first specified rate by operating pump 70 to withdraw cell culture (medium and cells) from bioreactor tank 10 via conduit 20 and by operating harvest pump 50 to withdraw solution through filter system 100. Method 300 may proceed from step 308 to step 310, where cells may be removed from the cell culture using discharge conduit 22 by discharge pump 40 at a second specified rate. Method 300 may proceed from step 310 to step 312, where one or both of fresh medium and nutrients may be introduced to the cell culture at a third specified rate using input conduits and feed pump 30 to keep the total input of medium and nutrients equal to the combined output of discharge pump 40 and harvest pump 50. The specified rate may be a set point or range of rates at which the pumps are operated and / or maintained. The specified speed may be determined by the controller 200 .

[0074] It is contemplated that each of steps 302 through 312 may be performed in any order, and in some cases may be performed simultaneously in real time via multiple feedback loops executed by controller 200.

[0075] Steps 308, 310, and 312 may be controlled by controller 200 based on data received from Raman probe 18 in step 304 and from scale 110 in step 306. The weight of bioreactor tank 10 (and cell culture 2 contained therein) may be controlled via a PID (proportional-integral-derivative) loop. Additionally, controller 200 may be configured to analyze Raman spectra obtained from Raman probe 18 to determine one or more process parameters, including, for example, glucose concentration, glutamine concentration, glutamate concentration, ammonia concentration, lactate concentration, total cell density, and viable cell density. Each of these variables may also be controlled by a negative feedback loop.

[0076] Examples of the present disclosure can provide an elegant, flexible, and inexpensive solution to existing control solutions, with relatively few missing data. The control strategy of the present disclosure can provide stable bioreactor level control. For example, level fluctuations have been reduced from + / - 0.5 L / day to + / - 0.01 L / day using the control system of the present disclosure. Improvements in weight fluctuations have also been achieved, for example, from 5-10% weight fluctuations using other systems, such as volumetric calibration, to 0.1-0.5% error using the control system disclosed herein. This improvement may be at least partially attributable to the system's change from volumetric calibration of the pump to a software-controlled version based on weight and other parameters. Furthermore, the control system of the present disclosure may be fully integrated with process information (PI) alarms (e.g., email alerts) and can be remotely accessed for shutdown. Furthermore, the systems and methods of the present disclosure can provide more reproducible and reliable results than conventional systems and methods.

[0077] Example 1 (FIGS. 4 and 5) The experiments described in Example 1 compare a perfusion bioreactor with a fed-batch bioreactor and show higher achieved viable cell concentrations and cell-specific productivity in the perfusion bioreactor versus the fed-batch bioreactor.

[0078] In one experiment, the cell line and medium were cultured in a 15 L bioreactor. Bioreactor set points included temperature (35.5°C), agitation (250 RPM), pH (controlled using CO2 and sodium bicarbonate) (6.85-7.15), and working volume (11 L). An ATF4 cell retention device equipped with a 0.2 μm hollow fiber filter was attached to the bioreactor. The hollow fiber filter retained the cells but allowed proteins and nutrients to pass through. Two reactor volumes (or 22 L of medium) were passed through the filter every 24 hours.

[0079] Both the bioreactor and ATF were placed on a scale. The weights of the bioreactor, cell culture, and ATF were sent via an Ethernet connection to a computer running control software. The weight was compared to a setpoint (11.0 kg, e.g., the working volume of the bioreactor) and a PID controller (designed in MATLAB but implemented via the control software SynTQ) determined whether to engage the feed pump. The harvest pump was set to a constant speed equivalent to the desired perfusion rate (2 reactor volumes per day). The feed and perfusion pumps were automatically controlled using SynTQ software, which broadcast an OPC signal to a Kepware server. The Kepware server then broadcast this signal via an Ethernet connection to a MODBUS analog output module, which converted the digital value to a hardware milliampere output of 4–20 mA.

[0080] Using this system, the bioreactor weight could be controlled to within + / - 10 g (0.09%) of the initial 11 kg weight. Prior to implementing this system, the bioreactor weight could not be controlled to within more than 0.5 kg (4.54%) overnight. Within the same system, a Kaiser Optical Raman probe was used to capture Raman spectra from the cell culture. The controller utilized models developed from previous batches to predict cell number, glucose, lactate, ammonia, and other nutrient concentrations. The Raman probe captures thousands of different spectra that are then analyzed by a computer program, such as SIMCA. Using multiple component analysis and offline data for a given parameter, the program creates a model across all probe measurements. This SIMCA model is then uploaded to SynTQ, which accesses it in real time each time the probe takes a reading (e.g., every 15 to 45 minutes).

[0081] Control of various nutrients using Raman probes allows for increased productivity of cell lines, increased viability in long-term cell culture, and improved quality of multiple aspects of proteins.

[0082] 4 shows the maximum viable cell concentration at day 37 of a batch of a perfusion bioreactor according to the present disclosure (Exhibit "Ex." 1), which is approximately twice the maximum viable cell concentration at day 7 of a batch of a comparably sized fed-batch reactor (Ex. 2). The fact that the maximum viable cell concentration was achieved at day 37 (as opposed to day 6 for the fed-batch bioreactor) demonstrates the robustness and longevity of the perfusion bioreactor process.

[0083] Figure 5 shows the cell-specific productivity (cSP) from days 1 to 12 of the fed-batch process (Ex. 2) versus the cSP from days 12 to 25 of the perfused batch process (Ex. 1). Similar productivity was achieved from days 25 to 37 of the perfused batch.

[0084] Perfusion batch VCC 50 x 10 6 To exceed cells / mL required a perfusion rate of 3 reactor volumes per day, which may be commercially prohibitive in many cases.

[0085] Optimization of the medium using a "push-to-low" strategy reduces the required perfusion rate. For example, 20 x 10 cells 6The cells are grown to 1000 cells / mL and maintained at steady state. The perfusion rate can be set at 2 reactor volumes / day for 5 days. On day 5, the perfusion rate can be reduced to 1.5 reactor volumes / day. If the cells persist, the perfusion rate can be reduced to 1 reactor volume / day after 5 days. If cells begin to die, amino acid analysis or other analysis can be used to determine how to better enrich the medium, for example, by supplementing nutrients in the medium or adjusting the nutrient concentration in the medium. In one non-limiting example, a strategy is described in "The Push to Low Approach for Optimization of High Density Perfusion Cultures of Animal Cells" by Bayer et al. (Adv. BioChem. Engin. / Biotechnol. 2006, 101:75-98.).

[0086] NOVA Flex data can be acquired, in this case by analyzing samples to obtain offline measurements. Previous NOVA data can be used to build a Raman model, at which point a probe can be inserted into the reactor. The model can provide VCC data every 15-45 minutes, 1 second, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours, rather than once a day as with NOVA, which requires manual sampling. Figure 5 shows day 20 of a given run. The first 20 days of the run were used to collect NOVA data, and this NOVA data was used to create the Raman model for the latter part of the run.

[0087] In the following examples, typical ranges for certain process parameters are as follows: pH: 6.85-7.40, dissolved oxygen: 30-60%, 35-55%, 40-50%, or 45%, temperature: 34-37.5°C, and agitation: 150-300 RPM, 175-275 RPM, 200-250 RPM, or 225 RPM on the benchtop.

[0088] Example 2 (Figs. 6 to 8) The experiment described in Example 2 presents data from a perfusion bioreactor without controlling VCC growth or glucose. VCC was observed to peak on day 7 as cells rapidly grew to high cell densities and then rapidly declined by day 11 upon depletion of nutrients in the medium ( FIG. 6 ). Controlling weight alone was insufficient to achieve steady-state VCC.

[0089] Additionally, glucose was not controlled during the perfusion run. Because the culture relied on glucose in the medium to nourish the cells during the perfusion run, this subsequently led to cell death during the culture. As the cells grew, glucose steadily decreased, while medium was constantly supplied (Figure 7). As shown in Figure 8, the sudden increase in glucose detection occurring after 10 days, as seen when monitoring cell viability, was due to complete cell death and therefore not due to glucose consumption.

[0090] In this experiment, a 15 L benchtop bioreactor was inoculated with Chinese hamster ovary (CHO) cells producing mAb1 at a given concentration. The cells were cultured at specific dissolved oxygen, temperature, agitation, and pH settings that were kept constant throughout the run. Fresh medium and nutrients were also provided to the cells in the form of a perfusion feed at a rate of twice the reactor volume per day. The reactor volume was kept constant by adding the same amount of feed removed from the perfusate to the reactor using a Repligen XCell ATF4 system. This was achieved by monitoring the weight of the bioreactor system and maintaining the weight within + / - 0.05 kg of a given target weight using a computer-assisted feedback loop control system. No Raman or release controls were provided to control the VCC or any other bioreactor parameters during this perfusion production run.

[0091] In a similar experiment (not shown), the flow rate was also set to feed two bioreactors per day, but the weight was not monitored, so pump fluctuations could not be adequately controlled.

[0092] In this analogous perfusion experiment, the feed and perfusate pumps were set to equivalent flow rates (determined by volumetric calibration of the pumps). This method did not provide flow rates accurate enough to match each other, and overnight (e.g., during a period when the bioreactor was not actively monitored), the feed pump added more medium to the reactor than the perfusate pump could remove. This resulted in reactor flooding and loss of culture.

[0093] Example 3 (Figs. 9 to 12) The experiment described in Example 3 presents data from a perfusion bioreactor equipped with VCC control. VCC control (Figure 9) resulted in stable steady states of viability (Figure 10), protein production (Figure 11), and nutrients (Figure 12).

[0094] In this experiment, a 15 L benchtop bioreactor was inoculated with CHO cells producing mAb2 at a given concentration. The cells were cultured at specific dissolved oxygen, temperature, agitation, and pH settings that were kept constant throughout the run. Fresh medium and nutrients were also provided to the cells in the form of a perfusion feed at a rate of twice the reactor volume per day. The reactor volume was kept constant by adding the same amount of feed removed from the perfusate using the ATF4 system. This was achieved by monitoring the system weight and maintaining the weight within a given target range of plus or minus 0.05 kg using a computerized control system. Raman control was not used in this run; the pump release rate was manually set after sampling the VCC. This process required multiple samples, and the release rate had to be adjusted multiple times per day.

[0095] The VCC was controlled during perfusion production culture, with a target VCC of 42.5 × 10 6 cells / mL(40~45×10 6 The release rate was 1.27 (cells / mL). Accordingly, when the VCC rose above the target, the release rate increased, and when the VCC fell below the target, the release rate decreased.

[0096] Example 4 (FIGS. 13 and 14) The experiments in this example compare perfusion culture (Figure 14) with fed-batch culture (Figure 13). Perfusion culture was able to achieve approximately four times the maximum cell number compared to fed-batch cell culture of the same protein under similar conditions (Figure 13). Fed-batch culture was performed at pilot scale, and perfusion experiments were performed at bench scale (15 L). The agitation and aeration strategies were scaled down to bench scale using a power per unit volume approach for agitation and a volume by volume matching strategy for aeration.

[0097] The perfusion culture method was able to produce 3.5 times the amount of protein compared to that produced in a fed-batch reactor in the same amount of time (Figure 14).

[0098] The perfusion culture method was performed by inoculating a 15 L benchtop bioreactor with a given concentration of CHO cells producing mAb2 (Ex. 5 and Ex. 7). The cells were cultured at specific dissolved oxygen, temperature, agitation, and pH settings that were kept constant throughout the run. Fresh medium and nutrients were also provided to the cells in the form of a perfusion feed at a rate of twice the reactor volume per day. The medium in this run was supplemented with increased concentrations of key nutrients compared to previous runs, allowing the cells to reach higher cell densities during the perfusion experiment. A weight control system was used to maintain the reactor volume constant by maintaining a given target weight within 0.05 kg. No Raman or release controls were provided during the perfusion production runs.

[0099] The fed-batch cell cultures (Ex.6 and Ex.8) were carried out under similar conditions (dissolved oxygen, temperature, agitation, and pH).

[0100] Example 5 (Figs. 15 to 18) Experiments described in Figures 15-18 were maintained at steady state for more than 30 days in this perfusion system, demonstrating beneficial results in viability, glucose and titer, as well as VCC (see, e.g., Figures 15-18).

[0101] A perfusion culture method with Raman, emission, and weight control was performed by inoculating a 15 L benchtop bioreactor with a given concentration of CHO cells producing mAb2. The cells were cultured at specific dissolved oxygen, temperature, agitation, and pH ranges that were kept constant throughout the run. The cells were also provided with fresh medium and nutrients in the form of a perfusion feed at a rate of twice the reactor volume per day. The medium in this run was supplemented with increased concentrations of key nutrients (compared to Examples 2 and 3), allowing the cells to be pushed to higher cell densities. A weight control system was used to add the same amount of feed removed from the perfusate using the ATF4 system to the reactor, monitor the system weight, and maintain the weight within plus or minus 0.05 kg of a given target using a computer feedback control system, thereby keeping the reactor volume constant.

[0102] Raman control and automatic emission control based on Raman feedback were used to control the VCC in this run (Figure 15). In the first experiment (Ex. 9), the Raman emission strategy was 6 The VCC was set to maintain 100 cells / mL. The VCC range was 35–45 × 10 6 cells / mL, which was slightly wider than the target generated by manual release in the previous experiment (Example 3), although the system was only sampled once per day and no adjustments were required in this perfusion run (compared to multiple adjustments multiple times per day with manual release as described in Example 3).

[0103] In the second experiment (Ex. 10), the VCC was increased to 10 × 10 with a perfusion rate of 1 reactor volume per day. 6 Conditions were similar to the first experiment, except that the concentration was set at 1000 cells / mL.

[0104] Example 6 (Figures 19 and 20) In one experiment, three different bioreactors were cultured using the cell line and medium. The bioreactor volumes were 3 L (Ex. 11), 15 L (Ex. 12), and 50 L (Ex. 13) (single-use bioreactors). The set points of the bioreactors included temperature (35.5°C), agitation (250 RPM), pH (controlled using CO2 and sodium bicarbonate) (6.85–7.15), and working volume (2 L, 10 L, and 35 L, respectively). All of these parameters were kept constant throughout the run. Each bioreactor was coupled to an ATF cell retention device (ATF2, ATF4, and ATF6, respectively) equipped with a 0.2-micron filter. The hollow fiber filter retained the cells, but the protein was allowed to pass through after 24 h.

[0105] At all three scales, perfusion culture was performed in each system using Raman, emission, and weight control. As in Example 5, the medium in this experiment was supplemented with additional nutrients. Weight in each system was controlled to + / - 0.05 kg for ATF4 and ATF6, and + / - 1 kg of a given target (due to the instrumental limitations of the scale itself).

[0106] Using Raman control and automatic release control based on Raman feedback, the VCC was set to 40 x 10^6 cells / mL in this run (Figure 19). This was observed in the ATF6 system, and we suspect this is because the Raman control model has not yet been optimized for large scale applications.

[0107] In all three of these runs, the perfusion rate was set between 1.8 and 2 RV / day, and all scale-up parameters were set using conventional methods.

[0108] This experiment resulted in comparable protein productivity (Figure 20) achieved in all three systems over a 5 day period.

[0109] Example 7 (Figure 21) In one experiment, a single bioreactor was cultured using the cell line and medium. The volume of the bioreactor was 15 L (Ex. 14). The set points for the bioreactor included temperature (35.5°C), agitation (250 RPM), and pH (controlled using CO2 and sodium bicarbonate) (6.85-7.15). All of these parameters were kept constant throughout the run. The bioreactor was coupled to an ATF4 cell retention device equipped with a 0.2 micron filter. The hollow fiber filter retained the cells, but the protein was allowed to pass through after 24 hours.

[0110] Perfusion culture was performed in a system using Raman, emission, and weight control. As in Example 5, the medium in this experiment was supplemented with additional nutrients. Weight in each system was controlled to + / - 0.05 kg for a given target ATF4.

[0111] Using Raman control and automatic release control based on Raman feedback, the VCC was set to 70x10^6 cells / mL in this run (see Figure 21).

[0112] The perfusion rate for this run was set at 2.5 RV / day to accommodate additional cells in the culture and ensure that medium depletion did not occur.

[0113] The reactor was able to maintain VCC at greater than 70 x 10^6 cells / mL for 7 days before an equipment failure terminated the batch. Viability remained above 90% during this time, indicating a healthy culture. Sustained production at such high densities was not possible prior to implementation of the control system of the present disclosure.

[0114] In particular, reference herein to "one embodiment" or "embodiments" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in, used in, and / or incorporated into one, some, or all embodiments of the present disclosure. The use or appearance of the phrase "in one embodiment" or "in another embodiment" herein does not necessarily refer to the same embodiment, nor does it necessarily refer to separate or alternative embodiments that are mutually exclusive with one or more other embodiments, nor is it limited to a single, exclusive embodiment. The same applies to the terms "embodiment" and "example." The present disclosure is not limited to any single aspect or embodiment thereof, nor to any combination and / or permutation of such aspects and / or embodiments. Furthermore, each aspect and / or embodiment of the present disclosure may be used alone or in combination with one or more of the other aspects and / or embodiments of the present disclosure. For simplicity, certain permutations and combinations are not otherwise described and / or illustrated herein.

[0115] Furthermore, as noted above, any embodiment or implementation described herein as "exemplary" should not be construed as preferred or advantageous over other embodiments or implementations, for example, but rather is intended to convey or illustrate that one or more embodiments are exemplary embodiment(s). (Addendum) As a preferred embodiment, the technical concept that can be understood from the above embodiment will be described below. [Item 1] providing a cell culture to a bioreactor, wherein conditions in the bioreactor enable the cell culture to produce a protein of interest (POI); measuring one or more process parameters (PP) of the culture in the bioreactor with a Raman probe, wherein the process parameters are selected from the group consisting of nutrient concentration, viable cell concentration, and protein attribute; weighing the bioreactor including the cell culture contents; removing cell-free spent medium from said cell culture at a first specified rate using a first discharge conduit; removing cells from said cell culture at a second specified rate using a second discharge conduit; introducing fresh medium or nutrients, or both, into said cell culture at a third specified rate using an input conduit; and 10. A method of controlling a bioreactor system, wherein the input and output conduits are adjusted based on the Raman probe measurements and the weight measurements of the bioreactor to (i) maintain one or more of the process parameters within predetermined ranges, (ii) maintain the weight of the bioreactor containing the cell culture within predetermined ranges, and (iii) maintain the third specified rate of the input conduit and the first and second specified rates of each of the output conduits within their respective predetermined ranges. [Item 2] 2. The method of claim 1, wherein the Raman measurement of the one or more process parameters of the culture in the bioreactor is performed at least once an hour. [Item 3] 10. The method of any one of the preceding items, wherein the method is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per mL for 30 days at steady state. [Item 4] 10. The method of any one of the preceding items, wherein the bioreactor has a volume of at least 10 L and the method is configured to maintain the weight of the bioreactor and the cell culture within a range of 20 g. [Item 5] 10. The method of any one of the preceding items, wherein the bioreactor has a volume of at least 10 L, and the method is configured to maintain the weight of the bioreactor with the cell culture within 0.1 percent of an initial weight of the bioreactor with the cell culture. [Item 6] 10. The method of any one of the preceding items, wherein if a process parameter deviates from a set-point value within its respective desired range, one or more of removal of cell-free medium, removal of cells, or introduction of either or both of fresh medium or nutrients is adjusted to reduce said deviation. [Item 7] 10. The method of any one of the preceding items, wherein at least two bioreactor volumes of spent medium per day are removed through the first discharge conduit. [Item 8] 10. The method of any one of the preceding items, wherein up to 3 bioreactor volumes of spent medium per day are removed through the first discharge conduit. [Item 9] 10. The method of any one of the preceding items, wherein the process parameters include a temperature of the cell culture and a pH of the cell culture, wherein the temperature is maintained at 35-36° C. and the pH is maintained at 6.85-7.15. [Item 10] 10. The method of any one of the preceding items, wherein the process parameters include a cell-specific productivity, and the method is configured to maintain cells in the cell culture at a cell-specific productivity of at least 15-25 pg / cell / day for at least 25-37 days. [Item 11] 10. The method of any one of the preceding items, wherein the process parameters include a glucose concentration, and the method is configured to maintain a glucose concentration between about 5 mM and about 85 mM, or between about 1 g / L and about 15.5 g / L. [Item 12] 10. The method of any one of the preceding items, wherein the process parameters include lactate concentration, and the method is configured to maintain a lactate concentration of less than about 60 mM, or less than about 6 g / L. [Item 13] 10. The method of any one of the preceding items, wherein the process parameters include an ammonia concentration, and the method is configured to maintain an ammonia concentration of less than about 15 mM. [Item 14] 10. The method of any one of the preceding items, wherein each of the removal of cell-free spent medium, removal of cells, and introduction of either or both of fresh medium or nutrients is controlled by a respective pump. [Item 15] 10. The method of any one of the preceding items, wherein the bioreactor comprises a filter configured to retain cells and allow liquid to pass through. [Item 16] providing a cell culture to a bioreactor; measuring one or more process parameters of the cell culture in the bioreactor with a Raman probe; removing cell-free spent medium from said cell culture at a first specified rate using a first discharge conduit; removing cells from said cell culture at a second specified rate using a second discharge conduit; introducing fresh medium or nutrients, or both, into said cell culture at a third specified rate using an input conduit; and A method of controlling a bioreactor system, comprising: varying one or more of the first specified rate, the second specified rate, or the third specified rate based on measurements from the Raman probe. [Item 17] a tank having an input conduit and at least one discharge conduit; at least one pump; a filter coupled to the tank; a Raman probe coupled to the tank; a controller coupled to the at least one pump and the Raman probe, the controller configured to control the at least one pump based on input from the Raman probe. [Item 18] Item 18. The bioreactor culture system of item 17, wherein the at least one discharge conduit comprises a first discharge conduit for connection to a second pump configured to control removal of fluid from the tank, and a second discharge conduit for connection to a third pump configured to control removal of cells from the tank. [Item 19] Item 19. The bioreactor culture system of item 17 or item 18, wherein the filter is configured to retain cells in the tank and allow liquid to pass through the filter. [Item 20] 20. The bioreactor culture system according to any one of items 17 to 19, wherein the Raman probe is disposed in the tank. [Item 21] 21. The bioreactor culture system according to any one of items 17 to 20, wherein the controller is coupled to the first pump, the second pump, and the third pump. [Item 22] 22. The bioreactor culture system of any one of items 17 to 21, further comprising a scale configured to measure a weight of the tank including the cell culture therein, wherein the controller is configured to receive weight data from the scale. [Item 23] 23. The bioreactor culture system of claim 22, wherein the controller is configured to compare the weight of the tank to a weight set point and adjust one or more of the outputs of the first pump, the second pump, and the third pump based on the comparison. [Item 24] 24. The bioreactor culture system of any one of items 17 to 23, wherein the controller is configured to receive spectral data from the Raman probe, determine a parameter of the cell culture based on the received spectral data, compare the determined parameter to a set point for the parameter, and adjust one or more of an output of the first pump, the second pump, or the third pump based on the comparison. [Item 25] 25. The bioreactor culture system of claim 24, wherein adjusting the output of one or more of the first pump, the second pump, and the third pump reduces a deviation between the determined parameter and the set point for the parameter or between the received weight and the set point for the weight. [Item 26] 26. The bioreactor culture system of any one of items 17 to 25, wherein the method is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per mL for 30 days at steady state. [Item 27] 27. The bioreactor culture system of any one of items 17 to 26, wherein the tank has a volume of at least 10 L and the method is configured to maintain the weight of the tank containing the cell culture within a range of 20 g. [Item 28] 28. The bioreactor culture system of any one of items 17 to 27, wherein the tank has a volume of at least 10 L, and the method is configured to maintain the weight of the bioreactor containing the cell culture within 0.1 percent of an initial weight of the tank containing the cell culture. [Item 29] The controller determining a plurality of parameters of the bioreactor culture based on the received spectral data; comparing each of the plurality of said parameters to a respective set point for each of the plurality of said parameters; 29. The bioreactor culture system of claim 28, configured to adjust an output of one or more of the first pump, the second pump, and the third pump based on the comparison to reduce a deviation between the determined parameter and the respective set point. [Item 30] 30. The bioreactor culture system of item 29, wherein the plurality of parameters comprises temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity. [Item 31] 31. The bioreactor culture system according to any one of items 17 to 30, wherein the filter is configured to retain cells and allow liquid to pass through. [Item 32] 32. The bioreactor culture system according to items 17 to 31, further comprising a scale, wherein the tank and the filter are mounted on the scale. [Item 33] 32. The bioreactor culture system according to any one of items 17 to 31, further comprising a scale, wherein the tank is placed on the scale. [Item 34] 32. The bioreactor culture system according to any one of items 17 to 31, further comprising a scale, wherein the tank is in physical contact with the scale. [Item 35] a tank having an input conduit and at least one discharge conduit; at least one pump; a filter coupled to the tank; scale in contact with the tank; a Raman probe coupled to the tank; a controller coupled to the at least one pump, the scale, and the Raman probe, the controller configured to control the at least one pump based on input from the Raman probe and input from the scale. [Item 36] a tank having an input conduit for connection to a first pump configured to control fluid delivery to the tank, a first output conduit for connection to a second pump configured to control fluid removal from the tank, and a second output conduit for connection to a third pump configured to control cell removal from the tank; a filter coupled to the reservoir, the filter configured to retain cells in the reservoir and allow liquid to pass through the filter; a scale configured to measure the weight of the tank including the cell culture therein; a Raman probe disposed within the tank; a controller coupled to the first pump, the second pump, the third pump, the scale, and the Raman probe, the controller comprising: receiving weight data from the scale; comparing the weight of the tank to a weight set point; receiving spectral data from the Raman probe; determining a parameter of the cell culture based on the received spectral data; comparing the determined parameter with a set point for the parameter; and the controller configured to adjust one or more of the throughputs of the first pump, the second pump, and the third pump based on the comparison; and A bioreactor culture system comprising: [Item 37] 37. The bioreactor culture system of claim 36, wherein adjusting the throughput of one or more of the first pump, the second pump, and the third pump reduces a deviation between the determined parameter and the set point for the parameter or between the received weight and the set point for the weight. [Item 38] 38. The bioreactor culture system of any one of items 36-37, wherein the method is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per mL for 30 days at steady state. [Item 39] 39. The bioreactor culture system of any one of items 36 to 38, wherein the tank has a volume of at least 10 L and the method is configured to maintain the weight of the tank containing the cell culture within a range of 20 g. [Item 40] 40. The bioreactor culture system of any one of items 36 to 39, wherein the tank has a volume of at least 10 L, and the method is configured to maintain the weight of the bioreactor containing the cell culture within 0.1 percent of the initial weight of the tank containing the cell culture. [Item 41] The controller determining a plurality of parameters of the bioreactor culture based on the received spectral data; comparing each of the plurality of said parameters to a respective set point for each of the plurality of said parameters; 41. The bioreactor culture system of any one of items 36 to 40, configured to adjust the throughput of one or more of the first pump, the second pump, and the third pump based on the comparison to reduce a deviation between the determined parameter and the respective set point. [Item 42] 42. The bioreactor culture system of claim 41, wherein the plurality of parameters comprises temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity. [Item 43] 43. The bioreactor culture system of any one of items 36 to 42, further comprising a filter configured to retain cells and allow liquid to pass through. [Item 44] Item 44. The bioreactor culture system of item 43, wherein the tank and the filter are mounted on the scale. [Item 45] 45. The bioreactor culture system according to any one of items 36 to 44, wherein the tank is placed on the scale. [Item 46] 46. ​​The bioreactor culture system according to any one of items 36 to 45, wherein the tank is in physical contact with the scale. [Item 47] a tank having an input conduit and at least one discharge conduit; at least one pump; a filter coupled to the tank; a Raman probe coupled to the tank; a controller coupled to the at least one pump and the Raman probe, the controller configured to control the at least one pump based on input from the Raman probe. [Item 48] a tank having an input conduit for connection to a first pump configured to control fluid delivery to the tank, a first output conduit for connection to a second pump configured to control fluid removal from the tank, and a second output conduit for connection to a third pump configured to control cell removal from the tank; a filter coupled to the reservoir, the filter configured to retain cells in the reservoir and allow liquid to pass through the filter; a scale configured to measure the weight of the tank including the cell culture therein; a Raman probe disposed within the tank; A bioreactor culture system comprising the first pump, the second pump, the third pump, and a controller coupled to the scale. [Item 49] The bioreactor system shown in Figures 1 and 2 of the present disclosure. [Item 50] The method of controlling a bioreactor system shown in FIG. 3 of the present disclosure.

Claims

1. 1. A bioreactor culture system comprising: a tank having an input conduit and at least one discharge conduit; at least one pump; a Raman probe connected to the tank; a controller connected to the at least one pump and the Raman probe, the controller configured to control the at least one pump based on input from the Raman probe; and A bioreactor culture system comprising:

2. the controller is connected to the first pump, the second pump, and the third pump; The bioreactor culture system of claim 1 .

3. further comprising a scale configured to measure the weight of the tank including the cell culture therein; the controller is configured to receive weight data from the scale. The bioreactor culture system of claim 2 .

4. The controller comparing the weight of the tank to a weight set point; and adjusting one or more of the outputs of the first pump, the second pump, and the third pump based on the comparison; configured to: The bioreactor culture system according to claim 3 .

5. The controller receiving spectral data from the Raman probe; determining a parameter of the cell culture based on the received spectral data; comparing the determined parameter to a set point for the parameter; and adjusting one or more of the outputs of the first pump, the second pump, or the third pump based on the comparison; configured to: The bioreactor culture system according to claim 3 .

6. adjusting the output of one or more of the first pump, the second pump, and the third pump reduces a deviation between the determined parameter and the set point for the parameter or reduces a deviation between the received weight and the set point for the weight; The bioreactor culture system according to claim 5 .

7. the controller is configured to maintain the cell culture in the tank at an average viable cell concentration of at least 30 million cells per mL for 30 days at steady state. The bioreactor culture system of claim 1 .

8. the tank has a volume of at least 10 L; the controller is configured to maintain the weight of the tank containing the cell culture within a range of 20 g. The bioreactor culture system of claim 1 .

9. the tank has a volume of at least 10 L; the controller is configured to maintain the weight of the tank containing the cell culture within 0.1 percent of an initial weight of the tank containing the cell culture. The bioreactor culture system of claim 1 .

10. The controller determining a plurality of parameters of the bioreactor culture based on the received spectral data; comparing each of the plurality of said parameters to a respective set point for each of the plurality of said parameters; and adjusting an output of one or more of the first pump, the second pump, and the third pump based on the comparison to reduce a deviation between the determined parameter and the respective set point; configured to: The bioreactor culture system according to claim 5 .

11. The plurality of parameters include temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity; The bioreactor culture system of claim 10.

12. The plurality of parameters comprises at least one parameter selected from temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity. The bioreactor culture system of claim 10.

13. the controller is configured to maintain a perfusion rate in the bioreactor culture system based on a constant perfusion rate setpoint; the perfusion rate defines an overall flow rate through the bioreactor culture system to maintain the overall flow rate substantially constant; The bioreactor culture system of claim 1 .

14. the at least one exhaust conduit includes a first exhaust conduit and a second exhaust conduit; The at least one pump a first pump connected to the input conduit, the controller configured to control the first pump at a first specified speed; a second pump connected to the first discharge conduit, the controller configured to control the second pump at a second specified speed; a third pump connected to the second discharge conduit, the controller configured to control the third pump at a third specified speed; Including, the controller is configured to adjust at least one of the specified speeds in response to a corresponding change in the other specified speeds; The bioreactor culture system of claim 1 .

15. 1. A bioreactor culture system comprising: a tank having an input conduit, a first discharge conduit, and a second discharge conduit; a first pump connected to the input conduit; a second pump connected to the first discharge conduit; a third pump connected to the second discharge conduit; a Raman probe connected to the tank; a controller connected to the first pump, the second pump, the third pump, and the Raman probe; Equipped with The controller: controlling the first pump at a first specified speed; controlling the second pump to a second specified speed; controlling the third pump to a third specified speed; configured to adjust at least one of the specified rates in response to corresponding changes in other specified rates to maintain a perfusion rate in the bioreactor culture system based on a constant perfusion rate set point; The perfusion rate defines an overall flow rate through the bioreactor culture system to maintain the overall flow rate substantially constant. Bioreactor culture system.

16. the controller is configured to adjust the first designated rate, the second designated rate, and / or the third designated rate based on measurements from the Raman probe to maintain one or more process parameters within predetermined ranges and to maintain the first designated rate of the input conduit and the second and third designated rates of the output conduit within their respective predetermined ranges.

16. The bioreactor culture system of claim 15.

17. further comprising a filter connected to the tank; the filter is a hollow fiber filter module configured to operate under alternating tangential flow; 16. The bioreactor culture system of claim 15.

18. The alternating tangential flow is a first flow of a fluid in a first direction parallel to a membrane surface of the hollow fiber; a second flow of fluid in a second direction perpendicular to the membrane surface of the hollow fiber; This is achieved by leading 18. The bioreactor culture system of claim 17.

19. 1. A bioreactor culture system comprising: a tank having an input conduit and at least one discharge conduit; at least one pump; scale in contact with the tank; a Raman probe connected to the tank; a controller connected to the at least one pump, the scale, and the Raman probe; Equipped with The controller is configured to maintain a perfusion rate in the bioreactor culture system based on a constant perfusion rate setpoint, the perfusion rate defining an overall flow rate through the bioreactor culture system so as to maintain the overall flow rate substantially constant. Bioreactor culture system.

20. further comprising a filter connected to the tank; the filter is a hollow fiber filter module configured to operate under alternating tangential flow; 20. The bioreactor culture system of claim 19.

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