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 Applications (if any) This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 572,918, filed on October 16, 2017, under 35 U.S.C. § 119, and the entire disclosure of this U.S. provisional patent application is incorporated herein by reference.

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

Background Art

[0003] Cell cultures can be maintained using bioreactors to produce biological substances such as proteins. In fed - batch bioreactors, during the culture, one or more nutrients are supplied to the bioreactor, and the biological substances remain in the bioreactor until the end of the batch. Perfusion bioreactors began to gain popularity 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 costs.

[0004] For example, in control solutions for perfusion reactors, attempts are made to calibrate the volumetric flow rates of the input and discharge supply pumps while dealing with pump bias flow and process variations. However, due to the inherent differences between any two given pumps (e.g., manufacturing variations) and the inability to achieve tight control, if the production run fails, the bioreactor can become overfilled or emptied. Also, existing control solutions lack the ability to measure other types of parameters such as ammonia, glucose, and protein quality attributes. Embodiments of the present disclosure address one or more of the limitations and drawbacks of existing perfusion bioreactors.

Summary of the Invention

[0005] Embodiments of the present disclosure relate, among other things, to a method of controlling a bioreactor and a bioreactor system that are useful for controlling a cell culture process for protein production. Each of the embodiments disclosed herein may include one or more of the features described in relation to any of the other embodiments.

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

[0007] One embodiment of the present disclosure is to supply a cell culture to a bioreactor, such that, depending on the conditions in the bioreactor, the cell culture can produce a protein of interest (POI); to measure process parameters (PP) of the culture in the bioreactor by Raman, wherein the process parameters are selected from the group consisting of nutrient concentration, viable cell concentration, and protein attributes; to measure the weight of the bioreactor containing the cell culture contents; to remove cell-free spent medium from the cell culture at a first specified rate using a first discharge conduit; to remove cells from the cell culture at a second specified rate using a second discharge conduit; to introduce one or both of fresh medium and nutrients into the cell culture at a third specified rate using an inlet conduit, and (i) to maintain one or more of the process parameters within a predetermined range, (ii) to maintain the weight of the bioreactor containing the cell culture within a predetermined range, and (iii) to maintain the third specified rate of the inlet conduit and the respective first and second specified rates of the discharge conduits within their respective predetermined ranges, for which purpose the inlet conduit and the discharge conduits are adjusted based on the Raman probe measurements and the weight measurements of the bioreactor, and relates to a method for controlling a bioreactor system.

[0008] In some embodiments, the measurement of one or more process parameters of the culture in the bioreactor by Raman is performed at regular intervals, for example, at least once per hour. In other embodiments, the method is configured to maintain the cell culture at a steady state for at least about 30 days at an average viable cell concentration of at least about 30 million cells per mL. 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 variation in 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 maintains the weight of the bioreactor and the cell culture within a variation within a weight range determined based on the initial weight of the bioreactor and the cell culture content, for example, within a variation in the range of about 20 ± 2 g. In some embodiments, the bioreactor controls 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 when the process parameters deviate from the setpoint values within their respective desired ranges, and as a result, the bioreactor is adjusted to reduce the deviation. At least two bioreactor volumes of spent medium per day are removed through the first discharge conduit. A maximum of three bioreactor volumes of spent medium per day are removed through the first discharge conduit. The process parameters include the temperature of the cell culture and the pH of the cell culture, the temperature is maintained at about 30 to 40 °C, about 32 to about 38 °C, or about 34 to about 38 °C, and 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.The process parameters include cell-specific productivity, and the method is configured to maintain cells in a cell culture at a cell-specific productivity of at least 25 to 37 days, at least about 15 to 60 pg / cell / day, about 15 to 25 pg / cell / day, at least about 17 to 23 pg / cell / day, or at least about 19 to 21 pg / cell / day. The process parameters include 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, 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 respective pumps. 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 that includes 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] The method according to the present disclosure is shown to include the following steps. Supplying a cell culture to a bioreactor (302), wherein the 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 (304), wherein the process parameters are selected from the group consisting of at least nutrient concentration, viable cell concentration, and protein attributes; 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); removing cells from the cell culture at a second specified rate using a second discharge conduit (310); introducing one or both of fresh medium and nutrients into the cell culture at a third specified rate using an input conduit, and (i) maintaining one or more of the process parameters within a predetermined range, (ii) maintaining the weight of the bioreactor containing the cell culture within a predetermined range, and (iii) maintaining the third specified rate of the input conduit and the respective first and second specified rates of the discharge conduits within their respective predetermined ranges, wherein the input conduit and the discharge conduits are adjusted based on Raman probe measurements and the weight measurement of the bioreactor (312).

[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 discharge 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, the controller being configured to control the at least one pump based on an input from the Raman probe.

[0012] At least one discharge conduit includes a first discharge conduit for connecting to a second pump configured to control the removal of fluid from the tank, and a second discharge conduit for connecting to a third pump configured to control the removal of cells from the tank. The filter is configured to retain cells within the tank and allow liquid to pass through the filter. The Raman probe is disposed within 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 the weight of the tank containing the cell culture, and the controller is configured to receive weight data from the scale. The controller is configured to compare the weight of the tank to a weight setpoint and, based on the comparison, adjust one or more of the outputs of the first pump, the second pump, and the third pump. The controller is configured to receive spectral data from the Raman probe, determine parameters of the cell culture based on the received spectral data, compare the determined parameters to parameter setpoints, and, based on the comparison, adjust one or more of the outputs of the first pump, the second pump, or the third pump. Adjusting one or more of the outputs of the first pump, the second pump, and the third pump reduces the deviation between the determined parameters and the parameter setpoints or 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 in a steady state. The tank has a volume of at least 10 L, and the method is configured to maintain fluctuations in the weight of the tank containing the cell culture within a range of 20 g. The tank has a volume of at least 10 L, and the method is configured to maintain fluctuations in the weight 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 with a respective setpoint of the plurality of parameters, and adjust one or more outputs of the first pump, the second pump, and the third pump based on the comparison to reduce the deviation between the determined parameters and the respective setpoints. The plurality of parameters includes temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity. The filter is configured to retain cells and allow liquid to pass through. The bioreactor includes a scale, and the tank and the filter are placed on the scale. The bioreactor includes a scale, and the tank is placed 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 inlet conduit and at least one outlet 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 another embodiment, the filter includes a mesh material. In some embodiments, the filter includes 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 inlet conduit and at least one outlet 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 an input from the Raman probe and an input from the scale.

[0015] In another embodiment, a bioreactor culture system is disclosed. The bioreactor culture system includes a tank having an inlet conduit configured to connect to a first pump configured to control the delivery of fluid to the tank, a first discharge conduit configured to connect to a second pump configured to control the removal of fluid from the tank, and a third discharge conduit configured to connect to a third pump configured to control the removal of cells from the tank; a filter coupled to the tank, the filter being configured to retain cells within the tank and allow liquid to pass through the filter; a scale configured to measure the weight of the tank including the cell culture within 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 being 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 a 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 the deviation between the determined parameter and the set point of the parameter, or the deviation between the received weight and the set point of the weight. The controller is configured to maintain the cell culture at an average viable cell concentration of at least 30 million cells per milliliter for 30 days in a steady state. The tank has a volume of at least 3 L, and the controller is configured to maintain the variation in the weight of the tank containing the cell culture within a range of 20 g. The tank has a volume of at least 3 L, and the controller is configured to maintain the variation in the weight 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 with the respective set points of 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 the deviation between the determined parameter and the respective set point. The plurality of parameters includes temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity. The bioreactor culture system includes a filter configured to retain cells and allow liquid to pass through. The tank and the filter are on a scale. The tank is on a scale.

[0017] In certain embodiments, the bioreactor culture system according to the present disclosure is exemplified as including the following elements. A tank (10) having an inlet conduit for connection to a first pump (30) configured to control fluid delivery to the tank, a first discharge conduit for connection to a second pump (40) configured to control removal of fluid from the tank, and a third discharge conduit for connection to a third pump (50) configured to control removal of cells from the tank; a filter (100) coupled to, connected to, or otherwise in fluid communication with the tank, the filter being configured to retain cells within 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 within the tank; 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 this 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 parameters of the cell culture based on the received spectral data, compare the determined parameters to parameter setpoints, and adjust one or more of the throughput of the first pump, the second pump, and the third pump based on the comparison.

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

[0020] Aspects of the present disclosure may be implemented in connection with the embodiments shown in the accompanying drawings. These drawings show different aspects of the present disclosure, and where appropriate, similar labels are given to reference numbers that indicate the same structure, component, material, and / or element in different figures. Various combinations of structures, components, and / or elements other than those specifically shown are contemplated, and it is understood that such combinations are within the scope of the present disclosure.

[0021] Furthermore, many embodiments are described and illustrated herein. The present disclosure is not limited to any single aspect or its embodiments, nor to any combination and / or permutation of such aspects and / or embodiments. Further, 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 the sake of simplicity, certain permutations and combinations are not separately described and / or illustrated herein. In particular, embodiments or aspects described herein as "exemplary" should not be construed as being, for example, more preferred or advantageous than other embodiments or aspects, but rather are intended to reflect or indicate that the embodiment(s) is / are an "example" of the embodiment(s).

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] Repeatedly, many embodiments are described and illustrated in this specification. The present disclosure is not limited to any single aspect, any of its embodiments, any combination and / or permutation of such aspects and / or embodiments. Each of the aspects and / or embodiments 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 those combinations and permutations are not separately described herein.

[0024] In particular, for the sake of brevity and clarity of the description, specific aspects of the figures are intended to show the general structure and / or construction method of various embodiments. Descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring other features. The elements of the figures are not necessarily drawn to scale. The dimensions of some characteristic elements may be exaggerated compared to other elements to enhance the understanding of the examples of the embodiments. For example, those skilled in the art will understand that cross-sectional views are not drawn to scale and should not be construed as representing the proportional relationships between various components. Cross-sectional views are provided to assist in the description of the various components of the illustrated assembly and to show their relative arrangement to each other.

[0025] Hereinafter, examples of the present disclosure will be described in detail with reference to the accompanying drawings that illustrate the present disclosure. As much as possible, the same reference numbers are used throughout the drawings to indicate the same or similar parts. In the following description, relative terms such as "about", "substantially", "approximately", etc. are used to indicate that there can be a variation of ±10% in the numerical values described. Further, in the claims, values, limitations, and / or ranges mean ±10% of the values, limitations, and / or ranges.

[0026] The term "conduit" refers to a flow path, tubing, connection, passage, etc. through which a fluid can move. By way of example, the conduit can include a BioPure thermoplastic tube manufactured by Watson-Marlow.

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

[0028] The terms "fed-batch cell culture" or "fed-batch culture" refer to batch cultures in which animal cells and a medium are initially supplied to a culture vessel and additional culture nutrients are provided to the culture in the vessel continuously or as discrete bolus additions, with or without periodic harvesting of cells and / or product before the end of the culture. Fed-batch cultures include "semicontinuous fed-batch cultures" in which the entire culture (which may include cells and medium) is periodically removed and replaced with fresh medium. Fed-batch cultures are distinguished from simple "batch cultures" by the addition (or removal) of components to the vessel during the culture. Fed-batch cultures can be further distinguished from perfusion cultures insofar as the medium is not exchanged during the fed-batch process, whereas in perfusion cultures, for example, all or some of the cells are retained in the culture by using a filter or cell retention device and the medium is supplied continuously or intermittently while growth-inhibiting by-products are constantly or periodically removed from the culture vessel. In a fed-batch process different from a perfusion process, the culture is continued until a maximum or otherwise determined working volume and / or protein production is reached, after which the fed-batch culture product is harvested.

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

[0030] The term "cell" includes any cell suitable for the 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, for example, 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: 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 cell comprises one or more viral genes (e.g., retinal cells expressing viral genes (e.g., PER.C6® cells)). In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.

[0031] "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 in the same sense as "cell population".

[0032] Assuming current state-of-the-art supply strategies, CHO cells are a standard industrial value for fed-batch culture of CHO cells, (after about one week) 10×10 6It has achieved a cell count exceeding cells / mL and a numerical value of the titration concentration of human IgG exceeding 2 g / L (harvested approximately two weeks later). See Kim, B J, 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 important industrial mammalian cell lines. See Omasa et al, Current Pharmaceutical Biotechnology,2010,11: 233-240.

[0033] The terms "cell culture medium" and "medium" generally refer to a nutrient solution used for the growth of mammalian cells that provides nutrients necessary to promote cell growth, such as a carbohydrate energy source, essential amino acids, trace elements, vitamins, etc. The cell culture medium may contain extracts that supply raw materials to assist cell growth, such as serum or peptone (hydrolysate). The medium may contain yeast-derived extracts or soybean extracts instead of animal-derived extracts. A chemically defined medium refers to a cell culture medium in which all chemical components are known. Chemically defined media contain no animal-derived components such as serum-derived peptone or animal-derived peptone. Also, the medium may not contain proteins. "Fresh medium" is medium that has not yet been introduced into the cell culture and / or has not yet been utilized by the cells of the cell culture. Fresh medium generally contains a high level of nutrients and may contain little or no waste. "Used medium" may mean the medium that has been used by the cells in the cell culture and generally contains a low level of nutrients (since these nutrients can be utilized by the cells in the cell culture) and a high level of waste compared to the levels present in fresh medium.

[0034] In a perfusion bioreactor, the culture medium can be continuously removed from the cell culture and replaced with fresh medium. By continuously adding fresh medium while removing waste products, the cells in the cell culture can be provided with the nutrients they need to achieve high cell densities. Unlike batch and fed-batch cultures, where conditions constantly change, the perfusion method provides a means to maintain the culture in a steady state. Generally, about one culture volume is exchanged per day, and the cell densities achieved by perfusion are usually two to ten times or more the densities achieved at the peak of batch or fed-batch cultures. By exchanging nutrients and / or removing apoptotic cells, cell viability can be maintained in a steady state for extended periods. In steady-state production, the quality attributes of the protein (or other target compound) produced at the beginning of the batch can be substantially the same as those of the protein (or other target compound) produced at the end of the batch. Proteins can be evaluated based on various post-translational modifications such as glycoforms, charge heterogeneity, aggregation, and various measures of purity. Since the cell culture conditions in such reactors are constantly changing, it is not possible to substantially equalize protein quality in a fed-batch reactor.

[0035] Depending on the culture conditions in the bioreactor, the cell culture can produce the protein of interest (POI) to provide a protein material with little variation. In some culture conditions of the cell culture, one or more process parameters can be selected at least 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 method of this bioreactor enables setting control over the flow of various constituent substances such as the culture medium (e.g., nutrients, etc.), proteins, and cells inside and outside the bioreactor. The method of this bioreactor includes removing cell-free used culture medium from the cell culture at a first specified flow rate using a first discharge conduit. The method includes removing cells from the cell culture at a second specified flow rate using a second discharge conduit. The method includes introducing one or both of fresh culture medium or nutrients into the cell culture at a third specified flow rate using an input conduit. Based on the 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. 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 so as to maintain one or more of the process parameters within a predetermined range. 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 so as to maintain the third specified flow rate of the input conduit and the respective first specified flow rate and second specified flow rate of the discharge conduits within their respective predetermined ranges.

[0037] The removal of cell-free used culture medium, the removal of cells, and the introduction of one or both of fresh culture medium or nutrients are each controlled by respective pumps. The bioreactor includes a filter configured to retain cells and allow the passage of liquid.

[0038] The methods and systems of the present disclosure include, among other reasons, methods for controlling the weight of a bioreactor and its contents for the purpose of adopting a stable production process. The method includes measuring the weight of a bioreactor containing a cell culture content. In a further embodiment, the method employs controlling the weight of the bioreactor in combination with the control of the flow rate as described above in connection with the conduits. The method includes measuring the weight of a bioreactor containing a cell culture content, and one or more of a first specified flow rate, a second specified flow rate, and a third specified flow rate are adjusted based on the measured weight. The first specified flow rate, the second specified flow rate, and the third specified flow rate are adjusted based on the measured weight so as to maintain the third specified flow rate of the input conduit and the respective first specified flow rate and second specified flow rate of the discharge conduits within their respective predetermined ranges. The first specified flow rate, the second specified flow rate, and / or the third specified flow rate are adjusted so as to maintain the weight of the cell culture and the bioreactor within a predetermined range. The measurement of the process parameter (PP) of the cell culture in the bioreactor by a Raman probe is performed at least once per hour. The method is configured to maintain the cell culture at a steady state for at least about 30 days at an average viable cell concentration of at least 30 million cells per mL. The bioreactor has a volume of at least 10 L, and the method is configured to maintain the variation in the weight of the bioreactor and the cell culture within a range of 20 g. The bioreactor has a volume of at least 10 L, and the method is configured to maintain the variation in the weight of the bioreactor containing the cell culture within 0.1 percent of the initial weight of the bioreactor containing the cell culture. When the process parameter deviates from the set point value within its 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 or nutrients are adjusted to reduce the deviation. For example, at least 2 bioreactor volumes of spent medium per day are removed through the first discharge conduit, or a maximum of 3 bioreactor volumes of spent medium per day are removed through the first discharge conduit.

[0039] In addition, one or more process parameters include the temperature of the cell culture and the pH of the cell culture, the temperature being maintained at 35 to 36 °C and the pH being maintained at 6.85 to 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] One or more process parameters include 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 to 25 pg / cell / day for at least 25 to 37 days.

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

[0042] 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] One or more process parameters include 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 the concentration of nutrients, process parameters, or quality attributes of a cell culture at an invariant level, a constant level, or a stable level. An invariant level, a constant level, or a stable level is understood to refer to a level within a predetermined set point or a predetermined set range. The set point, and thus the steady state level, may be shifted by an operator during the course of cell culture production. The set point or steady state level may also include a range of set values or thresholds.

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

[0046] Throughout the manufacturing process of a specific therapeutic protein product, product attributes or protein quality attributes that require control can be identified based on their potential impact on quality, particularly the clinical impact. Associated protein quality attributes can affect purity, safety, and / or efficacy. Quality attributes refer to the physical, chemical, biological, or microbiological properties or characteristics of the manufactured formulation that should be within appropriate limits, ranges, or distributions to ensure the desired product (protein) quality. For example, International Refer to the Council for Harmonization (ICH) Q8(R2) Pharmaceutical Development (ICH, August 2009). Quality attributes of protein products 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 cysteine, and host cell proteins, and may be considered attributes that have a major impact on product quality. Certain 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 (titration concentration), viable cell count (VCC), nutrient concentrations (such as glucose, phosphate, amino acids), ammonia, pH, lactate, etc. Formulations that are sensitive to specific process parameters during the manufacturing process may require appropriate control because they can cause changes in protein attributes that exceed or fall below the threshold values of those specific attributes. Therefore, process parameters may include those whose variation has the potential to affect the above quality attributes by more than the defined threshold values, and thus, the process parameters need to be monitored or controlled to ensure that the process produces a substance of the desired quality.

[0047] Terms such as "cell-specific productivity" and "cell-specific rate" refer to the rate of product expression in specific, for example, per cell, or in units of cell mass or volume. Cell-specific productivity is measured, for example, in grams of protein produced per cell per day.

[0048] The bioreactor system 1 may include a bioreactor tank 10, a supply storage tank 28, a supply pump 30, a discharge pump 40, and a harvest pump 50. The bioreactor system 1 may also include an ATF pump 70, a discharge tank 80, and a harvest tank 90. The pumps 30, 40, 50, and 70 may be operatively coupled to a controller 200. However, in some examples, the ATF pump 70 may be coupled to and controlled by a separate controller 102.

[0049] The bioreactor tank 10 can be a vat, barrel, container, flask, or other suitable receptacle made in a predetermined size to suit a number of operating 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 of metal (e.g., steel or stainless steel), metal alloy, glass, and / or polymer (e.g., 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 each other. 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. Supply reservoir 28 may include any suitable nutrient source for bioreactor tank 10, and the nutrient supply may be directed to bioreactor tank 10 by supply pump 30 via suitable conduits. The nutrient supply (culture medium) may include a carbon source (e.g., glucose), water, salts, an amino acid source, and / or other nutrients.

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

[0052] Aeration device 14 may be a sparger configured to supply oxygen and / or other gases to the cell culture within bioreactor tank 10. Aeration device 14 may be coupled to a source of oxygen or other gas, and the gas may be directed into the cell culture such that the gas bubbles in the cell culture, thereby aerating the cell culture. In some examples, a microporous sparger 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 can be driven from above or below by mechanical and / or magnetic means. A bottom-driven agitator can, for example, free up space within the lid 12 for measurement equipment such as temperature, pH, dissolved oxygen, foam, carbon dioxide, and other sensors, as well as for inlets for acids, alkalis, foam, fresh media, and outlet ports, etc., which may be desirable in some cases. The agitator 16 may include a radial agitator, an axial agitator, Rushton impellers, pitched blade impellers, marine blade impellers, etc.

[0054] For example, the Raman probe 18 may be an optical fiber Raman probe, for example, within a stainless steel enclosure and having a transparent window, for example, of sapphire or glass. The Raman probe 18 can be configured to enable Raman sampling of the cell culture 2. The Raman probe 18 can be configured to irradiate the cell culture 2 with monochromatic light (e.g., a laser of 785 nm or another suitable wavelength) and detect the scattered light from the cell culture 2.

[0055] Raman spectroscopy is a form of vibrational spectroscopy that provides information on molecular vibrations and can be used in situ for the identification and quantification of samples by inserting a Raman probe. In some embodiments, the monitoring of process variables is performed 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 with 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 the viability of cell cultures or the quality of proteins. In situ Raman analysis can provide real-time evaluation of one or more process variables in cell cultures. Manufacturers of Raman probes include, but are not limited to, tech5usa, Anton Paar, InPhotonics, Kaiser Optical Systems, Inc., and FiberTech Optica.

[0056] Bioreactor tank 10 may be connected to a filter system 100 having a hollow fiber filter therein. The hollow filter membrane (e.g., polysulfone) may include 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 is selected such that the size of the pores in the mesh is close to the diameter of the cells from cell culture 2, which can be effective in ensuring a high retention rate of cells 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 or other biologically derived substance of interest can be perfused or retained based on the pore size of the filter (e.g., 0.2 μm or 50 kD).

[0057] Via conduit 20 and pump 70, fluid from bioreactor tank 10 can be sent to filter system 100. Pump 70 may be reversible to allow fluid to flow back from filter system 100 to bioreactor tank 10. Filter system 100 can operate under cross-tangential flow. In one example, cross-tangential flow may mean that there is one flow in the same direction as the membrane surface of the hollow fiber (e.g., tangential direction) and that flow is going back and forth, and there is another flow in a direction substantially perpendicular to the filter surface described above. Cross-tangential flow can be achieved using a pump (e.g., pump 70) that circulates the cell culture across a filter module containing hollow fibers and another pump (e.g., pump 50) that removes the low cell density liquid prior to filter separation. Cross-tangential flow can be useful in preventing fouling and shear problems typical of other cell retention mechanisms.

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

[0059] The discharge pump 40 can be configured to remove cells from the bioreactor tank 10 via the conduit 22. The conduit 22 can be an immersion tube selected to avoid cell aggregation and clogging (which may occur, for example, if the conduit 22 is too narrow for the viscosity of the culture 2). The conduit 22 can include a thermoplastic elastomer tube (such as bioprene). The discharge pump 40 may be controlled, for example, by the processor 200. By cell discharge via the discharge pump 40, cells can be removed from the cell culture 2 in the bioreactor tank 10. The cell discharge rate (controlled by the discharge of 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, when a significant amount of the cell culture 2 is removed from the cell discharge along with the 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 send 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 storage container and the hollow fiber filter therein, can also be placed on the scale 110. The scale 110 can be any suitable scale or load cell configured to measure the weight of the components placed on the scale.

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

[0062] The controller 200 may be configured to receive raw spectral data from the Raman probe 18 and determine process parameters such as glucose concentration, glutamine concentration, glutamate concentration, ammonia concentration, lactate concentration, total cell density, titration concentration, and viable cell density. The controller 200 can establish a feedback loop that uses these determined process parameters to adjust one or more of the fluid flows through the supply pump 30, the discharge pump 40, and the harvest pump 50. That is, the controller 200 can set one or more set points for glucose concentration (e.g., from about 5 mM to about 85 mM, or from about 0.5 g / L to about 15.5 g / L, from about 1 g / L to about 15.5 g / L, from about 0.5 g / L to about 8 g / L, from about 2 g / L to about 6 g / L, or from about 3 g / L to about 5 g / L), 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), 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), 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, less than about 6 mM), lactate 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), and compare the values determined (based on the Raman spectrum from the Raman probe 18) to their respective set points.

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

[0064] For example, when the determined glucose concentration is lower than the setpoint glutamine concentration, the controller 200 may, for example, increase the discharge of the supply pump 30, increase the discharge of the release pump 40, and / or decrease the discharge of the harvest pump 50 to promote an increase in the glucose concentration, or the controller 200 may increase the discharge of the supply pump 30 and increase the discharge of the harvest pump 50. For example, when the determined glutamine concentration is lower than the setpoint glutamine concentration, the controller 200 may, for example, increase the discharge of the supply pump 30, increase the discharge of the release pump 40, and / or decrease the discharge of the harvest pump 50 to promote an increase in the glutamine concentration, or the controller 200 may increase the discharge of the supply pump 30 and increase the discharge of the harvest pump 50. For example, when the determined glutamate concentration is lower than the setpoint glutamate concentration, the controller 200 may, for example, increase the discharge of the supply pump 30, increase the discharge of the release pump 40, and / or decrease the discharge of the harvest pump 50 to promote an increase in the glutamate concentration, or the controller 200 may increase the discharge of the supply pump 30 and increase the discharge of the harvest pump 50. For example, when the determined lactate concentration is lower than the setpoint lactate concentration, the controller 200 may, for example, increase the discharge of the supply pump 30, increase the discharge of the release pump 40, and / or decrease the discharge of the harvest pump 50 to promote an increase in the lactate concentration, or the controller 200 may increase the discharge of the supply pump 30 and increase the discharge of the harvest pump 50. For example, when the determined total cell density is lower than the setpoint total cell density, the controller 200 may, for example, increase the discharge of the supply pump 30, decrease the discharge of the release pump 40, and / or increase the discharge of the harvest pump 50 to promote an increase in the total cell density, or the controller 200 may increase the discharge of the supply pump 30 and increase the discharge of the harvest pump 50.For example, when the determined viable cell density is smaller than the setpoint viable cell density, the controller 200 may, for example, increase the discharge of the supply pump 30, decrease the discharge of the release pump 40, and / or increase the discharge of the harvest pump 50 to promote an increase in 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 setpoint (the perfusion rate does not vary based on the concentration within the reactor). Similarly, the controller 200 may use a negative feedback loop to control the bioreactor weight (and the weight of cell culture 2).

[0066] It should be noted that the addition or subtraction of various nutrients introduced into the reactor can be combined with corresponding changes to other inputs to ensure that the total mass and / or total volume of the materials introduced into the reactor remains the same. That is, since the perfusion rate is maintained constant, an increase in one nutrient, for example, a glucose solution, glutamine, glutamate, etc., may occur in association with a decrease in the mass or volume of the corresponding initial nutrient feed stream.

[0067] In one embodiment, the system can include at least two feedback loops. One is for weight control and one is for control of process parameters (e.g., VCC, glucose, glutamine, glutamate, ammonia, lactate, etc.). In one example, the various inlet pumps and outlet pumps are not controlled by competing loops. For example, the perfusion rate can be set (e.g., 20 L / day), and then the Raman probe 18 measures one or more culture values, and the controller 200 can evaluate the steps to be taken based on the measured values from the Raman probe 18. For example, if the controller 200 determines that the VCC is too high, the controller 200 can initiate cell removal via the discharge pump 40 and at the same time decrease the flow rate of the harvest pump 50 to keep the total volume through the system constant. The additional steps that the controller 200 should take when 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 supply pump may be added to add glucose, lactose, glutamine, glutamate, etc. In an alternative embodiment, or in addition, the discharge can be adjusted to react to an increase in 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 on a server that is controlled via a network connection or some other connection such as a serial connection, for example. The controller 200 may create clones on one or more redundant servers in case of a failure in one or more of the servers.

[0070] The controller 200 may be configured to apply Kalman filtering, e.g., linear quadratic estimation (LQE), to Raman spectrum 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 an estimated value of an unknown variable that tends to be more accurate than one based on a single measurement only. Thus, the determined process parameters may 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 a PI (Process Information) historian or, in other cases, may be coupled to a PI (Process Information) historian. The PI historian may be an application with a time series database capable of recording data from the process control system. The PI historian enables a user to record, analyze, and monitor real-time information. The controller 200 can store, for example, weight values from the scale 110, spectral data from the Raman probe 18, and the pump speeds of the supply pump 30, the discharge pump 40, and the harvest pump 50 in the PI historian.

[0072] Figure 3 shows method 300 according to the present disclosure. One or more steps of method 300 may be performed in any order, simultaneously with other steps, or may be entirely omitted. Method 300 can start at step 302, where bioreactor system 1 can be assembled, cell culture 2 can be supplied into bioreactor tank 10, and a cell line can be inoculated into cell culture 2. Next, method 300 can proceed to step 304, where 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 can include any of the aforementioned parameters determined from Raman spectral data obtained by Raman probe 18. Method 300 can proceed to step 306, where the weight of bioreactor tank 10 (containing cell culture 2 therein) is measured by scale 110 and provided to processor 200.

[0073] Method 300 can proceed from step 306 to step 308, where the 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 also by operating harvest pump 50 to withdraw solution from filter system 100. Method 300 can proceed from step 308 to step 310, where cells can be removed from the cell culture at a second specified rate using discharge conduit 22 by discharge pump 40. Method 300 can proceed from step 310 to step 312, where one or both of fresh medium and nutrients can be introduced into the cell culture at a third specified rate using inlet conduit and supply pump 30 such that the total input of medium and nutrients is maintained equal to the combined discharge of discharge pump 40 and harvest pump 50. The specified rate may be a set value or a range of rates at which the pumps operate and / or are maintained. The specified rate may be determined by controller 200.

[0074] Each of steps 302 through 312 may be performed in any order and, in some cases, is contemplated to 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 the cell culture 2 contained therein) can be controlled via a PID (proportional-integral-derivative) loop. Additionally, controller 200 may be configured to analyze the Raman spectrum 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 can 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, and can reduce relatively few data losses. The control strategy of the present disclosure can exhibit stable bioreactor level control. For example, the level fluctuations were reduced from + / - 0.5 L / day to + / - 0.01 L / day using the control system of the present disclosure. Also, an improvement in weight fluctuations has been achieved, for example, from 5-10% weight fluctuations using other systems such as volume measurement calibration, to 0.1-0.5% error using the control system disclosed herein. This improvement may be due, at least in part, to changing the system from pump volume measurement calibration to a software control version based on weight and other parameters. Further, the control system of the present disclosure may be fully integrated with process information (PI) alarms (e.g., email alerts) and can be accessed remotely to shut down. Further, the systems and methods of the present disclosure can provide results that are more reproducible and reliable than conventional systems and methods.

[0077] Example 1 (Figures 4 and 5) The experiments described in Example 1 compared a perfusion bioreactor with a fed-batch bioreactor and showed higher achieved viable cell concentrations and cell-specific productivities in the perfusion bioreactor relative to the fed-batch bioreactor.

[0078] In one experiment, a 15 L volume bioreactor was cultured using a cell line and a medium. The bioreactor set points included temperature (35.5 degrees Celsius), 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 the ATF were placed on scale. The weights of the bioreactor, cell culture, and ATF were sent via an Ethernet (registered trademark) connection to a computer running control software. The weights were compared to a setpoint (11.0 kg, for example, the working volume of the bioreactor), and a PID controller (designed in MATLAB but executed via the control software SynTQ) determined whether to use the supply pump. The harvest pump was set to a constant rate corresponding to the desired perfusion rate (2 reactor volumes per day). The supply pump and perfusion pump were automatically controlled using SynTQ software that broadcasts OPC signals to a Kepware server. The Kepware server broadcasts this signal via an Ethernet (registered trademark) connection to a MODBUS analog output module, and this MODBUS analog output module converts the digital value to a milliamp output on hardware of 4 - 20 mA.

[0080] Using this system, the weight of the bioreactor could be controlled within + / - 10 g (0.09%) of the initial 11 kg weight. Prior to implementing this system, the weight of the bioreactor in the bioreactor could not be controlled within a range of more than 0.5 kg (4.54%) overnight. Within the same system, a Raman probe from Kaiser Optical was used to capture Raman spectra from the cell culture. The controller utilized a model developed in a previous batch to predict cell count, glucose, lactate, ammonia, and other nutrient concentrations. The Raman probe captures thousands of various spectra that are later analyzed with a computer program such as SIMCA. Using multivariate analysis and offline data of a given parameter, the program creates a model across all probe measurements. Next, this SIMCA model is uploaded to SynTQ and made accessible in real - time each time the probe makes a reading (for example, every 15 to 45 minutes, etc.).

[0081] By controlling various nutrients using a Raman probe, it becomes possible to improve the productivity of cell lines, increase the survival rate in long-term cell culture, and improve the quality of multiple aspects of proteins.

[0082] Figure 4 shows the maximum viable cell concentration on the 37th day of a perfusion bioreactor batch according to the present disclosure (separate sheet "Ex." 1), and this concentration is approximately twice the maximum viable cell concentration on the 7th day of a fed-batch reactor batch of the same size (Ex. 2). The fact that the maximum viable cell concentration was achieved on the 37th day (in contrast to the 6th day of the fed-batch bioreactor) reveals the robustness and long lifespan of the perfusion bioreactor process.

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

[0084] In the perfusion batch, to exceed 50×10 6 cells / mL, a perfusion rate of 3 reactor volumes per day was required, but this may often be commercially prohibited.

[0085] By optimizing the medium using a "push-to-low" strategy, the required perfusion rate is reduced. For example, when the cells are at 20×10 6Grow to a cell density of cells / mL and maintain in a steady state. The perfusion rate can be set at 2 reactor volumes per day for 5 days. On day 5, the perfusion rate can be decreased to 1.5 reactor volumes per day. If the cells can sustain it, the perfusion rate may decrease to 1 reactor volume per day after 5 days. If the cells start to die, amino acid analysis or other analysis can be used to determine ways to better fortify the medium, such as replenishing nutrients in the medium or adjusting the nutrient concentration in the medium. In one non-limiting example, the 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 which case offline measurements can be obtained by analyzing the samples. Using previous NOVA data, a Raman model can be constructed, and at that point, the probe can be inserted into the reactor. The model does not require manual sampling like NOVA once a day, but can provide VCC data every 15 - 45 minutes, every second, every minute, every 2 minutes, every 3 minutes, every 4 minutes, every 5 minutes, every 10 minutes, every 15 minutes, every 20 minutes, every 25 minutes, every 30 minutes, every 35 minutes, every 40 minutes, every 45 minutes, every 50 minutes, every 55 minutes, every hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, or every 6 hours. Figure 5 shows the 20th day 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 second half of the run.

[0087] In the following examples, the general ranges of specific 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 bench top.

[0088] Example 2 (Figs. 6 - 8) The experiments described in Example 2 show data for a perfusion bioreactor with uncontrolled VCC increase or glucose. It was observed that VCC reached a peak on day 7 because the cells grew rapidly to a large cell density and then decreased rapidly by day 11 as the nutrients in the medium were depleted (Fig. 6). It was insufficient to control only by weight to achieve a steady state of VCC.

[0089] Also, glucose was not controlled during the perfusion run. Since the culture fed the cells with nutrients depending on the glucose in the medium during the perfusion operation, this subsequently led to cell death during the culture. As the cells grew, glucose decreased steadily, but the medium was always supplied (Fig. 7). As shown in Fig. 8, as seen when monitoring cell viability, the sharp increase in glucose detection occurring after 10 days was due to complete cell death and thus not due to glucose consumption.

[0090] In this experiment, Chinese hamster ovary (CHO) cells that produce mAb1 were seeded at a given concentration in a 15 L bench-top bioreactor. The cells were cultured at specific dissolved oxygen, temperature, agitation, and pH that were kept constant during operation. Also, fresh medium and nutrients were provided to the cells in the form of a perfusion supply at a rate of two times the reactor volume per day. The volume of the reactor was kept constant by adding the same amount of supply removed from the perfusion fluid to the reactor using Repligen's XCell ATF4 system. This was achieved by monitoring the weight of the bioreactor system and using a computer-aided feedback loop control system to maintain the weight within + / - 0.05 kg of a given target weight. During this perfusion production run, no Raman control or other arbitrary bioreactor parameter control was provided for the VCC.

[0091] In a similar experiment (not shown in the figures), the flow rate was also set to supply the medium of two bioreactors per day, but since the weight was not monitored, the fluctuations of the pump could not be properly controlled.

[0092] In this similar perfusion experiment, the supply pump and the perfusion fluid pump were set to the same flow rate (determined by volumetric measurement calibration of the pump). In this method, it was not possible to provide a flow rate accurate enough to match each other, and during the night (for example, during the period when the bioreactor was not actively monitored), the supply pump added more medium to the reactor than the amount that the perfusion fluid pump could remove. As a result, the reactor overflowed and the culture was lost.

[0093] Example 3 (Figs. 9 - 12) The experiment described in Example 3 shows data for a perfusion bioreactor with VCC control. The VCC control (Fig. 9) resulted in stable steady states for viability (Fig. 10), protein production (Fig. 11), and nutrients (Fig. 12).

[0094] In this experiment, CHO cells that produce mAb2 were seeded at a given concentration in a 15 L bench-top bioreactor. The cells were cultured at specific dissolved oxygen, temperature, agitation, and pH that were kept constant during operation. Also, fresh medium and nutrients were provided to the cells in the form of perfusion supply at a rate of twice the reactor volume per day. The volume of the reactor was kept constant by adding the same amount of supply removed from the perfusate back to the reactor using an ATF4 system. This was achieved by monitoring the weight of the system and using a computer control system to maintain the weight within the range of plus 0.05 kg or minus 0.05 kg of a given target. Raman control was not used for this run, and the pump discharge rate was manually set after sampling of the VCC. Multiple samples were required for this process, and the discharge rate had to be adjusted multiple times a day.

[0095] VCC was controlled during perfusion production culture, and the target VCC was 42.5×10 6 cells / mL (40 - 45×10 6 cells / mL). Accordingly, when the VCC increased above the target, the discharge rate increased, and when the VCC dropped below the target, the discharge rate decreased.

[0096] Example 4 (Figures 13 and 14) In the experiment of this example, the perfusion culture method (Figure 14) was compared with the fed-batch culture method (Figure 13). The perfusion culture method was able to achieve approximately 4 times the maximum cell number compared to the fed-batch cell culture method of the same protein under similar conditions (Figure 13). The fed-batch culture was carried out at the pilot scale, and the perfusion experiment was carried out at the bench scale (15 L). The strategies for agitation and aeration were scaled down to the bench scale using the power approach per unit volume for agitation and the volume by the 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 the fed-batch reactor in the same time (Figure 14).

[0098] The perfusion culture method was carried out by providing a 15L bench-top bioreactor seeded 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 that were kept constant during the run. Also, fresh medium and nutrients were provided to the cells in the form of a perfusion supply at a rate of twice the reactor volume per day. The medium in this run was supplemented with key nutrients at increased concentrations compared to previous experiments, and as a result, the cells could be pushed to higher cell densities during the perfusion experiment. The reactor volume was kept constant by using a weight control system to maintain a given target weight within 0.05 kg. No Raman control or release control was provided during the production run of perfusion.

[0099] Fed-batch cell culture (Ex.6 and Ex.8) was carried out under similar conditions (dissolved oxygen, temperature, agitation, and pH).

[0100] Example 5 (Figs. 15 - 18) The experiments described in Figs. 15 - 18 were maintained in a steady state for more than 30 days with this perfusion system, showing beneficial results for viability, glucose, and titration concentrations, as well as VCC (see, for example, Figs. 15 - 18).

[0101] The perfusion culture method involving Raman, release, and weight management was carried out by providing a 15L benchtop bioreactor seeded with a given concentration of CHO cells producing mAb2. The cells were cultured in a specific range of dissolved oxygen, temperature, agitation, and pH that was kept constant during operation. Also, fresh medium and nutrients were provided to the cells in the form of perfusion supply at a rate of twice the reactor volume per day. The medium in this run was supplemented with important nutrients at increased concentrations (compared to Examples 2 and 3), and as a result, the cells could be pushed to a higher cell density. A weight control system was used to add to the reactor the same amount of feed removed from the perfusion fluid using the ATF4 system, monitor the weight of the system, and maintain the weight within the range of plus or minus 0.05 kg of a given target using a computer feedback control system, thus keeping the volume of the reactor constant.

[0102] The VCC was controlled in this run using Raman control and automatic release control based on Raman feedback (Figure 15). In the first experiment (Ex.9), the Raman release strategy was set to maintain a VCC of 40×10 6 cells / mL. The range of VCC was 35 - 45×10 6 cells / mL, which was slightly wider than the target that occurred with manual release in the previous experiment (Example 3). However, the system was sampled only once a day, and no adjustment was necessary in this perfusion run (compared to multiple adjustments per day by manual release as described in Example 3).

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

[0104] Example 6 (Figures 19 and 20) In one experiment, three different bioreactors were cultured using cell lines and media. The volumes of the bioreactors 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 degrees Celsius), 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 during operation. Each bioreactor was coupled with an ATF (ATF2, ATF4, and ATF6 respectively) cell retention device equipped with a 0.2 micron filter. The hollow fiber filter retained the cells while allowing the protein to pass through after 24 hours.

[0105] At all three scales, perfusion cultures were performed in each system using Raman, effluent, and weight control. Similar to Example 5, the media for this experiment was supplemented with additional nutrients. The weight within each system was controlled to + / - 0.05 kg for ATF4 and ATF6, and to + / - 1 kg for a given target (due to the limitations of the scale equipment itself).

[0106] Using Raman control and automatic effluent control based on Raman feedback, the VCC was set to 40 x 10^6 cells / mL in this run (Figure 19). Variations in the Raman probe were observed in the 50 L ATF6 system. This is presumably because the Raman control model has not yet been optimized for large scales.

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

[0108] As a result of this experiment, equivalent protein productivity (Figure 20) was achieved in all three systems over a period of 5 days.

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

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

[0111] Using Raman control and automatic efflux control based on Raman feedback, the VCC was set to 70×10^6 cells / mL in this run (see Figure 21).

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

[0113] The reactor was able to maintain the VCC at 70×10^6 cells / mL or higher for 7 days until the batch ended due to equipment failure. During this period, the viability was maintained above 90%, indicating a healthy culture. Prior to the implementation of the control system of the present disclosure, such continuous production at such a high density was not possible.

[0114] In particular, references to "one embodiment" or "an embodiment" in this specification mean that a particular feature, structure, or characteristic described in connection with the embodiment can be included in, used in, and / or incorporated into one, some, or all embodiments of the present disclosure. The use or occurrence of the expressions "in one embodiment" or "in another embodiment" in this specification does not refer to the same embodiment, nor do they necessarily have to be separate or alternative embodiments that are mutually exclusive with one or more other embodiments, nor are they 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 its embodiments, nor to any combination and / or permutation of such aspects and / or embodiments. Furthermore, each aspect and / or its embodiments of the present disclosure may be used alone or in combination with one or more of the other aspects and / or its embodiments of the present disclosure. For the sake of simplicity, specific permutations and combinations are not separately described and / or illustrated in this specification.

[0115] Furthermore, as described above, embodiments or aspects described as "exemplary" in this specification should not be construed as being more preferable or advantageous than, for example, other embodiments or aspects, but rather are intended to convey or indicate that one or more embodiments are exemplary embodiments. (Appendix) As a preferred embodiment, the technical idea that can be grasped from the above embodiments is described below. [Item 1] Supplying a cell culture to a bioreactor, such that the cell culture can produce a protein of interest (POI) under the conditions in the bioreactor. Measuring one or more process parameters (PP) of the culture in the bioreactor using a Raman probe, wherein the process parameter is selected from the group consisting of nutrient concentration, viable cell concentration, and protein attributes. Measuring the weight of the bioreactor containing the cell culture content; Using a first discharge conduit to remove cell-free used medium from the cell culture at a first specified rate; Using a second discharge conduit to remove cells from the cell culture at a second specified rate; Using an input conduit to introduce one or both of fresh medium or nutrients into the cell culture at a third specified rate, and (i) maintaining one or more of the process parameters within a predetermined range, (ii) maintaining the weight of the bioreactor containing the cell culture within a predetermined range, and (iii) maintaining the third specified rate of the input conduit and the respective first and second specified rates of the discharge conduits within their respective predetermined ranges of rates, wherein the input conduit and the discharge conduits are adjusted based on the Raman probe measurement values and the weight measurement value of the bioreactor, a method for controlling a bioreactor system. [Item 2] The method according to item 1, wherein the measurement of the one or more process parameters of the culture in the bioreactor by Raman is performed at least once per hour. [Item 3] The method according to 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 in a steady state. [Item 4] 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. The method according to any one of the preceding items. [Item 5] The bioreactor 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 bioreactor containing the cell culture, according to any one of the preceding items. [Item 6] If the process parameters deviate from the setpoint values within their respective desired ranges, one or more of the removal of cell-free medium, removal of cells, or introduction of one or both of fresh medium or nutrients are adjusted to reduce the deviation, according to any one of the preceding items. [Item 7] At least 2 bioreactor volumes of used medium are removed per day through the first discharge conduit, according to any one of the preceding items. [Item 8] A maximum of 3 bioreactor volumes of used medium are removed per day through the first discharge conduit, according to any one of the preceding items. [Item 9] The process parameters include the temperature of the cell culture and the pH of the cell culture, the temperature is maintained at 35 - 36 °C, and the pH is maintained at 6.85 - 7.15, according to any one of the preceding items. [Item 10] The process parameters include cell-specific productivity, and the method is configured to maintain the cells in the cell culture with a cell-specific productivity of at least 15 - 25 pg / cell / day for at least 25 - 37 days, according to any one of the preceding items. [Item 11] The process parameters include glucose concentration, and the method is configured to maintain the glucose concentration at about 5 mM to about 85 mM, or about 1 g / L to about 15.5 g / L, according to any one of the preceding items. [Item 12] 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, according to any one of the preceding items. [Item 13] The process parameter includes an ammonia concentration, and the method is the method according to any one of the preceding items, which is configured to maintain an ammonia concentration of less than about 15 mM. [Item 14] The method according to any one of the preceding items, wherein the removal of the cell-free used medium, the removal of cells, and the introduction of one or both of fresh medium or nutrients are each controlled by a respective pump. [Item 15] The bioreactor includes a filter configured to retain cells and allow liquid to pass through, and the method is the method according to any one of the preceding items. [Item 16] Supplying a cell culture to the bioreactor, Measuring one or more process parameters of the cell culture in the bioreactor by a Raman probe, Removing cell-free used medium from the cell culture at a first specified rate using a first discharge conduit, Removing cells from the cell culture at a second specified rate using a second discharge conduit, Introducing one or both of fresh medium or nutrients into the cell culture at a third specified rate using an inlet conduit, and A method for controlling a bioreactor system, including changing one or more of the first specified rate, the second specified rate, or the third specified rate based on the measurement value of the Raman probe. [Item 17] A tank having an inlet 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 bioreactor culture system comprising: at least one pump and a controller coupled to the Raman probe, wherein the controller is configured to control the at least one pump based on an input from the Raman probe. [Item 18] The bioreactor culture system according to item 17, wherein the at least one discharge conduit includes a first discharge conduit for connecting to a second pump configured to control removal of fluid from the tank and a second discharge conduit for connecting to a third pump configured to control removal of cells from the tank. [Item 19] The bioreactor culture system according to item 17 or item 18, wherein the filter is configured to retain cells within the tank and allow liquid to pass through the filter. [Item 20] The bioreactor culture system according to any one of items 17 to 19, wherein the Raman probe is disposed within the tank. [Item 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] The bioreactor culture system according to any one of items 17 to 21, further comprising a scale configured to measure the weight of the tank including the cell culture within the tank, wherein the controller is configured to receive weight data from the scale. [Item 23] The bioreactor culture system according to item 22, wherein the controller is configured to compare the weight of the tank to a setpoint of the weight and, based on the comparison, adjust one or more of the outputs of the first pump, the second pump, and the third pump. [Item 24] 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 set point of the parameter, and adjust one or more of the outputs of the first pump, the second pump, or the third pump based on the comparison. The bioreactor culture system according to any one of items 17 to 23. [Item 25] Adjusting one or more of the outputs of the first pump, the second pump, and the third pump reduces the deviation between the determined parameter and the set point of the parameter, or the deviation between the received weight and the set point of the weight. The bioreactor culture system according to item 24. [Item 26] 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 in a steady state. The bioreactor culture system according to any one of items 17 to 25. [Item 27] 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. The bioreactor culture system according to any one of items 17 to 26. [Item 28] 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. The bioreactor culture system according to any one of items 17 to 27. [Item 29] The controller is Based on the received spectral data, determine a plurality of parameters of the bioreactor culture Compare each of the plurality of parameters with each of the set points of the plurality of parameters, and Based on the comparison, it is configured to adjust the output of one or more of the first pump, the second pump, and the third pump to reduce the deviation between the determined parameter and each of the set points, the bioreactor culture system according to item 28. [Item 30] The plurality of parameters include temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity, the bioreactor culture system according to item 29. [Item 31] The filter is configured to retain cells and allow liquid to pass through, the bioreactor culture system according to any one of items 17 to 30. [Item 32] Further including a scale, with the tank and the filter placed on the scale, the bioreactor culture system according to items 17 to 31. [Item 33] Further including a scale, with the tank placed on the scale, the bioreactor culture system according to any one of items 17 to 31. [Item 34] Further including a scale, with the tank in physical contact with the scale, the bioreactor culture system according to any one of items 17 to 31. [Item 35] A tank having an inlet conduit and at least one discharge 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, A controller coupled to the at least one pump, the scale, and the Raman probe, the controller being configured to control the at least one pump based on an input from the Raman probe and an input from the scale, the bioreactor culture system comprising the controller. [Item 36] A tank having an inlet conduit for connection to a first pump configured to control fluid delivery to the tank, 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. A filter coupled to the tank, the filter being configured to retain cells within the tank and allow liquid to pass through the filter. A scale configured to measure the weight of the tank containing 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 being configured to: Receive weight data from the scale. Compare the weight of the tank to a setpoint of the weight. 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 setpoint of the parameter, and Adjust one or more of the throughput of the first pump, the second pump, and the third pump based on the comparison. A bioreactor culture system comprising the same. [Item 37] The bioreactor culture system according to Item 36, wherein adjusting one or more of the throughput of the first pump, the second pump, and the third pump reduces a deviation between the determined parameter and the setpoint of the parameter, or a deviation between the received weight and the setpoint of the weight. [Item 38] The bio-reactor culture system according to any one of items 36 to 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 in a steady state. [Item 39] The bio-reactor culture system according to 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] The bio-reactor culture system according to 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 is configured to determine a plurality of parameters of the bio-reactor culture based on the received spectral data, compare each of the plurality of parameters with their respective set points, and 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 the deviation between the determined parameter and its respective set point, according to the bio-reactor culture system of any one of items 36 to 40. [Item 42] The plurality of parameters include temperature, pH, nutrient concentration, lactate concentration, ammonia concentration, and cell-specific productivity, according to the bio-reactor culture system of item 41. [Item 43] The bio-reactor culture system according to any one of items 36 to 42, further comprising a filter configured to retain cells and allow liquid to pass through. [Item 44] The bioreactor culture system according to item 43, wherein the tank and the filter are placed on the scale. [Item 45] The bioreactor culture system according to any one of items 36 to 44, wherein the tank is placed on the scale. [Item 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 being configured to control the at least one pump based on an input from the Raman probe, the bioreactor culture system comprising the controller. [Item 48] A tank having an input conduit for connecting to a first pump configured to control fluid delivery to the tank, a first discharge conduit for connecting to a second pump configured to control removal of fluid from the tank, and a second discharge conduit for connecting to a third pump configured to control removal of cells from the tank, the tank, A filter coupled to the tank, the filter being configured to retain cells in the tank and allow liquid to pass through the filter, the filter, A scale configured to measure the weight of the tank containing 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 FIGS. 1 and 2 of the present disclosure. [Item 50] The method for controlling the bioreactor system shown in FIG. 3 of the present disclosure.

Claims

1. A method for controlling a bioreactor system, comprising: supplying a cell culture to a perfusion bioreactor, wherein the cell culture is enabled to produce a target protein according to conditions in the perfusion bioreactor; measuring one or more process parameters of the cell culture using a Raman probe; measuring the weight of the perfusion bioreactor containing the cell culture contents; removing cell-free used medium from the cell culture at a first specified rate using a first discharge conduit; removing cells from the cell culture at a second specified rate using a second discharge conduit; introducing one or both of fresh medium or nutrients into the cell culture at a third specified rate using an input conduit; and adjusting at least one of the specified rates in response to a change in another of the specified rates so as to maintain the perfusion rate of the perfusion bioreactor based on a constant perfusion rate set value, wherein the perfusion rate defines the overall flow rate through the perfusion bioreactor such that the overall flow rate remains substantially constant when at least one of the first specified rate, the second specified rate, and the third specified rate is adjusted, and the input conduit and the discharge conduits are adjusted based on the Raman probe measurement value of the perfusion bioreactor and the weight measurement value of the perfusion bioreactor to maintain the one or more process parameters within a predetermined range and to maintain the third specified rate of the input conduit and the respective first and second specified rates of the discharge conduits within predetermined ranges, respectively.

2. The method according to claim 1, wherein the measurement of the one or more process parameters of the cell culture in the perfusion bioreactor by Raman is performed at least once per hour.

3. The method according to claim 1, 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 in a steady state.

4. The perfusion bioreactor has a volume of at least 10 L, and the method further comprises: maintaining fluctuations in the weight of the perfusion bioreactor and the cell culture within a range of 20 g. The method according to claim 1.

5. The perfusion bioreactor has a volume of at least 10 L, The method maintaining the variation in the weight of the perfusion bioreactor containing the cell culture within 0.1 percent of the initial weight of the perfusion bioreactor containing the cell culture; The method according to claim 1, further comprising.

6. When the process parameters deviate from the setpoint values within their respective desired ranges, one or more of removal of cell-free medium, removal of cells, or introduction of one or both of fresh medium or nutrients are adjusted to reduce the deviation of the process parameters from the setpoint values. The method according to claim 1.

7. The method according to claim 1, wherein at least 2 bioreactor volumes and at most 3 bioreactor volumes of spent medium are removed per day through the first discharge conduit.

8. The process parameters include the temperature of the cell culture and the pH of the cell culture, the temperature is maintained at 35 to 36 °C, and the pH is maintained at 6.85 to 7.

15. The method according to claim 1.

9. The process parameters include cell-specific productivity, The method maintaining the cells in the cell culture with a cell-specific productivity of at least 15 pg / cell / day for at least 25 days; The method according to claim 1, further comprising.

10. The process parameters include glucose concentration, The method maintaining the glucose concentration at about 5 mM to about 85 mM, or about 1 g / L to about 15.5 g / L; The method according to claim 1, further comprising.

11. The process parameters include lactate concentration, The method maintaining a lactate concentration of less than about 60 mM, or less than about 6 g / L; The method according to claim 1, further comprising.

12. The process parameters include ammonia concentration, The method maintaining an ammonia concentration of less than about 15 mM; The method according to claim 1, further comprising.

13. Each of the removal of cell-free spent medium, removal of cells, and introduction of one or both of fresh medium or nutrients is controlled by a respective pump. The method according to claim 1.

14. The perfusion bioreactor according to claim 1, comprising a filter configured to retain cells and allow liquid to pass through.

15. The method according to claim 1, wherein the inlet conduit and the outlet conduit are adjusted based on the Raman probe measurement value of the perfusion bioreactor and the weight measurement value of the perfusion bioreactor so as to maintain the perfusion rate of the perfusion bioreactor based on the constant perfusion rate set value.

16. Establish a feedback loop for maintaining each of the one or more process parameters measured by the Raman probe within a respective predetermined range, Adjusting at least one of the first specified rate, the second specified rate, and the third specified rate using the feedback loop The method according to claim 1, comprising:

17. A method for controlling a bioreactor system, Supplying a cell culture to a perfusion bioreactor, Measuring one or more process parameters of the cell culture with a Raman probe, Measuring the weight of the perfusion bioreactor containing the cell culture contents, Using a first outlet conduit to remove cell-free spent medium from the cell culture at a first specified rate, Using a second outlet conduit to remove cells from the cell culture at a second specified rate, Using an inlet conduit to introduce one or both of fresh medium or nutrients into the cell culture at a third specified rate, and Determining a first change to one or more of the first specified rate, the second specified rate, and the third specified rate based on the Raman probe measurement value and the weight measurement value of the perfusion bioreactor, Applying a second change to the other specified rates to which the first change has not been applied among the first specified rate, the second specified rate, and the third specified rate, so that the overall input and overall output of the perfusion bioreactor remain substantially constant between the first change and the second change, applying in response to the first change so as to substantially maintain the perfusion rate, which is the rate of the overall flow through the perfusion bioreactor, comprising A method in which the input conduit and the discharge conduit are automatically adjusted by a controller that receives the Raman probe measurement values and the weight measurement value of the perfusion bioreactor to (i) maintain the one or more process parameters within a predetermined range, (ii) maintain each of the first specified rate and the second specified rate of the discharge conduit within a predetermined range, (iii) maintain the third specified rate of the input conduit within a predetermined range, and (iv) maintain the perfusion rate of the perfusion bioreactor based on a constant perfusion rate setting value.

18. The method according to claim 17, wherein the input conduit and the discharge conduit are automatically adjusted to maintain the weight of the perfusion bioreactor containing the cell culture within a predetermined range.

19. Establishing a feedback loop for maintaining the Raman probe measurement values within respective desired ranges, Applying the second change based on the feedback loop The method according to claim 17, comprising.

20. A method for controlling a bioreactor system, Supplying a cell culture to a perfusion bioreactor, Measuring one or more process parameters of the cell culture with a Raman probe, Measuring the weight of the perfusion bioreactor containing the cell culture contents, Using a first discharge conduit to remove cell-free spent medium from the cell culture at a first specified rate, Using a second discharge conduit to remove cells from the cell culture at a second specified rate, Using an input conduit to introduce one or both of fresh medium or nutrients into the cell culture at a third specified rate, Changing one or more of the first specified rate, the second specified rate, and the third specified rate based on the Raman probe measurement values and the weight measurement value of the perfusion bioreactor, wherein the perfusion rate, which is the rate of the overall flow through the perfusion bioreactor, is maintained based on a constant value, changing, and Maintain at least one of the process parameters within a predetermined range, maintain each of the first specified speed and the second specified speed of each of the discharge conduits within a predetermined range, maintain the third specified speed of the input conduit within a predetermined range, and maintain the perfusion rate of the perfusion bioreactor based on a constant value, and adjust the input conduit and the discharge conduit based on the Raman probe measurement value and the weight measurement value of the perfusion bioreactor. A method comprising.

21. The method according to claim 1, wherein the perfusion rate of the perfusion bioreactor is maintained within a variation of ±10% based on the constant perfusion rate set value.

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