Multi-chamber cell culture system and method

Abstract

Systems and methods for continuous culture include performing a first continuous culture of cells within a chamber of a first bioreactor under culture conditions that result in cell growth; moving a culture fluid containing the cells from the chamber of the first bioreactor, through one or more fluid conduits, into a chamber of a second bioreactor; and performing a second continuous culture of the cells within the chamber of the second bioreactor under culture conditions that produce at least one culture product. This process is carried out under conditions where the rate of cell growth in the first continuous culture is faster than the rate in the second continuous culture, and the production of the culture product in the first continuous culture is less than the production in the second continuous culture. TIFF2025522855000003.tif69170

Description

Technical Field

[0001] Statement Regarding Federally Sponsored Research This invention was made under Cooperative Research and Development Agreement (CRADA) FP00011895 between Pow Genetic Solutions, Inc. and The Regents of the University of California Ernest Orlando Lawrence Berkeley National Laboratory, which is operated by the U.S. Department of Energy. The U.S. government has certain rights in this invention.

[0002] Reference to Related Applications This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 356,974, filed June 29, 2022, entitled “Multi-Chamber Cell Culture System and Method,” the content of which is hereby incorporated by reference in its entirety.

Background Art

[0003] Background The rapidly growing biomanufacturing and synthetic biology industries rely on bioreactors (also called fermenters) to bioconvert raw materials into high-value products. Biomanufacturing is a newly emerging field that can be used to produce much of the world economy's physical materials with improved performance and an improved environmental footprint to combat climate change. Recent estimates project that the total annual economic impact of synthetic biology will reach $200 billion to $400 billion by 2040. Two-thirds of this impact will be in non-medical fields, including agriculture, consumer products, biomaterials, biochemicals, bioenergy, and many others. Among all potential applications of synthetic biology, the biomanufacturing of alternative proteins, biomaterials, biochemicals, and biofuels will experience the greatest growth over the next 10 to 20 years.

[0004] Furthermore, the petrochemical industry has enabled the production of ubiquitous products that form the backbone of modern society, from pharmaceuticals to household goods. However, petroleum-based processes are unsustainable and contribute to global climate change. Biomanufacturing with renewable alternatives has not been very competitive in terms of price because the underlying production processes, batch or fed-batch fermentation, have not changed in decades. Replacing common batch and fed-batch bioreactor systems with continuous-flow bioreactors would minimize equipment downtime, increase volumetric productivity, and reduce capital investment and operating costs. However, continuous-flow bioreactors are currently considered unreliable and are rarely used in biomanufacturing processes outside the pharmaceutical industry due to contamination and strain stability issues. Therefore, there is a need to develop reliable continuous-flow bioprocesses.

Brief Description of the Drawings

[0005] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments and, together with the detailed description, serve to enable those skilled in the relevant art to utilize these embodiments and other embodiments that will be apparent to those skilled in the art. The present invention will be described more specifically in conjunction with the following drawings.

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[0006] Summary Disclosed herein are multi-chamber cell culture systems and methods for producing a culture product. The multi-chamber cell culture system includes one or more first bioreactors in fluid communication with each other and one or more second bioreactors. Accordingly, one first bioreactor can supply a single second bioreactor; one first bioreactor can supply a plurality of second bioreactors; a plurality of first bioreactors can supply a single second bioreactor; or a plurality of first bioreactors can supply a plurality of second bioreactors.

[0007] In one aspect, the cells that produce the desired culture product are cultured in the chambers of one or more first bioreactors under conditions that maintain cell growth but produce little or no culture product (i.e., 5% or less of the amount of product produced in the second bioreactor). During the culture, the culture medium containing the growing cells is transferred directly to the chambers of one or more second bioreactors. The culture conditions in the second bioreactor are set to produce the culture product and to limit the growth rate of the cells therein. By limiting the growth rate of the cells in the second culture vessel, the generation and growth of mutant microorganisms that interfere with the production of the culture product can be suppressed. In some aspects, the cells in the second bioreactor are not growing and will have a very slow growth rate, for example, a doubling time of at least one week. The culture product is removed from the second bioreactor, for example, by withdrawing the cell culture medium and isolating the product therefrom. In some aspects, the cells removed with the cell culture medium are returned to the second culture vessel and recycled.

[0008] In another aspect, the cells that can produce the desired product are cultured in the first bioreactor under conditions that maintain cell density. Under steady state conditions, the cells under such culture will grow at a constant growth rate (e.g., logarithmic or exponential growth). In certain aspects, the culture conditions are set to result in a maximum or near-maximum growth rate. This can be achieved, for example, by supplying a cell culture medium that does not contain growth-limiting nutrients for the cells.

[0009] Growing cells from the first bioreactor are transferred through a fluidic conduit to each of one or more second bioreactors. The culture conditions within the second bioreactor(s) are maintained to produce (preferably optimally produce) one or more desired culture products. This includes culturing the cells transferred to the second bioreactor under non-growing or very slowly growing conditions. This can be achieved by a culture medium containing growth-limiting amounts of nutrients, such as micronutrients.

[0010] The culture broth from the second bioreactor is withdrawn and the culture product is isolated therefrom. In some embodiments, the cells in the effluent are returned to the second bioreactor for continuous production of the culture product.

[0011] A system for implementing these methods utilizes a feedback mechanism to maintain culture conditions and control the volume of the cell culture. This feedback mechanism includes a sensor that measures one or more culture parameters of the cell culture within each bioreactor, a computer module that calculates the culture parameter and determines whether it exceeds or falls below a predetermined set point of such a parameter, and an effector that adjusts the culture conditions towards the set point of the parameter. Such parameters include, for example, cell density, culture volume, temperature, pH, dissolved oxygen, and nutrient concentration. The effector can include, for example, a pump that moves liquid from a reagent reservoir to the bioreactor vessel, from the bioreactor vessel to another bioreactor vessel or to a collection vessel. Other elements include, for example, a temperature regulator that controls the temperature of the cell culture and an air pump that introduces oxygen into the cell culture. By utilizing such a feedback mechanism, it is possible to maintain various culture parameters over time.

Mode for Carrying Out the Invention

[0012] Detailed Description I. Continuous Culture The methods and systems provided herein use continuous cell culture to produce a desired culture product. It is possible to produce any suitable culture product, such as small molecules, proteins, polynucleotides, and / or cells.

[0013] Two major methods of culturing cells are batch culture and continuous culture. Batch culture is a closed system in which cell culture, such as fermentation, is carried out using a fixed amount of nutrients. Continuous culture is an open system in which nutrients are continuously added to the culture. A turbidostat, which is an example of continuous culture, is shown in Figure 1. Specifically, Figure 1 shows that cells are maintained in a steady state of growth over time. The cell concentration is indicated by "AU". The turbidostat maintains a constant turbidity of the culture, which reflects the cell density. Culture parameters are initially set and adjusted to reach a steady state. This may require, for example, 3 to 5 generations or more of cells. The turbidostat maintains the cell density by removing the culture medium containing cells from the bioreactor and replacing its volume with fresh medium. Thus, the growth rate is a function of the amount of culture medium removed over time when the culture is in a steady state. Batch culture is characterized by an initial rapid growth of cells, followed by reaching a plateau after the nutrients are exhausted and further growth is limited. Continuous culture typically features a stable growth rate and / or a stable culture output rate because nutrients are continuously added to the system and the cell density is maintained, for example, by removing excess cells from the culture. Further examples of continuous culture include chemostats and perfusion cultures.

[0014] The culture process encompasses any culturing of cells (i.e., bioproduction for producing bioproducts) for generating a culture output. Such processes include, but are not limited to, processes referred to as "fermentation" and "cell culture". "Fermentation" generally refers to the enzymatic conversion of one molecule to a different molecular species (e.g., the conversion of sugar to ethanol) by typically single-celled organisms. "Cell culture" generally refers to the culturing of single cells, such as mammalian cells or insect cells, for producing products such as polypeptides, valuable organic compounds, or biomass.For culturing, any suitable cells (archaea, prokaryotic cells, and / or eukaryotic cells) as follows can be used: fungal cells such as yeast (e.g., species of Saccharomyces spp, Pichia spp, Komagataella spp, Kuyveromyces spp, Aspergillus spp, Rhodoporidium spp, Lipolytica spp, Aspergillus spp, Neurospora spp, Trichoderma spp, Candida spp, or Penicillium); bacterial cells such as Escherichia coli, Bacillus spp, Costridia spp, Streptomyces spp, Pseudomonas spp, Ralstonia spp, Shewanella spp; animal cells such as arthropods (e.g., insects, shrimps, lobsters, crayfish, and crabs), chordates (fish, amphibians, reptiles, birds (e.g., chicken or pigeon), mammals (e.g., human, or non-human animals such as cows, sheep, goats, pigs, horses, dogs, cats, primates, etc.); cell lines such as CHO (Chinese hamster ovary cells), BHK21 (baby hamster kidney), NS0, Sp2 / 0 mouse cell line, insect cells (e.g., SP9, Sf9, sf21, S2), tobacco BY-2 cells, Oryza Sativa, or algal cells).

[0015] Furthermore, these processes include synthetic biology methods in which cells are genetically engineered to produce molecular outputs, for example, by intracellular expression of enzymes along synthetic pathways.

[0016] II. Multi-chamber cell culture system The multi-chamber cell culture system of the present disclosure includes one or more first bioreactors and one or more second bioreactors in fluid communication with the first bioreactors. As a result, the system can include a first bioreactor in fluid communication with a single second bioreactor via a fluid conduit, and further, a central first bioreactor in fluid communication with a plurality of second bioreactors.

[0017] In certain embodiments, the multi-chamber cell culture system further includes one or more reagent reservoirs fluidly connected to the first bioreactor and / or one or more second bioreactors. The reagent reservoirs can contain any suitable reagents such as growth medium, nutrient sources, carbon sources (e.g., glucose), buffers and / or one or more reagents for adjusting culture parameters (e.g., pH of the medium), and / or gases (e.g., oxygen).

[0018] In certain embodiments, the multi-chamber cell culture system further includes a reservoir that is fluidly connected to the second bioreactor and configured to receive a media effluent (preferably including culture products) from the second bioreactor. In certain embodiments, the media effluent includes one or more cells from the culture. In certain embodiments, the media effluent from the second bioreactor is passed through a filter that retains one or more cells (i.e., the retentate) in the media effluent while allowing the media (i.e., the permeate) including one or more culture products to pass through. In certain embodiments, the permeate includes fewer cells than the media effluent from the second bioreactor. In certain embodiments, the permeate contains no cells. In certain embodiments, the filter is at least about 50, 60, 70, 80, 85, 90, 95, 99, 99.5, 99.9, or 100% effective at retaining cells from the media effluent from the second bioreactor. In certain embodiments, the permeate contains at most about 50, 40, 30, 20, 15, 10, 5, 1, 0.6, 0.1, or 0% of the cells from the cell culture media effluent from the second bioreactor. In certain embodiments, one or more cells retained on the filter, i.e., the retentate, are returned to the second bioreactor (i.e., a cell recycle system).

[0019] In certain embodiments, the multi-chamber cell culture system further includes one or more feedback mechanisms and / or control systems for maintaining culture conditions and controlling the volume of the cell culture. This feedback mechanism includes one or more sensors that measure one or more process parameters of the bioreactor, a computer module that calculates the process parameter and determines whether it exceeds or falls below a predetermined setpoint of the parameter, and an effector that adjusts the process parameter towards the desired setpoint. In certain embodiments, the desired setpoint is input by an operator using a programmable interface. In certain embodiments, the setpoint is developed using one or more computer programs and one or more tests to obtain optimal results. Any suitable sensor can be used to measure process parameters, such as culture parameters such as cell density, culture volume, temperature, pH, dissolved oxygen, and / or nutrient concentration. Any suitable effector can be used, such as a liquid pump for moving liquid from a reservoir to a bioreactor vessel, from one bioreactor vessel to another bioreactor vessel, or to a collection vessel, a heat source and / or a cooling source for maintaining the temperature of the bioreactor, a mixing unit (e.g., an impeller, a pneumatic stirrer, a stirring bar and plate) for stirring the bioreactor, and / or an air pump for introducing a gas (e.g., oxygen or nitrogen) into the culture. Further examples of sensors and effectors are disclosed herein.

[0020] An exemplary explanatory diagram of a multi-chamber cell culture system is shown in FIG. 2. FIG. 2 shows a multi-chamber cell culture system including a first bioreactor (201, FIG. 2A) and a second bioreactor (208, FIG. 2B). The first bioreactor (FIG. 2A) is configured to contain a first cell culture (202) that can be mixed by a mixing unit (203). The first bioreactor further includes a first inflow conduit (204) connected to a first inflow port (205) that is in fluid communication with a first reagent reservoir. The first bioreactor further includes a first outflow conduit (206) (i.e., an outlet) connected to a first outflow port (207), where the first outflow conduit (206) and the outflow port (207) are in fluid communication with both the first cell culture (203) and the second bioreactor (208). The second bioreactor (208) is configured to contain a second cell culture (209) that can be mixed by a mixing unit (210). The second bioreactor (208) further includes a first inflow conduit (211) and a first inflow port (212) that are fluidly connected to the first outflow conduit (206) and the outflow port (207) of the first bioreactor, and these are configured to receive the first cell culture (203) from the first bioreactor (201). In certain embodiments, the first outflow conduit (206) of the first bioreactor (201) and the first inflow conduit (211) of the second bioreactor (208) are the same conduit. The second bioreactor (208) further includes a second inflow conduit (213) connected to a second inflow port (214) that is in fluid communication with a second reagent reservoir. The second bioreactor (208) further includes a first outflow conduit (215) connected to a first outflow port (216), where the first outflow conduit (215) and the outflow port (216) are in fluid communication with the second cell culture (209).

[0021] The second bioreactor can further include a cell recycling system (219, Figure 2C), which separates cells from their culture medium and is configured to return (e.g., recycle) the concentrated cell culture to the second bioreactor (208) through a third inflow conduit (217) connected to a third inflow port (218), or (2) transfer the cells to a reservoir (not shown). The cell recycling system includes a first inflow conduit (220) connected to a first inflow port (221), where the first inflow conduit (220) and inflow port (221) of the cell recycling system are in fluid communication with the first outflow conduit (215) and outflow port (216) of the second bioreactor and the cell culture (209). The cell recycling system further includes a first outflow conduit (222) connected to a first outflow port (223), both of which are in fluid communication with the third inflow conduit (217) and inflow port (218) of the second bioreactor, where the concentrated cell culture is transferred from the cell recycling system to the second bioreactor. The cell recycling system further includes a second outflow conduit (224) and a second outflow port (225) configured to transfer the cell-free medium to a downstream process or reservoir. In certain embodiments, the cell recycling system includes a filter, membrane, or hollow fiber cartridge operating as a tangential flow or alternating tangential flow filtration system. Any suitable mechanism for separating cells from the medium, such as a centrifuge, may be used. An exemplary hollow fiber cartridge is shown in Figure 6.

[0022] The first and second bioreactors can comprise any suitable number of connected conduits, ports, each of which can be connected to a pump as needed for the desired application. The ports can be attached to the lids and / or chambers of the bioreactors. The ports can optionally include a dip tube. The dip tube can be of any suitable length and diameter and may or may not be in contact with the cell culture. In either case, the ports and / or dip tubes can supply and / or remove one or more liquid and / or gas components from the bioreactor. Any suitable material can be used for the ports and / or conduits. In a preferred embodiment, the material is a biologically compatible material. In a particular embodiment, the material is a chemically resistant material. The first and second bioreactors can comprise any suitable number of sensors and / or effectors as needed for the desired application (disclosed herein).

[0023] In certain embodiments, a multi-chamber cell culture system includes a first bioreactor, one or more second bioreactors in fluid communication with the first bioreactor, one or more reagent reservoirs in fluid communication with the first bioreactor and optionally one or more second bioreactors, one or more pumps, and a control system. In certain embodiments, the first bioreactor includes a first chamber and one or more first sensors that provide measurements of at least cell density and cell culture volume within the first chamber. In certain embodiments, the one or more second bioreactors include a second chamber and one or more second sensors that provide measurements of at least culture conditions and cell culture volume within the one or more second chambers. In certain embodiments, the one or more pumps are configured to (1) move a liquid reagent from at least one reagent reservoir to the first chamber of the first bioreactor; (2) move a cell culture fluid from the first chamber to one or more second chambers; (3) move a cell culture fluid out of the one or more second chambers; (4) move a liquid reagent from at least one reagent reservoir to one or more second chambers; and / or (5) move a cell culture fluid from the second chamber of the second bioreactor to one or more reservoirs and / or a downstream process. In certain embodiments, the one or more pumps are configured for (1)-(3). In a preferred embodiment, the one or more pumps are configured for (1)-(4). In a more preferred embodiment, the one or more pumps are configured for (1)-(3), (5), and optionally (4). In certain embodiments, the control system is configured to (1) control the cell density of a cell culture within the first chamber of the first bioreactor using measurements from the one or more sensors; (2) move a cell culture fluid from the first chamber of the first bioreactor to one or more second chambers of the second bioreactor; (3) control the volume and culture conditions of a cell culture within the one or more second chambers of the second bioreactor using measurements from the one or more sensors; and / or (4) move a cell culture fluid from the second chamber of the second bioreactor to one or more reservoirs and / or a downstream process.In certain embodiments, the control system is configured for (1)-(3). In certain embodiments, the control system is configured for (1)-(4).

[0024] In certain embodiments, the multi-chamber cell culture system comprises a forward osmosis membrane or a reverse osmosis membrane.

[0025] A. Bioreactor Cell culture is performed within a bioreactor. A bioreactor is a system that supports and maintains a biologically active environment over a predetermined period of time. Such a system includes a container or chamber configured to contain a culture medium and components for maintaining or changing various culture parameters. The bioreactor can be configured to maximize any suitable parameter of cell culture. In certain embodiments, the bioreactor can be configured to maintain an environment that supports optimal cell growth for a desired application. In certain embodiments, the bioreactor can be configured to maintain a desired cell density for a desired application. In further embodiments, the bioreactor can be configured to maintain an environment that supports optimal production of one or more desired culture products.

[0026] Batch and continuous bioreactor systems are commercially available, for example, from Sartorius (Goettingen, Germany) and ThermoFisher (Waltham, MA).

[0027] Figures 3A and 3B show an exemplary bioreactor. Figure 3A shows an exemplary schematic and components of a bioreactor. Figure 3B shows a bioreactor connected to a computer, the computer having a user interface that displays culture parameters rather than receiving user instructions for controlling culture conditions. A reagent reservoir, such as a bottle, containing various types of nutrients is attached to the culture through a conduit, such as a tube. The reservoir can also receive excess medium from the culture and / or one or more culture products in the medium or culture broth.

[0028] A bioreactor is typically a system configured to grow a cell culture by controlling one or more culture conditions and / or process parameters. Any suitable bioreactor configuration can be used, such as batch and / or continuous culture modes. In a preferred embodiment, the bioreactor is operated in a continuous culture mode, for example, as follows: (1) Turbidostat mode: Dynamically adjust the flow rate of nutrient supply and periodically remove cells to keep the turbidity in the vessel constant; (2) Chemostat mode: Cell growth is restricted and controlled by the addition of nutrients (e.g., glucose, oxygen, glutamine), and the depleted medium containing growth inhibitors is removed at approximately the same rate; and (3) Perfusion mode: Cells are retained within the bioreactor or recycled back to the bioreactor, fresh medium is supplied, and cell-free supernatant is removed at the same rate.

[0029] 1. Culture Chamber The culture chamber of a bioreactor generally has a volume of about 50 mL to about 50 L. This includes, for example, a volume of about 1 L to about 10 L. Industrial-sized fermenters can be sized from about 50 L to about 1,000,000 L. In certain embodiments, the culture chamber has a volume of about 1,000 L to about 10,000 L. A multi-chamber cell culture system can include any suitable culture chamber volume for each bioreactor and any suitable combination of culture chamber volumes. In certain embodiments, the culture chambers of the first bioreactor and one or more second bioreactors have the same volume. In certain embodiments, the culture chambers of the first bioreactor and one or more second bioreactors have different volumes. For example, the volume of the first culture chamber can be made smaller than the volume of the second culture chamber. In certain embodiments, one or more of the culture chambers of one or more second bioreactors have different volumes.

[0030] The multi-chamber cell culture system can include any suitable number of first bioreactors, for example, any of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or 18, and / or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 first bioreactors, such as 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2. Usually, the multi-chamber cell culture system includes one first bioreactor. Additionally or alternatively, the multi-chamber cell culture system can include any suitable number of second bioreactors that are in fluid communication with one or more first bioreactors, for example, any of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or 18, and / or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 second bioreactors, such as 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2. In a preferred embodiment, the multi-chamber cell culture system includes at least one first bioreactor and at least one second bioreactor. In a more preferred embodiment, the multi-chamber cell culture system includes one first bioreactor and one or more second bioreactors.

[0031] In certain embodiments, the chamber includes one or more baffles. The baffles assist in stirring / agitating the cell culture and can, for example, aid in the distribution of nutrients, the mixing of one or more reagents, and / or aeration.

[0032] The culture chamber comprises one or more ports through which liquid and / or gas can be introduced (“inflowed”) into the culture chamber or removed (“outflowed”) from the culture chamber. Each chamber can include any suitable number and / or combination of ports as required for the desired application. Such ports can communicate with fluid lines / conduits that communicate with other containers, such as reagent reservoirs, other culture vessels, collection reservoirs, and / or gas sources. Such fluid lines can include tubes such as flexible tubing. Any suitable tube material can be used, for example, glass, metal, plastic, rubber, and / or halogenated polymers. Preferred materials include biocompatible materials and / or chemically resistant materials, such as silicone, stainless steel, neoprene, polypropylene, pharmed, and / or Tygon. The movement of fluid through such tubes can be controlled by valves such as pressure valves, one-way valves, two-way valves, three-way valves, shear valves, pinch valves, piezo valves, solenoid valves, and / or pumps such as peristaltic pumps, syringe pumps, piezo pumps, diaphragm pumps. Any suitable combination of pumps and / or valves can be used. Generally, the pump can be selected based on the minimum and maximum liquid volume transfer required, for example, the feed rate and / or the medium exchange rate, and thus based on flow rate requirements such as hundreds of μL / min to hundreds of L / min (depending on both the application and volume of the cell culture system).

[0033] In certain embodiments, the bioreactor and its chambers can comprise one or more feeds for adding nutrients and other molecules, as well as one or more effluents for removing the culture fluid from the bioreactor. In certain embodiments, the first bioreactor of the multi-chamber cell culture system comprises at least one nutrient feed and at least one effluent fluidly connected to one or more second bioreactors. In certain embodiments, the second bioreactor of the multi-chamber cell culture system comprises at least one feed of the cell culture from the first bioreactor, at least one nutrient feed, and at least one effluent.

[0034] B. Sensors and Effectors The multi-chamber cell culture system can further include one or more sensors for measuring various culture parameters (e.g., metabolic processes and process variables), and effectors for controlling these parameters. In a preferred embodiment, the multi-chamber cell culture system includes at least one sensor. In certain embodiments, the multi-chamber cell culture system can include sensors that (1) directly measure one or more metabolites (e.g., acetic acid and ammonia), (2) detect changes in a substrate (e.g., glucose), or (3) indirectly measure metabolism via cell respiration by detecting changes in the gas composition (e.g., oxygen and carbon dioxide). In certain embodiments, one or more culture parameters are measured and data is provided, in particular, to a feedback mechanism for instructing the feeding strategy and / or process conditions. Any suitable number and combination of sensors can be used.

[0035] In certain embodiments, the sensor is an "in-line" or "in situ" sensor, in which case the sensor shares a direct interface with a component of the culture or bioreactor. Data from the in-line sensor can be monitored continuously or intermittently and / or analyzed. The continuity of the measurements typically depends on the response time of the signal, the flow rate of the sampling procedure, and / or the needs of a particular application. In certain embodiments, the sensor is an "off-line" sensor, in which case the sample is collected manually or by an automated method such as an autosampler and analyzed in a laboratory, e.g., by high performance liquid chromatography (HPLC), flow cytometry, microscopy, etc. In certain embodiments, off-line measurements can be performed using robotics or microfluidic systems for analysis by instruments such as HPLC or gas chromatography (GC), either in-line or off-line.

[0036] a) Cell density In certain embodiments, one or more bioreactors of a multi-chamber cell culture system include a sensor capable of measuring the cell density of the cell culture or a proxy thereof. For example, the density of the culture is a function of the optical density of the culture medium. For example, OD can be measured using a UV-Vis spectrophotometer 600Optical density can be measured. Examples of other spectroscopic methods for measuring cell density include mid-infrared (MIR), ultraviolet-visible (UV-VIS), fluorescence, Raman, and dielectric spectroscopy. Further, cell density is also a function of the capacitance of the cell culture. Thus, the capacitance of the culture medium can be measured using a capacitance meter such as a Hamilton probe or a Chloris probe. Further, cell density can be measured using the acoustic resonance of the cell culture medium, where the biomass is determined using the specific gravity of the medium. In certain embodiments, the sensor can distinguish the density component of live and / or viable cells in the culture from the density component of dead cells in the culture and provide an indication of the density of metabolically active cells in the culture.

[0037] The cell density of the culture can be affected by adding liquid to the bioreactor vessel to dilute the cell culture. The dilution rate in continuous culture can be modeled using the equation D = F / V -1 where the dilution rate (D; [hr -1 -1]) is a function of the flow of medium into the bioreactor (F; [L hr -1 -1]) and the volume of culture in the bioreactor (V; [L]). In certain embodiments, to reach a steady state of the culture, the dilution rate (D) needs to be equal to the specific growth (μ) of the cell culture, thereby keeping the biomass concentration in the bioreactor constant.

[0038] As described herein, pumps and valves can be used as effectors to move liquid into and out of a bioreactor. In certain embodiments, a multi-chamber cell culture system includes sensors that can provide measurements of cell density to one or more feedback routines on a computer, in which case the computer actuates the effectors to adjust the cell density to a desired set point. As described herein, pumps and valves can be used as effectors to move liquid into and out of a bioreactor as described above.

[0039] b) Culture volume In certain embodiments, one or more bioreactors of a multi-chamber cell culture system can include sensors that can measure cell volume or a proxy thereof. One such proxy for volume is mass. Thus, to measure the weight of a bioreactor, the bioreactor can be placed on a scale. Additionally or alternatively, level sensors such as float valves or capacitance liquid sensors, and / or optical liquid sensors can also be used. In certain embodiments, the optical liquid sensor is coupled to a level gauge.

[0040] The cell culture volume can be reduced by removing the liquid culture from the bioreactor vessel and increased by adding liquid to the bioreactor vessel. As described herein, pumps and valves can be used as effectors to remove liquid from the culture vessel. Alternatively, an overflow tube can function as a sensor and as a pump such as a gravity pump. The overflow tube senses that the height of the culture has exceeded the upper end of the overflow tube and uses gravity to draw liquid from the vessel through the tube. In certain embodiments, both the volume and the cell density (as described above) are maintained within desired ranges. In certain embodiments, a multi-chamber cell culture system includes a sensor that can provide a measurement of the cell volume to one or more feedback routines on a computer, in which case the computer actuates an effector to adjust the cell volume to a desired set point.

[0041] c) pH In certain embodiments, one or more bioreactors of a multi-chamber cell culture system can include a sensor capable of measuring the pH of the cell culture. The pH can be measured using any suitable pH meter, such as a pH electrode, e.g., an Ag / AgCl half-cell or an Ion Selective Field Effect Transistor (ISFET), or an optical pH meter.

[0042] The pH of the cell culture can be adjusted using a pump system controlled by a feedback system connected to a pH sensor, where the pump system feeds in a calculated amount of a suitable acid or base from a reservoir as needed and / or titrates the amount of acid or base until the sensor reads the desired pH. In certain embodiments, a multi-chamber cell culture system includes a sensor that can provide a measurement of the pH to one or more feedback routines on a computer, in which case the computer actuates an effector to adjust the pH to a desired set point.

[0043] d) Dissolved oxygen and / or carbon dioxide In certain embodiments, one or more bioreactors of a multi-chamber cell culture system can include sensors capable of measuring dissolved oxygen (“DO”) and / or dissolved carbon dioxide (“dCO2”) in the culture medium. Both dissolved oxygen and dissolved carbon dioxide can be measured with optical sensors, galvanic cells, and / or polarographic cells.

[0044] Dissolved oxygen can be controlled using an aeration system that includes a pump and / or valve in communication with one or more gas sources (e.g., a canister or the atmosphere) that deliver air into the interior of one or more culture chambers. Dissolved carbon dioxide can be controlled by venting used gas containing CO2 when the aeration system delivers fresh gas to the culture chamber. In certain embodiments, the multi-chamber cell culture system includes sensors that can provide measured values of DO and / or dCO2 to one or more feedback routines on a computer, in which case the computer actuates effectors to adjust the concentration of gas within the bioreactor to a desired set point.

[0045] In certain embodiments, CO2 can constitute a carbon source for the culture, and thus controlling dCO2 in the culture can aid in growth and / or production of one or more culture products. In certain embodiments, the culture can be maintained in a hypoxic or anaerobic atmosphere.

[0046] e) Temperature In certain embodiments, one or more bioreactors of a multi-chamber cell culture system can include sensors capable of measuring the temperature of the culture medium. For this purpose, temperature sensors such as thermometers or thermistors can be used. In certain embodiments, the temperature sensor is installed within a thermowell where the temperature sensor can read the culture temperature without direct contact with the culture itself.

[0047] The temperature of the liquid culture within the culture chamber can be controlled using a temperature regulator such as a heating and / or cooling element. Non-limiting examples of heating elements include cartridge heaters, thin film resistance heaters, or thermoelectric coolers (TECs). Non-limiting examples of cooling elements include TECs or cooling jackets. The temperature of one or more bioreactors of a multi-chamber cell culture system can be maintained at any suitable temperature, such as at least about 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, or 85 °C, and / or at most 22, 24, 26, 28, 30, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, or 90 °C, for example 20 - 90 °C, preferably 24 - 50 °C, more preferably 25 - 40 °C. In certain embodiments, the multi-chamber cell culture system includes a sensor that can provide a measured temperature value to one or more feedback routines on a computer, in which case the computer actuates an effector to adjust the temperature within the bioreactor to a desired set point using a cooling and / or heating system.

[0048] f) Glucose concentration In certain embodiments, one or more bioreactors of a multi-chamber cell culture system can include a sensor capable of measuring any suitable chemical component of the culture medium. In preferred embodiments, the chemical component includes a nutrient. In even more preferred embodiments, the nutrient includes glucose, i.e., the concentration of glucose in the culture medium. The glucose concentration can be measured using an electrochemical glucose sensor and / or spectrophotometrically. It can also be measured biochemically.

[0049] The glucose concentration of the cell culture can be adjusted using a pump system controlled by a feedback system connected to a glucose sensor, where the pump system feeds in a calculated amount of glucose-containing liquid as needed and / or titrates the amount of glucose-containing liquid until the sensor reads the desired glucose concentration. In certain embodiments, the multi-chamber cell culture system includes a sensor that can provide measurements of glucose concentration to one or more feedback routines on a computer, in which case the computer actuates an effector to adjust the glucose concentration within the bioreactor to a desired set point.

[0050] g) Examples of other sensors and effectors As described above, by incorporating any suitable sensor, any suitable parameter (physical, chemical, and / or biological) can be measured as needed for the desired application. Table 1 shows a non-limiting and exemplary list of parameters and sensors. Non-limiting examples of additional parameters include foam, viscosity, pressure, agitation rate, pyruvate concentration, lactate concentration, acetate concentration, cell morphology, presence of protein, and / or concentration of metabolic products. Sensors for measuring these parameters can be configured to measure conductance (electrical conductivity), capacitance, optical density, torque, or other physical parameters. Further, the sensor can include spectroscopic, biochemical, optical, chromatographic, cytometry, microscopic, and / or electrochemical components for measuring the desired parameter. As an example, there is liquid chromatography with UV detection.

[0051] (Table 1) Examples of parameter and sensor types TIFF2025522855000002.tif108158

[0052] In certain embodiments, the bioreactor may include a foam control mechanism that includes a foam sensor and effectors (such as tubes and pumps) for reducing foam. In certain embodiments, the bioreactor may include a sparger and a mass flow controller that communicate with the interior of the chamber for introducing and / or replacing one or more gases and / or mixtures thereof. In certain embodiments, the bioreactor may include a mixing unit operably connected to a motor for mixing / stirring the cell culture. The bioreactor may also include a stirring device such as a paddle and ports for introducing nutrients and other chemicals and discharging the culture medium (which may contain cells).

[0053] C. First Bioreactor The first bioreactor can be any suitable bioreactor. In certain embodiments, the first bioreactor includes a first chamber. The first chamber of the first bioreactor can be configured to hold any suitable volume, such as, for example, a volume of about 50 mL to 50 L, a volume of about 1 L to about 10 L, a volume of about 5 L to 500 L, or a volume of 1,000 L to 10,000 L. In certain embodiments, the first chamber of the first bioreactor encompasses a volume of about 250 mL to about 10 L. The first bioreactor can have a volume that is the same as or less than the volume of the second bioreactor. For example, the first bioreactor can have a volume that is at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the volume of the second bioreactor. This includes, for example, any value between 10% and 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the volume of the second bioreactor.

[0054] In certain embodiments, the first bioreactor further includes one or more first sensors. The sensors can be any suitable sensors as disclosed herein. In a preferred embodiment, the first bioreactor includes a first sensor that provides at least a cell density measurement and a first sensor that provides a measurement of at least the cell culture volume within the first chamber. In certain embodiments, the first sensor that provides at least a cell density measurement includes an optical density sensor or a capacitance sensor. In certain embodiments, the first sensor that provides a measurement of at least the cell culture volume includes a scale that measures the mass of the bioreactor. In certain embodiments, the first bioreactor further includes one or more first sensors that measure temperature, pH, and / or dissolved oxygen. In certain embodiments, the first bioreactor includes a first sensor with a level sensor, such as an overflow tube, that is configured to move cell culture fluid from the first chamber when the height of the cell culture exceeds the upper end of the level sensor.

[0055] In certain embodiments, the first bioreactor comprises ports that communicate with a reagent reservoir via fluid conduits. Any suitable number and configuration (arrangement) of ports on the first bioreactor can be used. In certain embodiments, the first bioreactor comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, or 45 ports, and / or at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, or 50 ports, for example 1 - 50 ports, preferably 2 - 20 ports. In certain embodiments, fluid conduits such as tubes are connected to the ports. Any suitable number of reagent reservoirs can be connected to the first bioreactor.

[0056] In a preferred embodiment, the first bioreactor is configured to be in fluid communication with one or more second bioreactors.

[0057] In certain embodiments, the first chamber of the first bioreactor contains a cell culture, and one or more reagent reservoirs in fluid communication with the first chamber contain a growth medium, which growth medium contains one or more nutrients at a concentration that limits the growth rate of the cells in the cell culture.

[0058] An exemplary first bioreactor is shown in FIG. 2A. The first bioreactor is configured to contain a first cell culture (202) that can be mixed by a mixing unit (203). The first bioreactor further comprises a first inflow conduit (204) connected to a first inflow port (205) in fluid communication with the first reagent reservoir. The first bioreactor further comprises a first outflow conduit (206) (i.e., an outlet) connected to a first outflow port (207), and the first outflow conduit (206) and port (207) are in fluid communication with both the first cell culture (203) and the second bioreactor (208).

[0059] D. Second Bioreactor The second bioreactor can be any suitable bioreactor. In certain embodiments, the first bioreactor includes a second chamber. The second chamber of the first bioreactor can be configured to hold any suitable volume, such as, for example, a volume of about 50 mL to 50 L, a volume of about 1 L to about 10 L, a volume of about 5 L to 500 L, or a volume of 1,000 L to 10,000 L. In certain embodiments, the first chamber of the second bioreactor encompasses a volume of about 250 mL to about 10 L.

[0060] In certain embodiments, the second bioreactor further includes one or more second sensors. The sensor can be any suitable sensor as disclosed herein. In a preferred embodiment, the second bioreactor comprises a second sensor that provides a measurement of at least the cell culture volume within the second chamber. In certain embodiments, the second sensor that provides a measurement of at least the cell culture volume includes a scale that measures the mass of the bioreactor. In certain embodiments, the second bioreactor further includes one or more second sensors that measure temperature, pH, and / or dissolved oxygen. In certain embodiments, the second bioreactor includes a second sensor with a level sensor, such as an overflow tube, which is configured to move cell culture fluid from the second chamber when the height of the cell culture exceeds the upper end of the level sensor.

[0061] In certain embodiments, the second bioreactor comprises ports that communicate with a reagent reservoir via a fluid conduit and with each other. Any suitable number and configuration of ports on the second bioreactor can be used. In certain embodiments, the second bioreactor comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, or 45, and / or at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, or 50 ports, for example 1 - 50 ports, preferably 2 - 20 ports. In certain embodiments, a fluid conduit, such as a tube, is connected to the port. Any suitable number of reagent reservoirs can be connected to the second bioreactor.

[0062] Any suitable number of second bioreactors can be operably connected to the multi-chamber cell culture system. In certain embodiments, the multi-chamber cell culture system can include any suitable number of second bioreactors (i.e., a plurality of second bioreactors), for example, any of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or 18, and / or any of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 second bioreactors, for example 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2. In a preferred embodiment, one or more of the second bioreactors are configured to be in fluid communication with the first bioreactor. In a more preferred embodiment, each of one or more second bioreactors is configured to be in fluid communication with the first bioreactor.

[0063] The second bioreactor can include any suitable number of second chambers (i.e., a plurality of second chambers), for example, any of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, or 18, and / or any of at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 second bioreactors, for example 1 to 20, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2.

[0064] In certain embodiments, one or more of the second chambers of the second bioreactor have the same volume as the first chamber of the first bioreactor. In certain embodiments, one or more of the second chambers of the second bioreactor have a volume different from the volume of the first chamber of the first bioreactor. In certain embodiments, one or more of the second chambers of the second bioreactor have a volume that is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 times, and / or at most about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 times the volume of the first chamber of the first bioreactor, for example, from 0.1 to 100 times, preferably from 0.5 to 50 times, more preferably from 1 to 40 times. In certain embodiments, the second chamber of the second bioreactor has a volume greater than the volume of the first chamber of the first bioreactor. For example, the second chamber can have a volume from 2 to 100 times the volume of the first chamber of the first bioreactor. In certain embodiments, each of one or more of the second chambers of the second bioreactor has the same volume. In certain embodiments, one or more of the second chambers of the second bioreactor have a volume different from the remaining portions of one or more of the second chambers of the second bioreactor(s).

[0065] In certain embodiments, one or more of the second chambers of the second bioreactor contain a cell culture, and one or more reagent reservoirs in fluid communication with the second chamber contain a growth medium in which one or more nutrients are present at a concentration that limits the growth rate of the cells in the cell culture. In certain embodiments, the culture medium is formulated such that the nutrients are not limited in the first bioreactor, but one or more nutrients are present at a growth-limiting concentration in one or more of the second bioreactors.

[0066] An exemplary second bioreactor is shown in FIG. 2B. The second bioreactor (208) is configured to contain a second cell culture (209) that can be mixed by a mixing unit (210). The second bioreactor (208) further comprises a first inflow conduit (211) and a first inflow port (212) that are fluidly connected to the first outflow conduit (206) and the port (207) of the first bioreactor and configured to receive the first cell culture (203) from the first bioreactor (201). In certain embodiments, the first outflow conduit (206) of the first bioreactor (201) and the first inflow conduit (211) of the second bioreactor (208) are the same conduit. The second bioreactor (208) further comprises a second inflow conduit (213) connected to a second inflow port (214) that is in fluid communication with a second reagent reservoir. The second bioreactor (208) further comprises a second outflow conduit (215) connected to a second outflow port (216), where the second outflow conduit (215) and the port (216) are in fluid communication with the second cell culture (209).

[0067] In certain embodiments, one or more second bioreactors are in fluid communication with one or more collection vessels. In certain embodiments, one or more collection vessels are configured to receive a cell culture comprising both cells and a medium. In certain embodiments, one or more collection vessels are configured to receive a cell-free medium. In certain embodiments, one or more second bioreactors are in fluid communication with a cell recycle system (219, FIG. 2C), where the cell recycle system is configured to separate cells from their culture medium and (1) return the concentrated cell culture to the second bioreactor (208) through a third inflow conduit (217) connected to a third inflow port (218), or (2) transfer the cells to a reservoir (not shown). An exemplary cell recycle system comprises a first inflow conduit (220) connected to a first inflow port (221), where the first inflow conduit (220) and inflow port (221) of the cell recycle system are in fluid communication with a first outflow conduit (215) and outflow port (216) of the second bioreactor and the cell culture (209). The cell recycle system further comprises a first outflow conduit (222) connected to a first outflow port (223), both of which are in fluid communication with a third inflow conduit (217) and inflow port (218) of the second bioreactor, where the concentrated cell culture is transferred from the cell recycle system to the second bioreactor. The cell recycle system further comprises a second outflow conduit (224) and a second outflow port (225) configured to transfer the cell-free medium to a downstream process or reservoir.

[0068] E. Reagents, Fluid Conduits 1. Reagent Reservoir This system includes one or more reagent reservoirs. Each reagent reservoir contains a certain type of liquid reagent. This includes nutrients such as, for example, a carbon source, a nitrogen source, an acid source, and a base source. The reagent can include any suitable carbon source, such as sugars (e.g., glucose, xylose, sucrose, glycerol, acetate), molasses, malt extract, starch, dextrin, fruit pulp, CO or CO2). The reagent can include any suitable nitrogen source, such as amino acids or polypeptides, urea, ammonium salts (e.g., ammonium sulfate, ammonium phosphate or ammonia), corn steep liquor, yeast extract, peptone, and soybean meal).

[0069] In certain embodiments, the reagent reservoir can contain a reagent that includes one or more of the following: metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdates, selenites and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenic acid, aminobenzoic acids, thiamine, biotin, cyanocobalamin, folic acid), inducers, salts, phosphates, sulfates, chlorides, acetates, citrates and other anionic salts, magnesium, calcium, sodium, potassium, ammonium and other cationic salts, boric acid, choline, ascorbic acid, lipoic acid, nicotinic acid, inositol, antifoaming agents (e.g., Antifoam 204, Antifoam A, Antifoam C), amino acids (e.g., glutamic acid, leucine, and tryptophan), nucleobases (e.g., adenine, cytosine, thymine, uracil, and guanine), complex nutrients (e.g., yeast extract, peptone, tryptone, and casamino acids), macro-nutrients, micro-nutrients, and cell growth factors.

[0070] The reagent reservoir can contain any suitable combination of reagents, such as a growth medium containing a mixture of salts, vitamins, a carbon source, and a nitrogen source.

[0071] The reagent reservoir is in fluid communication with the first and / or second bioreactor vessels via a fluid conduit such as a tube. The conduit can engage ports on the reagent reservoir and the bioreactor vessels.

[0072] 2. Pumps, Valves, and Filters The multi-chamber cell culture system can include one or more pumps for pumping liquid from one container to another. This can include, for example, pumping liquid from a reagent reservoir to the first and / or second bioreactor vessels, and also between the vessels of the first and second bioreactors. The pumps can be under the control of a control system.

[0073] In certain embodiments, one or more pumps are configured to (1) move a liquid reagent from at least one reagent reservoir to the first chamber of the first bioreactor; (2) move a cell culture medium from the first chamber to one or more second chambers; (3) move a cell culture medium out of one or more second chambers; (4) move a liquid reagent from at least one reagent reservoir to one or more second chambers. In certain embodiments, one or more pumps are configured for (1), (2), and (3). In a preferred embodiment, one or more pumps are configured for (1)-(4).

[0074] Any suitable pump can be used, such as a peristaltic pump, diaphragm pump, piezo pump, gravity pump, and / or syringe pump. In one embodiment, one or more pumps are peristaltic pumps. The peristaltic pump is placed on the fluid conduit between the vessels and can induce liquid to move through the fluid conduit.

[0075] The multi-chamber cell culture system may further include one or more valves to prevent the flow of liquid and / or to direct the flow of liquid. Any suitable number and type of valves can be used. For example, the multi-chamber cell culture system can include a first pump and a first valve in fluid communication with the first reagent reservoir and the chamber of the bioreactor. When nutrients are consumed by the cell culture, the control system operates to open the valve so that the pump can transfer a certain amount of nutrients to the cell culture. After the transfer is completed, the control system can operate to close the valve and stop the pump.

[0076] In certain embodiments, the ports of the pump, valve, and / or chamber are provided with fittings for tubing. In certain embodiments, the tubing fittings provide fluid communication between two chambers, between a chamber and a reagent reservoir, or between an outflow conduit from a second chamber or between the second chamber and the outflow conduit.

[0077] Asepsis can be important for the operation of continuous bioreactors, such as multi-chamber cell culture systems. In certain embodiments, a filter is fluidly connected to the conduit such that the liquid flowing through the conduit passes through the filter and undesirable substances are removed. In certain embodiments, the filter removes biological contaminants from the fluid and prevents biological contaminants from entering the bioreactor. In certain embodiments, the filter has a pore size of 0.1 - 0.2 μm.

[0078] F. Control System In certain embodiments, the control system is configured to: (1) control cell growth, e.g., cell density, of the cell culture in the first chamber of the first bioreactor using measurements from one or more sensors; (2) transfer the cell culture medium from the first chamber of the first bioreactor to one or more second chambers of the second bioreactor; (3) control the volume and culture conditions of the cell culture in one or more second chambers of the second bioreactor using measurements from one or more sensors; and / or (4) transfer the cell culture medium from the second chambers of the second bioreactor to one or more reservoirs and / or downstream processes. In certain embodiments, the control system is configured for (1)-(3). In certain embodiments, the control system is configured for (1)-(4). Thus, the control system functions as a feedback system. Information from one or more sensors is processed by a computer to determine the state of one or more culture parameters. If a culture parameter deviates from a predetermined setpoint, the computer sends an instruction to an effector to adjust the parameter towards the setpoint.

[0079] In certain embodiments, the control system sets the dilution rate of a liquid reagent transferred into the first chamber and / or one or more second chambers to maintain the concentration of one or more nutrients. In certain embodiments, the control system maintains the cell density of the cell culture at or near a desired value (determined throughout the process or by an operator). In certain embodiments, the control system provides a measurement of the optical density of the cell culture and actuates one or more effectors to add a reagent to the cell culture and / or remove the cell culture from the chamber of the bioreactor. In certain embodiments, the control system includes a user programmable module that calculates the dilution rate as a function of the error between the target optical density and the measured optical density.

[0080] In certain embodiments, the control system sets the volume of the cell culture within the first chamber of the first bioreactor and / or one or more second chambers of the second bioreactor. In certain embodiments, the control system comprises a level sensor (e.g., an overflow tube) that moves cell culture fluid from the chamber when the height of the cell culture exceeds the upper end of the level sensor. In certain embodiments, the control system comprises a feedback routine that calculates the volume of the cell culture within one or more second chambers based on measurements received from one or more sensors and, if the volume exceeds a set point, actuates a pump to move liquid cell culture from the one or more second chambers.

[0081] In certain embodiments, one or more sensors provide measurements of one or more culture parameters in the first chamber and / or one or more second chambers to the control system. In certain embodiments, the multi-chamber cell culture system further comprises one or more effectors that affect changes in one or more culture parameters. In certain embodiments, the control system comprises one or more feedback routines that calculate one or more culture parameters based on the measurements and actuate one or more effectors to adjust the culture parameters towards a target level.

[0082] In certain embodiments, the multi-chamber cell culture system comprises one or more sensors that provide one or more measurements of the pH, temperature, and dissolved O2 of the cell culture within the first chamber and / or one or more second chambers. In certain embodiments, the system further comprises (1) one or more reagent reservoirs containing acids and bases that are in fluid communication with the first reservoir and / or one or more second reservoirs; (2) one or more temperature controllers that control the temperature of the cell culture within the first chamber and / or one or more second chambers; (3) one or more aerators for sending air to the cell culture within the first chamber and / or one or more second chambers. In certain embodiments, the control system comprises one or more feedback routines that calculate culture conditions including one or more of pH, temperature, dissolved O2, and nutrient concentration based on measurements received from one or more sensors, and operate one or more pumps to transfer an acid or base from the reagent reservoir into the second chamber to adjust the pH to a target pH; one or more temperature controllers to adjust the temperature to a target temperature; and / or one or more aerators to send air to the cell culture until a target dissolved oxygen level is reached.

[0083] 1. Computer The feedback routines provided herein may be executed by a programmable digital computer.

[0084] FIG. 4 shows an exemplary computer system. The computer system (401) includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") (405), which can be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system (401) also includes a memory or memory location (410) (e.g., random access memory, read-only memory, flash memory), an electronic storage device (415) (e.g., hard disk), a communication interface (420) (e.g., network adapter) for communicating with one or more other systems, and peripheral devices (425), such as cache, other memory, data storage devices, bioreactors, and / or electronic display adapters, etc. The computer-readable memory (410), storage device (415), interface (420), and peripheral devices (425) communicate with the CPU (405) via a communication bus (solid line), such as a motherboard. The storage device 415 can be a data storage device (or data repository) for storing data. The computer system (401) can be operably coupled to a computer network ("network") (430) with the aid of the communication interface (420). The network (430) can be the Internet, an intranet and / or an extranet, or an intranet and / or an extranet that communicates with the Internet. The network (430) can be, in some cases, a telecommunications and / or data network. The network (430) can include one or more computer servers that enable distributed computing, such as cloud computing.

[0085] The CPU (405) can execute a series of machine-readable instructions, which can be embodied in a program or software (code). The instructions may be stored in a memory location such as a computer-readable memory (410). The instructions can be sent to the CPU (405), and then the CPU (405) can be programmed or configured to execute the method of the present disclosure.

[0086] The storage device (415) can store files such as drivers, libraries, and stored programs. The storage device (415) can store user data, such as user preferences, log files, videos and other images, and user programs. The computer system (401) can optionally include one or more additional data storage devices, which are external to the computer system (401), such as being installed on a remote server that communicates with the computer system (401) via, for example, an intranet or the Internet.

[0087] The computer system (401) can communicate with one or more remote computer systems via the network (430).

[0088] The methods described herein can be executed by machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system (401), such as, for example, the computer-readable memory (410) or the electronic storage device (415). The machine executable code or machine readable code can be provided in the form of software. In use, the code can be executed by the processor (405). In some cases, the code is retrieved from the storage device (415) and stored in the memory 410 for immediate access by the processor (405). In some situations, the electronic storage device (415) can be excluded and the machine executable instructions are stored in the memory (410). The code can be used to communicate with and issue instructions to electronic devices on the apparatus, such as, for example, a circuit board (440), a module, or a subsystem.

[0089] The computer system (401) can communicate with one or more remote computer systems via a network (430).

[0090] The machine-executable code can be stored in an electronic storage device such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. The "memory" type of medium can include any or all of tangible memories such as computers, processors, or their associated modules (e.g., various semiconductor memories, tape drives, disk drives, etc.), which can always provide non-transitory storage for software programming. All or part of the software may sometimes be communicated via the Internet or other various electrical communication networks.

[0091] The computer system (401) can include, or communicate with, an electronic display (435) that includes a user interface (UI) (440) for providing, for example, input parameters of the methods described herein. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0092] In certain embodiments, the control system comprises a computer including a processor and a memory containing executable code that, when executed by the processor, performs one or more feedback routines to control parameters of the bioreactor. In certain embodiments, the parameters of the bioreactor include control of cell density in the first chamber, control of the volume of the cell culture in the first chamber and / or one or more second chambers, control of culture conditions in one or more second chambers, and control of cell density. In certain embodiments, one of the feedback routines calculates the cell density of the cell culture in the first chamber based on measurements received from one or more sensors and operates a pump to move a liquid reagent from a reagent reservoir to the first chamber to adjust the cell density to a setpoint. In certain embodiments, one of the feedback routines calculates the volume of the cell culture in the first chamber based on measurements received from one or more sensors and operates a pump to move the liquid cell culture from the first chamber to one or more second chambers if the volume exceeds a setpoint.

[0093] In another embodiment, the feedback routine measures parameters such as cell density in the first bioreactor, controls the addition of culture medium or removal of cell culture to maintain the cells in the growth phase, measures one or more parameters related to the culture conditions in the second bioreactor, and controls the addition of medium and / or nutrients and removal of cell culture to maintain the cells in the production phase.

[0094] 2. Machine Learning In certain embodiments, one or more data types from one or more parameters of a multi-chamber culture system are stored and can be used to identify improvements in process and / or culture parameters. In certain embodiments, a network is provided that includes a plurality of separate multi-chamber cell culture systems, where each of the systems transmits information regarding one or more aspects of one or more processes in that system to a central processing unit. In certain embodiments, one or more of the plurality of systems are spatially separated. The central processing unit can process the information and transmit an output, such as an output that causes a change in one or more of the multi-chamber cell culture systems, to one or more of the multi-chamber cell culture systems. In certain embodiments, the network includes at least 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 40, 50, 70, 100, 200, or 500 multi-chamber cell culture systems, and / or at most 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 40, 50, 70, 100, 200, 500, or 1000 multi-chamber cell culture systems. The central processing unit can be a single device or a plurality of devices, and can be distributed, such as in a cloud-based system. The processing device can learn from information provided by the various systems and can be configured to adjust conditions in one or more of the systems, at least in part, based on that learning, for example, by using a machine learning algorithm. Any suitable multi-chamber cell culture system can be networked.

[0095] In some embodiments, the input to the feedback mechanism can be derived using a method involving implementing machine learning techniques using the data types of the multi-chamber culture system to implement linear and non-linear models, such as processes like CART (classification and regression trees), artificial neural networks like back propagation networks, discriminant analysis (e.g., Bayesian classifier or Fisher analysis), logistic classifiers, and support vector classifiers (e.g., support vector machines).

[0096] Machine learning algorithms for identifying optimal culture conditions can utilize deep learning techniques. Deep learning methods utilize multiple layers in the learning process.

[0097] One deep learning method is reinforcement learning. Reinforcement learning is an aspect of machine learning where an agent learns to act in an environment by performing specific actions and observing the rewards / results obtained from those actions. The agent takes an action (a t ) with respect to its environment. This yields information regarding the state of the environment (S t ) and a reward (R t ) indicating whether the result is better than the previous result. The agent acts based on the hypothesis of maximizing the reward.

[0098] One version of reinforcement learning is called direct search. "Direct search" refers to the sequential consideration of trial solutions, including the comparison of each trial solution with the "best" obtained up to that point, along with a strategy for determining what the next trial solution will be (as a function of previous results).

[0099] Q-learning is a model-free reinforcement learning algorithm for learning the value of actions in a specific state.

[0100] An artificial neural network uses a collection of interconnected nodes. A neural network consists of node layers including an input layer, one or more hidden layers, and an output layer. Each node, i.e., artificial neuron, is connected to another node and has associated weights and thresholds. When the output of an individual node exceeds a specified threshold, that node is activated and sends data to the next layer of the network (which consists of node layers including an input layer, one or more hidden layers, and an output layer). Each node, i.e., artificial neuron, is connected to another node and has associated weights and thresholds. When the output of any individual node exceeds a specified threshold, that node is activated and sends data to the next layer of the network.

[0101] III. Method for Producing a Product The methods provided herein can use any suitable system, such as a multi-chamber cell culture system as described above.

[0102] This specification provides a method for producing a product in a multi-chamber cell culture system. This method includes culturing cells in a first bioreactor under conditions that maintain cell growth, for example, by maintaining a certain cell density. Under such a certain cell density condition, when the cells reach a steady state, they grow at a certain growth rate. In certain embodiments, the cells are cultured in a cell culture medium in which no nutrient is growth-limiting and other culture conditions such as temperature, pH, and dissolved oxygen are set to provide a maximum or near-maximum growth rate.

[0103] In certain embodiments, the cells in the first culture chamber are then automatically transferred to the chamber (or each of a plurality of second bioreactor chambers) of a second bioreactor in which the culture conditions are set to optimize the production of the desired culture product.

[0104] In certain embodiments, the culture fluid is removed from the second culture chamber, and the culture product is isolated from the culture fluid. In certain embodiments, the cells in the removed culture fluid are returned to the second culture chamber to continue product production.

[0105] In certain embodiments, provided herein is a method for: (1) performing a first continuous culture of cells in a first chamber of a first bioreactor; (2) during the first continuous culture, transferring a culture fluid containing the cells from the first chamber of the first bioreactor to a second chamber of at least one second bioreactor; (3) performing a second continuous culture of the cells transferred from the first bioreactor in the second chamber of the one or more second bioreactors to produce a culture product; and (4) recovering at least one culture product from the second chamber of the one or more second bioreactors. In preferred embodiments, the first bioreactor is operated under culture conditions that maintain cell growth by maintaining a constant cell density (e.g., turbidostat). In certain embodiments, the one or more second bioreactors are operated under certain culture conditions that are advantageous for the production of the culture product. In certain embodiments, the certain culture conditions in the one or more second bioreactors are optimized for the formation of the culture product (e.g., chemostat). Any suitable culture conditions can be used depending on the cells and the application. In certain embodiments, the method further comprises maintaining a constant cell culture volume within the chamber of the first bioreactor (as disclosed herein). In certain embodiments, the constant cell culture volume within the first bioreactor is maintained by transferring the culture fluid from the first chamber of the first bioreactor to the second chamber of the one or more second bioreactors, for example, using pumps and / or valves, when the volume for the first bioreactor exceeds a target volume. In certain embodiments, the cell density in the first and / or the one or more second bioreactors is maintained. Any suitable sensor for cell density can be used (as disclosed herein). In certain embodiments, the sensor is an optical density sensor, and the method comprises measuring the optical density of the cell culture and diluting the cell culture by adding a liquid such as a nutrient solution and / or withdrawing a portion of the cell culture fluid to maintain a target optical density. In certain embodiments, the culture conditions in the one or more second bioreactors are optimized with respect to the production of the one or more culture products.In certain embodiments, the method includes adding one or more inducer molecules to a culture of one or more second bioreactors to induce the activity of a biochemical pathway that produces a culture output (i.e., a culture product). In preferred embodiments, the inducer molecules are not added to the culture of the first bioreactor. In certain embodiments, the growth rate of the culture within the second bioreactor is limited by a nutrient, and the nutrient maintains a doubling rate that is at least 2, 3, 5, 10, 15, 20, 50, 100 times slower than that of the first bioreactor, preferably 10 to 100 times slower. In certain embodiments, the method further includes collecting / recovering one or more culture products from the culture broth.

[0106] A. Turbidostat and Chemostat There are two ways to operate a bioreactor in continuous culture: the turbidostat and the chemostat. In both cases, nutrients and other chemicals are added to the cell culture at a ratio called the dilution rate. The dilution rate is determined by feedback from sensors within the culture vessel to maintain the desired parameters.

[0107] A turbidostat (Figure 1) is a continuous bioreactor in which the cell density is maintained constant. Additionally or alternatively, the culture volume of the turbidostat can also be kept constant. The cell density or biomass is determined by proxy measures such as turbidity or capacitance. Once the cells reach a steady state, the growth rate becomes constant if the cell density is maintained. In this system, nutrients can be maintained in excess to achieve the maximum or desired growth rate. The maximum growth rate varies depending on the medium formulation and process conditions. In certain embodiments, while culturing a cell culture under turbidostat mode, the growth rate of the culture is maximized and / or set to a desired rate, and as a result, the bioproduction of the culture product is restricted. In certain embodiments, no culture product is produced while culturing a cell culture under turbidostat mode. In certain embodiments, the turbidostat is connected to a volume sensor, and when the volume exceeds the set point, the culture is withdrawn from the turbidostat to maintain both turbidity and volume constant.

[0108] A chemostat is a type of continuous bioreactor that maintains a steady state of culture conditions. The culture conditions are maintained by continuously adding medium to the culture to maintain the concentration of nutrients and other chemicals, and simultaneously removing the culture broth to keep the culture volume constant. The growth rate can be physically adjusted by changing the rate of addition of chemicals to the chemostat and also by changing the rate of removal of the culture broth. The culture conditions can be set to favor or optimize the production of the culture product. Such conditions generally divert the cell machinery away from cell growth and towards the reduction of the culture product.

[0109] In certain embodiments, the method includes operating a first bioreactor in turbidostat mode and one or more second bioreactors in chemostat mode.

[0110] B. Culture Conditions In either the turbidostat mode or the chemostat mode, it is possible to control many different culture conditions.

[0111] 1. Cell density Cell density can be maintained as follows. Select a set point for the cell density. Determine the cell density measurement value from a sensor within the bioreactor. For example, a spectrometer can measure the turbidity of a cell culture. If the cell density exceeds the set point, add culture medium to the culture chamber. If the cell density is below the set point, stop adding the culture medium until the cell density exceeds the set point due to cell growth. In certain embodiments, the OD of the first bioreactor is at least about 0.5, 0.6, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, or 200, and / or at most about 0.6, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, or 50, for example 1 - 50, 1 - 20, 1 - 10, 50 - 400, 50 - 300 or 100 - 200. In certain embodiments, the OD of the second bioreactor is at least about 0.5, 0.6, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, or 250, and / or at most about 0.6, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300, for example 1 - 300, preferably 50 - 300, more preferably 50 - 200.

[0112] Cell density can be affected by adding liquid to the bioreactor vessel to dilute the cell culture. The dilution rate for continuous culture can be modeled using the equation D = F / V -1 and is here, the dilution rate (D; [hr-1 ) is a function of the flow of the medium (F; [L / hr -1 ) into the bioreactor and the culture volume (V; [L]) in the bioreactor. In certain embodiments, in order to reach a steady state of the culture, the dilution rate (D) needs to be equal to the specific growth (μ) of the cell culture, thereby keeping the biomass concentration in the bioreactor constant. In certain embodiments, the method includes a control system with one or more sensors and effectors that actively maintain the cell density of one or more bioreactors. In certain embodiments, the cell density of the bioreactor is input into a user programmable module, and the control system actively maintains the cell density of one or more bioreactors. In certain embodiments, maintaining the cell density includes adding additional liquid, such as a nutrient solution, and / or removing the culture medium. In a preferred embodiment, maintaining the cell density includes both adding additional liquid and removing the cell culture. As described herein, a pump can be used as an effector to move liquid into and out of the bioreactor.

[0113] 2. Culture volume The culture volume can be maintained in one or both of the turbidostat mode and the chemostat mode.

[0114] Since the mass increases or decreases in response to the increase or decrease in volume, the mass of the bioreactor is a proxy for the volume of the cell culture. If the cell culture becomes too large, this system can reduce the volume of the cell culture by removing the cell culture from the bioreactor. This can be done using a pump or a mechanical device such as a top off tube.

[0115] In certain embodiments, the method includes a control system comprising one or more sensors and effectors that actively maintain the culture volume of one or more bioreactors. In certain embodiments, the culture volume of the bioreactor is input into a user programmable module, and the control system actively maintains the culture volume of one or more bioreactors. In certain embodiments, maintaining the culture volume includes adding additional liquid, such as a nutrient solution, and / or removing the culture fluid. In certain embodiments, maintaining the culture volume includes both adding additional liquid and removing the cell culture. As described herein, a pump can be used as an effector to move liquid into and out of the bioreactor.

[0116] 3. Temperature Temperature can affect both the growth rate of cells in the culture and their product production. Thus, temperature control is a culture condition that can be controlled using any suitable control system as described above in the methods described herein. The temperature of one or more bioreactors of a multi-chamber cell culture system is maintained at any suitable temperature, such as at least about 16, 18, 20, 22, 24, 26, 28, 30, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80 or 85 °C, and / or at most 22, 24, 26, 28, 30, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85 or 90 °C, such as 15 - 90 °C, preferably 20 - 50 °C, more preferably 25 - 40 °C.

[0117] 4. pH pH indicates the acidity or alkalinity of the culture. It can be controlled by adding a solution containing an acid or a base to the culture. The pH of a cell culture can be adjusted using a pump system controlled by a feedback system connected to a pH sensor, where the pump system delivers a calculated amount of the appropriate acid or base as needed and / or titrates the amount of acid or base until the sensor reads the desired pH.

[0118] 5. Dissolved O2 Dissolved oxygen is required for the growth of aerobic cells and microorganisms. It can be controlled using an aeration system that introduces air into the cell culture. In certain embodiments, the method involves controlling using an aeration system comprising a pump and / or valve in communication with one or more gas sources (e.g., canisters or the atmosphere) that deliver air into the interior of one or more culture chambers. Dissolved carbon dioxide can be controlled by discharging the used gas containing CO2 when the aeration system delivers fresh gas into the culture chamber.

[0119] In certain embodiments, the method involves maintaining the desired gas concentration of one or more gases. In certain embodiments, the gas concentration in the cell culture is measured using a suitable sensor, and a control system with a feedback mechanism operates one or more aeration systems comprising one or more gas pumps and / or valves in communication with one or more gas sources (e.g., canisters or the atmosphere), and the pump system delivers a calculated amount of gas into the interior of one or more culture chambers. In certain embodiments, one or more gases (e.g., CO2) are discharged when the aeration system delivers fresh gas into the culture chamber.

[0120] 6. Nutrient Concentration The concentration of nutrients can be extremely important for the growth rate. To maintain the maximum growth rate, the concentration of nutrients in the cell culture needs to be set so as not to limit the growth rate.

[0121] However, when producing a product, it is desirable to set culture conditions that prioritize product production. In such situations, it is desirable to lower the concentration of one or more nutrients. For example, cells cultured in turbidostat mode typically grow at a high growth rate and require high levels of nutrients. However, when they are transferred to a bioreactor operating in chemostat mode, it may be necessary to lower the concentration of one or more nutrients, such as micronutrients, so that cell physiology is directed towards the production of the culture product. In certain embodiments, the growth rate of the culture in the second bioreactor is maintained under conditions that limit growth (e.g., do not grow or grow very slowly). This can be, for example, a doubling rate less than any of 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 5 days, 6 days, 7 days, 2 weeks, or 1 month. In other embodiments, the culture is maintained at a doubling rate of at least any one of about 72, 48, 24, 12, 6, or 3 hours. In certain embodiments, the growth rate of the culture in the second bioreactor is maintained at a doubling rate that is at least 2, 3, 5, 10, 20, 50, or 100 times slower than the growth rate of the first bioreactor.

[0122] In certain embodiments, the method includes maintaining a desired nutrient concentration of one or more nutrients. In certain embodiments, the nutrient concentration in the cell culture is measured using a suitable sensor, and a control system with a feedback mechanism operates one or more pump systems that are in fluid communication with one or more nutrient reservoirs, and the pump systems deliver a calculated amount of nutrient solution as needed and / or titrate the amount of nutrient-containing liquid until the sensor reads the desired nutrient concentration.

[0123] C. Culture Product The culture output can be any measurable characteristic of the culture to be optimized. The culture output includes, for example, cell biomass, molecular products, and proxies for cell health based on product production or cell physiology. In certain embodiments, the method further includes collecting / recovering one or more culture products.

[0124] In certain embodiments, the culture output is the biomass of the cells themselves. In this case, the culture output to be optimized can be the cell growth rate. The cell growth rate can be measured as a function of the change in turbidity over time.

[0125] In certain embodiments, the culture output is a chemical product produced by the cells (a "culture product"). A culture product can be a molecular entity that is the product of fermentation or gene expression, which is typically a product that is collected from the culture and commercialized. Culture products contemplated herein include polypeptides, such as proteins, enzymes, antibodies (e.g., monoclonal antibodies), vaccines, extracellular vesicles, and recombinant pharmaceutical proteins (e.g., hormones, growth factors, enzymes, and cytokines). Culture products also include organic molecules that are the products of synthetic pathways in cells, mediated, for example, by enzymes. Such products include, for example, industrial chemicals. These include flavorings (e.g., vanillin); fragrances (e.g., aldehydes, coumarin, indole), amino acids, organic acids (e.g., citric acid, lactic acid, and acetic acid); alcohols (e.g., ethanol, isopropanol, ketones such as acetone); fatty acids (e.g., palmitic acid and oleic acid), but are not limited thereto. The product output can be measured directly as a concentration or as a function of an amount, for example, as a volumetric production rate, product titer, product yield, or specific production rate.

[0126] In certain embodiments, the culture output is a proxy for output generation (a "culture proxy"), e.g., based on cell physiology. A culture proxy is a measurable parameter indicative of the health and / or physiological function of a cell culture. These include, but are not limited to, specific CO2 production rate, specific O2 consumption rate, organic acid profile, metabolite profile, byproduct profile, production economics (cost to produce 1 kg of product under specific cell culture conditions). Specific rates can be defined as the rate of change of a compound per cell per hour, or volumetric production rate can be defined as the rate of change of a compound per liter of fermentation medium per hour.

[0127] D. Cell Growth in the First Bioreactor In the first bioreactor, the cells are cultured under culture conditions adapted for continuous cell growth and minimal or no production of the culture product.

[0128] The continuous growth rate can include a growth rate that is maintained constant over the period until the cells are transferred to the second culture vessel. The growth rate can be measured as a function of the doubling time. The growth rate can be the maximum growth rate. However, it may also be desirable to be less than the maximum growth rate. Thus, the growth rate can include any rate that is at least 1%, 2%, 4%, 8%, 16%, 32%, 50%, 60%, 70%, 80%, or 90% of the maximum growth rate, e.g., a rate from 10% to 100% of the maximum growth rate.

[0129] In certain embodiments, the cells are cultured at a constant density and can be cultured under conditions optimized for a high or desirable growth rate. This includes, for example, the use of a cell culture medium in which no nutrient is at a growth-limiting concentration.

[0130] In other embodiments, the culture medium contains one or more nutrients, such as micronutrients, that are present in an amount in the first bioreactor that allows cell growth, but do not support cell growth after transfer to one or more second production bioreactors. For example, the amount of nutrients present in the first bioreactor is such that after transfer, the remaining nutrients are rapidly consumed and the cells reach a steady state based on the amount of nutrients supplied to the second bioreactor.

[0131] In certain embodiments, the methods provided herein include maintaining a constant growth rate in cell culture. Any suitable method for maintaining a constant growth rate can be used, for example, one or more reagents / substrates / nutrients can be limited and / or the temperature can be adjusted. In certain embodiments, the growth rate is any of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% of the maximum cell growth rate. In certain embodiments, when one or more reagents / substrates / nutrients are limiting, the growth rate is at most 50, 60, 70, 80, 90, 95, or 100% of the maximum cell growth rate compared to a culture lacking one or more reagents / substrates / nutrients. In certain embodiments, the cell growth rate is maintained without nutrient limitation. In certain embodiments, the cell growth rate is neither limited nor controlled.

[0132] The production of the culture product in the first bioreactor can be made less than that in the second bioreactor. For example, the amount of the product produced in the first bioreactor per unit volume can be less than any of 50%, 40%, 30%, 20%, 10%, 5%, 3%, or 1% of the amount for the second bioreactor. "Minimal" production of the culture product means less than commercially viable production. For example, the cost to produce the product does not exceed twice the value of the product produced. This amount can be, for example, 5% of the amount when the cells are cultured under conditions optimized for the product, or less than 0.05 moles per liter per hour. The production of the culture product can be measured in grams of product per gram of dry cell weight per hour. The production of the culture product can be suppressed by appropriately setting the culture conditions. For example, if the product is produced via an induction process, the culture is not exposed to the inducer. Further, by maintaining low or no production, the provided method contributes to the stability of the strain by reducing the metabolic burden on the primary strain and decreasing the probability of the occurrence of mutant strains.

[0133] In certain embodiments, the culture broth from the first bioreactor containing cells is periodically transferred to the second bioreactor. This can be the case, for example, when the volume of the cell culture exceeds a set point. In that case, the liquid removed from the culture chamber is offset by the culture medium added to that chamber.

[0134] E. Cell Growth in the Second Bioreactor In the second bioreactor(s), the cells can be cultured under culture conditions for producing the desired culture product. Such conditions can be conditions that are favorable for the production of the culture product or conditions optimized with respect to the production of the culture product. The cells in the second continuous culture have a slower growth rate than in the first bioreactor. The growth rate of the cells in the second bioreactor can be at most 50%, 40%, 30%, 20%, 10%, 5%, 3%, 1%, 0.5%, or 0.1% of the growth rate for the first bioreactor.

[0135] The culture conditions can be growth - limiting. For example, the culture can contain growth - limiting concentrations of one or more nutrients. A growth - limiting concentration is a concentration that prevents cells from growing at their maximum growth rate. The growth - limiting doubling time of cells in the second bioreactor can be at least any of 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 2 weeks, or 1 month. Alternatively, the growth - limiting growth rate can be at most 50%, 40%, 30%, 20%, 10%, 5%, 3%, 1%, 0.5%, or 0.1% of the maximum growth rate of the cells. Under conditions that limit growth, the chance of occurrence and growth of unwanted mutant microorganisms is reduced. Thereby, the second bioreactor can continue to produce the product over a longer period. Further, by maintaining a slow growth rate in the second continuous culture, the provided method contributes to the stability of the strain by reducing the probability that a mutant strain or contaminant overgrows the parental strain.

[0136] In the second bioreactor(s), the cells are usually cultured under certain culture conditions that produce the desired culture product (e.g., optimized for its production). The optimal conditions for producing a certain product are specific to that product and the cells that produce it. Methods for determining the optimal culture conditions include, for example, varying culture parameters variously to identify the value or set point of the parameter that optimizes the production of the product. Culture parameters to be optimized include any of those described herein, such as temperature, pH, dissolved oxygen, and the concentration of nutrients (carbon source, nitrogen source, minerals, etc.), but are not limited thereto.

[0137] In certain embodiments, the methods provided herein include maintaining a constant growth rate in the second cell culture. Any suitable method for maintaining a constant growth rate can be used, for example, one or more reagents / substrates / nutrients can be limited and / or the temperature can be adjusted.

[0138] In the second bioreactor, one or more nutrients are typically at growth-limiting concentrations and are also at a concentration that is relatively lower than that in the first bioreactor. For example, the nutrient(s) can be present in the first bioreactor at a concentration (e.g., molar concentration) that is at least 2-fold, 4-fold, 10-fold, 25-fold, 50-fold, 100-fold, or 500-fold higher than the concentration of the nutrient(s) in the second bioreactor.

[0139] In one aspect, the relative concentration of the rate-limiting nutrient(s) can be normalized with respect to the respective amount of carbon in each of the first and second bioreactors. This can be useful when the concentration of carbon in the second bioreactor is not self-limiting. For example, the relative amount of carbon:rate-limiting nutrient (e.g., phosphorus) is 100:1 in the first bioreactor but 1000:1 in the second bioreactor, and the normalized ratio is 1:10. Thus, the normalized ratio of carbon:rate-limiting nutrient in the first continuous culture to carbon:rate-limiting nutrient in the second continuous culture can be any of at least 1:5, 1:10, 1:50, 1:100, 1:500, 1:1000, 1:5000, or 1:10,000.

[0140] In certain aspects, the methods provided herein include maximizing the bioproduction of one or more culture products in the second bioreactor. In certain aspects, the bioproduction occurs in one or more second bioreactors for any of at least about 10, 20, 30, 40, 80, 200, 250, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 hours, and / or for any of at most about 20, 30, 40, 80, 200, 250, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 hours, e.g., for 1 to 10,000 hours, preferably for 50 to 1,000 hours.

[0141] 1. Cell recycling The cell culture in one or more second bioreactors is typically maintained at a constant volume. Therefore, when other liquids such as the culture medium and reagent solution from the first bioreactor are added to the second bioreactor, it is necessary to remove the culture medium from the second bioreactor. The liquid thus removed contains the cells being cultured in the second bioreactor. In certain embodiments, the cells are returned to the second bioreactor. One way to do this is to use a filter such as a hollow fiber cartridge. The liquid that has flowed into one end of the hollow fiber cartridge is forced through the filter. However, the cells are too large to pass through the filter. As a result, the liquid containing the cells is returned to the second bioreactor. The cell-depleted liquid can be stored in a container for product collection.

[0142] F. Product Collection The culture medium in one or more (optionally, depending on the metabolic pathway of the cell culture, the first bioreactor) of the second bioreactors contains the culture products for collection. In certain embodiments, the method further includes collecting / recovering one or more culture products. In certain embodiments, when the culture medium is withdrawn from the chambers of one or more second bioreactors and the cells are separated from the culture medium, a cell-free broth is obtained. In certain embodiments, the cell-free broth is continuously supplied to a reservoir using a cell recycling system.

[0143] The isolation of the product from the culture medium depends on the nature of the product. For example, if the product is a protein, it can be isolated on an affinity column, including a solid support derivatized with an antibody that binds to the protein, for example. Any suitable chromatography and / or separation method can be used, for example, size exclusion, ion exchange, or affinity chromatography can be used. Small molecule products can be isolated from the culture medium using chromatography, for example, preparative HPLC, distillation, and / or crystallization. Any suitable method can be used.

[0144] Exemplary Embodiments 1. a) Under culture conditions that result in cell growth, performing a first continuous culture of cells in a chamber of at least one first bioreactor; b) During the first continuous culture, transferring the culture fluid containing the cells from the chamber of the at least one first bioreactor into the chamber of the at least one second bioreactor via one or more fluid conduits that place the chamber of the at least one first bioreactor in fluid communication with the chamber of the at least one second bioreactor; c) Under culture conditions that produce at least one culture product, performing a second continuous culture of the cells transferred from the at least one first bioreactor in the chamber of the at least one second bioreactor; A method comprising the steps above, wherein the rate of cell growth in the first continuous culture is faster than the rate in the second continuous culture, and the production of the culture product in the first continuous culture is less than the production in the second continuous culture.

[0145] 2. d) Recovering at least one culture product from the chamber of the at least one second bioreactor; The method of embodiment 1, further comprising this step.

[0146] 3. The method of embodiment 1, wherein the rate of cell growth in the first continuous culture is at least twice the rate for the second continuous culture, and the production of the culture product in the second continuous culture is at least twice the production for the first continuous culture.

[0147] 4. The method of embodiment 1, wherein the rate of cell growth in the first continuous culture is at least ten times the rate for the second continuous culture, and the production of the culture product in the second continuous culture is at least ten times the production in the first continuous culture.

[0148] 5. The method of aspect 1, wherein the rate of cell growth in the first continuous culture is at least 100 times the rate for the second continuous culture, and the production of the culture product in the second continuous culture is at least 100 times the production for the first continuous culture.

[0149] 6. The method of aspect 1, wherein the rate of cell growth in the first continuous culture is at least 80%, such as at least 90%, of the maximum growth rate.

[0150] 7. The method of aspect 1, wherein the production of the culture product in the second continuous culture is at least 80%, such as at least 90%, of the optimal culture product production rate.

[0151] 8. The method of aspect 1, comprising maintaining the cell density in the at least one first bioreactor.

[0152] 9. The method of aspect 8, wherein maintaining the cell density in the at least one first bioreactor comprises maintaining the optical density for the first continuous cell culture.

[0153] 10. The method of aspect 8, wherein maintaining the cell density comprises adding a nutrient solution to the first continuous culture.

[0154] 11. The method of aspect 8, wherein performing the first continuous culture further comprises maintaining a constant cell culture volume within the chamber of the at least one first bioreactor.

[0155] 12. The method of aspect 11, wherein maintaining the volume comprises transferring a culture solution containing the medium and cells from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor when the volume for the first continuous culture exceeds the target volume.

[0156] 13. The method of aspect 1, wherein moving the culture medium from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor includes pumping the culture medium out.

[0157] 14. The method of aspect 1, including growing the cells in the chamber of the at least one first bioreactor at a constant cell growth rate.

[0158] 15. The method of aspect 1, wherein the growth rate of the cells in the first continuous culture is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the maximum cell growth rate.

[0159] 16. The method of aspect 1, wherein none of the nutrients in the first continuous culture are growth-limiting.

[0160] 17. The method of aspect 1, wherein at least one nutrient, such as carbon or phosphorus, in the first continuous culture is at a growth-limiting concentration.

[0161] 18. The method of aspect 1, wherein the first continuous cell culture produces no culture product or produces a minimal amount of culture product.

[0162] 19. The method of aspect 1, wherein the production rate of the culture product per unit volume in the first continuous cell culture is less than any of 50%, 40%, 30%, 20%, 10%, 5%, 3%, or 1% of the production rate for the second continuous culture.

[0163] 20. The method of aspect 1, wherein the amount for the first continuous culture is less than the amount for the second continuous culture, for example, the ratio of the relative amounts is less than any of 1:1, 1:10, 1:100, and 1:1000, for example, about 1:10 to 1:50.

[0164] 21. The method of embodiment 1, wherein the volume for one or both of the first continuous culture and the second continuous culture is from about 50 mL to about 100,000 L, such as from about 500 mL to about 10 L.

[0165] 22. The method of embodiment 1, wherein the cells are transferred from the chamber of the at least one first bioreactor during growth, such as during logarithmic growth.

[0166] 23. The method of embodiment 1, wherein the culture fluid containing the cells is continuously transferred from the chamber of the at least one first bioreactor into the chamber of the at least one second bioreactor for a period of at least 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months.

[0167] 24. The method of embodiment 1, wherein the culture fluid containing the cells is transferred from the chamber of the at least one first bioreactor into the chamber of the at least one second bioreactor in a total volume of at least 0.01 liter, 0.1 liter, 1 L, 2 L, 5 L, 10 L, 50 L, 100 L, 500 L, 1,000 L, 2,000 L, 5,000 L, 10,000 L, 20,000 L, 50,000 L, 100,000 L, 500,000 L, 1,000,000 L, 5,000,000 L, 10,000,000 L, 50,000,000 L, 100,000,000 L and 1,000,000,000 L.

[0168] 25. The method of embodiment 1, wherein the culture fluid containing the cells is transferred from the chamber of the at least one first bioreactor into the chamber of the at least one second bioreactor when the optical density (O.D.) for the first continuous culture reaches at least one of 1, 5, 10, 25, 50, 100, 200, and 400.

[0169] 26. The method of embodiment 1, wherein the growth rate of the cells in the second continuous culture is at most 50%, 40%, 30%, 20%, 10%, 5%, 3%, or 1% of the growth rate for the first continuous culture.

[0170] 27. The method of embodiment 1, wherein the growth rate of the cells in the second continuous culture is at most 50%, 40%, 30%, 20%, 10%, 5%, 3%, or 1% of the maximum growth rate of the cells under non-limiting culture conditions.

[0171] 28. The method of embodiment 1, wherein the cells do not grow in the second continuous cell culture.

[0172] 29. The method of embodiment 1, wherein at least one nutrient in the second continuous culture is present at a concentration that limits the growth rate.

[0173] 30. The method of embodiment 29, wherein the concentration of the nutrient in the first continuous culture is at least 2-fold, 4-fold, 10-fold, 25-fold, 50-fold, 100-fold, or 500-fold higher than the concentration of the nutrient in the second continuous culture.

[0174] 31. The culture conditions of the second continuous culture include growth-limiting concentrations of one or more nutrients (e.g., metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdate, selenite, and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid(s), thiamine, biotin, cyanocobalamin, folic acid), salts, phosphates, sulfates, chlorides, acetates, citrates, and other anion salts, magnesium, calcium, sodium, potassium, ammonium, and other cation salts, boric acid, choline, ascorbic acid, lipoic acid, nicotinic acid, inositol, amino acids (e.g., glutamic acid, leucine, and tryptophan), nucleobases (e.g., adenine, cytosine, thymine, uracil, and guanine), or complex nutrients (e.g., yeast extract, peptone, tryptone, casamino acids, and corn steep liquor)) selected from the group consisting of:

[0175] 32. The method of embodiment 1, wherein the culture conditions in the second continuous culture are maintained constant for any one of at least 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

[0176] 33. The method of embodiment 1, wherein the second continuous culture is carried out for any one of at least about 10, 20, 30, 40, 80, 200, 250, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 hours, and / or for any one of at most about 20, 30, 40, 80, 200, 250, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 hours, for example, 1 to 10,000 hours, preferably 50 to 1,000 hours.

[0177] 34. The method of embodiment 1, including carrying out the second continuous culture in a plurality of different second bioreactors.

[0178] 35. The method of embodiment 1, wherein the at least one first bioreactor is a plurality of first bioreactors.

[0179] 36. The method of embodiment 1, wherein the culture conditions of the second continuous culture are optimized with respect to the production of at least one culture product.

[0180] 37. The method of embodiment 1, wherein the culture conditions of the second continuous culture are different from the culture conditions of the first continuous culture.

[0181] 38. The method of embodiment 1, including maintaining one or more of a target pH, a target temperature, a target dissolved oxygen content, a target carbon concentration, and a target nitrogen concentration in the first and / or second continuous culture.

[0182] 39. The method of aspect 1, wherein the culture conditions of the first and / or second continuous culture include maintaining a constant concentration of one or more nutrients in the culture.

[0183] 40. The method of aspect 1, wherein in the second continuous culture, the activity of a biochemical pathway that generates a culture output is induced, but not in the first continuous culture.

[0184] 41. The method of aspect 1, wherein the second continuous culture has an OD of at least one of 10, 50, 100, or 1000.

[0185] 42. The method of aspect 1, wherein the cells in the second continuous culture have a doubling time of at most one of 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 5 days, 6 days, 7 days, 2 weeks, or 1 month.

[0186] 43. The method of aspect 1, wherein the cells in the second continuous culture are maintained at a constant density.

[0187] 44. The method of aspect 1, wherein the cells removed from the chamber of the at least one second bioreactor are recycled to the chamber by a cell recycling device such as a hollow fiber filter.

[0188] 45. Performing either the first or the second continuous culture involves supplying one or more of the following: metal (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdate, selenite, and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid(s), thiamine, biotin, cyanocobalamin, folic acid), anti - contamination agents such as antibiotics or biocides, inducers, salts, phosphates, sulfates, chlorides, acetates, citrates, and other anionic salts, magnesium, calcium, sodium, potassium, ammonium, and other cationic salts, boric acid, choline, ascorbic acid, lipoic acid, nicotinic acid, inositol, antifoaming agents (e.g., Antifoam 204, Antifoam A, Antifoam C), amino acids (e.g., glutamic acid, leucine, and tryptophan), nucleobases (e.g., adenine, cytosine, thymine, uracil, and guanine), complex nutrients (e.g., yeast extract, peptone, tryptone, casamino acids, and corn steep liquor), macronutrients, micronutrients, and cell growth factors, according to the method of aspect 1.

[0189] 46. Performing either the first or the second continuous culture involves supplying a carbon source (e.g., sugars (e.g., glucose, xylose, sucrose, glycerol, or acetate), molasses, malt extract, starch, dextrin, pulp, CO or CO2), according to the method of aspect 1.

[0190] 47. Performing either the first or the second continuous culture involves supplying one or more of the following nitrogen sources: amino acids or polypeptides, urea, ammonium salts (e.g., ammonium sulfate, ammonium phosphate, or ammonia), corn steep liquor, yeast extract, peptone, and soybean meal, according to the method of aspect 1.

[0191] 48. Performing either the first or the second continuous culture involves controlling one or more of the nitrogen sparging rate, aeration rate, oxygen sparging rate, carbon dioxide sparging rate, culture agitation rate, CO2 concentration, carbon source concentration, nitrogen source concentration, metal, vitamin, salt concentration, and antibiotic concentration, according to the method of aspect 1.

[0192] 49. The step of recovering the at least one culture product includes withdrawing the cell culture fluid from the chamber of the at least one second bioreactor and separating the cells from the cell culture fluid to obtain a cell-free broth, according to the method of aspect 1.

[0193] 50. The culture product is selected from polypeptides (e.g., proteins, enzymes, antibodies), organic molecules that are products of synthetic pathways in cells (e.g., flavorings (e.g., vanillin), fragrances (e.g., aldehydes, coumarin, indole), amino acids, organic acids (e.g., citric acid, lactic acid, and acetic acid), alcohols (e.g., ethanol, isopropanol, ketones such as acetone), and industrial chemicals such as fatty acids (e.g., palmitic acid, and oleic acid)), according to the method of aspect 1.

[0194] 51. The cells include archaea, prokaryotic cells, and / or eukaryotic cells, according to the method of aspect 1.

[0195] 52. The method of embodiment 1, wherein the cell is a fungal cell (e.g., yeast (e.g., species of Saccharomyces spp, Pichia spp, Komagataella spp, Kuyveromyces spp, Aspergillus spp, Rhodoporidium spp, Lipolytica spp, Aspergillus spp, Neurospora spp, Trichoderma spp, Candida spp, or Penicillium)).

[0196] 53. The method of embodiment 1, wherein the cell is a bacterial cell (e.g., Escherichia coli, species of Bacillus spp, Costridia spp, Streptomyces spp, Pseudomonas spp, Ralstonia spp, Shewanella spp).

[0197] 54. The method of embodiment 1, wherein the cell comprises an insect cell, an animal cell or a plant cell.

[0198] 55. The method of embodiment 1, wherein the cell is an animal cell (e.g., an arthropod (e.g., an insect, a shrimp, a lobster, a crayfish, and a crab), a chordate (e.g., a fish, an amphibian, a reptile, a bird (e.g., a chicken or a pigeon), a mammal (e.g., a human, or a non-human animal such as a cow, a sheep, a goat, a pig, a horse, a dog, a cat, a primate, etc.)).

[0199] 56. The method of aspect 1, wherein the cells comprise a cell line (e.g., CHO (Chinese hamster ovary cells), BHK21 (baby hamster kidney), NS0, Sp2 / 0 mouse cell line, insect cells (e.g., SP9, Sf9, sf21, S2), tobacco BY-2 cells, Oryza Sativa, or algal cells).

[0200] 57. The method of aspect 1, wherein the cells are not photosynthetic cells.

[0201] 58. (a) (i) At least one first bioreactor comprising a first chamber, and (ii) one or more first sensors that provide measurements of at least cell density and cell culture volume within the first chamber ; and (b) (i) At least one second bioreactor comprising a second chamber, and (ii) one or more second sensors that provide measurements of at least culture conditions and cell culture volume within the one or more second chambers wherein the first chamber is in fluid communication with the one or more second chambers; (c) One or more reagent reservoirs containing a liquid reagent, the reagent reservoir being in fluid communication with the first chamber and optionally the one or more second chambers; (d) (i) Move the liquid reagent from at least one reagent reservoir to the first chamber; (ii) Move the cell culture from the first chamber to the one or more second chambers; (iii) Move the cell culture out of the one or more second chambers; (iv) Optionally, move the liquid reagent from at least one reagent reservoir to the one or more second chambers and one or more pumps configured to; (e) (i) Using the measurements from the one or more sensors, control the cell growth, e.g., cell density, of the cell culture in the first chamber at a higher rate than the ratio for the second chamber; (ii) Transfer the cell culture medium from the first chamber to the one or more second chambers; and (iii) Using the measurements from the one or more sensors, control the culture conditions in the one or more second chambers and the cell culture, e.g., volume and / or cell growth, to produce a culture product at a higher rate than the ratio for the first chamber A control system configured as such and A system comprising.

[0202] 59. The system of aspect 58, wherein the control system uses the measurements from the one or more sensors to establish that the cell growth rate in the first continuous culture is at least 2 times, at least 10 times, or at least 100 times the cell growth rate for the second continuous culture, and the production rate of the culture product in the second continuous culture is at least 2 times, at least 10 times, or at least 100 times the production rate for the first continuous culture.

[0203] 60. The system of aspect 58, further comprising one or more recovery containers in fluid communication with the one or more second chambers.

[0204] 61. The system of aspect 58, wherein the first chamber and the one or more second chambers have a volume of 250 ml to 1,000,000 liters.

[0205] 62. The system of aspect 58, wherein the control system sets the dilution rate of the liquid reagent transferred into the first chamber and / or the one or more second chambers to maintain the concentration of one or more nutrients.

[0206] 63. The system of aspect 58, wherein the first chamber and the one or more second chambers comprise ports that communicate with a reagent reservoir via fluid conduits and with each other.

[0207] 64. The system of aspect 58, wherein the one or more second chambers are a plurality of second chambers.

[0208] 65. The system of aspect 58, wherein one or more first or second sensors that provide volume measurements include a scale that measures the mass of the bioreactor.

[0209] 66. The system of aspect 58, wherein a sensor that provides a cell density measurement of the cell culture within the first chamber includes an optical density sensor or a capacitance sensor.

[0210] 67. The system of aspect 65, wherein controlling the cell density includes controlling the optical density of the cell culture.

[0211] 68. The system of aspect 66, including a user programmable module that calculates a dilution rate as a function of the error between a target optical density and a measured optical density.

[0212] 69. The system of aspect 58, wherein the one or more first sensors provide measurements of the temperature, pH, and dissolved oxygen of the cell culture within the first chamber.

[0213] 70. The system of aspect 58, wherein the one or more second sensors provide measurements of the temperature, pH, and dissolved oxygen of the cell culture within the second chamber.

[0214] 71. The system of aspect 58, wherein at least one pump is a peristaltic pump or a gravity pump.

[0215] 72. The system of aspect 70, wherein the peristaltic pump comprises a fitting for a tube that fluidly communicates between two chambers, between a chamber and a reagent reservoir, or between the second chamber and an outflow conduit.

[0216] 73. The system of aspect 58, wherein the control system for controlling the volume of cell culture in the first chamber or the one or more second chambers includes a level sensor (e.g., an overflow tube), and the level sensor moves cell culture fluid from the chamber when the height of the cell culture exceeds the upper end of the level sensor.

[0217] 74. The system of aspect 58, wherein the control system includes a computer comprising a processor and a memory containing executable code, and when the executable code is executed by the processor, one or more feedback routines for controlling cell growth, e.g., cell density, in the first chamber, controlling the volume of cell culture in the first chamber and / or one or more second chambers, and controlling culture conditions in the one or more second chambers. are performed.

[0218] 75. The system of aspect 73, wherein one of the feedback routines calculates the cell density of the cell culture in the first chamber based on measurements received from the one or more sensors and operates a pump to move a liquid reagent from a reagent reservoir to the first chamber to adjust the cell density to a set point.

[0219] 76. The system of aspect 73, wherein one of the feedback routines calculates the volume of the cell culture in the first chamber based on measurements received from the one or more sensors and operates a pump to move a liquid cell culture from the first chamber to the one or more second chambers if the volume exceeds a set point.

[0220] 77. (I) The one or more sensors provide measurements of one or more culture parameters in the first and / or one or more second chambers; (II) The system further includes one or more effectors that affect changes in the one or more culture parameters; (III) One or more of the feedback routines calculate one or more culture parameters based on the measurements and activate the one or more effectors to adjust the culture parameters toward a target level. The system of aspect 73.

[0221] 78. (I) The one or more sensors provide measurements of one or more of pH, temperature, and dissolved O2 of the cell culture in the first and / or one or more second chambers; (II) The system includes one or more reagent reservoirs containing acids and bases in fluid communication with the first and / or one or more second reservoirs; one or more temperature controllers for controlling the temperature of the cell culture in the first and / or one or more second chambers; one or more aerators for delivering air to the cell culture in the first chamber and / or one or more second chambers further including one or more of; (III) One or more of the feedback routines calculate culture conditions including one or more of pH, temperature, dissolved O2, and nutrient concentration based on the measurements received from the one or more sensors, one or more pumps for moving an acid or base from the reagent reservoir into the second chamber to adjust the pH to a target pH; one or more temperature controllers for adjusting the temperature to a target temperature; and / or one or more aerators for delivering air to the cell culture to a target dissolved oxygen level and activate The system of aspect 73.

[0222] 79. A system according to aspect 73, wherein a feedback routine calculates the volume of the cell culture in the one or more second chambers based on measurements received from the one or more sensors, and if the volume exceeds a set point, actuates a pump to move liquid cell culture from the one or more second chambers.

[0223] 80. A system according to aspect 58, further comprising a filter in fluid communication with the outlet of the second chamber, the filter configured to recycle cells that cannot pass through the membrane back to the second chamber and to pass cell-depleted liquid medium out of the cartridge and into a collection vessel.

[0224] 81. A system according to aspect 79, wherein the filter comprises a hollow fiber cartridge.

[0225] 82. A system according to aspect 58, wherein the first chamber contains a cell culture, the one or more reagent reservoirs in fluid communication with the first chamber contain a growth medium, and the medium contains one or more nutrients at a concentration that limits the growth rate of the cells in the cell culture.

[0226] 83. A system according to aspect 58, wherein the culture medium is formulated such that nutrients are not limited in the at least one first bioreactor, but one or more nutrients are present in the one or more second bioreactors at a concentration that limits growth.

[0227] 84. One or more control subsystems (A) a processor; (B) a memory coupled to the processor; and (C) computer-executable instructions that use measurements from the one or more sensors to calculate culture conditions in the first chamber and / or one or more second chambers and, based on the calculations, control pumps that move liquid into and out of the chambers A system according to aspect 58, comprising a computer having the same.

[0228] 85. · A temperature sensor and a temperature regulator; · A dissolved oxygen meter and an aeration system in communication with the interior of the first chamber and / or one or more second chambers; · Analytical equipment for measuring the concentration of nutrients in the cell culture within the chamber; · An impeller or pneumatic stirrer for mixing the liquid within the chamber, and a motor configured to operate the impeller or pneumatic stirrer; · An outflow portion in communication with the interior of the container, and an adjustable valve or pump for controlling the flow of liquid from the chamber; · A baffle within the chamber; · A sparger and a mass flow controller in communication with the interior of the chamber for introducing one or more gases and mixtures thereof; · A user interface for communicating commands with a computer; · A foam control device; · A hollow fiber membrane for cell recycling; and · A reverse osmosis membrane or a forward osmosis membrane The system of aspect 58, comprising one or more of the foregoing.

[0229] 86. The system of aspect 58, wherein the control system is configured to implement any of the methods of aspects 1 - 35.

[0230] 87. a) Performing a first continuous culture of cells within the chamber of a first bioreactor under culture conditions that maintain a constant cell density; b) During the first continuous culture, transferring the culture liquid containing cells from the chamber of the first bioreactor, through one or more fluid conduits that place the chamber of the first bioreactor in fluid communication with the chamber of at least one second bioreactor, into the chamber of the at least one second bioreactor; c) To produce at least one culture product, under certain culture conditions, in each chamber of the one or more second bioreactors, perform a second continuous culture of the cells transferred from the first bioreactor; and d) Recover at least one culture product from each chamber of the one or more second bioreactors A method comprising.

[0231] 88. (a) (i) A first chamber, and (ii) One or more first sensors that provide at least cell density measurements and cell culture volume measurements within the first chamber A first bioreactor equipped with; (b) (i) A second chamber, and (ii) One or more second sensors that provide at least culture condition measurements and cell culture volume measurements within the one or more second chambers One or more second bioreactors equipped with, wherein the first chamber is in fluid communication with the one or more second chambers; (c) One or more reagent reservoirs containing a liquid reagent, the reagent reservoir being in fluid communication with the first chamber and optionally the one or more second chambers; (d) (i) Move the liquid reagent from at least one reagent reservoir to the first chamber; (ii) Move the cell culture fluid from the first chamber to the one or more second chambers; (iii) Move the cell culture fluid outside the one or more second chambers; (iv) Optionally, move the liquid reagent from at least one reagent reservoir to the one or more second chambers One or more pumps configured to; (e) (i) Controlling cell growth, e.g., cell density, of the cell culture in the first chamber using measurements from the one or more sensors; (ii) Moving the cell culture medium from the first chamber to the one or more second chambers; and (iii) Controlling the culture conditions in the one or more second chambers and, e.g., the volume and / or cell growth of the cell culture using measurements from the one or more sensors A control system configured to A system comprising.

Example

[0232] Example 1: Production of a culture product using a multi-chamber cell culture system This example demonstrates the ability to produce a culture product using a multi-chamber cell culture system (Figure 5).

[0233] Preparation of Inoculum : Bacterial colonies were streaked from a glycerol stock stored at -80°C onto an LB agar plate containing the appropriate antibiotic and cultured under appropriate conditions until visible colonies formed. A single colony from the LB agar plate was inoculated into 5 mL of growth medium containing the appropriate antibiotic and cultured overnight at 37°C with shaking at 250 rpm. The next day, 3 mL of the turbid overnight culture was inoculated into 300 mL of growth medium and incubated at 37°C with shaking at 250 rpm. When the optical density of the culture reached approximately 1 at 600 nm, the cells were ready to be inoculated into the first bioreactor. A 10 mL aliquot of cells from the 300 mL culture was stored in 15% glycerol at -80°C in a freezer in preparation for future bioreactor operation.

[0234] The following bioreactor operation was performed using a Sartorius Biostat B 1.8 L bioreactor.

[0235] Assembly and Sterilization of Bioreactor: Two bioreactors were prepared. The reactor vessel headplate was fixed to the glass container, and the following components were inserted into the vessel head ports: (1) a condenser, (2) a sparger, (3) a four-way port assembly (one port for supply, two ports for acid / base, one port for addition), (4) a sampling port, (5) an effluent port, (6) a dissolved oxygen (DO) probe, and (7) a pH probe. This assembly, together with the supply reservoir bottle, the growth medium, and the supply tube, was autoclaved at 121 °C for 30 minutes. After autoclaving, the container was cooled to room temperature, and an OD probe was attached to the appropriate vessel head port for measuring the optical density. The bioreactor was placed on the scale, and the scale was zeroed after adding the batch medium (500 - 1000 L). The base bottle was connected to both tanks. The growth medium was set on the scale attached to the first bioreactor. The effluent port of the first bioreactor was connected to the second bioreactor. The glucose reservoir was connected to the second bioreactor. The outlet from the second bioreactor was connected to a 0.2 μm hollow fiber cell recycle system, and the maximum back and forward pressure was set to less than 30 psi. The filtrate end of the 0.2 μm hollow fiber cell recycle system was connected to the recovery reservoir on the scale, and the retentate end of the 0.2 μm hollow fiber cell recycle system was connected to the inlet of the second bioreactor.

[0236] Operation of Bioreactor: After assembly, any sterilized additional reagents were added to the growth medium. All parts of the bioreactor were connected to the Biostat-B control system, and a temperature probe was inserted. This enabled the Fermwork software to receive inputs from the temperature, DO, and pH probes and to control agitation, air flow, temperature, supply pump, and base pump. The supply line and the base line were connected from their respective reservoirs to a 3-way port assembly. The pump tube parts of the lines were inserted into the appropriate pump heads, and the lines were primed by manually operating the pumps until it was confirmed that liquid was entering the bioreactor, after which the pumps were switched to automatic control. The base reservoir contained 14 - 28% ammonium hydroxide.

[0237] Subsequently, the system was stabilized using the following parameters: 37 °C, pH: 6.8; DO: 30% of saturation. Once stabilized, the first bioreactor was inoculated with an initial OD 600Using a sterilized syringe, approximately 10 mL of the seed culture was inoculated into the addition port so that [[ID=]] becomes 0.05 to 0.3. After the cells reached the mid-log phase, the turbidostat program was initiated in the first bioreactor. This made it possible to maintain a constant biomass concentration in the first bioreactor and send excess cells to the second bioreactor. To maintain a constant volume by the effluent pump rate controlled by the weight of the protein mass, the mass_stat program was initiated for the first bioreactor. The second bioreactor was initiated as a standard fed-batch reactor. The feed is triggered by a hunger spike, which is a sharp increase in dissolved O2 indicating limitation of the carbon source. After the first and second bioreactors were stabilized, the pump that transfers the effluent from the second bioreactor to the hollow fiber cell recycle system was turned on, whereby the cells were concentrated and recycled to the second bioreactor. Next, the mass_stat program was initiated in the second bioreactor, where the effluent pump to the hollow fiber is controlled by the mass of the second bioreactor tank. Optionally, a purge tank is connected to the second bioreactor via a dip tube to collect the liquid and cells that exceeded the set volume in the second bioreactor from the second bioreactor. The purge tank is placed on a balance to measure the mass of the removed purge liquid.

[0238] For analysis, samples were taken periodically from the first bioreactor, the second bioreactor, the second bioreactor effluent tank, and the second bioreactor purge tank. The OD of the samples was measured spectrophotometrically, the supernatant of the culture product was analyzed by HPLC, and the mass of the tank was recorded to calculate the production of the culture product and the consumption of the carbon source.

[0239] As used herein, unless otherwise specified, the following meanings apply. The word "may" is used in a permissive sense (i.e., having the possibility of doing) rather than a mandatory sense (i.e., having to do). The words "include", "including", "includes", etc. mean including, but not limited to. The singular forms "a", "an", and "the" include plural referents. Thus, for example, a reference to "an element" includes combinations of two or more elements, even if other terms or phrases for one or more elements, such as "one or more", are used. The phrase "at least one" includes "one", "one or more", "one or plural", and "plural". The term "or" is non-exclusive, i.e., includes both "and" and "or", unless otherwise indicated. The term "any of" between a modifier and a sequence means that the modifier modifies each member of the sequence. Thus, for example, the expression "at least any of 1, 2 or 3" means "at least 1, at least 2, or at least 3". The term "about" refers to a range of plus or minus 5% from the specified numerical value within the context of a particular usage situation. Thus, for example, "about 100" means between 95 and 105. The term "consisting essentially of" refers to including the recited elements and other elements that do not materially affect the basic and novel characteristics of the claimed combination.

[0240] This specification and the drawings are not intended to limit the invention to the particular forms disclosed, but on the contrary, are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. It should be understood that further modifications and alternative aspects of various aspects of the invention will be apparent to those skilled in the art in view of this specification. Accordingly, this specification and the drawings are to be construed as illustrative only and are intended to teach those skilled in the art a general manner of implementing the invention. It should be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be used in place of those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all of which will be apparent to those skilled in the art after benefit of this description of the invention. Changes may be made to the elements described herein without departing from the spirit and scope of the invention as set forth in the following claims. The headings used herein are for organizational purposes only and are not used to limit the scope of this description.

[0241] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

**Claim 1** a) performing a first continuous culture of cells in a chamber of at least one first bioreactor under culture conditions that result in cell growth; b) during the first continuous culture, moving the culture fluid containing the cells from the chamber of the at least one first bioreactor through one or more fluid conduits that place the chamber of the at least one first bioreactor in fluid communication with the chamber of at least one second bioreactor into the chamber of the at least one second bioreactor; c) performing a second continuous culture of the cells transferred from the at least one first bioreactor in a chamber of the at least one second bioreactor under culture conditions that produce at least one culture product A method comprising: wherein the rate of cell growth in the first continuous culture is faster than the rate in the second continuous culture, and the production of the culture product in the first continuous culture is less than the production in the second continuous culture. **Claim 2** d) recovering at least one culture product from the chamber of the at least one second bioreactor The method according to claim 1, further comprising. **Claim 3** The method according to claim 1, wherein the rate of cell growth in the first continuous culture is at least twice the rate for the second continuous culture, and the production of the culture product in the second continuous culture is at least twice the production for the first continuous culture. **Claim 4** The method according to claim 1, wherein the rate of cell growth in the first continuous culture is at least ten times the rate for the second continuous culture, and the production of the culture product in the second continuous culture is at least ten times the production in the first continuous culture. **Claim 5** The method according to claim 1, wherein the rate of cell growth in the first continuous culture is at least one hundred times the rate for the second continuous culture, and the production of the culture product in the second continuous culture is at least one hundred times the production for the first continuous culture. **Claim 6** The method according to claim 1, wherein the rate of cell growth in the first continuous culture is at least 80%, for example at least 90%, of the maximum growth rate. **Claim 7** The method according to claim 1, wherein the production of the culture product in the second continuous culture is at least 80%, for example at least 90%, of the optimal culture product production rate. **Claim 8** The method according to claim 1, comprising maintaining the cell density in said at least one first bioreactor.

9. The method according to claim 8, wherein maintaining the cell density in said at least one first bioreactor comprises maintaining the optical density for a first continuous cell culture.

10. The method according to claim 8, wherein maintaining the cell density comprises adding a nutrient solution to said first continuous culture.

11. The method according to claim 8, wherein performing said first continuous culture further comprises maintaining a constant cell culture volume within a chamber of said at least one first bioreactor.

12. The method according to claim 11, wherein maintaining the volume comprises, when the volume for said first continuous culture exceeds a target volume, moving a culture solution comprising a medium and cells from a chamber of said at least one first bioreactor to a chamber of said at least one second bioreactor.

13. The method according to claim 1, wherein moving the culture solution from a chamber of said at least one first bioreactor to a chamber of said at least one second bioreactor comprises pumping out the culture solution.

14. The method according to claim 1, comprising growing cells within a chamber of said at least one first bioreactor at a constant cell growth rate.

15. The method according to claim 1, wherein the growth rate of cells in said first continuous culture is any one of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the maximum cell growth rate.

16. The method according to claim 1, wherein none of the nutrients in said first continuous culture are growth limiting.

17. The method according to claim 1, wherein at least one nutrient, such as carbon or phosphorus, in said first continuous culture is at a growth limiting concentration.

18. The method according to claim 1, wherein the first continuous cell culture produces no culture product or produces a minimal amount of culture product.

19. The method according to claim 1, wherein the production rate of the culture product per unit volume in the first continuous cell culture is less than any one of 50%, 40%, 30%, 20%, 10%, 5%, 3% or 1% of the production rate for said second continuous culture.

20. The amount for the first continuous culture is less than the amount for the second continuous culture, for example, the ratio of the relative amounts is less than any of 1:1, 1:10, 1:100, and 1:1000, for example, about 1:10 to 1:

50. The method according to claim 1.

21. The volume for one or both of the first continuous culture and the second continuous culture is about 50 mL to about 100,000 L, for example, about 500 mL to about 10 L. The method according to claim 1.

22. During growth, for example, during logarithmic growth, the cells are transferred from the chamber of the at least one first bioreactor. The method according to claim 1.

23. The culture solution containing cells is continuously transferred from the chamber of the at least one first bioreactor into the chamber of the at least one second bioreactor for a period of at least 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. The method according to claim 1.

24. The culture solution containing cells is transferred from the chamber of the at least one first bioreactor into the chamber of the at least one second bioreactor in a total volume of at least 0.01 liter, 0.1 liter, 1 L, 2 L, 5 L, 10 L, 50 L, 100 L, 500 L, 1,000 L, 2,000 L, 5,000 L, 10,000 L, 20,000 L, 50,000 L, 100,000 L, 500,000 L, 1,000,000 L, 5,000,000 L, 10,000,000 L, 50,000,000 L, 100,000,000 L and 1,000,000,000 L. The method according to claim 1.

25. When the optical density (O.D.) for the first continuous culture reaches at least any of 1, 5, 10, 25, 50, 100, 200, and 400, the culture solution containing cells is transferred from the chamber of the at least one first bioreactor into the chamber of the at least one second bioreactor. The method according to claim 1.

26. The method according to claim 1, wherein the growth rate of the cells in the second continuous culture is at most 50%, 40%, 30%, 20%, 10%, 5%, 3%, or 1% of the growth rate for the first continuous culture.

27. The method according to claim 1, wherein the growth rate of the cells in the second continuous culture is at most 50%, 40%, 30%, 20%, 10%, 5%, 3%, or 1% of the maximum growth rate of the cells under non-limiting culture conditions.

28. The method according to claim 1, wherein the cells do not grow in the second continuous cell culture.

29. The method according to claim 1, wherein at least one nutrient in the second continuous culture is present at a concentration that limits the growth rate.

30. The method according to claim 29, wherein the concentration of the nutrient in the first continuous culture is at least 2-fold, 4-fold, 10-fold, 25-fold, 50-fold, 100-fold, or 500-fold higher than the concentration of the nutrient in the second continuous culture.

31. The method according to claim 1, wherein the culture conditions of the second continuous culture include a growth-limiting concentration of one or more nutrients (e.g., metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdate, selenite, and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid(s), thiamine, biotin, cyanocobalamin, folic acid), salts, phosphates, sulfates, chlorides, acetates, citrates, and other anion salts, magnesium, calcium, sodium, potassium, ammonium, and other cation salts, boric acid, choline, ascorbic acid, lipoic acid, nicotinic acid, inositol, amino acids (e.g., glutamic acid, leucine, and tryptophan), nucleobases (e.g., adenine, cytosine, thymine, uracil, and guanine), or complex nutrients (e.g., yeast extract, peptone, tryptone, casamino acids, and corn steep liquor)) selected from.

32. The method according to claim 1, wherein the culture conditions in the second continuous culture are maintained constant for any of at least 1 hour, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 4 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

33. The method according to claim 1, wherein the second continuous culture is carried out for any one of at least about 10, 20, 30, 40, 80, 200, 250, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 hours, and / or for any one of at most about 20, 30, 40, 80, 200, 250, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 hours, for example, 1 to 10,000 hours, preferably 50 to 1,000 hours.

34. The method according to claim 1, comprising carrying out the second continuous culture in a plurality of different second bioreactors.

35. The method according to claim 1, wherein the at least one first bioreactor is a plurality of first bioreactors.

36. The method according to claim 1, wherein the culture conditions of the second continuous culture are optimized with respect to the production of at least one culture product.

37. The method according to claim 1, wherein the culture conditions of the second continuous culture are different from the culture conditions of the first continuous culture.

38. The method according to claim 1, comprising maintaining one or more of a target pH, a target temperature, a target dissolved oxygen content, a target carbon concentration, and a target nitrogen concentration in the first and / or second continuous culture.

39. The method according to claim 1, wherein the culture conditions of the first and / or second continuous culture include maintaining a constant concentration of one or more nutrients in the culture.

40. The method according to claim 1, wherein in the second continuous culture, the activity of a biochemical pathway that generates a culture output is induced, but not in the first continuous culture.

41. The method according to claim 1, wherein the second continuous culture has an OD of at least 10, 50, 100, or 1000.

42. The method according to claim 1, wherein the cells in the second continuous culture have a doubling time of at most 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 5 days, 6 days, 7 days, 2 weeks, or 1 month.

43. The method according to claim 1, wherein the cells in the second continuous culture are maintained at a constant density.

44. The method according to claim 1, wherein cells taken out from the chamber of the at least one second bioreactor are recycled to the chamber by a cell recycling device such as a hollow fiber filter.

45.

45. Performing either the first or second continuous culture involves supplying one or more of the following: metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdates, selenites, and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid(s), thiamine, biotin, cyanocobalamin, folic acid), anti - contamination agents such as antibiotics or biocides, inducers, salts, phosphates, sulfates, chlorides, acetates, citrates, and other anionic salts, magnesium, calcium, sodium, potassium, ammonium, and other cationic salts, boric acid, choline, ascorbic acid, lipoic acid, nicotinic acid, inositol, antifoaming agents (e.g., Antifoam 204, Antifoam A, Antifoam C), amino acids (e.g., glutamic acid, leucine, and tryptophan), nucleobases (e.g., adenine, cytosine, thymine, uracil, and guanine), complex nutrients (e.g., yeast extract, peptone, tryptone, casamino acids, and corn steep liquor), macronutrients, micronutrients, and cell growth factors. The method according to claim 1.

46. Performing either the first or second continuous culture involves supplying a carbon source (e.g., sugar (e.g., glucose, xylose, sucrose, glycerol, or acetate), molasses, malt extract, starch, dextrin, pulp, CO or CO 2 ) is the method according to claim 1, which includes supplying

47.

47. Performing either the first or second continuous culture involves supplying one or more of the following nitrogen sources: amino acids or polypeptides, urea, ammonium salts (e.g., ammonium sulfate, ammonium phosphate, or ammonia), corn steep liquor, yeast extract, peptone, and soybean meal. The method according to claim 1.

48. Performing either the first or second continuous culture involves controlling one or more of the nitrogen sparging rate, aeration rate, oxygen sparging rate, carbon dioxide sparging rate, culture agitation rate, concentration of CO 2 , concentration of carbon source, concentration of nitrogen source, concentration of metal, vitamin, salt, and concentration of antibiotic. The method according to claim 1.

49. The step of recovering the at least one culture product includes withdrawing a cell culture solution from the chamber of the at least one second bioreactor and separating cells from the cell culture solution to obtain a cell - free broth. The method according to claim 1.

50. The method according to claim 1, wherein the culture product is selected from polypeptides (e.g., proteins, enzymes, antibodies), organic molecules that are products of synthetic pathways in cells (such as flavorings (e.g., vanillin), fragrances (e.g., aldehydes, coumarin, indole), amino acids, organic acids (e.g., citric acid, lactic acid, and acetic acid), alcohols (e.g., ethanol, isopropanol, ketones such as acetone), and industrial chemicals such as fatty acids (e.g., palmitic acid, and oleic acid)).

51. The method according to claim 1, wherein the cells comprise archaea, prokaryotic cells and / or eukaryotic cells.

52. The method according to claim 1, wherein the cells comprise fungal cells (e.g., yeast (e.g., species of Saccharomyces spp, Pichia spp, Komagataella spp, Kuyveromyces spp, Aspergillus spp, Rhodoporidium spp, Lipolytica spp, Aspergillus spp, Neurospora spp, Trichoderma spp, Candida spp, or Penicillium)).

53. The method according to claim 1, wherein the cells comprise bacterial cells (e.g., Escherichia coli, species of Bacillus spp, Costridia spp, Streptomyces spp, Pseudomonas spp, Ralstonia spp, Shewanella spp).

54. The method according to claim 1, wherein the cells comprise insect cells, animal cells or plant cells.

55. The method according to claim 1, wherein the cells are animal cells (for example, arthropods (such as insects, shrimps, lobsters, crayfish, and crabs), chordates (such as fish, amphibians, reptiles, birds (such as chickens or pigeons), mammals (such as humans, or non-human animals such as cows, sheep, goats, pigs, horses, dogs, cats, and primates))).

56. The method according to claim 1, wherein the cells include cell lines (for example, CHO (Chinese hamster ovary cells), BHK21 (baby hamster kidney), NS0, Sp2 / 0 mouse cell line, insect cells (such as SP9, Sf9, sf21, S2), tobacco BY-2 cells, Oryza Sativa, or algal cells).

57. The method according to claim 1, wherein the cells are not photosynthetic cells.

58. （a） (i) a first chamber, and (ii) at least one first sensor that provides measurements of at least cell density and cell culture volume within the first chamber at least one first bioreactor comprising; （b） (i) a second chamber, and (ii) at least one second sensor that provides measurements of at least culture conditions and cell culture volume within the one or more second chambers at least one second bioreactor, wherein the first chamber is in fluid communication with the one or more second chambers; (c) one or more reagent reservoirs containing liquid reagents, the reagent reservoirs being in fluid communication with the first chamber and optionally the one or more second chambers; （d） (i) moving a liquid reagent from at least one reagent reservoir to the first chamber; (ii) moving the cell culture from the first chamber to the one or more second chambers; (iii) moving the cell culture out of the one or more second chambers; (iv) optionally, moving a liquid reagent from at least one reagent reservoir to the one or more second chambers one or more pumps configured to; （e） (i) using the measurements from the one or more sensors, controlling the cell growth, such as cell density, of the cell culture within the first chamber to a higher ratio than the ratio for the second chamber; (ii) moving the cell culture from the first chamber to the one or more second chambers; and (iii) To produce a culture product at a higher rate than the rate for the first chamber, using the measurements from the one or more sensors, control the culture conditions in the one or more second chambers and, for example, the volume and / or cell growth of the cell culture A control system configured as A system comprising. **Claim 59** The system according to claim 58, wherein the control system uses the measurements from the one or more sensors to establish that the cell growth rate in the first continuous culture is at least 2 times, at least 10 times, or at least 100 times the cell growth rate for the second continuous culture, and that the production rate of the culture product in the second continuous culture is at least 2 times, at least 10 times, or at least 100 times the production rate for the first continuous culture. **Claim 60** The system according to claim 58, further comprising one or more recovery vessels in fluid communication with the one or more second chambers. **Claim 61** The system according to claim 58, wherein the first chamber and the one or more second chambers have a volume of 250 ml to 1,000,000 liters. **Claim 62** The system according to claim 58, wherein the control system sets the dilution rate of a liquid reagent transferred into the first chamber and / or one or more second chambers to maintain the concentration of one or more nutrients. **Claim 63** The system according to claim 58, wherein the first chamber and the one or more second chambers comprise ports in communication with a reagent reservoir and with each other via fluid conduits. **Claim 64** The system according to claim 58, wherein the one or more second chambers are a plurality of second chambers. **Claim 65** The system according to claim 58, wherein one or more first or second sensors providing volume measurements comprise a scale measuring the mass of the bioreactor. **Claim 66** The system according to claim 58, wherein the sensor providing the cell density measurement of the cell culture in the first chamber comprises an optical density sensor or a capacitance sensor. **Claim 67** The system according to claim 65, wherein controlling the cell density comprises controlling the optical density of the cell culture. **Claim 68** The system according to claim 66, comprising a user programmable module that calculates the dilution rate as a function of the error between the target optical density and the measured optical density.

69. The system according to claim 58, wherein the one or more first sensors provide measurements of the temperature, pH, and dissolved oxygen of the cell culture within the first chamber.

70. The system according to claim 58, wherein the one or more second sensors provide measurements of the temperature, pH, and dissolved oxygen of the cell culture within the second chamber.

71. The system according to claim 58, wherein at least one pump is a peristaltic pump or a gravity pump.

72. The system according to claim 70, wherein the peristaltic pump comprises a fitting for a tube that fluidly communicates between the two chambers, between a chamber and a reagent reservoir, or between the second chamber and an outflow conduit.

73. The system according to claim 58, wherein the control system for controlling the volume of the cell culture within the first chamber or the one or more second chambers includes a level sensor (e.g., an overflow tube), and when the height of the cell culture exceeds the upper end of the level sensor, the level sensor moves the cell culture fluid out of the chamber.

74. The control system includes a computer comprising a processor and a memory containing executable code, and when the executable code is executed by the processor, one or more feedback routines for controlling cell growth, e.g., cell density, in the first chamber, controlling the volume of the cell culture in the first chamber and / or one or more second chambers, and controlling the culture conditions in the one or more second chambers are performed. The system according to claim 58.

75. One of the feedback routines calculates the cell density of the cell culture within the first chamber based on measurements received from the one or more sensors and operates a pump to move a liquid reagent from a reagent reservoir to the first chamber to adjust the cell density to a set point. The system according to claim 73.

76. One of the feedback routines calculates the volume of the cell culture within the first chamber based on measurements received from the one or more sensors and operates a pump to move a liquid cell culture from the first chamber to the one or more second chambers if the volume exceeds a set point. The system according to claim 73.

77. (I) The one or more sensors provide measurements of one or more culture parameters in the first and / or one or more second chambers; (II) The system further comprises one or more effectors that affect changes in the one or more culture parameters; (III) One or more of the feedback routines calculate one or more culture parameters based on the measurements and activate the one or more effectors to adjust the culture parameters toward a target level. The system according to claim 73.

78. (I) The one or more sensors provide one or more measured values of pH, temperature, and dissolved O 2 in the cell culture within the first and / or one or more second chambers; 2 of; (II) The system comprises one or more reagent reservoirs containing acids and bases, in fluid communication with the first and / or one or more second reservoirs; one or more temperature controllers for controlling the temperature of the cell culture in the first and / or one or more second chambers; one or more aerators for delivering air to the cell culture in the first chamber and / or one or more second chambers and further comprises one or more of; (III) One or more of the feedback routines calculate culture conditions including one or more of pH, temperature, dissolved O 2 , and nutrient concentration, based on measurements received from the one or more sensors, one or more pumps for moving an acid or a base from a reagent reservoir into the second chamber to adjust the pH to a target pH; one or more temperature controllers for adjusting the temperature to a target temperature; and / or one or more aerators for delivering air to the cell culture to a target dissolved oxygen level. Activating The system according to claim 73.

79. One feedback routine calculates the volume of the cell culture in the one or more second chambers based on measurements received from the one or more sensors and, if the volume exceeds a set point, activates a pump to move liquid cell culture from the one or more second chambers. The system according to claim 73.

80. The system according to claim 58, further comprising a filter in fluid communication with the outlet of the second chamber, the filter configured to recycle cells that cannot pass through the membrane back to the second chamber and allow cell-depleted liquid medium to exit the cartridge and pass to a collection vessel.

81. The system according to claim 79, wherein the filter comprises a hollow fiber cartridge.

82. The system according to claim 58, wherein the first chamber contains a cell culture, the one or more reagent reservoirs in fluid communication with the first chamber contain a growth medium, and the medium contains one or more nutrients at a concentration that limits the growth rate of the cells in the cell culture.

83. The system according to claim 58, wherein the culture medium is formulated such that nutrients are not limited in the at least one first bioreactor, but one or more nutrients are present at a concentration that limits growth in the one or more second bioreactors.

84. One or more control subsystems include (A)a processor; (B)a memory coupled to the processor; and (C)computer-executable instructions that use measurements from the one or more sensors to calculate culture conditions in the first chamber and / or one or more second chambers, and based on the calculations, control pumps that move liquid to and from the chambers. The system according to claim 58, comprising a computer having the above.

85. ・A temperature sensor and a temperature regulator; ・A dissolved oxygen meter and an aeration system in communication with the interior of the first chamber and / or one or more second chambers; ・Analytical equipment for measuring the concentration of nutrients in the cell culture within the chamber; ・An impeller or a pneumatic stirrer for mixing the liquid within the chamber, and a motor configured to operate the impeller or the pneumatic stirrer; ・An outflow portion in communication with the interior of the container, and an adjustable valve or pump for controlling the flow of liquid from the chamber; ・A baffle within the chamber; ・A sparger and a mass flow controller in communication with the interior of the chamber for introducing one or more gases and mixtures thereof; ・A user interface for communicating with the computer to convey instructions; ・A foam control device; ・A hollow fiber membrane for cell recycling; and ・A reverse osmosis membrane or a forward osmosis membrane The system according to claim 58, including one or more of the above.

86. The system according to claim 58, wherein the control system is configured to perform any of the methods according to claims 1 to 35.

87. a)Performing a first continuous culture of cells in the chamber of the first bioreactor under culture conditions that maintain a constant cell density; b) During the first continuous culture, transferring the culture fluid containing cells from the chamber of the first bioreactor into the chamber of at least one second bioreactor through one or more fluid conduits that put the chamber of the first bioreactor in fluid communication with the chamber of the at least one second bioreactor; c) Performing a second continuous culture of the cells transferred from the first bioreactor in the respective chambers of the one or more second bioreactors under certain culture conditions to produce at least one culture product; and d) Recovering at least one culture product from the respective chambers of the one or more second bioreactors A method comprising.

88. （a） (i) A first chamber, and (ii) One or more first sensors that provide at least measurements of cell density and cell culture volume within the first chamber A first bioreactor equipped with; （b） (i) A second chamber, and (ii) One or more second sensors that provide at least measurements of culture conditions and cell culture volume within the one or more second chambers One or more second bioreactors equipped with, wherein the first chamber is in fluid communication with the one or more second chambers; (c) One or more reagent reservoirs containing liquid reagents, the reagent reservoirs being in fluid communication with the first chamber and optionally the one or more second chambers; （d） (i) Transferring the liquid reagent from at least one reagent reservoir to the first chamber; (ii) Transferring the cell culture fluid from the first chamber to the one or more second chambers; (iii) Transferring the cell culture fluid out of the one or more second chambers; (iv) Optionally, transferring the liquid reagent from at least one reagent reservoir to the one or more second chambers One or more pumps configured to; （e） (i) Controlling cell growth, such as cell density, of the cell culture in the first chamber using the measurements from the one or more sensors; (ii) Transferring the cell culture fluid from the first chamber to the one or more second chambers; and (iii) Controlling the culture conditions in the one or more second chambers and, for example, the volume and / or cell growth of the cell culture using the measurements from the one or more sensors A control system configured to A system including

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