Multi-chamber cell culture system and method

Abstract

Systems and methods for continuous culture comprising performing a first continuous culture of cells in a chamber of a first bioreactor under culture conditions that cause cell growth; moving a culture fluid comprising cells from the chamber of the first bioreactor into the chamber of the second bioreactor through one or more fluid conduits; and performing a second continuous culture of the cells in the chamber of the second bioreactor under culture conditions producing at least one culture product. The method is carried out under conditions in which the cell growth rate in the first continuous culture is greater than that in the second continuous culture and the production of the culture product in the first continuous culture is less than that in the second continuous culture.

Description

[0001] Statement Regarding Federally Funded Research

[0002] This invention was made under CRADA FP00011895 between Pow Genetic Solutions, Inc. and the Directors of the University of California Ernest Orlando Lawrence Berkeley National Laboratory operated for the United States Department of Energy. The Government has certain rights in this invention.

[0003] Citation of Related Applications

[0004] This application claims the benefit of the priority date of U.S. Provisional Patent Application No. 63 / 356,974 (“Multi-chamber Cell Culture System and Method”), filed on June 29, 2022, the contents of which are incorporated herein by reference in their entirety.

[0005] background

[0006] The booming biomanufacturing and synthetic biology industries rely on bioreactors (also known as fermenters) to transform raw biological materials into value-added products. Biomanufacturing is an emerging industry that can be used to produce a large portion of the physical materials in the global economy, not only with improved properties but also with a better environmental footprint to combat climate change. Recent estimates suggest that the total annual economic impact of synthetic biology will reach $2-4 trillion by 2040. Two-thirds of this impact is in agriculture, consumer products, biomaterials, biochemicals, bioenergy, and many other areas outside of healthcare. Of all the potential applications of synthetic biology, biomanufacturing of alternative proteins, biomaterials, biochemicals, and biofuels will account for the largest growth over the next 10 to 20 years.

[0007] In addition, the petrochemical industry makes it possible to manufacture ubiquitous products (ranging from medicines to daily household items) that constitute the backbone of modern society. However, oil-based methods are unsustainable and lead to global climate change. The biomanufacturing of renewable alternatives cannot compete in price to a large extent because the basic production method (batch or fed-batch fermentation) has not changed for decades. Replacing common batch and fed-batch bioreactor systems with continuous flow bioreactors will minimize the equipment downtime, increase volumetric productivity, and reduce capital investment and operating costs. However, due to pollution and strain stability problems, continuous flow bioreactors are currently considered to be unreliable and are rarely used in biomanufacturing processes outside the pharmaceutical industry. Therefore, there is a demand for developing reliable continuous flow bioprocesses. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable those skilled in the relevant art to make and use these embodiments and other embodiments that will be apparent to those skilled in the art. The present invention will be described in more detail with reference to the following drawings, in which:

[0010] Figure 1 Shown are results from a turbidostat where temperature and pH were maintained constant over time and cells were maintained at a constant concentration / density (AU).

[0011] Figure 2 The invention shows a method including a second bioreactor ( Figure 2 B) is in fluid communication, optionally with a cell recovery system ( Figure 2 C) a first bioreactor in fluid communication ( Figure 2 A) An exemplary multi-chamber cell culture system.

[0012] Figure 3 A and Figure 3 B shows an exemplary bioreactor.

[0013] Figure 4 An exemplary computer system is shown.

[0014] Figure 5 An exemplary method for producing a culture product using a multi-chamber cell culture system is shown.

[0015] Figure 6An exemplary hollow fiber cartridge for recycling cells to a second continuous cell culture is shown. The cartridge 601 includes a feed port 607 connected to the effluent of a second bioreactor containing a second continuous culture, a retentate port 609 for returning cell feed to the second continuous culture, hollow fibers 605 forming a semipermeable membrane (and medium containing culture products can pass through the semipermeable membrane but cells cannot), and a permeate port 603 (through which medium containing culture products leaves the cartridge).

[0016] Overview

[0017] Disclosed herein are multi-chamber cell culture systems and methods for producing culture products. The multi-chamber cell culture system includes one or more first bioreactors and one or more second bioreactors in fluid communication with each other. Thus, the first bioreactor can feed a single second bioreactor; the first bioreactor can feed more than one second bioreactor; more than one first bioreactor can feed a single second bioreactor; or more than one first bioreactor can feed more than one second bioreactor.

[0018] In one embodiment, cells producing a culture product of interest are grown in the chamber of one or more first bioreactors under conditions that maintain cell growth but produce little or no culture product (i.e., no more than 5% of the amount of the product produced in the second bioreactor). During the culture period, the culture fluid containing the growing cells is directly transferred to the chamber of one or more second bioreactors. The culture conditions in the second bioreactor are set to produce the culture product and limit the growth rate of the cells therein. Limiting the growth rate of the cells in the second culture container can inhibit the production and growth of mutant microorganisms that interfere with the production of the culture product. In some embodiments, the cells that do not grow in the second bioreactor will have a very slow growth rate, such as a doubling time of at least one week. Take out the culture product from the second bioreactor, for example, by taking out the cell culture medium and separating the product for sale therefrom. In some embodiments, the cells taken out with the cell culture medium are recycled back into the second culture container.

[0019] In another embodiment, the cells that can produce the product of interest are grown in the first bioreactor under conditions that maintain cell density. Under steady state, the cells in such a culture will grow at a constant growth rate (e.g., logarithmic phase or exponential growth). In certain embodiments, the culture conditions are set to produce a maximum or near-maximum growth rate. This can be achieved, for example, by providing a cell culture medium in which there is no nutrient that limits cell growth.

[0020] The cells from the growth of the first bioreactor are transferred to each of the one or more second bioreactors via a fluid conduit. The culture conditions in the one or more second bioreactors are maintained to produce (preferably optimally produce) one or more desired culture products. This may include growing the transferred cells in the second bioreactor under conditions of no growth or very low growth. This may be achieved with a culture medium comprising a growth-limiting amount of nutrients such as micronutrients.

[0021] The culture fluid from the second bioreactor is removed and the culture product is separated therefrom. In some embodiments, the cells in the effluent are returned to the second bioreactor to continue producing the culture product.

[0022] The system that carries out these methods adopts feedback mechanism to maintain culture condition and regulate the volume of cell culture.Feedback mechanism comprises the sensor of one or more culture parameters of cell culture in each bioreactor, calculates culture parameters and determines that they are higher than or lower than the computer module of the predetermined set point of these parameters and the effector of adjusting culture condition towards parameter set point.Such parameter comprises for example cell density, culture volume, temperature, pH, dissolved oxygen and nutrient concentration.The effector comprises for example pump, to move liquid in bioreactor container and move in other bioreactor containers or move in collecting container from reagent reservoir.Other factors can comprise for example temperature regulator that regulates cell culture temperature and the air pump that oxygen is introduced in cell culture.By using such feedback mechanism, various culture parameters can be maintained over time.

[0023] Details

[0024] I. Continuous Culture

[0025] The methods and systems provided herein use continuous culture of cells to produce desired culture products. Any suitable culture product can be produced, such as small molecules, proteins, polynucleotides and / or cells.

[0026] The two main 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 with a fixed amount of nutrients. Continuous culture is an open system in which nutrients are added continuously to the culture. An example of a continuous culture, a turbidostat, is shown in Figure 1 middle. Specifically, Figure 1The maintenance of cells in a stable growth state over time is shown. Cell concentration is indicated by "AU". The turbidostat maintains a constant turbidity of the culture, and the turbidity reflects the cell density. The culture parameters are initially set and allowed to reach a steady state. This may require, for example, 3 to 5 or more generations of cells. The turbidostat maintains cell density by removing the culture medium containing the cells from the bioreactor and replacing the volume with fresh culture medium. Therefore, when the culture is in a steady state, the growth rate is a function of the amount of the culture medium removed and time. Batch culture is characterized by the initial rapid growth of cells, which then enters a plateau phase after nutrients are exhausted, which limits further growth. Continuous culture is generally characterized by a stable growth rate and / or a stable culture output rate, because nutrients are continuously added to the system, and, for example, by removing excess cells from the culture to maintain cell density. Other examples of continuous culture include chemostat and perfusion culture.

[0027] Cultivation processes include any cell culture for producing a culture output (i.e., for bioproduction to produce a bioproduct). These processes include, but are not limited to, processes referred to as "fermentation" and "cell culture." "Fermentation" generally refers to the enzymatic conversion of molecules into different molecular species, typically by a unicellular organism (e.g., the conversion of sugars into ethanol). "Cell culture" generally refers to the cultivation of a single cell (e.g., a mammalian cell or an insect cell) for the production of a product (such as a polypeptide or a value-added organic compound or biomass). Any suitable cell may be used for the culture (archaea, prokaryotes and / or eukaryotes), such as fungal cells, e.g., yeast (e.g., Saccharomyces spp, Pichia spp, Komagataella spp, Kuyveromyces spp, Aspergillus spp, Rhodoporidium spp, Lipolyticaspp, Aspergillus spp, Neurospora spp, Trichoderma spp, Candida spp, or Penicillium), bacterial cells, e.g., Escherichia coli, Bacillus spp. spp), Clostridia spp, Streptomyces spp, Pseudomonas spp, Ralstonia spp, Shewanella spp, animal cells, such as arthropods (e.g., insects, shrimp, lobster, crayfish, and crab); chordates (e.g., fish, amphibians, reptiles, birds (e.g., chicken or turkey); mammals (e.g., humans or non-human animals, such as bovines, lambs, goats, pigs, horses, dogs, cats, primates)), cell lines, such as CHO (Chinese hamster ovary cells), BHK21 (baby hamster kidney), NS0, Sp2 / 0 mouse cell lines, insect cells (e.g., SP9, Sf9, sf21, S2), tobacco BY-2 cells, rice (Oryza Sativa), or algae cells.

[0028] Processes also include synthetic biology approaches, where cells are engineered to produce a molecular output, for example by expressing enzymes within the cell along a synthetic pathway.

[0029] II. Multi-chamber cell culture system

[0030] 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 bioreactor. Thus, the system may include a first bioreactor in fluid communication with a single second bioreactor via a fluid conduit, and more, may include a central first bioreactor in fluid communication with more than one second bioreactor.

[0031] In certain embodiments, the multi-chamber cell culture system further comprises one or more reagent reservoirs fluidly connected to the first bioreactor and / or one or more second bioreactors. The reagent reservoirs may contain any suitable reagents, such as growth medium, nutrient sources, carbon sources (e.g., glucose), buffers, and / or one or more reagents and / or gases (e.g., oxygen) for adjusting culture parameters (e.g., pH of the culture medium).

[0032] In certain embodiments, the multi-chamber cell culture system also includes a reservoir, which is fluidically connected to the second bioreactor and is configured to receive the culture medium effluent from the second bioreactor, preferably comprising a culture product. In certain embodiments, the culture medium effluent comprises one or more cells from the culture. In certain embodiments, the culture medium effluent from the second bioreactor is made to flow through a filter, wherein the filter retains one or more cells in the culture medium effluent (i.e., retentate), while allowing the culture medium (i.e., filtrate) comprising one or more culture products to pass through. In certain embodiments, the filtrate comprises cells less than the culture medium effluent from the second bioreactor. In certain embodiments, the filtrate does not contain cells. In certain embodiments, the filter retains cells in 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9% or 100% at least about any one effective from the culture medium effluent from the second bioreactor. In certain embodiments, the filtrate has no more than about any of 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, 0.6%, 0.1%, or 0% of the cells of the cell culture medium 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., the cell recovery system).

[0033] In certain embodiments, the multichamber cell culture system also includes one or more feedback mechanisms and / or control systems to maintain culture conditions and regulate the volume of the cell culture. The feedback mechanism includes one or more sensors that measure one or more process parameters of the bioreactor, a computer module that calculates process parameters and determines whether they are higher than or lower than the predetermined set point of these parameters, and an effector that adjusts the process parameters toward the set point of expectation. In certain embodiments, the set point of expectation is input by the operator using a programmable interface. In certain embodiments, one or more computer programs and one or more tests are used to develop the set point to produce optimal results. Any suitable sensor can be used to measure process parameters, such as culture parameters, for example 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 into a bioreactor vessel, from one bioreactor vessel to other bioreactor vessels, or into a collection vessel, a heat source and / or cooling source for maintaining the temperature of the bioreactor, a mixing unit for agitating the bioreactor (e.g., an impeller, a pneumatic stirrer, a stir bar and plate), and / or an air pump for introducing a gas (e.g., oxygen or nitrogen) into the culture. Additional examples of sensors and effectors are disclosed herein.

[0034] An exemplary diagram of a multi-chamber cell culture system is shown in Figure 2 middle. Figure 2 The invention shows a method comprising a first bioreactor (201, Figure 2 A) and the second bioreactor (208, Figure 2 B) multi-chamber cell culture system. The first bioreactor ( Figure 2A) is configured to include a first cell culture (202) that can be mixed by a mixing unit (203). The first bioreactor also includes a first inlet conduit (204) connected to a first inlet port (205) that is in fluid communication with a first reagent reservoir. The first bioreactor also includes a first effluent conduit (206) (i.e., an outlet) connected to a first effluent port (207), wherein the first effluent conduit (206) and the 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 include a second cell culture (209) that can be mixed by a mixing unit (210). The second bioreactor (208) also includes a first inlet conduit (211) and a first inlet port (212) that are fluidly connected to the first effluent conduit (206) and the port (207) of the first bioreactor, and is configured to receive the first cell culture (203) from the first bioreactor (201). In certain embodiments, the first effluent conduit (206) of the first bioreactor (201) and the first inlet conduit (211) of the second bioreactor (208) are the same conduit. The second bioreactor (208) further comprises a second inlet conduit (213) connected to a second inlet port (214) in fluid communication with a second reagent reservoir. The second bioreactor (208) further comprises a first effluent conduit (215) connected to a first effluent port (216), wherein the first effluent conduit (215) and the port (216) are in fluid communication with a second cell culture (209).

[0035] The second bioreactor may further include a cell recovery system (219, Figure 2C), wherein the cell recovery system is configured to separate the cells from their culture medium and (1) return (e.g., recover) the concentrated cell culture to the second bioreactor (208) via a third inlet conduit (217) connected to a third inlet port (218), or (2) transfer the cells to a reservoir (not shown). The cell recovery system includes a first inlet conduit (220) connected to a first inlet port (221), wherein the first inlet conduit (220) and port (221) of the cell recovery system are in fluid communication with a first effluent conduit (215) and port (216) of the second bioreactor and the cell culture (209). The cell recovery system also includes a first effluent conduit (222) connected to a first effluent port (223), wherein both the first effluent port (223) and the first effluent conduit (222) are in fluid communication with a third inlet conduit (217) and port (218) of the second bioreactor (208), wherein the concentrated cell culture is transferred from the cell recovery system to the second bioreactor. The cell recovery system also includes a second effluent conduit (224) and a second effluent port (225) configured to transfer the cell-free culture medium to a downstream process or storage. In certain embodiments, the cell recovery system includes a filter, membrane, or hollow fiber cartridge that is a tangential flow or alternating tangential flow filtration system. Any suitable mechanism for separating cells from the culture medium can be used, such as a centrifuge. An exemplary hollow fiber cartridge is depicted in FIG. Figure 6 middle.

[0036] The first bioreactor and the second bioreactor can include any suitable number of connected conduits, ports, each of which can be connected to a pump as required for the desired application. The port can be attached to the cover and / or chamber of the bioreactor. The port can optionally include a dip tube. The dip tube can include any suitable length and diameter, and is in contact with or not in contact with the cell culture. In either case, the port and / or dip tube can deliver and / or remove one or more liquid and / or gas components from the bioreactor. Any suitable material can be used for the port and / or conduit. In a preferred embodiment, the material is a biocompatible material. In certain embodiments, the material is a chemically resistant material. The first bioreactor and the second bioreactor can include any suitable number of sensors and / or effectors required for the desired application (as disclosed herein).

[0037] In certain embodiments, the multi-chamber cell culture system comprises 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 with one or more second bioreactors, one or more pumps and a control system. In certain embodiments, the first bioreactor comprises a first chamber and one or more first sensors, the one or more first sensors providing at least a measure of the cell density in the first chamber and a measure of the volume of the cell culture. In certain embodiments, the one or more second bioreactors comprises a second chamber and one or more second sensors, the one or more second sensors providing at least a measure of the culture conditions in the one or more second chambers and a measure of the volume of the cell culture. In certain embodiments, one or more pumps are configured to (1) move a liquid reagent from at least one reagent reservoir to a first chamber of a 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 a second chamber of a second bioreactor to one or more reservoirs and / or downstream processes. In certain embodiments, one or more pumps are configured for (1)-(3). In preferred embodiments, one or more pumps are configured for (1)-(4). In more preferred embodiments, one or more pumps are configured for (1)-(3), (5), and optionally (4). In some embodiments, the control system is configured to (1) use measurements from one or more sensors to control the cell density of the cell culture in the first chamber of the first bioreactor; (2) move the cell culture fluid from the first chamber of the first bioreactor to one or more second chambers of the second bioreactor; (3) use measurements from the one or more sensors to control the volume and culture conditions of the cell culture in the one or more second chambers of the second bioreactor; and / or (4) move the cell culture fluid from the second chamber of the second bioreactor to one or more reservoirs and / or downstream processes. In some embodiments, the control system is configured for (1)-(3). In some embodiments, the control system is configured for (1)-(4).

[0038] In certain embodiments, the multi-chamber cell culture system comprises a forward or reverse osmosis membrane.

[0039] A. Bioreactor

[0040] The cultivation of cells is carried out in a bioreactor. A bioreactor is a system that supports and maintains a biologically active environment within a predetermined amount of time. Such a system includes a container or chamber configured to contain a culture medium, and elements that maintain or change various culture parameters. The bioreactor can be configured to maximize any suitable parameter of the cell culture. In certain embodiments, the bioreactor can be configured to maintain an environment for the optimal cell growth of the desired application. In certain embodiments, the bioreactor can be configured to maintain the desired cell density for the desired application. In other embodiments, the bioreactor can be configured to maintain an environment for the optimal production of the culture product that supports one or more desired products.

[0041] Batch and continuous bioreactor systems are commercially available from, for example, Sartorius (Goettingen, Germany) and ThermoFisher (Waltham, MA).

[0042] Figure 3 A and Figure 3 B shows an exemplary bioreactor. Figure 3 A shows an exemplary schematic diagram and components of a bioreactor. Figure 3 B shows a bioreactor attached to a computer having a user interface that indicates culture parameters rather than accepting user instructions for controlling culture conditions. Reagent reservoirs (e.g., bottles) containing various types of nutrients are attached to the culture via conduits (e.g., tubes). The reservoirs may also receive excess culture medium from the culture and / or one or more culture products in the culture medium or culture fluid.

[0043] A bioreactor is a system configured to grow a cell culture, typically by adjusting one or more culture conditions and / or process parameters. Any suitable bioreactor configuration may be used, such as batch and / or continuous culture modes. In preferred embodiments, the bioreactor operates in a continuous culture mode, for example: (1) a turbidostat mode: dynamically adjusting the flow rate of nutrient delivery and periodically removing cells to keep the turbidity inside the container constant; (2) a chemostat mode: limiting and controlling cell growth by nutrient addition (e.g., glucose, oxygen, glutamine), and removing depleted culture medium with growth inhibitors at approximately the same rate; and (3) a perfusion mode: cells are retained in the bioreactor or recycled back into the bioreactor, and fresh culture medium is provided and cell-free supernatant is removed at the same rate.

[0044] 1. Culture room

[0045] The culture chamber of a bioreactor generally has a volume between about 50mL and about 50L. This includes, for example, a volume between about 1L and about 10L. An industrial size fermenter can have a size between about 50L and about 1,000,000L. In certain embodiments, the culture chamber can have a volume between about 1,000L and about 10,000L. A multi-chamber cell culture chamber system can include any suitable combination of any suitable culture chamber volume and culture chamber volume for each bioreactor. In certain embodiments, the culture chambers of a first bioreactor and one or more second bioreactors include the same volume. In certain embodiments, the culture chambers of a first bioreactor and one or more second bioreactors include different volumes. For example, the volume of the first culture chamber can be less than the volume of the second culture chamber. In certain embodiments, one or more culture chambers of one or more second bioreactors include different volumes.

[0046] The multi-chamber cell culture system can include any suitable number of first bioreactors, such as at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 or 18 and / or no more than any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 first bioreactors, for example 1-20, preferably 1-10, more preferably 1-5, even more preferably 1-2. Typically, 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 in fluid communication with one or more first bioreactors, such as at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 or 18 and / or no more than any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 second bioreactors, for example 1-20, preferably 1-10, more preferably 1-5, even more preferably 1-2. In a preferred embodiment, the multi-chamber cell culture system includes at least 1 first bioreactor and at least one second bioreactor. In an even more preferred embodiment, the multi-chamber cell culture system includes one first bioreactor and one or more second bioreactors.

[0047] In certain embodiments, the chamber includes one or more baffles. The baffles can help stir / agitation the cell culture, which can help, for example, with nutrient distribution, mixing of one or more reagents, and / or aeration.

[0048] The culture chamber includes one or more ports from which liquids and / or gases can be introduced ("input") into the culture chamber or removed ("effluent") 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 be connected to a fluid line / conduit, which is connected to other containers (such as reagent reservoirs, other culture containers, collection reservoirs and / or gas sources). Such fluid lines can include tubes, such as flexible tubes. Any suitable tube material can be used, such as glass, metal, plastic, rubber and / or halogenated polymers. Preferred materials include biocompatible and / or chemically resistant materials, such as silicone, stainless steel, neoprene, polypropylene, pharmed and / or Tygon. The movement of fluids 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, piezoelectric valves, solenoid valves, and / or controlled by pumps, such as peristaltic pumps, syringe pumps, piezoelectric pumps, diaphragm pumps. Any suitable pump and / or valve combination may be used. Typically, a pump may be selected based on the minimum and maximum volume transfer required (e.g., feed rate and / or medium exchange rate and therefore flow requirements), which will depend on both the application and volume of the cell culture system, such as 100 uL / min to 100 L / min.

[0049] In certain embodiments, the bioreactor and its chambers may contain one or more feeds for adding nutrients and other molecules and one or more effluents for removing culture fluid from the bioreactor. In certain embodiments, a first bioreactor of a multi-chamber cell culture system contains at least one feed for nutrients and at least one effluent that is fluidly connected to one or more second bioreactors. In certain embodiments, a second bioreactor of a multi-chamber cell culture system contains at least one feed for cell culture, at least one feed for nutrients, and at least one effluent from a first bioreactor.

[0050] B. Sensors and Effectors

[0051] The multi-chamber cell culture system can also include one or more sensors for measuring various culture parameters (e.g., metabolic processes and process variables), and effectors for adjusting these parameters. In preferred embodiments, 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., acetate and ammonia), (2) detect changes in substrates (e.g., glucose), (3) or indirectly measure metabolism through cellular respiration by detecting changes in gas composition (e.g., oxygen and carbon dioxide). In certain embodiments, one or more culture parameters are measured and the data is provided to a feedback mechanism to guide, among other things, feeding strategies and / or process conditions. Any suitable number and combination of sensors can be used.

[0052] In certain embodiments, the sensor can be an "online" or "in-situ" sensor, wherein the sensor has a direct interface with the components of the culture or bioreactor. The data from the online sensor can be monitored and / or analyzed continuously or in an intermittent manner. The continuity of the measurement generally depends on the response time of the signal, the flow rate of the sampling procedure and / or the needs of a specific application. In certain embodiments, the sensor can be an "offline" sensor, wherein samples are collected manually or in an automated manner (such as an automatic sampler), and analyzed in a laboratory, such as high pressure liquid chromatography (HPLC), flow cytometry or microscopy analysis. In certain embodiments, off-line measurements can be performed with a robot or microfluidic system for online or offline analysis with an instrument such as HPLC or gas chromatography (GC).

[0053] a) Cell density

[0054] In certain embodiments, one or more bioreactors in a multi-chamber cell culture system include a sensor that can measure 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 fluid. For example, the OD can be measured using a UV-Vis spectrophotometer. 600 The optical density at .Other examples of spectra for measuring cell density include mid-infrared (MIR), UV-VIS, fluorescence, Raman and dielectric spectroscopy. Additionally, cell density is also a function of the capacitance of the cell culture. Therefore, a capacitance meter such as a Hamilton probe or a Chloris probe can be used to measure the capacitance of the culture fluid. In addition, cell density can be measured using the acoustic resonance of the cell culture medium, wherein the specific gravity of the culture medium is used to determine biomass. In certain embodiments, the sensor is capable of distinguishing the density component of live cells and / or surviving cells from the density component of dead cells in the culture to provide an indication of the density of metabolically active cells in the culture.

[0055] The cell density of the culture can be affected by adding a flow to the bioreactor vessel to dilute the cell culture. The dilution rate of a continuous culture can be calculated using the formula D = FV -1 To model the dilution rate (D; [hr -1 ]) is the medium flow rate into the bioreactor (F; [L hr -1 ]) and the culture volume (V; [L]) in the bioreactor. In certain embodiments, in order to achieve a steady-state culture, the dilution rate (D) should be equal to the specific growth (μ) of the cell culture, thereby maintaining a constant biomass concentration in the bioreactor.

[0056] As discussed herein, pumps and valves can be used as effectors to move liquids into and out of a bioreactor. In certain embodiments, a multichamber cell culture system includes a sensor that can provide a measurement of cell density to one or more feedback routines on a computer, wherein the computer actuates the effector to adjust the cell density to a desired set point. As discussed herein, pumps and valves can be used as effectors to move liquids into and out of a bioreactor as discussed above.

[0057] b) Culture volume

[0058] In some embodiments, one or more bioreactors in a multi-chamber cell culture system can include a sensor that can measure the volume of cells or a representation thereof. One such representation of volume is mass. Thus, the bioreactor can be placed on a scale to measure the weight of the bioreactor. Additionally or alternatively, a level sensor, such as a float valve or a capacitive fluid sensor, and / or an optical fluid sensor can be used. In some embodiments, the optical fluid sensor is coupled to a level gauge.

[0059] The cell culture volume can be reduced by taking out the liquid culture from the bioreactor container, or increased by adding liquid into the bioreactor container. As discussed herein, pumps and valves can be used as effectors to take out liquid from the culture container. Alternatively, an overflow tube can be used as a sensor and a pump, such as a gravity pump. The overflow tube senses when the height of the culture exceeds the top of the overflow tube, and uses gravity to pull the liquid through the tube and pull it out of the container. In certain embodiments, both volume and cell density (as described above) are maintained within a desired range. In certain embodiments, the multichamber cell culture system includes a sensor that can provide a measurement of cell volume to one or more feedback routines on a computer, wherein the computer actuates the effector to adjust the cell volume to a desired set point.

[0060] c) pH

[0061] In certain embodiments, one or more bioreactors in a multi-chamber cell culture system may include a sensor that can measure 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.

[0062] The pH of the cell culture can be adjusted using a pumping system controlled by a feedback system connected to a pH sensor, wherein the pumping system delivers calculated volumes of a suitable acid or base and / or titrates the amount of acid or base as needed from a reservoir until the sensor reads the desired pH. In certain embodiments, the multi-chamber cell culture system includes a sensor capable of providing a measurement of pH to one or more feedback routines on a computer, wherein the computer actuates an effector to adjust the pH to a desired set point.

[0063] d) Dissolved oxygen and / or carbon dioxide

[0064] In certain embodiments, one or more bioreactors in a multi-chamber cell culture system may include sensors that can measure dissolved oxygen ("DO") and / or dissolved carbon dioxide ("dCO2") in the culture fluid. Both dissolved oxygen and dissolved carbon dioxide can be measured using optical sensors, galvanic cells, and / or polarographic cells.

[0065] Dissolved oxygen can be controlled using a ventilation system including a pump and / or valve in communication with one or more gas sources (e.g., a canister or atmospheric air) that delivers air to the interior of one or more culture chambers. Dissolved carbon dioxide can be controlled by venting waste gas containing CO2 as the ventilation system delivers new gas to the culture chamber. In certain embodiments, the multi-chamber cell culture system includes a sensor capable of providing a measurement of DO and / or dCO2 to one or more feedback routines on a computer, wherein the computer actuates effectors to adjust the gas concentration in the bioreactor to a desired set point.

[0066] In certain embodiments, CO2 may comprise a carbon source for the culture, thus regulating dCO2 in the culture facilitates growth and / or production of one or more culture products. In certain embodiments, the culture may be maintained in an anoxic or anaerobic atmosphere.

[0067] e) Temperature

[0068] In certain embodiments, one or more bioreactors in a multi-chamber cell culture system can include a sensor that can measure the temperature of the culture fluid. A temperature sensor such as a thermometer or thermistor can be used for this purpose. In certain embodiments, the temperature sensor is placed in a thermowell, where the temperature sensor is able to read the culture temperature without direct contact with the culture itself.

[0069] The temperature of the fluid culture in the culture chamber can be regulated using a temperature regulator such as a heating element and / or a cooling element. Non-limiting examples of heating elements include cartridge heaters, thin film resistor heaters, or thermoelectric coolers (TECs). Non-limiting examples of cooling elements include TECs or cooling jackets. The temperature of one or more bioreactors in a multi-chamber cell culture system can be maintained at any suitable temperature, such as 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, or 85°C. At least about any and / or no more than 22° C., 24° C., 26° C., 28° C., 30° C., 30° C., 32° C., 34° C., 36° C., 38° C., 40° C., 42° C., 44° C., 46° C., 48° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C. or 90° C., e.g., 20° C.-90° C., preferably 24° C.-50° C., more preferably 25° C.-40° C. In certain embodiments, the multi-chamber cell culture system comprises a sensor capable of providing a measure of temperature to one or more feedback routines on a computer, wherein the computer actuates an effector to adjust the temperature in the bioreactor to a desired set point using a cooling and / or heating system.

[0070] f) Glucose concentration

[0071] In certain embodiments, one or more bioreactors in the multi-chamber cell culture system may include a sensor that can measure any suitable chemical composition of the culture fluid. In a preferred embodiment, the chemical composition includes nutrients. In an even more preferred embodiment, the nutrients include glucose, i.e., the concentration of glucose in the culture fluid. Glucose concentration can be measured with an electrochemical glucose sensor and / or spectroscopy. Glucose concentration can also be biochemically measured.

[0072] The glucose concentration of the cell culture can be adjusted using a pumping system controlled by a feedback system connected to a glucose sensor, wherein the pumping system delivers a calculated volume of a fluid containing glucose and / or titrates an amount of a fluid containing glucose as needed until the sensor reads a desired glucose concentration. In certain embodiments, the multi-chamber cell culture system includes a sensor capable of providing a measurement of the glucose concentration to one or more feedback routines on a computer, wherein the computer actuates an effector to adjust the glucose concentration in the bioreactor to a desired set point.

[0073] g) Other examples of sensors and effectors

[0074] As mentioned above, any suitable sensor can be incorporated to measure any suitable parameter (physical, chemical and / or biological) required for the desired application. Table 1 shows a non-limiting exemplary list of parameters and sensors. Non-limiting examples of other parameters include foam, viscosity, pressure, stirring speed, pyruvic acid concentration, lactate concentration, acetate concentration, cell morphology, protein presence and / or metabolite concentration. The sensor measuring these parameters can be configured to measure conductivity, capacitance, optical density, torque or any other physical parameter. Other sensors can include spectroscopy, biochemistry, optics, chromatography, cytometry, microscope and / or electrochemical components to measure the desired parameters. An example includes liquid chromatography with UV detection.

[0075] Table 1: Parameter and sensor type examples

[0076]

[0077] In certain embodiments, the bioreactor may include a foam control mechanism including a foam sensor and an effector, such as a pipe and a pump, for reducing foam. In certain embodiments, the bioreactor may include an ejector and a mass flow controller communicated with the interior of the chamber for inputting and / or displacing 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 an impeller and a port for inputting nutrients and other chemicals and for outputting culture medium (which may include cells).

[0078] c. First bioreactor

[0079] 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 accommodate any suitable volume, such as a volume between about 50mL and 50L, for example, a volume between about 1L and about 10L, a volume between about 5L and 500L, or a volume between 1,000L and 10,000L. In certain embodiments, the first chamber of the first bioreactor includes a volume between about 250mL and about 10L. The first bioreactor can have a volume equal to or less than the volume of the second bioreactor. For example, the first bioreactor can have a volume that is no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or any one of 90% of the volume of the second bioreactor. This includes, for example, between 10% and 20%, 30%, 40%, 50%, 60%, 70%, 80% or any one of 90% of the volume of the second bioreactor.

[0080] In certain embodiments, the first bioreactor further comprises one or more first sensors. The sensor can be any suitable sensor as disclosed herein. In a preferred embodiment, the first bioreactor comprises a first sensor that provides at least a measurement of cell density and a first sensor that provides at least a measurement of the volume of the cell culture in the first chamber. In certain embodiments, the first sensor that provides at least a measurement of cell density comprises an optical density sensor or a capacitive sensor. In certain embodiments, the first sensor that provides at least a measurement of the volume of the cell culture comprises a scale that measures the mass of the bioreactor. In certain embodiments, the first bioreactor further comprises one or more first sensors that measure temperature, pH and / or dissolved oxygen. In certain embodiments, the first bioreactor comprises a first sensor that comprises a liquid level sensor, such as an overflow tube, which is configured to remove the cell culture fluid from the first chamber when the height of the cell culture exceeds the top of the liquid level sensor.

[0081] In certain embodiments, the first bioreactor comprises a port that is communicated with a reagent reservoir and is communicated with each other by a fluid conduit. Any suitable number and the port of configuration on the first bioreactor can be used. In certain embodiments, the first bioreactor comprises at least about any one and / or no more than about any one of 2,3,4,5,6,7,8,9,10,12,14,16,18,20,22,24,26,28,30,35,40 or 45 ports, for example 1-50 port, preferably 2-20 port. In certain embodiments, a fluid conduit (such as a pipe) is connected to a port. Any suitable number of reagent reservoirs can be connected to the first bioreactor.

[0082] In a preferred embodiment, a first bioreactor is configured to be in fluid communication with one or more second bioreactors.

[0083] In certain embodiments, the first chamber of the first bioreactor comprises a cell culture, and wherein the one or more reagent reservoirs in fluid communication with the first chamber comprise a growth medium in which one or more nutrients are present at a concentration that limits the growth rate of cells in the cell culture.

[0084] An exemplary first bioreactor is shown in Figure 2 A. The first bioreactor is configured to include a first cell culture (202) that can be mixed by a mixing unit (203). The first bioreactor also includes a first inlet conduit (204) connected to a first inlet port (205) in fluid communication with a first reagent reservoir. The first bioreactor also includes a first effluent conduit (206) (i.e., an outlet) connected to a first effluent port (207), wherein the first effluent conduit (206) and the port (207) are in fluid communication with both the first cell culture (203) and the second bioreactor (208).

[0085] D. Second bioreactor

[0086] 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 accommodate any suitable volume, such as a volume between about 50mL and 50L, for example a volume between about 1L and about 10L, a volume between about 5L and 500L, or a volume between 1,000L and 10,000L. In certain embodiments, the first chamber of the second bioreactor includes a volume between about 250mL and about 10L.

[0087] In certain embodiments, the second bioreactor further comprises 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 at least a measurement of the volume of the cell culture in the second chamber. In certain embodiments, the second sensor that provides at least a measurement of the volume of the cell culture comprises a scale that measures the mass of the bioreactor. In certain embodiments, the second bioreactor further comprises one or more second sensors that measure temperature, pH and / or dissolved oxygen. In certain embodiments, the second bioreactor comprises a second sensor that comprises a liquid level sensor, such as an overflow tube, that is configured to remove the cell culture fluid from the second chamber when the height of the cell culture exceeds the top of the liquid level sensor.

[0088] In certain embodiments, the second bioreactor comprises a port that is communicated with a reagent reservoir and is communicated with each other by a fluid conduit. Any suitable number and the port of configuration can be used on the second bioreactor. In certain embodiments, the second bioreactor comprises at least about any one and / or no more than about any one of 2,3,4,5,6,7,8,9,10,12,14,16,18,20,22,24,26,28,30,35,40 or 45 ports, for example 1-50 port, preferably 2-20 port. In certain embodiments, a fluid conduit (such as a pipe) is connected to a port. Any suitable number of reagent reservoirs can be connected to the second bioreactor.

[0089] The second bioreactor of any suitable number can be operably connected to the multi-chamber cell culture system. In certain embodiments, the multi-chamber cell culture system can include the second bioreactor of any suitable number (that is, more than one second bioreactor), such as 1,2,3,4,5,6,7,8,9,10,12,14,16 or 18 at least any one and / or no more than 2,3,4,5,6,7,8,9,10,12,14,16,18 or 20 second bioreactors any one, for example 1-20, preferably 1-10, more preferably 1-5, even more preferably 1-2. In a preferred embodiment, one or more of the second bioreactors are configured to be in communication with the first bioreactor fluid. In a preferred embodiment, each of the one or more second bioreactors is configured to be in communication with the first bioreactor fluid.

[0090] The second bioreactor may comprise any suitable number of second chambers (i.e., more than one second chamber), such as at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 or 18 and / or no more than any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 second bioreactors, for example 1-20, preferably 1-10, more preferably 1-5, even more preferably 1-2.

[0091] In certain embodiments, one or more second chambers of the second bioreactor have the same volume as the first chamber of the first bioreactor. In certain embodiments, one or more second chambers of the second bioreactor include a volume different from the first chamber of the first bioreactor. In certain embodiments, one or more second chambers of the second bioreactor are 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 of the volume of the first chamber of the first bioreactor at least about any one and / or no more than 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× about any one, for example 0.1-100×, preferably 0.5-50×, more preferably 1-40×. 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 between twice and 100 times the volume of the first chamber of the first bioreactor. In certain embodiments, each of the one or more second chambers of the second bioreactor has the same volume. In certain embodiments, one or more of the second chambers of the second bioreactor includes a volume different from the rest of the one or more second chambers of the one or more second bioreactors.

[0092] In certain embodiments, one or more second chambers of the second bioreactor include a cell culture, and one or more reagent reservoirs in fluid communication with the second chamber include a growth medium in which one or more nutrients are present at a concentration that limits the growth rate of cells in the cell culture. In certain embodiments, the medium is formulated so that nutrients are not limited in the first bioreactor, but one or more nutrients are present at a concentration that limits growth in the one or more second bioreactors.

[0093] An exemplary second bioreactor is shown in Figure 2B. The second bioreactor (208) is configured to include a second cell culture (209) that can be mixed by a mixing unit (210). The second bioreactor (208) also includes a first inlet conduit (211) and a first inlet port (212) that are fluidically connected to the first effluent conduit (206) and port (207) of the first bioreactor, and is configured to receive the first cell culture (203) from the first bioreactor (201). In certain embodiments, the first effluent conduit (206) of the first bioreactor (201) and the first inlet conduit (211) of the second bioreactor (208) are the same conduit. The second bioreactor (208) also includes a second inlet conduit (213) connected to a second inlet port (214) that is fluidically connected to a second reagent reservoir. The second bioreactor (208) also includes a second effluent conduit (215) connected to a second effluent port (216), wherein the second effluent conduit (215) and the port (216) are fluidically connected to the second cell culture (209).

[0094] In some embodiments, one or more second bioreactors are in fluid communication with one or more collection containers. In some embodiments, one or more collection containers are configured to receive a cell culture comprising both cells and cell culture medium. In some embodiments, one or more collection containers are configured to receive a cell-free culture medium. In some embodiments, one or more second bioreactors are in fluid communication with a cell recovery system (219, Figure 2 C) fluid communication, wherein the cell recovery system is configured to separate the cells from their culture medium and (1) return the concentrated cell culture to the second bioreactor (208) via a third inlet conduit (217) connected to a third inlet port (218), or (2) transfer the cells to a reservoir (not shown). An exemplary cell recovery system includes a first inlet conduit (220) connected to a first inlet port (221), wherein the first inlet conduit (220) and port (221) of the cell recovery system are in fluid communication with a first effluent conduit (215) and port (216) of the second bioreactor and the cell culture (209). The cell recovery system also includes a first effluent conduit (222) connected to a first effluent port (223), wherein both the first effluent port (223) and the first effluent conduit (222) are in fluid communication with a third inlet conduit (217) and port (218) of the second bioreactor (208), wherein the concentrated cell culture is transferred from the cell recovery system to the second bioreactor. The cell recovery system also includes a second effluent conduit (224) and a second effluent port (225) configured to transfer the cell-free culture medium to a downstream process or storage.

[0095] E. Reagent and fluid conduits

[0096] 1. Reagent storage

[0097] The system includes one or more reagent reservoirs. Each reagent reservoir contains a liquid reagent of a certain class. This can include, for example, nutrients, such as carbon sources, nitrogen sources, acid sources and / or alkaline sources. Reagents can include any suitable carbon source, such as sugar (for example, glucose, xylose, sucrose, glycerol or acetate), molasses, malt extract, starch, dextrin, pulp, CO or CO 2. Reagents can include any suitable nitrogen source, such as amino acids or polypeptides, urea, ammonium salts (for example, ammonium sulfate, ammonium phosphate or ammonia), corn steep liquor, yeast extract, peptone and soybean meal.

[0098] In certain embodiments, the reagent reservoir may include reagents including one or more of the following: metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdate, selenite and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid, 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, niacin, inositol, antifoaming agents (e.g., Antifoam 204, Antifoam A, Antifoam C), amino acids (e.g., glutamic acid, leucine and tryptophan), nucleic acid bases (e.g., adenine, cytosine, thymine, uracil and guanine), complex nutrients (e.g., yeast extract, peptone, tryptone and casamino acids), macronutrients, micronutrients and cell growth factors.

[0099] The reagent reservoir may include any suitable combination of reagents, such as a growth medium comprising a mixture of salts, vitamins, carbon source, and nitrogen source.

[0100] The reagent reservoir is in fluid communication with the first and / or second bioreactor container via a fluid conduit (eg, a tube). The conduit may engage ports in the reagent reservoir and the bioreactor container.

[0101] 2. Pumps, Valves and Filters

[0102] The multi-chamber cell culture system may include one or more pumps to pump liquid from one container to another. This includes, for example, pumping liquid from a reagent reservoir to the first and / or second bioreactor container and between the first and second bioreactor containers. The pump may be under the control of a control system.

[0103] In certain embodiments, one or more pumps are configured to (1) move a liquid reagent from at least one reagent reservoir to a first chamber of a 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 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 preferred embodiments, one or more pumps are configured for (1)-(4).

[0104] Any suitable pump can be used, such as a peristaltic pump, a diaphragm pump, a piezoelectric pump, a gravity pump and / or a syringe pump. In one embodiment, one or more pumps are peristaltic pumps. The peristaltic pump can be positioned on the fluid conduit between the containers to induce liquid to move through the fluid conduit.

[0105] The multi-chamber cell culture system may also include one or more valves to prevent liquid flow and / or direct fluid flow. Any suitable number and type of valves may be used. For example, the multi-chamber cell culture system may include a first pump and a first valve in fluid communication with a first reagent reservoir and a chamber of a bioreactor. When the cell culture uses nutrients, the control system may actuate the valve to open and enable the pump to transfer the value of the nutrients to the cell culture. After the transfer is complete, the control system may actuate the valve to close and disable the pump.

[0106] In certain embodiments, pumps, valves and / or chamber ports include fittings for tubes. In certain embodiments, fittings for tubes provide fluid communication between two chambers, a chamber and a reagent reservoir, or an effluent conduit from a second chamber, or a second chamber and an effluent conduit.

[0107] Sterility can be important for the operation of continuous bioreactors including multi-chamber cell culture systems. In certain embodiments, the filter fluid is connected to a conduit so that the liquid flowing through the conduit passes through the filter to remove any undesirable material. In certain embodiments, the filter removes contaminating biological materials from the fluid to prevent contaminating biological materials from entering the bioreactor. In certain embodiments, the filter comprises a pore size of 0.1-0.2um.

[0108] F. Control system

[0109] In certain embodiments, the control system is configured to (1) use measurements from one or more sensors to control cell growth, such as cell density, of a cell culture in a first chamber of a first bioreactor; (2) move a cell culture fluid from the first chamber of the first bioreactor to one or more second chambers of a second bioreactor; (3) use measurements from one or more sensors to control the volume and culture conditions of a cell culture in one or more second chambers of a second bioreactor; and / or (4) move a cell culture fluid from a second chamber of a 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 is used 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. In the event that a parameter deviates from a selected set point, the computer sends instructions to the effector to adjust the parameter toward the set point.

[0110] In certain embodiments, the control system sets the dilution rate of the liquid reagent moved to 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 cell culture at or near the expected value (determined by process or by operator). In certain embodiments, the control system is provided with the measurement of the optical density of cell culture and actuates one or more effectors to add reagent to cell culture and / or take out cell culture from the chamber of bioreactor. In certain embodiments, the control system includes a user programmable module, and the user programmable module calculates the function of the error between the optical density of the target optical density and the measured optical density.

[0111] In some embodiments, the control system sets the volume of the cell culture in the first chamber of the first bioreactor and / or one or more second chambers of the second bioreactor. In some embodiments, the control system includes a level sensor (e.g., an overflow tube) that removes the cell culture fluid from the chamber when the height of the cell culture exceeds the top of the level sensor. In some embodiments, the control system includes a feedback routine that calculates the volume of the cell culture in the one or more second chambers based on measurements received from one or more sensors, and actuates a pump to remove liquid cell culture from one or more second chambers if the volume is above a set point.

[0112] In certain embodiments, one or more sensors provide a measurement of one or more culture parameters in the first chamber and / or one or more second chambers to a control system. In certain embodiments, the multi-chamber cell culture system further comprises one or more effectors to affect a change 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 measurement and actuate one or more effectors to adjust the culture parameters toward a target level.

[0113] In some embodiments, the multi-chamber cell culture system includes one or more sensors to provide a measurement of one or more of pH, temperature, and dissolved O2 of the cell culture in the first chamber and / or one or more second chambers. In some embodiments, the system also includes one or more of the following: (1) one or more reagent reservoirs containing acids and bases in fluid communication with the first reservoir and / or one or more second reservoirs; (2) one or more temperature controllers to control the temperature of the cell culture in the first chamber and / or one or more second chambers; (3) one or more aerators for aerating the cell culture in the first chamber and / or one or more second chambers. In some embodiments, the control system includes 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 actuates one or more pumps to move acid or base from the reagent reservoir into the second chamber to adjust the pH to a target pH, actuates one or more temperature controllers to adjust the temperature to a target temperature; and / or actuates one or more aerators to aerate the cell culture to a target dissolved oxygen level.

[0114] 1. Computer

[0115] The feedback routines provided herein may be performed by a programmable digital computer.

[0116] Figure 4An exemplary computer system is shown. The computer system (401) includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") (405), which can be a single-core or multi-core processor or more than one processor 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 unit (415) (e.g., a hard disk), a communication interface (420) for communicating with one or more other systems (e.g., a network adapter), and peripheral devices (425), such as a cache, other memory, data storage, a bioreactor, and / or an electronic display adapter. The computer readable memory (410), storage unit (415), interface (420), and peripheral devices (425) communicate with the CPU (405) via a communication bus (solid line), such as a motherboard. The storage unit 415 can be a data storage unit (or data repository) for storing data. The computer system (401) can be operatively coupled to a computer network ("network") (430) by means of a communication interface (420). The network (430) can be the Internet, an intranet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. In some cases, the network (430) is a telecommunications and / or data network. The network (430) can include one or more computer servers, which can implement distributed computing, such as cloud computing.

[0117] The CPU (405) can execute a series of machine-readable instructions, which can be embodied in a program or software (code). The instructions can be stored in a memory location, such as a computer-readable memory (410). The instructions can be directed to the CPU (405), which can then program or otherwise configure the CPU (405) to implement the methods of the present disclosure.

[0118] The storage unit (415) may store files, such as drivers, libraries, and saved programs. The storage unit (415) may store user data, such as user preferences, log files, videos or other images, and user programs. In some cases, the computer system (401) may include one or more additional data storage units that are external to the computer system (401), such as located on a remote server that communicates with the computer system (401) via an intranet or the Internet.

[0119] The computer system (401) may communicate with one or more remote computer systems via a network (430).

[0120] The methods described herein may be implemented by means of machine (e.g., computer processor) executable code stored in an electronic storage location of a computer system (401), such as, for example, a computer readable memory (410) or an electronic storage unit (415). The machine executable code or machine readable code may be provided in the form of software. During use, the code may be executed by the processor (405). In some cases, the code may be retrieved from the storage unit (415) and stored in the memory 410 for immediate access by the processor (405). In some cases, the electronic storage unit (415) may not be included, and the machine executable instructions may be stored in the memory (410). The code may be used to communicate and issue instructions to an electronic device (e.g., a circuit board (440), a module, or a subsystem) on the instrument.

[0121] The computer system (401) may communicate with one or more remote computer systems via a network (430).

[0122] Machine executable code may be stored in an electronic storage unit such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media may include any or all tangible memories of a computer, processor, etc. or its associated modules, such as a variety of semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory storage for software programming at any time. All or part of the software may sometimes be communicated via the Internet or other communication networks.

[0123] The computer system (401) may include or communicate with an electronic display (435) that includes a user interface (UI) (440) for providing, for example, input parameters for the methods described herein. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0124] In certain embodiments, control system comprises computer, and described computer comprises processor and memory, and described memory comprises executable code, and described executable code carries out one or more feedback routines to control the parameter of bioreactor when being executed by processor.In certain embodiments, the parameter of bioreactor comprises the cell density in the first chamber, the volume of the cell culture in the first chamber and / or one or more second chambers and the culture condition and cell density in one or more second chambers.In certain embodiments, one in feedback routine calculates the cell density of the cell culture in the first chamber based on the measurement received from one or more sensors, and actuates pump to move liquid reagent to the first chamber from reagent reservoir to adjust cell density to set point.In certain embodiments, one in feedback routine calculates the volume of the cell culture in the first chamber based on the measurement received from one or more sensors, and if volume is higher than set point, actuates pump to move liquid cell culture from the first chamber to one or more second chambers.

[0125] In another embodiment, the feedback routine measures parameters in the first bioreactor, such as cell density, and adjusts the addition of media or removal of cell culture to maintain the cells in the growth phase; and measures one or more parameters related to the culture conditions in the second bioreactor and adjusts the addition of media and / or nutrients and the removal of cell culture to maintain the cells in the production phase.

[0126] 2. Machine Learning

[0127] In some embodiments, one or more data types from one or more parameters of a multi-chamber culture system can be stored and used to identify improvements in processes and / or culture parameters. In some embodiments, a network is provided that includes more than one separate multi-chamber cell culture system, wherein each of the systems sends information about one or more aspects of one or more processes at the system to a central processing unit. In some embodiments, one or more of the more than one systems are spatially separated. The central processing unit can process information and send outputs to one or more of the multi-chamber cell culture systems, such as outputs that cause changes in 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 no more than 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 unit or more than one unit, and can be, for example, distributed, such as a cloud-based system. The processing unit can be configured to learn from information provided by the various systems, and at least in part based on learning, for example, by using a machine learning algorithm to adjust conditions at one or more systems. Any suitable multi-chamber cell culture system can be networked.

[0128] In some variations, the input to the feedback mechanism can be derived using methods that include implementing machine learning techniques, including linear and nonlinear models (e.g., processes such as CART classification and regression trees), artificial neural networks (such as back-propagation networks), discriminant analysis (e.g., Bayesian classifiers or Fischer analysis), logistic classifiers, and support vector classifiers (e.g., support vector machines) using data types from the multi-chamber culture system.

[0129] Machine learning algorithms used to identify optimal culture conditions can leverage deep learning techniques, which utilize multiple layers in the learning process.

[0130] One deep learning technique is reinforcement learning. Reinforcement learning is an aspect of machine learning where an agent learns to behave in an environment by taking certain actions to observe the rewards / results it gets from those actions. The agent takes an action (at) on its environment. This produces information about the state of the environment (St) and a reward (Rt) that indicates whether the outcome is better than the previous outcome. The agent works under the assumption of maximizing the reward.

[0131] One form of reinforcement learning is called direct search. “Direct search” refers to the sequential examination of trial solutions, including comparing each trial solution to the “best” solution obtained up to that point, and a policy for determining (as a function of earlier results) what the next trial solution will be.

[0132] Q-learning is a model-free reinforcement learning algorithm that learns the value of an action in a particular state.

[0133] Artificial neural networks use a collection of interconnected nodes. Neural networks consist of layers of nodes, including an input layer, one or more hidden layers, and an output layer. Each node or artificial neuron is connected to another node or artificial neuron and has an associated weight and threshold. If the output of any individual node is above a specified threshold, the node is activated, sending data to the next layer of the network.

[0134] III. Methods of Producing Products

[0135] The methods provided herein can use any suitable system, such as a multi-chamber cell culture system as described above.

[0136] Provided herein is a method for preparing a product in a multi-chamber cell culture system. The method includes culturing cells in a first bioreactor under conditions that maintain cell growth, for example by maintaining a constant cell density. Under such constant cell density conditions, when the cells reach a steady state, they grow at a constant growth rate. In certain embodiments, the cells grow in a nutrient medium in which there is no cell culture medium that limits growth, and other culture conditions (such as temperature, pH, and dissolved oxygen) are set to provide a maximum or near-maximum growth rate.

[0137] In certain embodiments, the cells in the first culture chamber are then automatically moved to a second bioreactor chamber (or each of more than one second bioreactor chambers) where culture conditions are set to optimize production of a desired culture product.

[0138] In certain embodiments, the culture fluid is removed from the second culture chamber, and the culture product is separated from the fluid. In certain embodiments, the cells in the removed culture fluid are returned to the second culture chamber to continue the production of the product.

[0139] In certain embodiments, provided herein are methods for: (1) performing a first continuous culture of cells in a first chamber of a first bioreactor; (2) during the first continuous culture, moving a culture fluid containing cells from the first chamber of the first bioreactor to the second chamber of at least one second bioreactor; (3) performing a second continuous culture of cells moved from the first bioreactor in the second chamber of one or more second bioreactors to produce a culture product; and (4) collecting at least one culture product from the second chamber of one or more second bioreactors. In a preferred embodiment, the first bioreactor is operated under culture conditions that maintain cell growth by maintaining a constant cell density (e.g., a turbidostat). In certain embodiments, one or more second bioreactors are operated under constant culture conditions that are conducive to the production of culture products. In certain embodiments, the constant culture conditions in one or more second bioreactors are optimized for forming a culture product (e.g., a chemostat). Depending on the cells and the application, any suitable culture conditions can be used. In certain embodiments, the method further comprises maintaining a constant volume of cell culture (as disclosed herein) in the chamber of the first bioreactor. In certain embodiments, when the volume of the first bioreactor exceeds the target volume, the cell culture of constant volume in the first bioreactor is maintained by using, for example, a pump and / or a valve to move the culture fluid from the first chamber of the first bioreactor to the second chamber of one or more second bioreactors. In certain embodiments, the cell density of the first and / or one or more second bioreactors is maintained. Any suitable sensor (as disclosed herein) for cell density can be used. In certain embodiments, the sensor includes optical density, and the method includes measuring the optical density of the cell culture and diluting the cell culture by adding fluid (such as nutrient solution), and / or taking out a part of the cell culture fluid to maintain the target optical density. In certain embodiments, the culture conditions of one or more second bioreactors are optimized for the production of one or more culture products. In certain embodiments, the method includes adding one or more inducer molecules to the culture of one or more second bioreactors to induce the activity of the biochemical pathways producing the culture output (i.e., culture product). In a preferred embodiment, the inducer molecule is not added to the culture of the first bioreactor. In some embodiments, the growth rate of the culture in the second bioreactor is limited by a doubling rate of nutrient maintenance, which is at least any one of 2, 3, 5, 10, 15, 20, 50, 100 times slower than the first bioreactor, preferably 10-100 times slower. In some embodiments, the method further comprises collecting / recovering one or more culture products from the culture fluid.

[0140] A. Turbidistat and Chemostat

[0141] Two methods of operating bioreactors in continuous culture include turbidostats and chemostat. In both cases, nutrients and other chemicals are added to the cell culture at a rate called the dilution rate. The dilution rate is determined by feedback from sensors in the culture vessel to maintain the desired parameters.

[0142] Turbidistat Figure 1 ) is a continuous bioreactor in which cell density remains constant. Additionally or alternatively, the culture volume of the turbidostat can be kept constant. Cell density or biomass is determined by representative measurements such as turbidity or capacitance. After the cell reaches a steady state, if the cell density is maintained, the growth rate will be constant. In this system, nutrients can be maintained in excess to achieve a maximum or desired growth rate. The maximum growth rate varies according to culture medium formulation and process conditions. In certain embodiments, when culturing cells under turbidostat mode, the growth rate of the culture is maximized and / or set to a desired rate, and therefore, the biological production of the culture product is limited. In certain embodiments, when culturing cells under turbidostat mode, no culture product is produced. In certain embodiments, the turbidostat is coupled to a volume sensor to maintain constant turbidity and volume by removing the culture from the turbidostat after the volume exceeds the set point.

[0143] A chemostat is a continuous bioreactor that maintains steady-state culture conditions. Culture conditions are maintained by continuously adding culture medium to the culture to maintain the concentration of nutrients and other chemicals, while also removing culture fluid to keep the culture volume constant. Growth rate can be physically regulated by changing the rate at which chemicals are added to the chemostat and by changing the rate at which culture fluid is removed. Culture conditions can be set to favor or optimize the production of culture products. Such conditions typically divert cellular mechanisms away from cell growth and toward a reduction in culture products.

[0144] In certain embodiments, the method comprises operating a first bioreactor in turbidostat mode and operating one or more second bioreactors in chemostat mode.

[0145] B. Culture Conditions

[0146] In turbidostat mode or chemostat mode, many different culture conditions can be adjusted.

[0147] 1. Cell Density

[0148] Cell density can be maintained as follows. A set point for cell density is selected. A measure of cell density is determined from a sensor in the bioreactor. For example, a spectrometer can measure the turbidity of the cell culture. If the cell density is above the set point, medium is added to the culture chamber. If the cell density is below the set point, the addition of medium is stopped until the cells grow so that the cell density is above the set point. In certain embodiments, the OD of the first bioreactor is at least about any of 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 no more than about any of 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, e.g., 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 any of 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 no more than about any of 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, e.g., 1-300, preferably 50-300, more preferably 50-200.

[0149] Cell density can be affected by adding flow to the bioreactor vessel to dilute the cell culture. The dilution rate of a continuous culture can be calculated using the formula D = FV -1 To model the dilution rate (D; [hr -1 ]) is the medium flow rate into the bioreactor (F; [L hr -1]) and the culture volume (V; [L]) in the bioreactor. In certain embodiments, in order to achieve a steady-state culture, the dilution rate (D) should be equal to the specific growth (μ) of the cell culture, thereby maintaining a constant biomass concentration in the bioreactor. In certain embodiments, the method includes a control system, which includes 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 cell density includes adding additional fluids, such as nutrient solutions, and / or removing culture fluids. In preferred embodiments, maintaining cell density includes both adding additional fluids and removing cell cultures. As discussed herein, a pump can be used as an effector to move liquids into and out of a bioreactor.

[0150] 2. Culture volume

[0151] The culture volume may be maintained in either or both turbidostat mode and chemostat mode.

[0152] The mass of the bioreactor is a proxy for the volume of the cell culture, since mass will increase or decrease as volume increases or decreases. When the cell culture becomes too large, the system can reduce the volume of the cell culture by removing the cell culture from the bioreactor. This can be done with a pump or mechanical device such as a top shut-off tube.

[0153] In certain embodiments, method comprises control system, and described control system comprises one or more sensors and effector of the culture volume that actively maintains one or more bioreactors.In certain embodiments, the culture volume of bioreactor is input in user programmable module, and control system actively maintains the culture volume of one or more bioreactors.In certain embodiments, maintaining culture volume comprises adding other fluid, for example nutrient solution, and / or taking out culture fluid.In certain embodiments, maintaining culture volume comprises adding other fluid and taking out cell culture.As discussed herein, pump can be used as effector so that liquid is moved into and out of bioreactor.

[0154] 3. Temperature

[0155] Temperature can affect both the growth rate of cells in culture and the production of their products. Therefore, temperature regulation is a culture condition that can be regulated using any suitable control system as described above in the methods herein. The temperature of one or more bioreactors in a multi-chamber cell culture system can be maintained at any suitable temperature, such as 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C or 85°C. At least about any one of and / or no more than 22°C, 24°C, 26°C, 28°C, 30°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C, for example 15°C-90°C, preferably 20°C-50°C, more preferably 25°C-40°C.

[0156] 4. pH

[0157] The pH indicates the acidity or alkalinity of the culture. The pH can be adjusted by adding a solution containing an acid or base to the culture. The pH of the cell culture can be adjusted using a pumping system controlled by a feedback system connected to a pH sensor, wherein the pumping system delivers a calculated volume of a suitable acid or base and / or the amount of titrated acid or base as needed until the sensor reads the desired pH.

[0158] 5. Dissolved O2

[0159] Dissolved oxygen is necessary for aerobic cells and microbial growth. Dissolved oxygen can use a ventilation system to introduce air into the cell culture to regulate. In certain embodiments, the method comprises using a ventilation system comprising a pump and / or a valve communicated with one or more gas sources (e.g., canister or atmospheric air) to control, and the pump and / or valve deliver air to the inside of one or more culturing chambers. Dissolved carbon dioxide can be controlled by evacuating the waste gas comprising CO when the ventilation system delivers new gas to the culturing chamber 2.

[0160] In certain embodiments, method comprises 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 including a feedback mechanism actuates one or more ventilation systems including one or more gas pumps and / or valves connected to one or more gas sources (e.g., canisters or atmospheric air), wherein the pumping system delivers the calculated volume of air to the inside of one or more culture chambers.In certain embodiments, one or more gases (e.g., CO2) are evacuated when the ventilation system delivers new gas to the culture chamber.

[0161] 6. Nutrient concentration

[0162] The concentration of nutrients can be critical to the growth rate. In order to maintain maximum growth rate, the concentration of nutrients in the cell culture should be set so as not to limit the growth rate.

[0163] In some embodiments, the production of product may be performed under the condition of culture conditions. However, for the production of product, it may be desirable to set culture conditions to produce the product of preference. In such a case, it may be desirable to reduce the concentration of one or more nutrients. For example, cells grown in turbidostat mode usually grow at a high growth rate and need high-level nutrients. However, when they are moved to the bioreactor operated in chemostat mode, it may be necessary to reduce the concentration of one or more nutrients (such as micronutrients) so that cell physiology becomes the production of culture product. In certain embodiments, the growth rate of culture in the second bioreactor is maintained under growth restriction (for example, no growth or growth is very slow) conditions. This can be, for example, less than any one doubling rate in 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 5 days, 6 days, 7 days, 2 weeks or 1 month. In other embodiments, culture is maintained with at least about any one doubling rate in 72,48,24,12,6 or 3 hours. In certain embodiments, the growth rate of the culture in the second bioreactor is maintained at at least any of a doubling rate that is 2, 3, 5, 10, 20, 50, or 100X slower than the growth rate in the first bioreactor.

[0164] In some embodiments, the method includes maintaining a desired nutrient concentration of one or more nutrients. In some embodiments, the nutrient concentration in the cell culture is measured using a suitable sensor, and a control system including a feedback mechanism actuates one or more pumping systems in fluid communication with one or more nutrient reservoirs, wherein the pumping system delivers a calculated volume of nutrient solution and / or titrates the amount of fluid containing the nutrient as needed until the sensor reads the desired nutrient concentration.

[0165] C. Culture products

[0166] Culture output can be any measurable characteristic of the culture to be optimized. Culture output includes, for example, cell biomass, molecular products, and product production based on cell physiology or a representation of cell health. In certain embodiments, the method further comprises collecting / recovering one or more culture products.

[0167] 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 and time.

[0168] In certain embodiments, the culture output is a chemical product ("culture product") produced by the cell. The culture product can be a molecular entity that is a product of fermentation or gene expression and is typically a product harvested and commercialized from the culture. 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 products of synthetic pathways (e.g., mediated by enzymes) in cells. Such products include, for example, industrial chemicals. These include, but are not limited to, flavorings (e.g., vanillin); spices (e.g., aldehydes, coumarins, indoles), 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). Product output can be measured directly as a concentration or as a function of quantity, for example, as a volumetric production rate, product titer, product yield, or specific production rate.

[0169] In certain embodiments, culture output is a representation ("culture representation") generated based on outputs such as cell physiology. A culture representation is a measurable parameter that indicates the health and / or physiology of a cell culture. These include, but are not limited to, specific CO2 generation rate, specific O2 consumption rate, organic acid spectrum, metabolite spectrum, byproduct spectrum, production economics (cost of producing 1 kg of product under specific cell culture conditions). Specific rate 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.

[0170] D. Cell Growth in the First Bioreactor

[0171] In the first bioreactor, the cells are grown under culture conditions suitable for continued growth of the cells and production of minimal or no culture product.

[0172] The continuous growth rate can include a constant growth rate over the time period during which the cells are transferred to the second culture container. The growth rate can be measured as a function of the doubling time. The growth rate can be a maximum growth rate. However, it can be desirable to be less than the maximum growth rate. Therefore, the growth rate can include at least any one of 1%, 2%, 4%, 8%, 16%, 32%, 50%, 60%, 70%, 80% or 90% of the maximum growth rate, such as between 10% and 100% of the maximum growth rate.

[0173] In certain embodiments, cells can be grown at a constant density, typically under conditions optimized for a high or desired growth rate. This would include, for example, the use of a cell culture medium in which nutrients are not present at growth-limiting concentrations.

[0174] In other embodiments, the culture medium comprises one or more nutrients, such as micronutrients, that are present in an amount that allows the cells to grow in the first bioreactor but does not support cell growth after transfer to one or more second production bioreactors. For example, the nutrients may be present in the first bioreactor in an amount such that any remaining nutrients are rapidly consumed after transfer, and the cells reach a steady state based on the amount of nutrients fed to the second bioreactor.

[0175] In certain embodiments, the method provided herein includes maintaining a constant growth rate in cell culture. Any suitable method for maintaining a constant growth rate can be used, such as limiting one or more reagents / substrates / nutrients and / or adjusting the temperature. In certain embodiments, the growth rate is at least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the maximum cell growth rate. In certain embodiments, wherein one or more reagents / substrates / nutrients are restrictive, compared with a culture that does not lack one or more reagents / substrates / nutrients, the growth rate is no more than 50%, 60%, 70%, 80%, 90%, 95% or 100% of the maximum cell growth rate. In certain embodiments, the cell growth rate is maintained without being limited by nutrients. In certain embodiments, the cell growth rate is not limited or controlled.

[0176] The production of the culture product in the first bioreactor can be less than the production of the culture product in the second bioreactor. For example, the amount of the product produced per unit volume in the first bioreactor can be less than any one of 50%, 40%, 30%, 20%, 10%, 5%, 3% or 1% of the amount of the product of the second bioreactor. The "minimum" production of the culture product means less than commercially feasible, for example, the cost of producing the product does not exceed twice the value of the product produced. The amount can be, for example, 5% of the amount when the cell is cultured under product optimization conditions, or less than 0.05 mol / l / hour. The production of the culture product can be measured in gm product per gram of cell dry weight per hour. By appropriately setting the culture conditions, the production of the culture product can be suppressed. For example, if the product is produced via an induction process, the culture is not exposed to an inducer. In addition, by maintaining little to no production, the method provided contributes to strain stability by reducing the metabolic load on the primary strain, thereby reducing the probability of developing mutant strains.

[0177] In certain embodiments, the culture fluid from the first bioreactor containing cells is periodically moved to the second bioreactor. For example, this can be when the volume of the cell culture exceeds a set point. In this case, the liquid removed from the culture chamber will be offset by the culture medium added to the chamber.

[0178] E. Cell Growth in the Second Bioreactor

[0179] In one or more second bioreactors, cells can be grown under culture conditions that produce a desired culture product. Such conditions can be conducive to or optimize the production of the culture product. The cells in the second continuous culture have a growth rate that is less than the growth rate of the first bioreactor. The cell growth rate in the second bioreactor may be no more than any one of 50%, 40%, 30%, 20%, 10%, 5%, 3%, 1%, 0.5% or 0.1% of the growth rate of the first bioreactor.

[0180] The culture conditions can be growth-restrictive. For example, the culture can include one or more nutrients of growth-restrictive concentration. Growth-restrictive concentration is a concentration that prevents cells from growing at a maximum growth rate. The growth-restrictive doubling time of cells in the second bioreactor can be at least any one 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-restrictive growth rate can be no more than any one of 50%, 40%, 30%, 20%, 10%, 5%, 3%, 1%, 0.5% or 0.1% of the maximum growth rate of the cell. Under restrictive growth conditions, the chance of undesirable mutant microorganism development and growth is reduced. This allows the second bioreactor to continue to produce products in a longer period of time. In addition, by maintaining a slow growth rate in the second continuous culture, the method provided helps strain stability by reducing the probability that mutant strains or pollutants will exceed the primary strain.

[0181] In one or more second bioreactors, cells are typically grown under constant culture conditions that produce (e.g., optimized for producing a desired culture product). The optimal conditions for producing a product are specific to the product and the cells that produce them. Methods for determining optimal culture conditions include, for example, changing culture parameters when identifying the values ​​or set points of parameters that optimize product production. Culture parameters to be optimized include any culture parameters described herein, including but not limited to temperature, pH, dissolved oxygen, and nutrients such as carbon sources, nitrogen sources, minerals, etc.

[0182] 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, such as limiting one or more reagents / substrates / nutrients and / or adjusting temperature.

[0183] In the second bioreactor, one or more nutrients are typically at a growth-limiting concentration and are also at a relative concentration that is less than the concentration in the first bioreactor. For example, the nutrient may be present in the first bioreactor at a concentration (e.g., molar concentration) that is at least any of 2, 4, 10, 25, 50, 100, or 500 times greater than the nutrient concentration in the second bioreactor.

[0184] In one embodiment, the relative concentration of the rate-limiting nutrient can be normalized for the amount of carbon in the first and second bioreactors, respectively. This can be useful when the carbon concentration in the second bioreactor is not limiting itself. For example, the relative amount of carbon: rate-limiting nutrients (e.g., phosphorus) can be 100:1 in the first bioreactor, but can be 1000:1 in the second bioreactor, with a normalized ratio of 1:10. Therefore, the normalized ratio of carbon: rate-limiting nutrients in the first continuous culture to carbon: rate-limiting nutrients in the second continuous culture can be 1:5, 1:10, 1:50, 1:100; 1:500; 1:1000; 1:5000 or 1:10,000.

[0185] In certain embodiments, the methods provided herein include maximizing the biological production of one or more culture products in the second bioreactor. In certain embodiments, biological production is carried out in one or more second bioreactors for 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 at least about any one and / or no more than 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 about any one, for example 1-10,000 hours, preferably 50-1,000 hours.

[0186] 1. Cell Recovery

[0187] The cell culture in one or more second bioreactors is usually maintained at a constant volume. When the culture fluid and other fluids (such as reagent fluid) from the first bioreactor are added to the second bioreactor, this needs to take out the culture fluid from the second bioreactor. Such taken-out fluid includes the cells grown in the second bioreactor. In certain embodiments, the cells can be returned to the second bioreactor. A method of doing so is to use a filter, such as a hollow fiber box. The fluid entering one end of the hollow fiber box is forced to pass through the filter. However, the cell is too large to pass through the filter. Therefore, the fluid containing the cell is returned to the second bioreactor. The fluid exhausted of the cell can be deposited in a container for product harvesting.

[0188] F. Product Harvest

[0189] The culture fluid in one or more second bioreactors (and optionally the first bioreactor, depending on the metabolic pathways in the cell culture) comprises a culture product for harvesting. In certain embodiments, the method also includes collecting / recovering one or more culture products. In certain embodiments, the culture fluid from one or more second bioreactors is taken out from the chamber, and cells are separated from the culture fluid to produce a cell-free culture fluid. In certain embodiments, the cell-free culture fluid is continuously provided to a reservoir using a cell recovery system.

[0190] Separation of the product from the culture fluid depends on the properties of the product. For example, if the product is a protein, it can be separated on an affinity column, which for example comprises a solid support derived from an antibody bound to the protein. Any suitable chromatography and / or separation method can be used, such as size exclusion, ion exchange or affinity chromatography. Small molecule products can be separated from the culture fluid using chromatography, such as preparative HPLC, distillation and / or crystallization. Any suitable method can be used.

[0191] Exemplary embodiments

[0192] 1. A method comprising:

[0193] a) carrying out a first continuous culturing of cells in at least one chamber of a first bioreactor under culture conditions resulting in growth of the cells;

[0194] b) during the first continuous culturing, moving a culture fluid comprising cells from a chamber of the at least one first bioreactor to a chamber of at least one second bioreactor via one or more fluid conduits, the one or more fluid conduits placing the chamber of the at least one first bioreactor in fluid communication with the chamber of the at least one second bioreactor; and

[0195] c) performing a second continuous culture of cells removed from the at least one first bioreactor in a chamber of the at least one second bioreactor under culture conditions for producing at least one culture product;

[0196] The cell growth rate in the first continuous culture is greater than the cell growth rate in the second continuous culture, and the production of the culture product in the first continuous culture is less than that in the second continuous culture.

[0197] 2. The method according to embodiment 1 further comprises:

[0198] d) collecting at least one culture product from the chamber of the at least one second bioreactor.

[0199] 3. A method according to embodiment 1, wherein the cell growth rate in the first continuous culture is at least twice that of the second continuous culture, and the production of the culture product in the second continuous culture is at least twice that of the first continuous culture.

[0200] 4. A method according to embodiment 1, wherein the cell growth rate in the first continuous culture is at least 10 times that of the second continuous culture, and the production of culture products in the second continuous culture is at least 10 times that of the first continuous culture.

[0201] 5. A method according to embodiment 1, wherein the cell growth rate in the first continuous culture is at least 100 times that of the second continuous culture, and the production of culture products in the second continuous culture is at least 100 times that of the first continuous culture.

[0202] 6. A method according to embodiment 1, wherein the cell growth rate in the first continuous culture is at least 80%, such as at least 90%, of the maximum growth rate.

[0203] 7. The method according to embodiment 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.

[0204] 8. The method according to embodiment 1 comprises maintaining the cell density in the at least one first bioreactor.

[0205] 9. The method of embodiment 8, wherein maintaining the cell density in the at least one first bioreactor comprises maintaining the optical density of the first continuous cell culture.

[0206] 10. The method of embodiment 8, wherein maintaining cell density comprises adding a nutrient solution to the first continuous culture.

[0207] 11. The method according to embodiment 8, wherein performing the first continuous culture further comprises maintaining a constant volume of cell culture in the chamber of the at least one first bioreactor.

[0208] 12. A method according to embodiment 11, wherein maintaining the volume includes moving the culture fluid containing culture 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 of the first continuous culture exceeds the target volume.

[0209] 13. The method of embodiment 1, wherein moving the culture fluid from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor comprises pumping the culture fluid with a pump.

[0210] 14. The method according to embodiment 1 comprises growing the cells at a constant cell growth rate in the chamber of the at least one first bioreactor.

[0211] 15. A method according to embodiment 1, wherein the cell growth rate in the first continuous culture is at least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the maximum cell growth rate.

[0212] 16. A method according to embodiment 1, wherein no growth-limiting nutrients are present in the first continuous culture.

[0213] 17. A method according to embodiment 1, wherein at least one nutrient in the first continuous culture, such as carbon or phosphorus, is at a growth-limiting concentration.

[0214] 18. A method according to embodiment 1, wherein the first continuous cell culture produces no culture product or produces a minimal amount of culture product.

[0215] 19. A method according to embodiment 1, wherein the production rate of 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 that in the second continuous culture.

[0216] 20. A method according to embodiment 1, wherein the volume of the first continuous culture is smaller than the volume of the second continuous culture, for example, wherein the relative volume ratio is less than any one of 1:1, 1:10, 1:100 and 1:1000, for example, between about 1:10 and 1:50.

[0217] 21. A method according to embodiment 1, wherein the volume of either or both of the first continuous culture and the second continuous culture is between about 50 ml and about 100,000 L, such as between about 500 ml and about 10 L.

[0218] 22. A method according to embodiment 1, wherein cells are removed from the chamber of the at least one first bioreactor during growth, such as during logarithmic growth.

[0219] 23. A method according to embodiment 1, wherein the culture fluid containing cells is continuously moved from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor within a time period of at least any one of 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.

[0220] 24. A method according to embodiment 1, wherein the culture fluid containing cells is moved from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor in a total volume of at least any one of 0.01 L, 0.1 L, 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.

[0221] 25. A method according to embodiment 1, wherein when the optical density (OD) of the first continuous culture reaches at least any one of 1, 5, 10, 25, 50, 100, 200 and 400, the culture fluid containing cells is moved from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor.

[0222] 26. A method according to embodiment 1, wherein the cell growth rate in the second continuous culture does not exceed any one of 50%, 40%, 30%, 20%, 10%, 5%, 3% or 1% of the growth rate of the first continuous culture.

[0223] 27. A method according to embodiment 1, wherein the growth rate of the cells in the second continuous culture does not exceed any one of 50%, 40%, 30%, 20%, 10%, 5%, 3% or 1% of the maximum growth rate of the cells under non-restrictive culture conditions.

[0224] 28. A method according to embodiment 1, wherein the cells in the second continuous cell culture do not grow.

[0225] 29. A method according to embodiment 1, wherein at least one nutrient in the second continuous culture is present at a growth rate limiting concentration.

[0226] 30. A method according to embodiment 29, wherein the concentration of the nutrient in the first continuous culture is at least any one of 2 times, 4 times, 10 times, 25 times, 50 times, 100 times or 500 times greater than the concentration of the nutrient in the second continuous culture.

[0227] 31. A method according to embodiment 1, wherein the culture conditions of the second continuous culture include growth-limiting concentrations of one or more nutrients (e.g., selected from metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdate, selenite and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid, thiamine, biotin, cyanocobalamin, folic acid), 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, amino acids (e.g., glutamic acid, leucine and tryptophan), nucleic acid bases (e.g., adenine, cytosine, thymine, uracil and guanine) or complex nutrients (e.g., yeast extract, peptone, tryptone, casamino acids and corn steep liquor)).

[0228] 32. A method according to embodiment 1, wherein the culture conditions in the second continuous culture are maintained constant for at least any one of 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.

[0229] 33. A method according to embodiment 1, wherein the second continuous culture is carried out for at least about any one of 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 no more than about any one of 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, 1-10,000 hours, preferably 50-1,000 hours.

[0230] 34. The method according to embodiment 1 comprises performing the second continuous culture in more than one different second bioreactor.

[0231] 35. The method of embodiment 1, wherein the at least one first bioreactor is more than one first bioreactor.

[0232] 36. A method according to embodiment 1, wherein the culture conditions of the second continuous culture are optimized to produce at least one culture product.

[0233] 37. A method according to embodiment 1, wherein the culture conditions of the second continuous culture are different from the culture conditions of the first continuous culture.

[0234] 38. The method according to embodiment 1 includes 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 continuous culture and / or the second continuous culture.

[0235] 39. A method according to embodiment 1, wherein the culture conditions of the first continuous culture and / or the second continuous culture include maintaining a constant concentration of one or more nutrients in the culture.

[0236] 40. The method of embodiment 1, comprising inducing the activity of a biochemical pathway that produces a culture output in the second continuous culture but not in the first continuous culture.

[0237] 41. A method according to embodiment 1, wherein the second continuous culture has an OD of at least any one of 10, 50, 100 or 1000.

[0238] 42. A method according to embodiment 1, wherein the cells in the second continuous culture have a doubling time of no more than any one of 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 5 days, 6 days, 7 days, 2 weeks or 1 month.

[0239] 43. A method according to embodiment 1, wherein the cells in the second continuous culture are maintained at a constant density.

[0240] 44. A method according to embodiment 1, wherein cells removed from the chamber of the at least one second bioreactor are recovered into the chamber using a cell recovery device such as a hollow fiber filter.

[0241] 45. The method according to embodiment 1, wherein performing the first continuous culture or the second continuous culture comprises providing one or more of the following: metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdate, selenite and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid, thiamine, biotin, cyanocobalamin, folic acid), pollution control agents such as antibiotics or biocides, inducers, salts, phosphates, sulfates, chlorides, acetates, citrates and the like. anionic salts, magnesium, calcium, sodium, potassium, ammonium and other cationic salts, boric acid, choline, ascorbic acid, lipoic acid, niacin, inositol, antifoaming agents (e.g., Antifoam 204, Antifoam A, Antifoam C), amino acids (e.g., glutamic acid, leucine and tryptophan), nucleic acid bases (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.

[0242] 46. ​​A method according to embodiment 1, wherein conducting the first continuous culture or the second continuous culture includes providing a carbon source (e.g., a sugar (e.g., glucose, xylose, sucrose, glycerol or acetate), molasses, malt extract, starch, dextrin, pulp, CO or CO2).

[0243] 47. A method according to embodiment 1, wherein performing the first continuous culture or the second continuous culture includes providing one or more of the following: a nitrogen source (e.g., an amino acid or a polypeptide, urea, an ammonium salt (e.g., ammonium sulfate, ammonium phosphate or ammonia), corn steep liquor, yeast extract, peptone and soybean meal).

[0244] 48. A method according to embodiment 1, wherein conducting the first continuous culture or the second continuous culture includes adjusting one or more of the following: nitrogen retention rate, ventilation rate, oxygen sparge rate, carbon dioxide retention rate, culture agitation speed, CO2 concentration, carbon source concentration, nitrogen source concentration, metal, vitamin, salt concentration and antibiotic concentration.

[0245] 49. A method according to embodiment 1, wherein collecting the at least one culture product comprises removing a cell culture fluid from a chamber of the at least one second bioreactor and separating cells from the cell culture fluid to produce a cell-free culture fluid.

[0246] 50. A method according to embodiment 1, wherein the culture product is selected from polypeptides (e.g., proteins, enzymes, antibodies), organic molecules that are products of synthetic pathways in the cell (e.g., industrial chemicals such as flavorings (e.g., vanillin)); flavors (e.g., aldehydes, coumarins, indoles), amino acids, organic acids (e.g., citric acid, lactic acid, and acetic acid); alcohols (e.g., ethanol, isopropanol, ketones such as acetone); and fatty acids (e.g., palmitic acid and oleic acid).

[0247] 51. A method according to embodiment 1, wherein the cells include archaea, prokaryotes and / or eukaryotes.

[0248] 52. The method of embodiment 1, wherein the cell comprises a fungal cell (e.g., a yeast (e.g., a Saccharomyces spp., a Pichia spp., a Komagataella spp., a Kuyveromyces spp., an Aspergillus spp., a Rhodoporidium spp., a Lipolytica spp., an Aspergillus spp., a Neurospora spp., a Trichoderma spp., a Candida spp., or a Penicillium spp.).

[0249] 53. The method of embodiment 1, wherein the cells comprise bacterial cells (e.g., Escherichia coli, Bacillus spp, Clostridia spp, Streptomyces spp, Pseudomonas spp, Ralstonia spp, Shewanella spp).

[0250] 54. The method of embodiment 1, wherein the cell comprises an insect cell, an animal cell or a plant cell.

[0251] 55. A method according to embodiment 1, wherein the cell comprises an animal cell (e.g., an arthropod (e.g., insects, shrimp, lobster, crayfish, and crab); a chordate (e.g., fish, amphibians, reptiles, birds (e.g., chicken or turkey); a mammal (e.g., human or non-human such as bovines, lambs, goats, pigs, horses, dogs, cats, primates)).

[0252] 56. A method according to embodiment 1, wherein the cell comprises a cell line (e.g., CHO (Chinese Hamster Ovary), BHK21 (Baby Hamster Kidney), NSO, Sp2 / 0 mouse cell line, insect cells (e.g., SP9, Sf9, sf21, S2), tobacco BY-2 cells, rice (Oryza Sativa), or algae cells).

[0253] 57. The method of embodiment 1, wherein the cell is not a photosynthetic cell.

[0254] 58. A system comprising:

[0255] (a) at least one first bioreactor, the at least one first bioreactor comprising:

[0256] (i) the first chamber; and

[0257] (ii) one or more first sensors providing at least a measure of the cell density in the first chamber and a measure of the volume of the cell culture;

[0258] (b) at least one second bioreactor, the at least one second bioreactor comprising:

[0259] (i) the second chamber; and

[0260] (ii) one or more second sensors that provide at least a measure of culture conditions and a measure of the volume of the cell culture in the one or more second chambers;

[0261] wherein the first chamber is in fluid communication with the one or more second chambers;

[0262] (c) one or more reagent reservoirs containing liquid reagents, wherein the reagent reservoirs are in fluid communication with the first chamber, and optionally with the one or more second chambers;

[0263] (d) one or more pumps configured to:

[0264] (i) moving a liquid reagent from at least one reagent reservoir to the first chamber;

[0265] (ii) moving cell culture fluid from the first chamber to the one or more second chambers;

[0266] (iii) removing the cell culture fluid from the one or more second chambers;

[0267] (iv) optionally moving a liquid reagent from at least one reagent reservoir to the one or more second chambers; and

[0268] (e) a control system, the control system being configured to:

[0269] (i) using measurements from said one or more sensors to control said

[0270] cell growth, e.g., cell density, of the cell culture in the first chamber;

[0271] (ii) moving cell culture fluid from the first chamber to the one or more second chambers; and

[0272] (iii) using measurements from the one or more sensors to control culture conditions and, for example, volume and / or cell growth, of the cell culture in the one or more second chambers to produce a culture product at a rate greater than the rate in the first chamber.

[0273] 59. A method according to embodiment 58, wherein the control system uses 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 that of 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 that of the first continuous culture.

[0274] 60. The system of embodiment 58 further comprises one or more collection containers, wherein the one or more collection containers are fluidly connected to the one or more second chambers.

[0275] 61. The system of embodiment 58, wherein the first chamber and the one or more second chambers have a volume between 250 mL and 1,000,000 liters.

[0276] 62. A system according to embodiment 58, wherein the control system sets the dilution rate of the liquid reagent moved into the first chamber and / or one or more second chambers to maintain the concentration of one or more nutrients.

[0277] 63. A system according to embodiment 58, wherein the first chamber and the one or more second chambers include ports that are connected to the reagent reservoir and to each other through fluid conduits.

[0278] 64. The system of embodiment 58, wherein the one or more second chambers are more than one second chamber.

[0279] 65. A system according to embodiment 58, wherein the one or more first sensors or second sensors providing a measure of volume include a scale that measures the mass of the bioreactor.

[0280] 66. A system according to embodiment 58, wherein the sensor that provides a measure of the cell density of the cell culture in the first chamber includes an optical density sensor or a capacitive sensor.

[0281] 67. A system according to embodiment 65, wherein controlling cell density includes controlling the optical density of the cell culture.

[0282] 68. A system according to embodiment 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.

[0283] 69. A system according to embodiment 58, wherein the one or more first sensors provide measurements of temperature, pH, and dissolved oxygen of the cell culture in the first chamber.

[0284] 70. A system according to embodiment 58, wherein the one or more second sensors provide measurements of temperature, pH, and dissolved oxygen of the cell culture in the second chamber.

[0285] 71. The system of embodiment 58, wherein at least one pump is a peristaltic pump or a gravity pump.

[0286] 72. A system according to embodiment 70, wherein the peristaltic pump includes fittings for tubing for fluid communication between the two chambers, between a chamber and a reagent reservoir, or between the second chamber and an effluent conduit.

[0287] 73. A system according to embodiment 58, wherein the control system for controlling the volume of the cell culture in the first chamber or the one or more second chambers includes a liquid level sensor (e.g., an overflow tube) that removes cell culture fluid from the chamber when the height of the cell culture exceeds the top of the liquid level sensor.

[0288] 74. A system according to embodiment 58, wherein the control system comprises a computer, the computer comprising a processor and a memory, the memory comprising executable code, which when executed by the processor performs one or more feedback routines to control cell growth (e.g., cell density) in the first chamber, control the volume of cell culture in the first chamber and / or one or more second chambers, and control culture conditions in the one or more second chambers.

[0289] 75. A system according to embodiment 73, wherein one of the feedback routines calculates a cell density of the cell culture in the first chamber based on measurements received from the one or more sensors, and actuates a pump to move liquid reagent from a reagent reservoir to the first chamber to adjust the cell density to a set point.

[0290] 76. A system according to embodiment 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 if the volume is above a set point, actuates a pump to move liquid cell culture from the first chamber to the one or more second chambers.

[0291] 77. The system of embodiment 73, wherein:

[0292] (I) the one or more sensors provide a measurement of one or more culture parameters in the first chamber and / or one or more second chambers;

[0293] (II) the system further comprises one or more effectors to affect a change in the one or more culture parameters;

[0294] (III) one or more of the feedback routines calculates one or more culture parameters based on the measurements, and actuates one or more effectors to adjust the culture parameters towards a target level.

[0295] 78. The system of embodiment 73, wherein:

[0296] (I) the one or more sensors provide a measurement of one or more of pH, temperature, and dissolved O2 of the cell culture in the first chamber and / or one or more second chambers;

[0297] (II) The system further comprises one or more of the following:

[0298] one or more reagent reservoirs containing an acid and a base in fluid communication with the first reservoir and / or one or more second reservoirs;

[0299] one or more temperature controllers that control the temperature of the cell culture in the first chamber and / or one or more second chambers;

[0300] one or more aerators for aerating the cell culture in the first chamber and / or one or more second chambers;

[0301] (III) one or more of the feedback routines calculates culture conditions including one or more of pH, temperature, dissolved O2, and nutrient concentration based on measurements received from the one or more sensors, and actuates:

[0302] one or more pumps to move acid or base from the reagent reservoir into the second chamber to adjust the pH to a target pH;

[0303] one or more temperature controllers to adjust the temperature to a target temperature; and / or

[0304] One or more aerators to aerate the cell culture to a target dissolved oxygen level.

[0305] 79. A system according to embodiment 73, wherein one of the feedback routines 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 is above a set point, actuates a pump to remove liquid cell culture from the one or more second chambers.

[0306] 80. The system according to embodiment 58 further includes a filter, which is connected to the outlet fluid of the second chamber, and the filter is configured to recover cells that cannot pass through the membrane back to the second chamber, and transfer the liquid culture medium depleted of cells from the box to the collection container.

[0307] 81. The system of embodiment 79, wherein the filter comprises a hollow fiber cartridge.

[0308] 82. A system according to embodiment 58, wherein the first chamber comprises a cell culture, and wherein the one or more reagent reservoirs in fluid communication with the first chamber comprise a growth medium in which one or more nutrients are present in a concentration that limits the growth rate of the cells in the cell culture.

[0309] 83. A system according to embodiment 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 a concentration that limits growth in the one or more second bioreactors.

[0310] 84. The system of embodiment 58, wherein the one or more control subsystems comprises a computer comprising:

[0311] (A) Processor;

[0312] (B) a memory coupled to the processor, and

[0313] (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 the pump to move liquid into and out of the chambers.

[0314] 85. The system of embodiment 58, comprising one or more of the following:

[0315] ●Temperature sensor and temperature regulator;

[0316] a dissolved oxygen meter and a ventilation system in communication with the interior of the first chamber and / or one or more second chambers;

[0317] ●Analyzers for measuring nutrient concentrations in cell cultures in chambers;

[0318] an impeller or pneumatic agitator for mixing the liquid in the chamber, and a motor configured to actuate the impeller or pneumatic agitator;

[0319] • an effluent communicating with the interior of the container and an adjustable valve or pump for regulating the flow of fluid from the chamber;

[0320] ● baffles in the chamber;

[0321] An ejector and a mass flow controller in communication with the interior of the chamber for inputting one or more gases and mixtures thereof;

[0322] ● a user interface for communicating instructions to the computer;

[0323] ●Foam control;

[0324] ●Hollow fiber membranes for cell recovery; and

[0325] ●Reverse or forward osmosis membrane.

[0326] 86. The system of embodiment 58, wherein the control system is configured to perform any of the methods described in embodiments 1-35.

[0327] 87. A method comprising:

[0328] a) carrying out a first continuous culturing of cells in a chamber of a first bioreactor under culture conditions maintaining a constant cell density;

[0329] b) during the first continuous culturing period, moving a culture fluid containing cells from a chamber of the first bioreactor to a chamber of at least one second bioreactor via one or more fluid conduits, the one or more fluid conduits placing the chamber of the first bioreactor in fluid communication with the chamber of the at least one second bioreactor;

[0330] c) performing a second continuous culture of the cells removed from the first bioreactor in a chamber of each of the one or more second bioreactors under constant culture conditions to produce at least one culture product; and

[0331] d) collecting the at least one culture product from the chamber of each of the one or more second bioreactors.

[0332] 88. A system comprising:

[0333] (a) a first bioreactor, the first bioreactor comprising:

[0334] (i) the first chamber; and

[0335] (ii) one or more first sensors providing at least a measure of the cell density in the first chamber and a measure of the volume of the cell culture;

[0336] (b) one or more second bioreactors, the one or more second bioreactors comprising:

[0337] (i) the second chamber; and

[0338] (ii) one or more second sensors that provide at least a measure of culture conditions and a measure of the volume of the cell culture in the one or more second chambers;

[0339] wherein the first chamber is in fluid communication with the one or more second chambers;

[0340] (c) one or more reagent reservoirs containing liquid reagents, wherein the reagent reservoirs are in fluid communication with the first chamber, and optionally with the one or more second chambers;

[0341] (d) one or more pumps configured to:

[0342] (i) moving a liquid reagent from at least one reagent reservoir to the first chamber;

[0343] (ii) moving cell culture fluid from the first chamber to the one or more second chambers;

[0344] (iii) removing the cell culture fluid from the one or more second chambers;

[0345] (iv) optionally moving a liquid reagent from at least one reagent reservoir to the one or more second chambers; and

[0346] (e) a control system, the control system being configured to:

[0347] (i) using measurements from one or more sensors to control cell growth of the cell culture in the first chamber;

[0348] long, such as cell density;

[0349] (ii) moving cell culture fluid from the first chamber to the one or more second chambers; and

[0350] (iii) using measurements from the one or more sensors to control culture conditions and, for example, volume and / or cell growth of the cell culture in the one or more second chambers. Example

[0351] Example 1: Production of culture products using a multi-chamber cell culture system

[0352] This example demonstrates the ability to produce culture products using a multi-chamber cell culture system. Figure 5 ).

[0353] Inoculum preparation : Bacterial colonies from glycerol stocks stored at -80°C were streaked onto LB agar plates with appropriate antibiotics and grown under suitable conditions until visible colonies formed. 5 mL of growth medium with appropriate antibiotics was inoculated with a single colony from the LB agar plate and grown overnight at 37°C with shaking at 250 rpm. The next day, 300 mL of growth medium was inoculated with 3 mL of turbid overnight culture and incubated at 37°C with shaking at 250 rpm. When the optical density of the culture at 600 nm reached approximately 1, the cells were ready to be inoculated into the first bioreactor. 10 mL of cell aliquots from the 300 mL culture were stored in 15% glycerol in a -80°C refrigerator for future bioreactor runs.

[0354] The following bioreactor runs were performed using a Sartorius Biostat B 1.8 L bioreactor.

[0355] Bioreactor assembly and sterilization: Prepare two bioreactors. The reactor vessel top plate is fixed to a glass container, and the following components are inserted into the top ports of the container: (1) condenser, (2) ejector, (3) 4-way port assembly (1 port for feed, 2 ports for acid / base, and 1 port for addition), (4) sampling port, (5) effluent port, (6) dissolved oxygen (DO) probe, and (7) pH probe. The assembly is autoclaved at 121°C for 30 minutes together with the feed storage bottle, growth medium and feed tube. After autoclaving and cooling the container to room temperature, the OD probe is installed to the appropriate container top port to measure optical density. The bioreactor is placed on a scale and the scale is zeroed after placing the batch culture medium (500-1000L). The bottom bottle is connected to the two tanks. The growth medium is placed on a scale attached to the first bioreactor. The effluent port of the first bioreactor is connected to the second bioreactor. The glucose reservoir is connected to the second bioreactor. The effluent from the second bioreactor was connected to a 0.2um hollow fiber cell recovery system and the maximum reverse and forward pressures were set to <30 psi. The filtrate end of the 0.2um hollow fiber cell recovery system was connected to a collection reservoir on a scale and the retentate end of the 0.2um hollow fiber cell recovery system was connected to the inlet of the second bioreactor.

[0356] Bioreactor Operation :After assembly, any post-sterilization additives are added to the growth medium. All components of the bioreactor are connected to the Biostat-B control system and the temperature probe is plugged in. This allows the Fermwork software to receive input from the temperature, DO, and pH probes and control agitation, airflow, temperature, and the feed and basal pumps. The feed and basal lines are connected from their respective reservoirs to the 3-way port assembly. The pump tubing segments of the lines are inserted into the appropriate pump heads and the lines are primed by manually running the pumps until liquid is seen entering the bioreactor and then switching the pumps back to automatic control. The basal reservoir contains 14%-28% ammonium hydroxide.

[0357] The system was then allowed to stabilize with the following parameters: 37°C, pH: 6.8; DO: 30% of saturation. After stabilization, the first bioreactor was inoculated to a starting OD of 0.05-0.3 by injecting ~10 mL of seed culture into the addition port using a sterile syringe. 600. After the cells reach mid-logarithmic phase, a turbidostat program is started in the first bioreactor. This allows the biomass concentration in the first bioreactor to be constant, and additional cells are pumped to the second bioreactor. The mass_stat program on the first bioreactor is started to maintain a constant volume, wherein the effluent pump speed is controlled by the weight of the tank mass. The second bioreactor is started as a standard fed batch reactor. The feed is triggered by a starvation spike, which is a sharp rise in dissolved O2 indicating carbon source limitation. After the first and second bioreactors are stable, the pump that moves the effluent from the second bioreactor to the hollow fiber cell recovery system is then opened, which allows cells to be enriched and recovered in the second bioreactor. The mass_stat program is then started on the second bioreactor, wherein 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 fluid and cells from the second bioreactor that are higher than a set volume in the second bioreactor. The purge tank is placed on a balance to measure the mass of the purge fluid taken out.

[0358] Samples were periodically collected from the first bioreactor, the second bioreactor, the second bioreactor effluent tank, and the second bioreactor purge tank for analysis. The OD of the samples was measured by spectroscopy, and the culture product of the supernatant was analyzed by HPLC and the quality of the tank was recorded to calculate the culture product production and carbon source consumption.

[0359] Unless otherwise specified, the following meanings used herein apply. The word "may" is used in a permissive sense (i.e., it means having the potential), rather than in a mandatory sense (i.e., it means that it must be used). The word "includes" and other meanings mean including but not limited to. The singular forms "one", "an" and "the" include plural referents. Thus, for example, referring to "element" includes a combination of two or more elements, although other terms and phrases such as "one or more" are used for one or more elements. The phrase "at least one" includes "one", "one or more", "one or more than one" and "more than one". Unless otherwise specified, the term "or" is non-exclusive, i.e., includes both "and" and "or". The term "any one" between a modifier and a sequence refers to each member of the modifier-modified sequence. Thus, for example, the phrase "at least 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 listed numerical value within the context of a particular use. Thus, for example, "about 100" means between 95 and 105. The term "consisting essentially of" means comprising the recited elements and other elements that do not materially affect the basic and novel characteristics of the claimed combination.

[0360] It should be understood that the description and the accompanying drawings are not intended to limit the present invention to the specific form disclosed, but on the contrary, the present invention will cover all modifications, equivalents and / or alternatives that fall within the spirit and scope of the present invention as defined by the appended claims. In view of this description, further modifications and alternative embodiments of various aspects of the present invention will be obvious to those skilled in the art. Therefore, the description and the accompanying drawings are only interpreted as illustrative, and are for the purpose of teaching those skilled in the art to implement the general mode of the present invention. It should be understood that the forms of the present invention shown and described herein will serve as examples of embodiments. Those elements and materials illustrated and described herein can be replaced, parts and processes can be reversed or omitted, and certain features of the present invention can be used independently, all of which will be obvious to those skilled in the art who benefit from the description of the present invention. Without departing from the spirit and scope of the present invention as described in the following claims, the elements described herein may be changed. The titles used herein are only for organizational purposes and are not meant to limit the scope of this specification.

[0361] All publications, patents, and patent applications mentioned in this specification are herein 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

1. A method comprising: a) carrying out a first continuous culturing of cells in at least one chamber of a first bioreactor under culture conditions resulting in growth of the cells; b) during the first continuous culturing, moving a culture fluid comprising cells from a chamber of the at least one first bioreactor to a chamber of at least one second bioreactor via one or more fluid conduits, the one or more fluid conduits placing the chamber of the at least one first bioreactor in fluid communication with the chamber of the at least one second bioreactor; and c) performing a second continuous culture of cells removed from the at least one first bioreactor in a chamber of the at least one second bioreactor under culture conditions for producing at least one culture product; The cell growth rate in the first continuous culture is greater than the cell growth rate in the second continuous culture, and the production of the culture product in the first continuous culture is less than that in the second continuous culture.

2. The method according to claim 1, further comprising: d) collecting at least one culture product from the chamber of the at least one second bioreactor.

3. The method according to claim 1, wherein the cell growth rate in the first continuous culture is at least twice that of the second continuous culture, and the production of the culture product in the second continuous culture is at least twice that of the first continuous culture.

4. The method according to claim 1, wherein the cell growth rate in the first continuous culture is at least 10 times that of the second continuous culture, and the production of the culture product in the second continuous culture is at least 10 times that of the first continuous culture.

5. The method according to claim 1, wherein the cell growth rate in the first continuous culture is at least 100 times that of the second continuous culture, and the production of the culture product in the second continuous culture is at least 100 times that of the first continuous culture. The method according to claim 1 , wherein the growth rate of the cells in the first continuous culture is at least 80%, such as at least 90%, of the maximum growth rate.

7. The method according to claim 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.

8. The method of claim 1, comprising maintaining the cell density in the at least one first bioreactor.

9. The method of claim 8, wherein maintaining the cell density in the at least one first bioreactor comprises maintaining the optical density of the first continuous cell culture.

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

11. The method of claim 8, wherein conducting the first continuous culture further comprises maintaining a constant volume of cell culture in a chamber of the at least one first bioreactor.

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

13. The method of claim 1, wherein moving the culture fluid from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor comprises pumping the culture fluid with a pump.

14. The method of claim 1, comprising growing the cells in the chamber of the at least one first bioreactor at a constant cell growth rate.

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

16. The method of claim 1, wherein a growth-limiting nutrient is absent in the first continuous culture.

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

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

19. The method of 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 that in the second continuous culture.

20. The method of claim 1, wherein the volume of the first continuous culture is smaller than the volume of the second continuous culture, for example, wherein the relative volume ratio is less than any one of 1:1, 1:10, 1:100 and 1:1000, for example between about 1:10 and 1:

50.

21. The method of claim 1, wherein the volume of either or both of the first continuous culture and the second continuous culture is between about 50 ml and about 100,000 L, such as between about 500 ml and about 10 L.

22. The method according to claim 1, wherein cells are removed from the chamber of the at least one first bioreactor during growth, such as during logarithmic growth.

23. The method of claim 1, wherein the culture fluid comprising cells is continuously moved from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor over a period of at least any of 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.

24. The method of claim 1, wherein the culture fluid comprising cells is moved from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor in a total volume of at least any one of 0.01 L, 0.1 L, 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.

25. The method of claim 1, wherein the culture fluid containing cells is moved from the chamber of the at least one first bioreactor to the chamber of the at least one second bioreactor when the optical density (OD) of the first continuous culture reaches at least any one of 1, 5, 10, 25, 50, 100, 200 and 400.

26. The method of claim 1, wherein the growth rate of cells in the second continuous culture is no more than any one of 50%, 40%, 30%, 20%, 10%, 5%, 3% or 1% of the growth rate of the first continuous culture.

27. The method of claim 1, wherein the growth rate of the cells in the second continuous culture is no more than any one of 50%, 40%, 30%, 20%, 10%, 5%, 3% or 1% of the maximum growth rate of the cells under non-restrictive culture conditions.

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

29. The method of claim 1, wherein at least one nutrient in the second continuous culture is present at a growth rate limiting concentration.

30. The method of claim 29, wherein the concentration of the nutrient in the first continuous culture is at least any one of 2 times, 4 times, 10 times, 25 times, 50 times, 100 times, or 500 times greater than the concentration of the nutrient in the second continuous culture.

31. The method of claim 1, wherein the culture conditions of the second continuous culture comprise growth-limiting concentrations of one or more nutrients (e.g., selected from metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdate, selenite and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid, thiamine, biotin, cyanocobalamin, folic acid), 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, amino acids (e.g., glutamic acid, leucine and tryptophan), nucleic acid bases (e.g., adenine, cytosine, thymine, uracil and guanine) or complex nutrients (e.g., yeast extract, peptone, tryptone, casamino acids and corn steep liquor)).

32. The method of claim 1, wherein the culture conditions in the second continuous culture are maintained constant for at least any one of 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 of claim 1, wherein the second continuous culturing is performed for at least about any one of 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 no more than about any one of 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., 1-10,000 hours, preferably 50-1,000 hours.

34. The method of claim 1, comprising conducting the second continuous culturing in more than one different second bioreactor.

35. The method of claim 1, wherein the at least one first bioreactor is more than one first bioreactor.

36. The method according to claim 1, wherein the culture conditions of the second continuous culture are optimized to produce at least one culture product. 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 of 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 continuous culture and / or the second continuous culture.

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

40. The method of claim 1, comprising inducing activity of a biochemical pathway that produces a culture output in the second continuous culture but not in the first continuous culture.

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

42. The method of claim 1, wherein the cells in the second continuous culture have a doubling time of no more than any one of 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 5 days, 6 days, 7 days, 2 weeks, or 1 month.

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

44. The method of claim 1, wherein cells removed from a chamber of the at least one second bioreactor are recovered into the chamber using a cell recovery device such as a hollow fiber filter.

45. The method of claim 1, wherein conducting the first continuous culture or the second continuous culture comprises providing one or more of: metals (e.g., iron, zinc, cobalt, copper, nickel, manganese, molybdate, selenite and other transition metals), vitamins (e.g., niacin, pyridoxine, riboflavin, pantothenate, aminobenzoic acid, thiamine, biotin, cyanocobalamin, folic acid), pollution control agents such as antibiotics or biocides, inducers, salts, phosphates, sulfates, chlorides, acetates, citrates, and the like. anionic salts, magnesium, calcium, sodium, potassium, ammonium and other cationic salts, boric acid, choline, ascorbic acid, lipoic acid, niacin, inositol, antifoaming agents (e.g., Antifoam 204, Antifoam A, Antifoam C), amino acids (e.g., glutamic acid, leucine and tryptophan), nucleic acid bases (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.

46. ​​The method of claim 1, wherein conducting the first continuous culture or the second continuous culture comprises providing a carbon source (e.g., a sugar (e.g., glucose, xylose, sucrose, glycerol, or acetate), molasses, malt extract, starch, dextrin, pulp, CO, or CO2).

47. The method of claim 1, wherein performing the first continuous culture or the second continuous culture comprises providing one or more of the following: a nitrogen source (e.g., an amino acid or polypeptide, urea, an ammonium salt (e.g., ammonium sulfate, ammonium phosphate or ammonia), corn steep liquor, yeast extract, peptone and soybean meal).

48. The method of claim 1, wherein performing the first continuous culture or the second continuous culture comprises adjusting one or more of the following: nitrogen retention rate, aeration rate, oxygen sparge rate, carbon dioxide retention rate, culture agitation speed, CO2 concentration, carbon source concentration, nitrogen source concentration, metals, vitamins, salt concentrations, and antibiotic concentrations.

49. The method of claim 1, wherein collecting the at least one culture product comprises removing a cell culture fluid from a chamber of the at least one second bioreactor and separating cells from the cell culture fluid to produce a cell-free culture fluid.

50. The method of claim 1, wherein the culture product is selected from polypeptides (e.g., proteins, enzymes, antibodies), organic molecules that are products of synthetic pathways in the cell (e.g., industrial chemicals such as flavoring agents (e.g., vanillin)); flavors (e.g., aldehydes, coumarins, indoles), amino acids, organic acids (e.g., citric acid, lactic acid, and acetic acid); alcohols (e.g., ethanol, isopropanol, ketones such as acetone); and fatty acids (e.g., palmitic acid and oleic acid).

51. The method of claim 1, wherein the cells comprise archaea, prokaryotes and / or eukaryotes.

52. The method of claim 1, wherein the cell comprises a fungal cell (e.g., a yeast (e.g., a Saccharomyces spp., Pichia spp., Komagataella spp., Kuyveromyces spp., Aspergillus spp., Rhodoporidium spp., Lipolytica spp., Aspergillus spp., Neurospora spp., Trichoderma spp., Candida spp., or Penicillium spp.).

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

54. The method of claim 1, wherein the cell comprises an insect cell, an animal cell, or a plant cell.

55. The method of claim 1, wherein the cell comprises an animal cell (e.g., an arthropod (e.g., insect, shrimp, lobster, crayfish, and crab); a chordate (e.g., fish, amphibian, reptile, bird (e.g., chicken or turkey); a mammal (e.g., human or non-human such as bovine, lamb, goat, pig, horse, dog, cat, primate)).

56. The method of claim 1, wherein the cell comprises a cell line (e.g., CHO (Chinese Hamster Ovary), BHK21 (Baby Hamster Kidney), NSO, Sp2 / 0 mouse cell line, insect cells (e.g., SP9, Sf9, sf21, S2), tobacco BY-2 cells, rice (Oryza Sativa), or algae cells).

57. The method of claim 1, wherein the cell is not a photosynthetic cell.

58. A system comprising: (a) at least one first bioreactor, the at least one first bioreactor comprising: (i) the first chamber; and (ii) one or more first sensors providing at least a measure of the cell density in the first chamber and a measure of the volume of the cell culture; (b) at least one second bioreactor, the at least one second bioreactor comprising: (i) the second chamber; and (ii) one or more second sensors that provide at least a measure of culture conditions and a measure of the volume of the cell culture in 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 liquid reagents, wherein the reagent reservoirs are in fluid communication with the first chamber, and optionally with the one or more second chambers; (d) one or more pumps configured to: (i) moving a liquid reagent from at least one reagent reservoir to the first chamber; (ii) moving cell culture fluid from the first chamber to the one or more second chambers; (iii) removing the cell culture fluid from 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; and (e) a control system, the control system being configured to: (i) controlling cell growth, such as cell density, of a cell culture in the first chamber at a rate greater than a rate in the second chamber using measurements from the one or more sensors; (ii) moving cell culture fluid from the first chamber to the one or more second chambers; and (iii) using measurements from the one or more sensors to control culture conditions and, for example, volume and / or cell growth, of the cell culture in the one or more second chambers to produce a culture product at a rate greater than the rate in the first chamber.

59. A method according to claim 58, wherein the control system uses 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 that of 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 that of the first continuous culture.

60. The system of claim 58, further comprising one or more collection containers in fluid communication with the one or more second chambers.

61. The system of claim 58, wherein the first chamber and the one or more second chambers have a volume between 250 mL and 1,000,000 liters.

62. The system of claim 58, wherein the control system sets a dilution rate of the liquid reagent moved into the first chamber and / or one or more second chambers to maintain a concentration of one or more nutrients.

63. The system of claim 58, wherein the first chamber and the one or more second chambers include ports that communicate with reagent reservoirs and with each other through fluid conduits.

64. The system of claim 58, wherein the one or more second chambers is more than one second chamber.

65. The system of claim 58, wherein the one or more first sensors or second sensors that provide a measure of volume include a scale that measures the mass of the bioreactor.

66. The system of claim 58, wherein the sensor that provides a measure of the cell density of the cell culture in the first chamber comprises an optical density sensor or a capacitive sensor.

67. The system of claim 65, wherein controlling cell density comprises controlling the optical density of the cell culture.

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

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

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

72. The system of claim 70, wherein the peristaltic pump includes fittings for tubing for fluid communication between the two chambers, a chamber and a reagent reservoir, or the second chamber and an effluent conduit.

73. The system of claim 58, wherein the control system for controlling the volume of the cell culture in the first chamber or the one or more second chambers comprises a level sensor (e.g., an overflow tube) that removes cell culture fluid from the chamber when the height of the cell culture exceeds the top of the level sensor.

74. The system of claim 58, wherein the control system comprises a computer comprising a processor and a memory, the memory comprising executable code that, when executed by the processor, performs one or more feedback routines to control cell growth, such as cell density, in the first chamber, control the volume of cell culture in the first chamber and / or one or more second chambers, and control culture conditions in the one or more second chambers.

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

76. A system according to claim 73, wherein one of the feedback routines calculates the volume of cell culture in the first chamber based on measurements received from the one or more sensors, and if the volume is above a set point, actuates a pump to move liquid cell culture from the first chamber to the one or more second chambers.

77. The system of claim 73, wherein: (I) the one or more sensors provide a measurement of one or more culture parameters in the first chamber and / or one or more second chambers; (II) the system further comprises one or more effectors to affect a change in the one or more culture parameters; (III) one or more of the feedback routines calculates one or more culture parameters based on the measurements, and actuates the one or more effectors to adjust the culture parameters towards a target level.

78. The system of claim 73, wherein: (I) the one or more sensors provide a measurement of one or more of pH, temperature, and dissolved O2 of the cell culture in the first chamber and / or one or more second chambers; (II) The system further comprises one or more of the following: one or more reagent reservoirs containing an acid and a base in fluid communication with the first reservoir and / or one or more second reservoirs; one or more temperature controllers that control the temperature of the cell culture in the first chamber and / or one or more second chambers; one or more aerators for aerating the cell culture in the first chamber and / or one or more second chambers; (III) one or more of the feedback routines calculates culture conditions including one or more of pH, temperature, dissolved O2, and nutrient concentration based on measurements received from the one or more sensors, and actuates: one or more pumps to move 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 aerate the cell culture to a target dissolved oxygen level.

79. A system according to claim 73, wherein one of the feedback routines 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 actuates a pump to remove liquid cell culture from the one or more second chambers if the volume is above a set point.

80. The system of claim 58, further comprising a filter in fluid communication with the outlet of the second chamber, the filter being configured to recover cells that are unable to pass through the membrane back into the second chamber and to transfer cell-depleted liquid culture medium from the cartridge to a collection container.

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

82. The system of claim 58, wherein the first chamber comprises a cell culture, and wherein the one or more reagent reservoirs in fluid communication with the first chamber comprise 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.

83. The system of claim 58, wherein the culture medium is formulated such that nutrients are not limiting in the at least one first bioreactor, but one or more nutrients are present at concentrations that limit growth in the one or more second bioreactors.

84. The system of claim 58, wherein the one or more control subsystems comprises a computer comprising: (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 the pump to move liquid into and out of the chambers.

85. The system of claim 58, comprising one or more of the following: ●Temperature sensor and temperature regulator; a dissolved oxygen meter and a ventilation system in communication with the interior of the first chamber and / or one or more second chambers; ●Analyzers for measuring nutrient concentrations in cell cultures in chambers; an impeller or pneumatic agitator for mixing the liquid in the chamber, and a motor configured to actuate the impeller or pneumatic agitator; • an effluent communicating with the interior of the container and an adjustable valve or pump for regulating the flow of fluid from the chamber; ● baffles in the chamber; An ejector and a mass flow controller in communication with the interior of the chamber for inputting one or more gases and mixtures thereof; ● a user interface for communicating instructions to the computer; ●Foam control; ●Hollow fiber membrane for cell recovery; and ●Reverse or forward osmosis membrane.

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

87. A method comprising: a) carrying out a first continuous culturing of cells in a chamber of a first bioreactor under culture conditions maintaining a constant cell density; b) during the first continuous culturing period, moving a culture fluid containing cells from a chamber of the first bioreactor to a chamber of at least one second bioreactor via one or more fluid conduits, the one or more fluid conduits placing 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 removed from the first bioreactor in a chamber of each of the one or more second bioreactors under constant culture conditions to produce at least one culture product; and d) collecting the at least one culture product from the chamber of each of the one or more second bioreactors.

88. A system comprising: (a) a first bioreactor, the first bioreactor comprising: (i) the first chamber; and (ii) one or more first sensors providing at least a measure of the cell density in the first chamber and a measure of the volume of the cell culture; (b) one or more second bioreactors, the one or more second bioreactors comprising: (i) the second chamber; and (ii) one or more second sensors that provide at least a measure of culture conditions and a measure of the volume of the cell culture in 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 liquid reagents, wherein the reagent reservoirs are in fluid communication with the first chamber, and optionally with the one or more second chambers; (d) one or more pumps configured to: (i) moving a liquid reagent from at least one reagent reservoir to the first chamber; (ii) moving cell culture fluid from the first chamber to the one or more second chambers; (iii) removing the cell culture fluid from 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; and (e) a control system, the control system being configured to: (i) using measurements from the one or more sensors to control cell growth, such as cell density, of a cell culture in the first chamber; (ii) moving cell culture fluid from the first chamber to the one or more second chambers; and (iii) using measurements from the one or more sensors to control culture conditions and, for example, volume and / or cell growth of the cell culture in the one or more second chambers.

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