System and method for biomass monitoring of cell culture bioreactors

Abstract

A method of monitoring biomass during cell culture of cells in a bioreactor is provided. The method comprises: culturing the cells in the bioreactor using a cell culture medium perfused through the bioreactor; measuring at least one of cellular nutrients and cellular byproducts in the cell culture medium; determining at least one of a rate of depletion of the cellular nutrients and a rate of accumulation of the cellular byproducts; and predicting the number of cells within the bioreactor at a specified culture time based on at least one of the depletion rate and the accumulation rate.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 63 / 436,310, filed on December 30, 2022, the contents of which are relied upon and incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure generally relates to systems and methods for monitoring cell cultures in bioreactor systems. Specifically, the present disclosure relates to methods, protocols, systems, and models for biomass monitoring of cell cultures within bioreactor systems. Background Art

[0004] In the bioprocessing industry, large-scale cell culture is required to produce hormones, enzymes, antibodies, vaccines, therapeutic proteins, and cell therapies. The cell and gene therapy market is growing rapidly, with promising treatments entering clinical trials and rapidly moving toward commercialization. However, a single cell therapy dose may require billions of cells or trillions of viruses. Therefore, being able to provide large quantities of cell products in a short period of time is crucial for clinical success.

[0005] Most cells used in bioprocessing are anchorage-dependent, meaning that the cells need to be attached to a surface in order to grow and function. Traditionally, adherent cells are cultured on two-dimensional (2D) cell-attaching surfaces incorporated into one of a variety of container formats, such as T-flasks, petridishes, cell factories, cell stacks, spinner bottles, and other multilayer containers (e.g., the 2D Cell Attachment Containers from Corning Inc.). These methods can have significant disadvantages, including the difficulty in achieving sufficiently high cell densities to make them useful for large-scale production of therapeutics or cells.

[0006] Alternative methods have been proposed to increase the volume density of cultured cells. These methods include microcarrier culture in the following: stirred tanks; hollow fiber bioreactors, where cells can form larger three-dimensional aggregates when they proliferate in the spaces between the fibers; and packed bed bioreactors. In a packed bed or fixed bed bioreactor, a packed or fixed cell substrate is used to provide an attachment surface for adherent cells. Culture medium is perfused along the surface or through the semi-porous substrate to provide nutrients and oxygen required for cell growth. For example, U.S. Patents No. 4,833,083; No. 5,501,971; and No. 5,510,262 have previously disclosed packed bed bioreactor systems containing a packed bed of a support or matrix system for retaining cells. The packed bed matrix is typically made of porous particles or non-woven polymer microfibers as a substrate.

[0007] One of the important problems of traditional fixed-bed bioreactors is the uneven distribution of cells in the bed. For example, the packed bed can act as a depth filter, where cells are mainly trapped in the inlet area, or other areas with relatively low flow and / or high substrate density, resulting in a gradient distribution of cells during the inoculation step. In addition, due to the random fiber packing, the flow resistance and cell retention efficiency of the packed bed cross section are uneven. For example, the culture medium flows quickly through an area with low cell packing density and slowly flows through an area with higher resistance due to a larger number of trapped cells. This creates a channeling effect, where nutrients and oxygen are more efficiently delivered to areas with lower volume cell density, and areas with higher cell density remain under suboptimal culture conditions.

[0008] Another significant disadvantage of conventional packed bed systems disclosed in the prior art is the inability to effectively collect intact viable cells at the end of the culture process. If the end product is cells, or if the bioreactor is used as part of a "seed train," where a cell population is grown in one container and then transferred to another container for further population growth, the collection of cells is important. U.S. Patent No. 9,273,278 discloses a bioreactor design for improving the efficiency of recovering cells from a packed bed during the cell collection step. The design is based on relaxing the packed bed matrix and stirring or agitating the packed bed particles to allow the porous matrix to collide and thus detach the cells. However, this method is time-consuming and labor-intensive and may result in significant cell damage, thereby reducing overall cell viability.

[0009] Furthermore, due to the random arrangement of fibers in conventional packed or fixed-bed substrates, bioreactor users can have difficulty predicting cell culture performance, as substrate arrangement and / or packing can vary from culture to culture. Monitoring the health or progress of the cell culture can also be difficult. For example, the presence of a fixed bed itself limits options for effectively monitoring the health of the culture and biomass production. Furthermore, because cells are believed to be trapped in the packed bed, the packed substrates of conventional fixed-bed bioreactors make efficient cell collection extremely difficult or impossible, further hindering understanding of cell culture performance.

[0010] Regardless of the platform used, the early stages of process development require users to have information to better understand cell behavior, virus production, and culture progress. Upstream bioreactor process development requires the identification of critical parameters and quality characteristics, along with parameter definitions and their relationship to the final product. Understanding these parameters and how they scale with higher densities or larger systems is crucial for process development and efficiency.

[0011] Upstream bioprocess production also complies with good manufacturing process (GMP) regulations and requirements involving process analytical technology (PAT). PAT is considered a tool for the design, analysis and control of production processes. Final product quality can be ensured by measuring process parameters and product characteristics. This can include a large amount of online culture process monitoring, which provides a useful tool for process characterization and detection of process changes. The relevant parameters for packed bed bioreactor process characterization and control are pH, temperature, dissolved oxygen or oxygen delivery (DO2) and carbon dioxide (CO2). However, one of the identified disadvantages of packed bed bioreactors is the difficulty in obtaining substrate samples to directly assess the status of cells and overall cell culture progress. Obtaining substrate samples risks contaminating the entire culture or, in the case of an inhomogeneous platform, provides misleading or inaccurate data.

[0012] Fixed-bed bioreactors have been increasingly used to expand production in adherent cell cultures. When it comes to cell culture, biomass monitoring is an important tool for designing, analyzing, and controlling pharmaceutical manufacturing processes. For suspension cell cultures, biomass monitoring can be achieved using optical or electrical methods. However, for adherent cell cultures using fixed-bed bioreactors, no validated method or sensor has been used to satisfactorily monitor biomass online. Historically, it has been proposed that substrate (e.g., glucose) consumption rates can be used to understand the kinetics of cell growth. However, since glucose consumption rate (GCR) is affected by many factors other than the number of viable cells, there is no established scheme, method, and model for obtaining and using effective substrate consumption rate and metabolite accumulation rate to calculate and predict biomass.

[0013] While it is possible to manufacture viral vectors for early clinical trials using existing platforms, a platform that can produce high-quality products in larger quantities to reach later commercial manufacturing scale is needed. Furthermore, systems and methods are needed that enable the collection of specific measurable parameters from cell cultures during bioreactor operations to better control various aspects of the culture process in real time and to detect and diagnose abnormal culture conditions. Summary of the Invention

[0014] According to an embodiment of the present disclosure, a method for monitoring the biomass of cells during cell culture in a bioreactor is provided. The method comprises: culturing the cells in the bioreactor using a cell culture medium perfused through the bioreactor; measuring at least one of cell nutrients and cell byproducts in the cell culture medium; determining at least one of the consumption rate of the cell nutrients and the accumulation rate of the cell byproducts; and predicting the number of cells within the bioreactor at a specified culture time based on at least one of the consumption rate and the accumulation rate. According to one aspect of the embodiment, the bioreactor is a fixed bed bioreactor having a substrate configured to culture cells attached to a surface of the substrate. At least one cell nutrient may be glucose or glutamine; and at least one cell byproduct may be lactate or ammonia. According to one aspect of the embodiment, the cell culture medium may be a cell culture medium enriched with glucose or glutamine.

[0015] According to aspects of the embodiment, measuring at least one of the cell nutrients and the cell byproducts in the cell culture medium comprises performing multiple measurements of the cell nutrients or the cell byproducts, the multiple measurements being spaced apart by a time interval that is less than the doubling time of the cells in the cell culture. The measurement interval is greater than or equal to a minimum time interval, the minimum time interval being the time at which the change in the level of the cell nutrients or the cell byproducts is greater than a measurement tolerance for measuring the cell nutrients or the cell byproducts. In aspects of the embodiment, the minimum time interval is greater than or equal to about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours. Measuring the cell nutrients in the cell culture medium can comprise measuring the cell nutrients in the cell culture multiple times per day. Measuring the cell byproducts in the cell culture medium can comprise measuring the cell byproducts in the cell culture multiple times per day.

[0016] According to aspects of the embodiments, predicting the cell number comprises calculating the biomass at the specified culture time using a mathematical model. The measuring may comprise using an inline sensor in a perfusion line of the cell culture medium. The measuring may further comprise using an offline measurement of a sample of the cell culture medium.

[0017] According to aspects of the embodiment, the method further includes: after determining the consumption rate and the accumulation rate, comparing at least one of the consumption rate of the first cell nutrient and the accumulation rate of the first cell byproduct with at least one of the consumption rate of the second cell nutrient and the accumulation rate of the second cell byproduct. The comparison may include comparing the consumption rate of glucose with the consumption rate of glutamine, comparing the consumption rate of glucose with the accumulation rate of ammonia, comparing the accumulation rate of lactate with the consumption rate of glutamine, and / or comparing the accumulation rate of lactate with the accumulation rate of ammonia. The method may further include determining an abnormality in the cell culture based on the comparison. The method may further include inoculating cells in the bioreactor at an inoculation density. The method may further include supplying fresh cell culture medium to the bioreactor, and the measuring may include a first measurement, which is performed at least one hour after supplying the fresh cell culture medium. According to aspects of the embodiment, the number of cells N is predicted. t This involves using the following equation:

[0018] N t =N 接种 e kt

[0019] where N 接种 is the number of cells inoculated into the bioreactor, k is the cell growth rate, and t is the time at which the cell number is predicted. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic illustration of a cell culture system according to one or more embodiments.

[0021] Figure 2 Operations for controlling the perfusion flow rate of a cell culture system are demonstrated, according to one or more embodiments.

[0022] Figure 3 For use according to one or more embodiments Figure 1 Graphs of bioreactor perfusion flow rate and oxygen concentration over time during an exemplary bioreactor run for a bioreactor system.

[0023] Figure 4A For Figure 3Graph of dissolved oxygen concentration over time during bioreactor operation.

[0024] Figure 4B For Figure 3 Graph of pH over time during bioreactor operation.

[0025] Figure 4C For Figure 3 Graph of media conditioning temperature over time during a bioreactor run.

[0026] Figure 5 For Figure 3 A graph of the oxygen consumption of a packed bed cell culture over time during bioreactor operation, including the slope α of the curve.

[0027] Figure 6 is a graph of slope α versus cell seeding density for a bioreactor according to one or more embodiments.

[0028] Figure 7 is a graph of slope α versus cell culture substrate surface area according to one or more embodiments.

[0029] Figure 8 is a graph of cell collection yield versus slope α according to one or more embodiments.

[0030] Figure 9 Graph showing the distribution of glucose concentration during four days of experimental culture according to the embodiment.

[0031] Figure 10 Graph showing the distribution of lactate concentration during four days of experimental culture according to the embodiment.

[0032] Figure 11 is the total amount of glucose consumed per day over time according to an example embodiment, V(C0-C t ) diagram.

[0033] Figure 12 is a graph of the total glucose consumption rate rN0 per day as an exponential function of culture duration, according to an example embodiment.

[0034] Figure 13 Schematic diagram of an experimental setup according to an embodiment.

[0035] Figure 14A FIG2 is a graph showing the total glucose consumption rate rN0 per day as an exponential function of the culture duration according to an embodiment.

[0036] Figure 14BFIG4 is a graph showing the total glutamine consumption rate rN0 per day as an exponential function of culture duration according to an embodiment.

[0037] Figure 14C Graph showing the total lactate accumulation rate rN0 per day as an exponential function of culture duration according to the Examples.

[0038] Figure 14D Graph showing the total ammonia accumulation rate rN0 per day as an exponential function of culture duration according to an embodiment.

[0039] Figure 15 is a graph showing a graph of glucose consumption over time when fitted to an exponential model according to an embodiment. DETAILED DESCRIPTION

[0040] Various embodiments of the present disclosure will be described in detail with reference to the accompanying drawings (if any). The various embodiments mentioned do not limit the scope of the invention, which is limited only by the scope of the appended claims. In addition, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments of the claimed invention.

[0041] Embodiments of the present disclosure include systems and methods for monitoring and controlling cell culture. This disclosure describes systems and methods for collecting specific signal signatures during bioreactor operation to provide better real-time control of key aspects and detect and diagnose abnormal culture conditions. The identified signature parameters of cell culture described in this disclosure can be used as tools for implementing process analytical techniques and online monitoring of upstream processes. Thus, optimized cell culture production processes can be established by developing routines and reproducibility for signature operating parameters.

[0042] According to embodiments of the present disclosure, bioreactor systems and methods for monitoring the status of a cell culture in a bioreactor system during a cell culture run are provided. Specifically, the embodiments describe a bioreactor system having an outlet sensor at the outlet of a cell culture bioreactor or vessel, a system capable of real-time signal collection and processing of signals from this and / or other sensors, and a cell culture method using such a system. For example, methods include using the sensor signals as triggers for important cell culture process steps, or for predicting the expected health of a cell culture over time for a specific bioreactor size or inoculation density, or for assessing its current health. Advantages of these systems and methods include the ability to proactively monitor bioreactor status in real time, without requiring physical sampling of the packed bed substrate for offline analysis. Continuous monitoring of bioreactor status also allows end users to proactively adjust bioprocess steps that depend on the progress of the culture process within the packed bed bioreactor. The ability to characterize and record the progress of a bioprocess run further allows end users to monitor and document the consistency of the process between batches. This type of tracking of the progress and consistency of cell culture runs can be highly advantageous.

[0043] In conventional large-scale cell culture bioreactors, different types of packed bed bioreactors have been used. Typically, these packed beds contain a porous matrix to retain adherent or suspended cells and support growth and proliferation. The packed bed matrix provides a higher surface area to volume ratio, so cell density can be higher than in other systems. However, the packed bed typically acts as a depth filter, where cells are physically trapped or entangled in the fibers of the matrix. Therefore, due to the linear flow of the cell inoculum through the packed bed, the cells experience uneven distribution within the packed bed, resulting in cell density varying across the depth or width of the packed bed. For example, the cell density may be higher at the inlet area of the bioreactor, while the cell density closer to the outlet of the bioreactor is significantly lower. In another example, the unevenness in the packed bed creates a channeling effect, where the cell culture medium preferentially flows through certain areas of the bed while being restricted from reaching other areas of the bed, similarly leading to uneven cell distribution and uneven or inconsistent culture medium or nutrient distribution. This uneven distribution of cells within the packed bed severely hinders the scalability and predictability of the bioreactor in bioprocess manufacturing and may even lead to reduced cell growth or viral vector production efficiency per unit surface area or volume of the packed bed.

[0044] Another problem encountered in packed-bed bioreactors disclosed in the prior art is the channeling effect, as described above. Due to the random nature of the nonwoven fibers in the packing, the local fiber density at any given cross-section of the packed bed is nonuniform. The medium flows faster in areas of low fiber density (high bed permeability) and much slower in areas of high fiber density (lower bed permeability). The resulting uneven perfusion of medium across the packed bed produces a channeling effect, which manifests itself as significant nutrient and metabolite gradients, thereby negatively impacting overall cell culture and bioreactor performance. Cells located in areas of low medium perfusion will starve and often die from nutrient depletion or metabolite poisoning. Cell collection is another problem encountered when using bioreactors filled with nonwoven fiber scaffolds. Because the packed bed acts as a depth filter, cells released at the end of the cell culture process are trapped within the packed bed, and cell recovery is extremely low. This significantly limits the use of such bioreactors in bioprocesses with living cells as products. Consequently, heterogeneity results in different areas being exposed to different flow and shear forces, which significantly reduces the available cell culture area, causes culture inhomogeneities, and interferes with transfection efficiency and cell release.

[0045] To address these and other problems with existing cell culture protocols, embodiments of the present disclosure provide bioreactor systems, cell growth substrates, matrices of the substrates, and methods of using the bioreactor systems and substrates that enable efficient and high-yield cell culture of anchorage-dependent cells and production of cell products (e.g., proteins, antibodies, viral particles). Embodiments include porous cell culture substrates made from an ordered and regular array of porous substrate materials that enable uniform cell seeding and perfusion of culture medium / nutrients, as well as efficient cell collection. Embodiments also enable scalable cell culture protocols in which substrates and bioreactors are capable of cell seeding and growth and / or cell product collection at process development scale to full production scale without sacrificing the uniform performance of the embodiments. For example, in some embodiments, the bioreactor can be easily scaled up from process development scale to product scale, with the number of viral genomes per unit substrate surface area (VG / cm2) being constant throughout the production scale. 2 ). The collectability and scalability of the embodiments herein enable these embodiments to be used for efficient train seeding to grow cell populations at multiple scales on the same cell substrate. Additionally, the embodiments herein provide a cell culture substrate with a high surface area, which, combined with the other features described, enables high-throughput cell culture protocols. In some embodiments, for example, the cell culture substrates and / or bioreactors discussed herein can produce 10 16 to 10 18 A viral genome (VG).

[0046] Embodiments of the present disclosure can achieve a viral vector platform of practical size that can be produced in batches greater than about 10 14 Viral genomes, each batch is greater than about 10 15 Viral genomes, each batch is greater than about 10 16 Viral genomes, each batch is greater than about 10 17 viral genomes or up to or greater than about 10 per batch 16 In some embodiments, the yield is about 10 per batch. 15 to about 10 18 For example, in some embodiments, the viral genome yield can be about 10 15 to about 10 16 viral genomes or batches, or approximately 10 per batch 16 to about 10 19 viral genomes, or about 10 per batch 16 to 10 18 viral genomes, or about 10 per batch 17 to about 10 19 viral genomes, or about 10 per batch 18 to about 10 19 viral genomes, or about 10 per batch 18 or more viral genomes.

[0047] In addition, the embodiments disclosed herein not only enable cells to attach and grow on cell culture substrates, but also enable the collection of live cultured cells. The inability to collect live cells is a significant drawback of current platforms, and it makes it difficult to construct and maintain a sufficient number of cells to achieve production capacity. According to one aspect of the embodiments of the present disclosure, live cells can be collected from cell culture substrates, including between 80% and 100% live cells, or between about 85% and about 99% live cells, or between about 90% and about 99% live cells. For example, of the collected cells, at least 80% are live cells, at least 85% are live cells, at least 90% are live cells, at least 91% are live cells, at least 92% are live cells, at least 93% are live cells, at least 94% are live cells, at least 95% are live cells, at least 96% are live cells, at least 97% are live cells, at least 98% are live cells or at least 99% are live cells. Trypsin, TrypLE TM (manufactured by Thermo Fisher Scientific) or (manufactured by Innovative Cell Technologies) to release cells from cell culture substrates.

[0048] According to one or more embodiments, a cell culture bioreactor can include a cell culture substrate within a bioreactor vessel. The substrate can be deployed in a packed bed bioreactor configuration or other configuration within a three-dimensional culture chamber of the bioreactor vessel. Due to contamination concerns, the vessel can be a disposable container that can be discarded after use.

[0049] like Figure 1 As shown, embodiments of the present disclosure include a bioreactor system 100 for culturing cells in a cell culture container 100. The cell culture container includes an inlet 112 and an outlet 114 that are fluidically connected to an internal reservoir 111 of the cell culture container 110. The internal reservoir 111 contains space for accommodating and culturing cells and may also include a cell culture substrate (not shown) on which adherent cells can be cultured. In some embodiments, the inlet 112 is located at one end of the cell culture container 110 for inputting culture medium, cells, and / or nutrients into the cell culture container 110, and the outlet 114 is located at the opposite end for removing culture medium, cells, or cell products from the cell culture container 110. The substrate within the internal reservoir can take many forms, some of which are discussed herein by way of example. Some embodiments may use one or both of the inlet 112 and outlet 114 to allow culture medium, cells, or other contents to flow in and out of the cell culture container 110. For example, the inlet 112 can be used to allow culture medium or cells to flow into the cell culture container 110 during the cell seeding, perfusion and / or culture phases, but can also be used to remove one or more of culture medium, cells, or cell products through the inlet 112 during the collection phase. Therefore, the terms "inlet" and "outlet" are not intended to limit the function of these openings, but should generally be understood to mean ports for introducing and removing fluids, respectively, during the normal process of cell growth. An outlet sensor 118 is disposed at the outlet 114 of the cell culture container 110. As used herein, "at the outlet" can mean a sensor disposed in series on a fluid flow path that receives culture medium from the outlet 114 and returns the culture medium to another part of the system (e.g., a culture medium conditioning container), or it can mean a sensor disposed within the cell culture container 110, but preferably after the cell culture substrate, packed bed, or other cell culture area within the cell culture container 110. In this way, the outlet sensor 114 can detect characteristics of the culture medium after the culture medium passes through the packed bed, cell culture substrate, or other cell culture area.

[0050] The system further includes a medium conditioning vessel (MCV) 120 that can hold and condition cell culture medium 122. Fluid flow paths 142, 144 transport conditioned medium 112 from the MCV 120 to the cell culture vessel 110 and return spent medium from the cell culture vessel 110 to the MCV 120. The MCV 120 can be coupled to a plurality of sensors and / or conditioning components 124a, 124b, 124c, 124d for sensing properties of the cell culture medium and adjusting or conditioning the medium as needed during cell culture. These sensors and conditioning components include, but are not limited to, dissolved gas (e.g., O2, air, CO2, N2) sensors and supplies, pH sensors, oxygenators / gas sparging units, temperature probes and temperature control devices, and nutrient and substrate addition ports. The gas mixture supplied to the sparging unit can be controlled by gas flow controllers for N2, O2, and CO2 gases. The medium conditioning vessel 120 may also contain an impeller for mixing the medium.

[0051] The system may also include a culture medium conditioning control unit 130 operably connected to the plurality of sensors and / or conditioning components 124a, 124b, 124c, 124d to process signals detected from these sensors and / or control the conditioning components to regulate the culture medium 122 within the MCV 120. The culture medium conditioning control unit 130 may also be operably connected to a pump 150 to control the pump 150 and, therefore, the rate of fluid flow through the fluid flow paths 142, 144 and perfusion through the cell culture vessel 110. Alternatively, the pump 150 and the outlet sensor 118 may be connected directly or through a perfusion control unit that is separate from the culture medium conditioning control unit 130. In some embodiments herein, a peristaltic pump is used, but other types of pumps may also be used. Figure 1 As shown, the medium conditioning container 120 is provided as a container separate from the bioreactor container 110. This has the advantage of being able to separate medium conditioning from cell culture and then supply the conditioned medium to the cell culture space. However, in some embodiments, medium conditioning can be performed within the bioreactor container 110.

[0052] In some embodiments, the medium conditioning control unit 130 can be used to maintain stable or desired levels of various parameters of the cell culture medium 122 within the MCV 120, thereby maintaining the bulk culture medium 122 at a specific temperature, oxygen saturation level, pH, and CO2 concentration. For example, for a given cell line or stage of the cell culture process, the cell culture medium 122 may be required to have a certain temperature, pH, dissolved gas content, or nutrient level to achieve optimal cell health and / or growth. The medium 122 from the medium conditioning container 120 is delivered to the cell culture container 110 through an inlet 112, which may also include an injection port for inoculating a cell inoculum and initiating cell culture. The cell culture container 110 may also include an outlet 114 through which the cell culture medium 122 exits the container 110. Additionally, cells or cell products may be output through the outlet 114. An outlet sensor 118 is provided to analyze the composition of the effluent from the cell culture container 110. As described above, the medium conditioning control unit 130 can receive a signal from the outlet sensor 118 (e.g., an O2 sensor) and, based on the signal, adjust the flow of fluid through the cell culture container 110 by sending a signal to the pump 150 (e.g., a peristaltic pump) upstream of the inlet 112 of the cell culture container 110. Thus, based on one or a combination of factors measured by the outlet sensor 118, the pump 150 can control the flow into the cell culture container 110 to achieve desired cell culture conditions. Because the cell culture medium 122 within the MCV 120 can be maintained at a desired state, changes in the flow rate can effectively address any needs of the cells within the cell culture container 110. For example, because the cell culture medium 122 exiting the MCV 120 is conditioned for optimal performance, the culture medium entering through the inlet 112 should meet the optimal requirements of the culture medium. If outlet sensor 118 detects a less-than-optimal level in the cell culture medium exiting cell culture vessel 110 at outlet 114, this may mean, for example, that the cells in the culture have consumed a certain amount of dissolved gas (e.g., oxygen) or cell nutrients in the medium and that at least some cells (i.e., cells near the outlet where medium consumption is greatest) are not being cultured optimally. Thus, for example, if the dissolved oxygen level in the cell culture medium at outlet sensor 118 is below an optimal level (e.g., for a given cell type, culture stage, etc.), the perfusion flow rate may be increased to supply a higher rate of conditioned medium, which should then result in all cells (even cells near the outlet) being cultured under optimal conditions.

[0053] The medium perfusion rate is controlled by the medium conditioning control unit 130, which collects and compares sensor signals from sensors 124a to 124d in the medium conditioning vessel 120 and MCV 120, as well as the outlet sensor 118. Due to the packing flow nature of the medium perfusion through the packed bed substrate in the cell culture vessel 110, nutrient, pH, and oxygen gradients are formed along the packed bed. The perfusion flow rate of the bioreactor can be automatically controlled by the medium conditioning control unit 130, which is operably connected to the pump 150. This control scheme is Figure 2 In the flowchart of Figure 2 In the illustrated sensing and control process 200, at step 202, optimal conditions are predetermined through a bioreactor optimization run. These optimal conditions include a minimum pH, a minimum oxygen level, and nutrients (e.g., glucose) at the outlet sensor 118, as well as the pH, oxygen level, and nutrients (e.g., glucose) in the MCV 120. These parameters are provided as examples, and one of ordinary skill in the art will appreciate that other parameters may also be relevant to a given application (e.g., temperature). The pH and oxygen level in the MCV 120 are independently controlled based on inputs from corresponding sensors located in the MCV 120. The nutrient (e.g., glucose) level in the MCV 120 is maintained, in part based on a signal from the outlet sensor 118, such that the nutrient level in the MCV 120 remains greater than the nutrient level detected by the outlet sensor 118. During the cell culture run, steps 204 and 206 are performed in parallel. In step 204, the outlet sensor 118 is used to measure the conditions (e.g., pH, O2, and glucose) at the outlet 114 of the cell culture vessel 110. In step 206, sensors 124a through 124d are used to measure conditions (e.g., pH, O2, and glucose) in MCV 120. In step 208, perfusion pump 150 is controlled by the control unit based on inputs from both steps 202 and 204. In step 210, a determination is made as to whether the pH at outlet sensor 118 is greater than the minimum pH determined in step 202; whether the oxygen at outlet sensor 118 is greater than the minimum oxygen level determined in step 202; and whether the nutrient level in MCV 120 is greater than the nutrient level at outlet sensor 118 and the nutrient level at outlet sensor 118 is greater than the minimum level determined in step 202. If all of these conditions are met, perfusion of the pump is continued at the current flow rate (step 212). If these conditions are not met, step 214 inquires whether the current perfusion rate is less than or equal to the maximum flow rate. If not, the system reassesses the minimum pH, O2, and glucose at outlet 114 or increases the nutrient level in MCV 120. However, if the current irrigation rate is less than the maximum flow rate, then step 218 directs increasing the irrigation flow rate. The sensing and control scheme 200 returns to Figure 2 Steps 204 and 206 are performed at the top of the diagram.

[0054] According to embodiments of the present disclosure, it is therefore possible to directly measure the nutrient and / or oxygen consumption of cells within a cell culture vessel and respond in a manner that maintains the desired conditions for the cells. For example, during a cell culture run, the medium conditioning control unit 130 is preprogrammed to maintain a specific level of oxygen saturation in the bulk culture volume relative to atmospheric saturation, where the level in the MCV is measured by sensors 124a to 124d. A second sensor (outlet sensor 118) is placed at the bioreactor outlet 114 to measure the oxygen saturation level in the culture medium just as the culture medium leaves the cell culture vessel. Using these sensors and controllers, a constant level of oxygen consumption can be maintained within physiological conditions by automatically adjusting the perfusion flow rate.

[0055] According to some embodiments of the present disclosure, a system and method for improved process monitoring is provided, which can accelerate the process development of cell culture schemes and improve the efficiency and reproducibility of cell culture processes. The ability to characterize and record the progress of these bioprocess operations will allow end users to monitor and record the consistency between batches of processes. The relevant parameters used for process characterization are cell growth, cell quality, culture medium conditions (temperature, pH, pO2 and pCO2) and metabolite concentrations (glucose, lactate, glutamine and ammonium). The temperature, pH, pO2 and pCO2 of the main culture medium are routinely controlled online in cell culture, but these and other process parameters are not currently monitored online in dynamic systems. Therefore, embodiments of the present disclosure provide systems and methods for obtaining, for example, oxygen consumption parameters in packed bed perfusion bioreactors, and demonstrate that this parameter is a feature of a given bioprocess and therefore can be used as a marker parameter for a given bioprocess.

[0056] As mentioned above, Figure 1 A schematic diagram of a bioreactor system (e.g., a fixed bed perfusion bioreactor) is presented. Cell culture medium entering the cell culture vessel 110 through the inlet 112 can have 100% saturation with atmospheric oxygen. Alternatively, according to Henry's law, the concentration of a gas in the liquid phase is equal to the Henry's law constant (k) multiplied by the partial pressure of the gas in the gas phase, so oxygen saturation can be expressed as the oxygen concentration in the cell culture medium and is equal to 204 μM at 100% saturation at standard atmospheric pressure. During the passage of the culture medium through the cell culture vessel, dissolved oxygen is used by the immobilized cells and its concentration in the cell culture medium decreases. Different cell types have different oxygen consumption rates. However, a bioreactor system having the sensing and control system of the present disclosure allows the user to run the process with a specified oxygen concentration at the bioreactor outlet, which is measured by the outlet sensor 118 and the culture medium conditioning and perfusion control system adjusts the oxygen concentration accordingly. Figure 2 The logical operations are presented in a flowchart.

[0057] To illustrate this sensing and control of a bioreactor system, some examples will be presented. Specifically, Figure 3 A typical diagram shows the percentage of dissolved oxygen (302) over time during bioreactor operation as measured by the outlet sensor 118 and the corresponding perfusion rate (304) of the culture medium in the system (ml / min). The flow rate is automatically controlled by the peristaltic perfusion flow control unit. In this example, the bioreactor is inoculated with cells at time 0:00 hours and the user sets the minimum oxygen saturation level of the culture medium at the outlet sensor 118 to 30%. The initial culture medium perfusion flow rate is set to 33 ml / min. The inoculated cells are then provided to the bioreactor system and begin to attach to the packed bed substrate and proliferate. As a result, oxygen consumption increases and the saturation level of the culture medium at the outlet decreases to approximately 30% at 26 hours after inoculation. Therefore, the control system automatically increases the perfusion flow rate to maintain a minimum oxygen saturation level of 30% at the bioreactor outlet 114. 72 hours after inoculation, the user reduces the setting of the minimum outlet oxygen saturation level from 30% to 15%, and the cell culture is carried out in automatic mode. It should be noted that the culture medium conditions are maintained independently in the culture medium conditioning container by the culture medium conditioning control unit. Examples of media conditioning vessel parameters are given in Figure 4A 、 4B and 4C. Specifically, Figure 4A The oxygen percentage in the MCV culture medium over time is shown. Figure 4B shows the pH of the culture medium over time, and Figure 4C The temperature of the culture medium over time is shown.

[0058] As described above, embodiments include real-time processing of signals and control of bioreactor systems, and the development of signatures of specific bioreactor runs that can be used as analytical tools to compare and validate independent bioreactor runs. Thus, signatures can be used to assess the health of cells cultured within a cell culture vessel and make decisions about subsequent process steps during a bioreactor run. For example, as described above, Figure 3 1 shows the recorded oxygen saturation concentration at the bioreactor outlet 114 over time during the cell culture process. The oxygen concentration level at the bioreactor outlet dropped from about 82% at time point 0 hours to about 30% during the first 26 hours of bioreactor operation. While this drop occurred, the oxygen concentration at the bioreactor inlet 112 remained constant, as shown in FIG. Figure 4A The values detected by the MCV120 are shown in Figure 2. Using the oxygen concentrations at the inlet and outlet, it is possible to use

[0059] Equation 1 determines the oxygen consumption rate of cells in culture:

[0060]

[0061] This oxygen consumption rate is shown in Figure 5 The results are expressed as %as / min and as time passes (in hours). Figure 5 Also shown is a dashed line representing the approximate slope α of the line, which can be used as a characteristic signal marker for the operation of the bioreactor. In other words, Figure 5 The slope α of the data in the graph directly reflects the cell culture progress within the bioreactor system. This value can be used as a process analysis tool to control and describe the upstream bioprocess. The following example shows that the data from a similar Figure 5 The slope of the graph, α, is directly related to the health of the cell culture and the biomass within the packed bed matrix.

[0062] To illustrate the use of parameter α, multiple cell culture runs were performed using bioreactor systems with different cell culture substrate sizes, bed heights, and cell seeding densities. Table 1 summarizes the parameters of the seven cell culture runs used.

[0063]

[0064] Table 1 .7 Overview of the bioreactor system operation, including packed bed height (cm), total number of cells inoculated (millions of cells), total packed bed surface area (cm 2 ), seeding density (cells / cm 2 ), total number of cells collected (billion cells), viability of collected cells (%), collection density (cells / cm 2 ), maximum perfusion flow rate (ml / min), maximum O2 consumption (AU) and slope α.

[0065] As shown in Table 1, multiple bioreactors were inoculated with different numbers of cells ranging from 151 to 453 million cells per bioreactor. Three identical bioreactors (bioreactors #1, #2, and #3) had the same packed bed height (2.7 cm), were inoculated with the same number of cells (151 million cells per bioreactor), and had the same total packed bed surface area (6780 cm). 2 ) and seeding density (22,222 cells / cm 2 Three other identical bioreactors (#4, #5, and #6) had the same packed bed height (5.4 cm), were inoculated with the same number of cells (302 million cells per bioreactor), and had the same total packed bed surface area (13,560 cm). 2) and seeding density (22,227 cells / cm 2 The final bioreactor (#7) had an increased bed height (8.1 cm), total number of cells inoculated (453 million cells), and packed bed surface area (20,340 cm 2 ), but with similar seeding density (22,222 cells / cm 2 During the five-day culture process, the main culture medium conditions (pH, DO2, temperature and CO2) are maintained in an automatic mode by a control system according to one or more embodiments described herein. The control system operates the culture medium conditioning vessel to maintain the culture medium conditions, wherein Figures 4A to 4C Represents a typical measurement result of a controlled culture medium. The culture medium perfusion flow rate of the bioreactor system is automatically maintained to maintain the DO2 at the bioreactor outlet at a specific saturation level. Again, Figure 3 The diagrams shown in FIG are typical perfusion flow rates and medium outlet DO2 found during these experiments. Figure 3 The diagram in derives a value for total oxygen consumption similar to Figure 5 The slopes of the linear curve fits of these plots were determined for each bioreactor run (e.g. Figure 5 ) and is presented in the last column of Table 1 (slope α). The value of slope α determined as described above can be used as a process analysis tool to control and describe upstream bioprocesses and predict biomass yield within the packed bed matrix.

[0066] For example, Figure 6 、 7 8 plot the α values from Table 1 against the number of cells seeded, the surface area of the packed bed, and the collection density, respectively. The linearity of these plots can be used to predict cell culture responses based on various cell culture system parameters. For example, Figure 6 The linearity of the graph in indicates that the upstream process developed for small-scale bioreactors #1 and #2 in Table 1 can be scaled up 2-fold and 3-fold for bioreactors #4 to 7. Therefore, continuous monitoring and recording of the slope α value can be used to determine the scalability of the upstream process. An alternative way to verify process scalability is to plot the slope α against the surface area of the bioreactor, as Figure 7 shown. Figure 6 and 7 The orange data points in correspond to failed bioreactor #3 from Table 1 (discussed below).

[0067] Monitoring the slope α value during the bioreactor run serves as a characteristic signal marker reflecting the health and expansion of the cell culture. For example, as shown in Table 1, bioreactors #1, 2, and 3 were seeded with the same number of cells. The characteristic signal marker (slope α) was measured for all bioreactors. Figure 8It was shown that real-time monitoring of the slope α can be used to compare the performance of identical bioreactors and predict bioreactor productivity. Figure 8 As can be seen, bioreactor #3 is operating under suboptimal conditions resulting in the lowest cell productivity. Therefore, monitoring the value of slope α during a bioprocess run can be used as a characteristic signal marker for a given process and can detect any process deviations if the predetermined value is not within the range defined during process development and optimization.

[0068] According to some embodiments, the medium conditioning vessel is controlled by a controller to provide appropriate temperature, pH, O2, and nutrients. Although in some embodiments, the bioreactor may also be controlled by the controller, in other embodiments, the bioreactor is disposed in a separate perfusion circuit, wherein a pump is used to control the flow rate of the medium through the perfusion circuit based on the detection of O2 at or near the bioreactor outlet.

[0069] Depending on the desired system, the cell culture substrate can be arranged in a variety of configurations within the culture chamber. For example, in one or more embodiments, the system includes one or more substrate layers whose width extends across the width of the cell culture space determined in the culture chamber. Multiple layers of substrate can be stacked to a predetermined height in this manner. The substrate layers can be arranged so that the first side and the second side of one or more layers are perpendicular to the overall flow direction of the culture medium flowing through the culture space determined in the culture chamber, or the first side and the second side of one or more layers may be parallel to the overall flow direction. In one or more embodiments, the cell culture substrate includes one or more substrate layers having a first orientation relative to the overall flow, and one or more other layers having a second orientation different from the first orientation. For example, each layer may have a first side and a second side that are parallel to or perpendicular to the overall flow direction, or at an angle between the first side and the second side.

[0070] In one or more embodiments, the cell culture system includes a plurality of discrete cell culture substrate blocks in a packed bed configuration, wherein the length and / or width of each substrate block is relatively small relative to the culture chamber. As used herein, each substrate block is considered to have a relatively small length and / or width relative to the culture chamber when the length and / or width of the substrate block is about 50% or less of the length and / or width of the culture space. Thus, the cell culture system can include a plurality of substrate blocks filled in the culture space in a desired arrangement. The arrangement of the substrate blocks can be random or semi-random, or can have a predetermined order or arrangement, such as the substrate blocks being oriented in a substantially similar orientation (e.g., horizontally, vertically, or at an angle between 0° and 90° relative to the overall flow direction).

[0071] As used herein, a "defined culture space" refers to the space within a culture chamber that is occupied by a cell culture matrix and in which cell seeding and / or culture will occur. The defined culture space may substantially fill the entire culture chamber, or may occupy a portion of the space within the culture chamber. As used herein, a "bulk flow direction" is defined as the direction of the overall mass flow of fluid or culture medium through or across the cell culture matrix during cell culture and / or during flow of culture medium into or out of the culture chamber.

[0072] In some embodiments of the present disclosure, there is only a single overall flow direction within a defined culture space, packed bed, and / or bioreactor container, such that the liquid or culture medium flow flows primarily in one direction from the bioreactor inlet through the packed bed to the bioreactor outlet. The liquid or culture medium flow is not interrupted by any complex flow paths within the packed bed space and primarily travels in one direction through the packed bed. This avoids the complex flow paths used in some conventional bioreactors, in which flow spacers, baffles, or channels are used to help distribute the cell culture medium through the cell culture substrate, which is typically due to the inherent heterogeneity of the bioreactor or cell culture substrate. However, in embodiments of the present disclosure, these complex flow paths are not required, and the culture medium flow can be maintained in a single direction from the bioreactor inlet to the bioreactor outlet. The foregoing is not intended to exclude the use of flow distributor plates at the inlet and outlet of the bioreactor plate, which can be used to distribute fluid across the width of the bioreactor container and / or control the pressure differential within the reactor, but will not otherwise affect the overall flow direction within the cell culture space through the packed bed and / or bioreactor container.

[0073] The packed bed cell culture substrate of one or more embodiments may include a substrate material configured to have a uniform and ordered porous structure. The substrate may be referred to as a "structurally determined" substrate, which means that the substrate has a physical structure that is not random, but ordered according to determined parameters. In one or more embodiments, the structurally determined substrate includes a plurality of openings that define the porosity of the substrate, and the plurality of openings are arranged in a regular or uniform pattern in each substrate block or layer. In one or more embodiments, the packed bed cell culture substrate may include a woven cell culture mesh substrate, without any other form of cell culture substrate disposed in or interspersed in the cell culture substrate. That is, the woven cell culture mesh substrate of the embodiments of the present disclosure is an effective cell culture substrate that does not require an irregular non-woven substrate of the type used in existing solutions. This makes it possible to simplify the design and construction of the cell culture system while providing a high-density cell culture substrate with other advantages discussed herein regarding flow uniformity, collectability, etc.

[0074] In one or more embodiments, a matrix is provided that has a structurally defined surface area for adherent cell attachment and proliferation, has good mechanical strength, and forms a highly uniform, multi-interconnected fluid network when assembled in a packed bed or other bioreactor. In certain embodiments, a mechanically stable, non-degradable woven mesh can be used as a substrate to support adherent cell production. The cell culture matrix disclosed herein supports the attachment and proliferation of anchorage-dependent cells at high volume densities. Such a matrix can achieve uniform cell seeding and efficient collection of cells or other products from the bioreactor. In addition, embodiments of the present disclosure support cell culture to provide uniform cell distribution during the seeding step and achieve monolayer or multilayer confluence of adherent cells on the disclosed matrix, while avoiding the formation of large and / or uncontrollable 3D cell aggregates that restrict nutrient diffusion and increase metabolic product concentrations. Thus, the matrix eliminates diffusion limitations during operation of the bioreactor. Furthermore, the matrix enables easy and efficient cell collection from the bioreactor. The structurally defined matrix of one or more embodiments enables complete cell recovery and continuous cell collection from the packed bed of the bioreactor.

[0075] By using a culture matrix with a structure that is sufficiently rigid, high flow resistance uniformity is achieved throughout the matrix or packed bed. According to various embodiments, the matrix can be deployed in a single layer or multilayer form. This flexibility eliminates diffusion limitations and allows for uniform delivery of nutrients and oxygen to cells attached to the matrix. In addition, the open matrix does not have any cell retention zones in the packed bed configuration, thereby allowing cells with high viability to be fully collected at the end of the culture. The matrix also achieves uniform filling of the packed bed and enables it to be directly expanded from a process development unit to a large-scale industrial bioprocessing unit. The ability to collect cells directly from the packed bed eliminates the need to resuspend the matrix in a stirring or mechanical oscillation container, which would increase complexity and potentially cause harmful shear stress to the cells. In addition, the high packing density of the cell culture matrix can achieve high bioprocess productivity in a manageable volume at an industrial scale.

[0076] Compared to existing cell culture substrates used in cell culture bioreactors (i.e., nonwoven substrates formed of random ordered fibers), embodiments of the present disclosure include cell culture substrates with a defined and ordered structure. The defined and ordered structure allows consistent and predictable cell culture results to be obtained. In addition, the substrate has an open porous structure that prevents cell entrapment and enables cells to flow evenly through the packed bed. This structure enables improved cell inoculation, nutrient delivery, cell growth, and cell collection. According to one or more specific embodiments, the matrix is formed by a substrate material having a thin sheet-like structure having a first side and a second side spaced apart with a relatively small thickness, so that the thickness of the sheet is small relative to the width and / or length of the first side and the second side of the substrate. In addition, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings has a certain size and geometry, thereby allowing cells to adhere to the surface of the substrate material as if it were an approximately two-dimensional (2D) surface, while also allowing sufficient fluid to flow around the substrate material and through the openings. In some embodiments, the substrate is a polymer-based material and can be in the form of a molded polymer sheet; a polymer sheet with openings punched through its thickness; a plurality of filaments fused into a mesh layer; a 3D-printed substrate; or a plurality of filaments woven into a mesh layer. The physical structure of the matrix has a high surface area to volume ratio for culturing anchorage-dependent cells. According to various embodiments, the matrix can be arranged or packed in a bioreactor in some of the manners discussed herein for uniform cell seeding and growth, uniform medium perfusion, and efficient cell collection.

[0077] According to one or more embodiments, the cell culture substrate can be one of the cell culture substrates and / or substrate materials disclosed in the following documents: U.S. patent application Ser. Nos. 16 / 781,685; 16 / 781,723; 16 / 781,764; 16 / 781,807; 16 / 781,847; 16 / 781,883; and 16 / 765,722, all of which are incorporated herein by reference in their entirety.

[0078] According to some embodiments, a method for cell culture using a bioreactor with a matrix is also provided for bioprocessing and producing therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0079] The provided cell culture substrates and bioreactor systems offer many advantages. For example, embodiments of the present disclosure can support the production of any of a variety of viral vectors, such as AAV (all serotypes) and lentiviruses, and can be used for in vivo and ex vivo gene therapy applications. Uniform cell seeding and distribution maximizes viral vector yield per container, and the design enables the collection of viable cells, which is very useful for train-style seeding consisting of multiple amplification stages performed using the same platform. In addition, the embodiments herein can be scaled up from process development scale to production scale, ultimately saving development time and costs. The methods and systems disclosed herein also allow for automation and control of the cell culture process to maximize vector yield and improve reproducibility. Finally, compared to other cell culture protocols, achieving viral vector production-grade scale (e.g., 10 per batch) is simpler and more efficient. 16 to 10 18 The number of containers required for each AAV VG can be significantly reduced.

[0080] Embodiments of the present disclosure relate to scalable bioreactor systems, including large-scale adherent cell culture fixed-bed bioreactors. Fixed-bed bioreactors have been increasingly used for scaled-up production of cells, viral vectors, extracellular vesicles, and therapeutic proteins. Scalability is a key aspect of advancing a process from the development phase to production scale. It is highly desirable to have the ability to monitor all relevant parameters with the same measurement type at each process scale to maintain high product quality and product quantity and within GMP compliance. The United States Food and Drug Administration (FDA) recommends the use of process analytical technology (PAT) as a mechanism for designing, analyzing, and controlling pharmaceutical manufacturing processes by measuring key process parameters that affect critical quality attributes.

[0081] Viable cell concentration (VCC), also known as biomass, is one of the most important key performance indicators during upstream technology in mammalian cell culture. For suspension cell cultures, biomass monitoring can be achieved using optical or electrical methods. However, for adherent cell cultures using fixed-bed bioreactors, there are no validated methods or sensors available for online monitoring of biomass. In fact, for adherent cell cultures, biomass is typically measured by off-line microscopy after staining. Historically, it has been proposed that substrate (e.g., glucose) consumption rates can be used to understand the kinetics of cell growth. However, since substrate consumption rates are affected by many factors other than viable cell number, there are no established protocols, methods, and / or models for obtaining and using effective substrate consumption rates and metabolite accumulation rates for biomass calculation and prediction.

[0082] However, embodiments of the present disclosure disclose methods and schemes for obtaining effective glucose consumption rates, glutamine consumption rates, lactate accumulation rates, and / or ammonia accumulation rates, and the use of at least one of these rates for monitoring biomass of adherent cell cultures in fixed bed bioreactors based on mathematical models. Embodiments of the methods include (1) using a culture medium rich in glucose and / or glutamine, (2) measuring glucose, glutamine, lactate, and / or ammonia concentrations multiple times (e.g., at least twice per day), (3) determining the rates of glucose and glutamine consumption and the rates of lactate and / or ammonia accumulation, and (4) using mathematical models to calculate and predict the biomass of the cells at a specific time of culture. Given the availability of online glucose sensors, the methods can be used for online biomass prediction and calculation, which can be used as a guide for, for example, transfection protocols and cell collection. Because culture medium samples can be collected using an offline sampling device (e.g., a syringe), aspects of the embodiments of the methods of the present disclosure can also be performed offline using a multiplexed analyzer (e.g., a Flex II analyzer from Nova Biomedical).

[0083] Aspects of the presently disclosed embodiments describe methods for identifying any anomalies in adherent cell culture in a fixed-bed bioreactor. Aspects of the embodiments include comparing the glucose consumption rate or lactate accumulation rate with the glutamine consumption rate or ammonia accumulation rate. According to certain aspects of the embodiments, any large difference between the glucose / lactate rate and the glutamine / ammonia rate indicates a cell culture anomaly, such as when cells become overly confluent and undergo apoptosis. Conversely, good agreement between the glucose / lactate rate and the glutamine / ammonia rate indicates healthy cell growth.

[0084] According to embodiments, the culture medium used in the cell culture preferably contains a high concentration of glucose (e.g., 2 g / L, 3 g / L, 4 g / L, 5 g / L or even 10 g / L) and / or a high concentration of glutamine (e.g., 2 mM, 3 mM, 4 mM, 5 mM or even 10 mM). In various aspects of the embodiments, at least when the viable cell concentration becomes high in the later stages of the cell culture, the cell culture medium is replenished or replaced daily so that the glucose concentration and / or glutamine concentration never falls below a threshold value. This threshold value may depend on the specific details of the cell culture, but may be, for example, 0.25 g / L for glucose and / or 0.25 mM for glutamine. It is believed that this has an impact on the measurement of the effective substrate consumption rate and the metabolite accumulation rate because the glucose consumption rate will vary depending on the initial glucose concentration at a given cell density for a given culture time.

[0085] As one aspect of the embodiment, the cell seeding density is predetermined and optimized based on the cell culture setup. For example, in a certain cell culture setup, the cell seeding density may be 20,000 cells / cm2 surface area. However, this number is provided for example only, as it will be understood that the cell seeding needs to be optimized so that the majority of cells (e.g., >90%) adhere to the fixed bed substrate surface.

[0086] Cell culture medium substrates and metabolites can be measured using online sensors or using offline sensors or analyzers after sample collection. According to embodiments, cell culture medium substrates and metabolites can be measured daily, including, for example, at least twice daily. The two measurements can be separated by at least a specific time interval, which can be, for example, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or even 20 hours. The first measurement can be performed at least one hour after the medium exchange (if any) is completed.

[0087] Depending on the bioreactor setup, appropriate mathematical models are used to calculate glucose / glutamine consumption rates and lactate / ammonia accumulation rates and to predict and calculate biomass for a specific culture duration.

[0088] Examples

[0089] In experiments conducted based on the embodiments of the present disclosure, a fixed bed bioreactor system as described herein was used in a perfusion loop with a medium conditioning vessel (MCV) and a pump. Two 1 mL syringes were attached to the perfusion loop tubing just upstream of the inlet 112 and downstream of the outlet 114 of the bioreactor vessel (see Figure 1 ). A syringe was used to collect culture medium samples every 30 minutes over an 8-hour period each day. In the early morning of each day, half of the culture medium in the MCV was drained and replaced with the same volume plus 40 mL of additional culture medium to replenish the previous day's sampling with fresh warm culture medium. All collected samples were analyzed offline using a FlexII analyzer from Nova Biopharmaceuticals. HEK293T cells were used. The cell seeding density was 22,000 cells / cm2. Cell attachment was found to be complete within 3.5 hours, with >95% of the cells attached.

[0090] like Figure 9 As shown in Figure 2, glucose concentration decreased over time each day. Similar results were obtained for glutamine. In contrast, lactate concentration increased over time each day, as shown in Figure 2. Figure 10(Similar results were obtained for ammonia.) However, the differences between samples collected upstream or downstream of the bioreactor at a given culture time were very small, even on the last day of cell culture, suggesting that when culture medium passes through the bioreactor only once, the amount of glucose or glutamine consumed is relatively small and therefore may not be used to obtain an effective rate of substrate consumption or metabolite accumulation. Therefore, according to embodiments of the present disclosure, only the concentration of substrates or metabolites from samples collected upstream of the bioreactor inlet can be used for biomass analysis.

[0091] The cell growth rate, k, varies with many factors, including medium composition and concentration, temperature, and other environmental factors such as pH, dissolved oxygen, and CO2. Assuming these environmental factors can be controlled so that the cell growth rate remains constant, the total cell count, N(t), can be integrated over the entire surface area, A:

[0092] N(t)=N0e kt Equation (2)

[0093] Where N0 is the initial cell number.

[0094] According to the mass balance perfusion reactor model, the cell culture medium substrate concentration gradient C 入 -C 出 is a function of the medium substrate consumption rate per cell, r, the total number of cells N, and the flow rate Q:

[0095]

[0096] In equation (3), C 入 is the concentration of the cell culture substrate entering the bioreactor, and C 出 is the concentration of the cell culture substrate leaving the bioreactor, and Q is the flow rate. As described above, in this experimental setup, the flow rate was approximately 50 mL / min, so that when the culture medium first passes through the bioreactor, the cell culture substrate concentration shows little or very little change. Therefore, it is difficult or impossible to calculate biomass directly using these concentration gradients. In order to calculate biomass using this parameter, it is necessary to significantly reduce the flow rate, or temporarily stop the flow (e.g., 1 minute, 2 minutes, 10 minutes, etc.) so that the sensor can detect the C of a given analyte. 入 and C 出 For example, for HEK293T cell culture, when 1m 2 The number of cells in the bioreactor was 1×10 9For example, when 100 mL of culture medium is required to fill the entire bioreactor, assuming a glucose consumption rate of approximately 0.001 mg / min / million cells, the flow would need to be stopped for 10 minutes to effectively detect the 0.1 g / L of glucose consumed. Such low flow rates or periods of stopped flow are not only difficult to achieve in practice, but also result in temporarily hypoxic culture conditions.

[0097] According to the continuous stirred tank reactor model of an MCV coupled to a bioreactor, the total amount of cell culture medium substrate consumed during a given time period is a function of the cell consumption rate r and the total cell number N:

[0098]

[0099] Where V is the total volume of the culture medium in the MCV. After rearrangement:

[0100]

[0101] Since kt is small enough (<0.5) within the maximum 8-hour sampling window per day,

[0102]

[0103] V(C0-C(t))=rN0t Equation (6)

[0104] Therefore, plotting equation (6) (V(C0-C) versus t) with the fitted line (time shifted to the first meaningful sampling of each day) yields the slope rN0, where N0 is the total cell count at the first meaningful sampling of each day. Furthermore, plotting rN0 versus t and fitting to an exponential curve provides:

[0105] rN0(t)=rN 接种 e kt Equation (7)

[0106] The slope is the cell growth rate k, and the intercept is r*N 接种 .

[0107] Figure 11 ] shows the total amount of glucose consumed per day (C0-C0) over time according to an example embodiment. t ) diagram. Figure 12 A graph showing the total glucose consumption rate rN0 per day as an exponential function of the culture duration according to an example embodiment is shown. Using glucose consumption as an example, Figure 11 As shown in Figure 2, the total amount of glucose consumed increases linearly over time within each day. However, the slope rN0 increases each day and is well fitted by an exponential function ( Figure 12 ), and we get 0.000391min -1The cell growth rate k and the intercept r*N of 0.212549 接种 These results indicate that the cell doubling time is approximately 29.5 hours and the glucose consumption rate is 0.0009531 mg / min / million cells. Therefore, the collection time t h =The number of cells at 5669 minutes is:

[0108]

[0109] The above number of 2046M is higher than the actual total number of cells collected, which was found to be 1790M.

[0110] Similarly, the profiles of glutamine, lactate and ammonia were also obtained and used to analyze cell growth and predict the cell number when collected (Table 2). These results show that in all four analytes, there is good consistency in the cell number when cell growth rate is collected. Interestingly, glucose and lactate produce almost identical cell growth parameters, while glutamine and ammonia have similarities. It is believed that this is because lactate is a cellular metabolite of glucose, and ammonia is a cellular metabolite of glutamine. It is also worth noting that the glucose / lactate analysis produces a higher predicted cell number compared to the glutamine / ammonia analysis.

[0111]

[0112] Table 2. Comparison of cell growth parameters obtained using four different analytes.

[0113] Additional experiments were performed using an alternative bioreactor system setup including refeed bottles, e.g. Figure 13 A syringe is connected to the tubing just below the bioreactor inlet, where the substrate concentration is called C t The second syringe is connected to the contents of the refeed bottle, where the substrate concentration is referred to as C 2,t . Evaluated with 5m 2 Two separate bioreactors with a substrate surface area of 22,000 cells / cm 2as the inoculation density and for 4 days of culture for HEK293T cells. The MCV contained 1800 mL of culture medium, while the refeed loop contained 3 L of culture medium. For cell inoculation, 300 mL of cell solution was used. The results showed that the cells reached approximately 95% attachment within 3.5 hours. Every day, 2 ml samples were collected from the lower port of the reactor and the refeed bottle at least twice. On day 2, one medium change was performed in the afternoon; on day 3, two medium changes were performed, one in the early morning and the other in the late afternoon. Cells were collected in the early morning of day 4 using an automated collection protocol. All collected samples were analyzed using a Flex II analyzer from Nova Biopharmaceuticals.

[0114] Since this bioreactor setup involves a third refeed bottle, a modified model was used to analyze the profile. V2 and C2 are the medium volume and glucose concentration in the refeed bottle, and Q2 is the flow rate between the MCV and the refeed bottle, then for the MCV:

[0115]

[0116] For refeed bottles:

[0117]

[0118] Substituting this into the MCV equation:

[0119]

[0120] Then the solution is:

[0121] V(C0-C(t))+V2(C 2,0 -C2(t))=rN0t

[0122] Glucose, glutamine, lactate and ammonia profiles were also obtained and used to analyze cell growth and predict cell number at harvest. Figures 14A to 14D ), for all four analyte profiles, their corresponding consumption rates or accumulation rates were well fitted with exponential functions, consistent with the exponential growth pattern of typical adherent cell cultures. Further analysis also showed that for all four analytes, there was good consistency between the cell growth rate and the number of cells at the time of collection (Table 3). Similarly, glucose and lactate produced almost identical cell growth parameters, while glutamine shared similarities with ammonia. The different cell growth parameters obtained may be related to slightly different culture conditions or cell culture variability between bioreactors (for example, compare the results of Table 3 for the first bioreactor with the results of Table 4 for the second bioreactor). In general, the cell parameters predicted using the ammonia accumulation rate were lower than those for all other three analytes, which may be due to the unique properties of ammonia. Some of the ammonia produced is known to escape from the cell culture medium and enter the headspace of the MCV.

[0123]

[0124] Table 3. The first 5m 2 Comparison of cell growth parameters obtained in bioreactors using four different analytes.

[0125]

[0126] Table 4. The second 5m 2 Comparison of cell growth parameters obtained in bioreactors using four different analytes.

[0127] As an alternative method, according to some embodiments, a metabolite kinetic model can be used. The above metabolite kinetic model requires that the V(C0-C(t))=rN0t or V(C0-C(t))+V2(C 2,0 For each data set where -C(t) = rN0t, no medium addition or exchange occurred during the time span of the data set. The advantage of this kinetic model is that the operator does not need to know the operation history of the entire culture process, as long as no medium addition or exchange occurred during the time span of each collected data set.

[0128] However, in practice, it is not uncommon to perform only a limited number of samplings over a span of several days, during which the culture medium is added or replaced, sometimes at a high frequency. For example, in the later stages of cell culture, the cell confluence may become high, so a fixed amount of culture medium is often added regularly (e.g., every hour) to maintain a relatively constant culture medium composition, thereby increasing the productivity of the culture. In this case, an alternative method is provided, as follows:

[0129]

[0130] Where Δ(VC) 调整 represents the change in the amount of metabolite in the MCV from t = 0, adjusted for any medium addition or change events. This model is called the accumulation model.

[0131] Table 5 provides an example. Assume that there are 7 samplings starting from t=0, and there is a culture medium addition of volume Va after t2, and a culture medium replacement of volume Ve after t4. Δ(VC) is calculated as described above, but needs to be adjusted for all samplings after any culture medium addition / replacement event. For example, for culture medium addition after t2, for all samplings starting from t3, Δ(VC) adds Va*Ca (Ca is the fresh culture medium concentration). Similarly, for culture medium replacement after t4, starting from t5, Δ(VC) needs to be adjusted for Ve*(Ca-Ce), where Ce is the concentration of the culture medium removed. In order to avoid the need to sample the removed culture medium, it is recommended to perform culture medium replacement immediately after regular sampling (in this case, immediately after t4) so that Ce=C4.

[0132]

[0133] Table 5. Examples of data preprocessing for surrogate models.

[0134] In the refeed bottle experiment above, there is a medium exchange from the system. Using the above method, the data can be prepared for the model:

[0135]

[0136] Note that in this case, Δ(VC) 调整 Has substantial meaning of total glucose consumed.

[0137] Figure 15 The total glucose consumption (related to the total cell number) was well fitted by the exponential function in the model. The predicted cell growth constant k was 5.44e-04min. -1 (doubling time 21 hours). The predicted cell yield was 19,893 M, which was in good agreement with the collected data of 19,175 M.

[0138] As demonstrated, this accumulation model has the advantage of being applicable to a wide range of cell culture protocols and metabolite data collection schemes, including infrequent sampling over longer time intervals and frequent media manipulation. However, this model requires tracking the entire history of cell culture manipulation, in particular any media additions, removals, or changes.

[0139] Illustrative embodiments

[0140] The following is a description of various aspects of the embodiments of the disclosed subject matter. Each aspect may include one or more of the various features, characteristics, or advantages of the disclosed subject matter. The embodiments are intended to illustrate several aspects of the disclosed subject matter and should not be considered as a comprehensive or exhaustive description of all possible embodiments.

[0141] Aspect 1 relates to a method for monitoring biomass during cell culture of cells in a bioreactor, the method comprising: culturing the cells in the bioreactor using a cell culture medium perfused through the bioreactor; measuring at least one of cell nutrients and cell byproducts in the cell culture medium; determining at least one of a consumption rate of the cell nutrients and an accumulation rate of the cell byproducts; and predicting a cell number within the bioreactor at a specified culture time based on at least one of the consumption rate and the accumulation rate.

[0142] Aspect 2 relates to the method of aspect 1, wherein the bioreactor is a fixed bed bioreactor comprising a substrate configured for culturing cells attached to a surface of the substrate.

[0143] Aspect 3 relates to the method of aspect 1 or aspect 2, wherein the at least one cell nutrient is glucose or glutamine.

[0144] Aspect 4 relates to the method of any one of aspects 1 to 3, wherein the at least one cellular byproduct is lactate or ammonia.

[0145] Aspect 5 relates to the method of any one of aspects 1 to 4, wherein the cell culture medium is a cell culture medium enriched with glucose or glutamine.

[0146] Aspect 6 relates to the method of any one of aspects 1 to 5, wherein measuring at least one of the cell nutrients and the cell byproducts in the cell culture medium comprises performing multiple measurements of the cell nutrients or the cell byproducts, the multiple measurements being spaced apart by a time interval that is less than a doubling time of the cells in the cell culture.

[0147] Aspect 7 relates to the method of aspect 6, wherein the measurement interval time is greater than or equal to a minimum interval time, the minimum interval time being the time when the change in the level of the cell nutrient or the cell byproduct is greater than the measurement tolerance of measuring the cell nutrient or the cell byproduct.

[0148] Aspect 8 relates to the method of Aspect 7, wherein the minimum interval time is greater than or equal to about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours or 20 hours.

[0149] Aspect 9 relates to the method of any one of aspects 1 to 8, wherein measuring the cell nutrients in the cell culture medium comprises measuring the cell nutrients in the cell culture multiple times per day.

[0150] Aspect 10 relates to the method of any one of aspects 1 to 9, wherein measuring the cellular byproduct in the cell culture medium comprises measuring the cellular byproduct in the cell culture medium multiple times per day.

[0151] Aspect 11 relates to the method of any one of aspects 1 to 10, wherein predicting the cell number comprises calculating the biomass at the specified culture time using a mathematical model.

[0152] Aspect 12 relates to the method of any one of aspects 1 to 11, wherein the measuring comprises using an inline sensor in the perfusion line of the cell culture medium.

[0153] Aspect 13 relates to the method of any one of aspects 1 to 11, wherein the measuring comprises using an off-line measurement of a sample of the cell culture medium.

[0154] Aspect 14 relates to the method of any one of aspects 1 to 13, further comprising, after determining the consumption rate and the accumulation rate, comparing at least one of the consumption rate of the first cell nutrient and the accumulation rate of the first cell byproduct to at least one of the consumption rate of the second cell nutrient and the accumulation rate of the second cell byproduct.

[0155] Aspect 15 relates to the method of aspect 14, wherein the comparison comprises comparing the consumption rate of glucose to the consumption rate of glutamine.

[0156] Aspect 16 relates to the method of aspect 14 or aspect 15, wherein the comparison comprises comparing the rate of glucose consumption to the rate of ammonia accumulation.

[0157] Aspect 17 relates to the method of any one of aspects 14 to 16, wherein the comparison comprises comparing the rate of accumulation of lactate to the rate of consumption of glutamine.

[0158] Aspect 18 relates to the method of any one of aspects 14 to 17, wherein the comparison comprises comparing the rate of accumulation of lactate to the rate of accumulation of ammonia.

[0159] Aspect 19 relates to the method of any one of aspects 14 to 18, further comprising determining an abnormality in the cell culture based on the comparison.

[0160] Aspect 20 relates to the method of any one of aspects 1 to 19, further comprising inoculating cells in the bioreactor at an inoculation density.

[0161] Aspect 21 relates to the method of any one of aspects 1 to 20, further comprising supplying fresh cell culture medium to the bioreactor.

[0162] Aspect 22 relates to the method of aspect 21, wherein the measuring comprises a first measurement, the first measurement being performed at least one hour after supplying the fresh cell culture medium.

[0163] Aspect 23 relates to the method of any one of aspects 1 to 22, wherein the number of cells N is predicted t This involves using the following equation:

[0164] N t =N 接种 e kt

[0165] where N 接种 is the number of cells inoculated into the bioreactor, k is the cell growth rate, and t is the time at which the cell number is predicted.

[0166] definition

[0167] "Fully synthetic" or "completely synthetic" refers to a cell culture article, such as a microcarrier or the surface of a culture vessel, that is composed entirely of synthetically derived materials and does not contain any animal-derived or animal-sourced materials. The disclosed fully synthetic cell culture articles eliminate the risk of xenogeneic contamination.

[0168] The terms "include," "includes," or similar terms mean including but not limited to, ie, inclusive rather than exclusive.

[0169] "User" refers to one who uses the systems, methods, articles, or kits disclosed herein, and includes one who cultures cells to collect cells or cell products, or one who uses cells or cell products cultured and / or collected according to the embodiments herein.

[0170] The use of "about" in describing embodiments of the present disclosure to modify, for example, the amount of an ingredient in a composition, concentration, volume, processing temperature, processing time, yield, flow rate, pressure, viscosity, and the like, and ranges thereof, or the size of a component, and the like, and ranges thereof, refers to variations in the numerical amount that may occur due, for example, to typical measuring and handling procedures used to prepare materials, compositions, compounds, concentrates, components, articles, or formulations for use; inadvertent errors in such procedures; differences in the manufacture, origin, or purity of starting materials or ingredients used to practice the methods; and similar considerations. The term "about" also encompasses amounts that vary due to aging of a composition or formulation having a particular initial concentration or mixture, as well as amounts that vary due to mixing or processing a composition or formulation having a particular initial concentration or mixture.

[0171] "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0172] As used herein, the indefinite articles "a" or "an" and their corresponding definite articles "the" mean at least one, or one or more, unless stated otherwise.

[0173] Abbreviations well known to those of ordinary skill in the art may be used (e.g., "h" or "hr" for one or more hours, "g" or "gm" for one or more grams, "mL" for milliliters, and "rt" for room temperature, "nm" for nanometers, and similar abbreviations).

[0174] The specific and preferred values disclosed for components, ingredients, additives, sizes, conditions, and the like, and their ranges are for illustration only; they do not exclude other defined values or other values within the defined ranges. The systems, kits, and methods of the present disclosure may include any value described herein, or any combination of values, specific values, more specific values, and preferred values, including explicit or implicit intermediate values and ranges.

[0175] Unless otherwise expressly stated, it is not intended that any method described herein be construed as requiring that its steps be performed in a specific order. Therefore, if a method claim does not actually recite the order in which its steps are to be followed, or if the claims or description do not otherwise specifically state that the steps are to be limited to a specific order, no specific order is intended to be inferred.

[0176] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the disclosed embodiments. Since those skilled in the art may envision modifications, combinations, sub-combinations, and variations of the disclosed embodiments that incorporate the spirit and substance of the embodiments, the disclosed embodiments should be construed to include all contents within the scope of the appended claims and their equivalents.

Claims

1. A method for monitoring biomass during cell culture of cells in a bioreactor, the method comprising: culturing the cells in the bioreactor using a cell culture medium perfused through the bioreactor; measuring at least one of a cell nutrient and a cell byproduct in the cell culture medium; determining at least one of a rate of consumption of a nutrient for the cell and a rate of accumulation of a byproduct of the cell; Based on at least one of the consumption rate and the accumulation rate, a cell number within the bioreactor at a specified culture time is predicted. 2 . The method of claim 1 , wherein the bioreactor is a fixed bed bioreactor comprising a substrate configured for culturing cells attached to a surface of the substrate.

3. The method of claim 1 or claim 2, wherein at least one cell nutrient is glucose or glutamine.

4. The method of any one of claims 1 to 3, wherein at least one cellular by-product is lactate or ammonia. The method according to any one of claims 1 to 4, wherein the cell culture medium is a cell culture medium enriched with glucose or glutamine.

6. The method of any one of claims 1 to 5, wherein measuring at least one of the cell nutrients and the cell byproducts in the cell culture medium comprises performing multiple measurements of the cell nutrients or the cell byproducts, wherein the multiple measurements are separated by a measurement interval that is less than a doubling time of cells in the cell culture.

7. The method of claim 6, wherein the measurement interval is greater than or equal to a minimum interval, the minimum interval being a time at which a change in the level of the cell nutrient or the cell byproduct is greater than a measurement tolerance for measuring the cell nutrient or the cell byproduct.

8. The method of claim 7, wherein the minimum interval time is greater than or equal to about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours.

9. The method of any one of claims 1 to 8, wherein measuring cell nutrients in the cell culture medium comprises measuring cell nutrients in the cell culture multiple times per day.

10. The method of any one of claims 1 to 9, wherein measuring cellular byproducts in the cell culture medium comprises measuring cellular byproducts in the cell culture medium multiple times per day.

11. The method according to any one of claims 1 to 10, wherein predicting the cell number comprises calculating the biomass at the specified culture time using a mathematical model.

12. The method according to any one of claims 1 to 11, wherein the measuring comprises using an inline sensor in a perfusion line of the cell culture medium.

13. The method according to any one of claims 1 to 11, wherein the measuring comprises using an off-line measurement of a sample of the cell culture medium.

14. The method of any one of claims 1 to 13, further comprising: after determining the consumption rate and the accumulation rate, comparing at least one of the consumption rate of a first cell nutrient and the accumulation rate of a first cell byproduct with at least one of the consumption rate of a second cell nutrient and the accumulation rate of a second cell byproduct.

15. The method of claim 14, wherein the comparing comprises comparing the consumption rate of glucose to the consumption rate of glutamine.

16. The method of claim 14 or claim 15, wherein the comparison comprises comparing the rate of glucose consumption to the rate of ammonia accumulation.

17. The method of any one of claims 14 to 16, wherein the comparison comprises comparing the rate of accumulation of lactate to the rate of consumption of glutamine.

18. The method of any one of claims 14 to 17, wherein the comparing comprises comparing the rate of accumulation of lactate to the rate of accumulation of ammonia.

19. The method of any one of claims 14 to 18, further comprising determining an abnormality in the cell culture based on the comparison.

20. The method of any one of claims 1 to 19, further comprising seeding the bioreactor with cells at a seeding density.

21. The method of any one of claims 1 to 20, further comprising supplying fresh cell culture medium to the bioreactor.

22. The method of claim 21, wherein the measuring comprises a first measurement, the first measurement being performed at least one hour after supplying the fresh cell culture medium.

23. The method according to any one of claims 1 to 22, wherein the number of cells N is predicted t This involves using the following equation: N t =N 接种 e kt where N 接种 is the number of cells inoculated into the bioreactor, k is the cell growth rate, and t is the time at which the cell number is predicted.

24. The method of any one of claims 1 to 23, wherein measuring at least one of the cell nutrients and the cell byproducts in the cell culture medium comprises measuring at least one of glucose, lactate, glutamine, and ammonia concentrations at least twice daily for at least three days during the cell culturing, wherein the measurements are separated by predetermined time intervals.

25. The method of claim 24, wherein the measuring comprises measuring at least two of glucose, lactate, glutamine, and ammonia concentrations.

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