WO2024052679A1 - System for bioprocessing - Google Patents

Abstract

The present disclosure provides systems and methods for bioprocessing. The system can comprise (a) one or more reactant tanks, wherein the one or more reactant tanks comprise reagents; (b) one or more vessels fluidly connected to the one or more reactant tanks; and (c) one or more waste tanks fluidically connected to the one or more vessels; wherein at least one of the one or more vessels comprises one or more sub-vessels, wherein the dimensions of the one or more sub-vessels are identical.

Description

SYSTEM FOR BIOPROCESSING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/404,502, filed September 7, 2022, which is herein entirely incorporated by reference.

BACKGROUND

[0002] During process development, scientists design experiments based on initial knowledge of what could constitute critical process parameters. For example, these process parameters can include cell seeding density, types of reagents, reagent replacement rate, CO2 or O2 concentration, pH, or temperature. Based on these parameters, scientists can need to run several experiments with slightly different parameter spaces to understand the impact of these parameters on their cells. Each experiment will typically be run at least in triplicates to obtain statistics on measurements values. Often, the number of experiments that can be carried out is simultaneously limited by the capacity of the research team, the lab space, or analytical capabilities. This process is highly iterative, with often hundreds of experiments being carried out before a process can move to the clinic and continues for many years in clinical stages to generate further data and optimizations.

[0003] This setup can result in a series of fundamental issues in cell and gene therapy research. Although processes steps are typically well documented, manual operations create variability due to the large number of undocumented degrees of freedom in a given process: pipetting technique, mixing method, speed of execution in the hood, human errors can all be source of variability in the final cell product. Cell culture vessels can also create variability within the product, for example how well spread the cells are within a flask, or how well mixed a bag is. These sources of variability make it difficult to separate the unavoidable variability from the starting cell material from the rest. This results in a poorly characterized process, and a poor understanding of cell behavior. Later, this can translate into unwanted variability in the final cell product, which can lead to batch rejection, longer process times, sub-optimal cell potency.

[0004] Due to the burden of sampling, running the analysis and extracting data, scientists often have a sparse view of their process parameters and cell health. For example, continuous monitoring of basic parameters such as pH and dissolved oxygen are only available on a handful of highly expensive research devices. Another example, cell counting, can be difficult to implement at the research scale, because the sampled cells represent a non-negligible amount compared to the total number of cells in culture. This lack of measurement prevents scientists from gaining a deep understanding of their process parameters and the impact on their cells. This later leads to poorly defined and controlled processes; which in turn leads to non-conform cell product, variability in final cell product, or suboptimal process efficiency.

[0005] The mixture of limited capacity due to lengthy and numerous manual operations (for processing and analytics), additional variability from operators and cell culture vessels, sterility issues, space limitations, sparse and dispersed analytical tools, combined with the vast parameter space to explore, leads to process development times that can span years. The pace at which experiments can be carried out becomes proportional to the size of the team and capital available, which is often a limiting factor for biotech companies. Even when many experiments can be carried out, the lack of data available, or that can be exploited and correlated, contributes to unsolved issues (e.g., high percentage of non-conformity of final product) that requires even longer development times or lead to high cost of goods later on.

[0006] Therefore, there remains a need for an automated research and process development system that can run a large number of end-to-end experiments with different process parameters at the same time, while capturing key data about cell health and process parameters

SUMMARY

[0007] The present disclosure provides a bioprocessing system with end-to-end automation along with the ability to run a large number of experiments with different process parameters at the same time and maintain sterility when connecting/disconnecting cell culture vessels and while running experiments.

[0008] In an aspect, provided herein is a bioprocessing system, comprising: (a) one or more mixing tanks, wherein the one or more reactant tanks comprise reagents; (b) one or more vessels fluidically connected to the one or more reactant tanks; and (c) one or more waste tanks fluidically connected to the one or more vessels; wherein at least one of the one or more vessels comprises one or more sub-vessels, and wherein the dimensions of the one or more sub-vessels are identical.

[0009] In some cases, a first reactant tank of the one or more reactant tanks is kept at a different temperature than a second reactant tank of the one or more reactant tanks. In some cases, a reactant tank of the one or more reactant tanks is kept at ambient temperature. In some cases, a reactant tank of the one or more reactant tanks is kept at a temperature of about 20 C In some cases, a reactant tank of the one or more reactant tanks is kept at a temperature of about 4 C. In some cases, a reactant tank of the one or more reactant tanks is kept at a temperature of about -20 °C.

[0010] In some cases, the one or more vessels are fluidically connected to the one or more reactant tanks or the one or more waste tanks through a connector system comprising one or more pumps, one or more valves, and tubing. In some cases, the system further comprises a steam sterilizer. In some cases, the steam sterilizer generates steam that flows through the one or more pumps, the one or more valves, or the tubing at a temperature at or above 121 °C at a pressure of 2 bars in absolute value. In some cases, the system further comprises one imaging system. In some cases, the one imaging system is configured to move among the one or more vessels, and wherein the imaging system is configured to capture images of individual vessels of the one or more vessels. In some cases, the one or more vessels are configured to move to the imaging system, and wherein the imaging system is configured to capture images of individual vessels of the one or more vessels.

[0011] In some cases, a vessel of the one or more vessels comprises a pH sensor configured to measure pH of the fluid in the vessel. In some cases, a vessel of the one or more vessels comprises a dissolved oxygen sensor configured to measure a level of dissolved oxygen in the vessel. In some cases, a vessel of the one or more vessels comprises a temperature sensor configured to measure a temperature of the vessel. In some cases, the one or more sub-vessels each comprise one or more bioprocessing chambers.

[0012] In some cases, the one or more bioprocessing chambers comprise a volume of less than 10 mL, 7 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, 0.5 mL, or 0.1 mL. In some cases, the one or more bioprocessing chambers comprise a cell culturing surface of less than 300 cm², 200 cm², 100 cm², 90 cm², 80 cm², 70 cm², 60 cm², 50 cm², 40 cm², 30 cm², 20 cm², 10 cm², 6 cm², 5 cm², or 1 cm². In some cases, the one or more bioprocessing chambers comprise a biocompatible material.

[0013] The system of claim 19, wherein the biocompatible material is a U.S. Pharmacopeia Convention (USP) Class VI material. In some cases, the one or more bioprocessing chambers are sterile. In some cases, the one or more bioprocessing chambers comprise a plurality of cells. In some cases, each of the one or more bioprocessing chambers comprise at least 50,000 cells/mL, 100,000 cells/mL, 500,000 cells/mL, 1 million cells/mL, 5 million cells/mL, 10 million cells/mL, 20 million cells/mL, 40 million cells/mL, 60 million cells/mL, or 100 million cells/mL. In some cases, each of the one or more bioprocessing chambers comprises at least 0.05 million, 0.25 million, 0.5 million, 1 million, 10 million, 50 million, 100 million, 150 million, or 500 million cells.

[0014] In some cases, a vessel of the one or more vessels is configured to have at least one process parameter that differs from another vessel of the one or more vessels. In some cases, the at least one process parameter is temperature. In some cases, the at least one process parameter is pH. In some cases, the at least one process parameter is dissolved oxygen concentration. In some cases, the at least one process parameter is reagent concentration. In some cases, the at least one process parameter is reagent composition. In some cases, the at least one of the one or more vessels comprising the one or more sub-vessels comprises cells. In some cases, the one or more sub-vessels comprise the same concentration of cells. In some cases, the method further comprises an inline metabolite analyzer fluidically connected to the one or more vessels.

INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0017] FIG. 1 schematically illustrates a process flow diagram of a research and process development, in accordance with some embodiments.

[0018] FIG. 2 schematically illustrates another process flow diagram of a research and process development, in accordance with some embodiments.

[0019] FIG. 3 illustrates a rendering of a research and process development device, in accordance with some embodiments.

[0020] FIG. 4 illustrates a rendering of a reagent and cell loading bay, in accordance with some embodiments.

[0021] FIGs. 5A and 5B illustrate outputs of a real time cell imaging and count system, in accordance with some embodiments.

[0022] FIG. 6 schematically illustrates a top view of a cell culture vessel comprising four subculture vessels, in accordance with some embodiments.

[0023] FIG. 7 shows an inline metabolite analyzer utilizing a robot arm for automated supernatant sampling and analysis, in accordance with some embodiments.

[0024] FIG. 8 schematically illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.

DETAILED DESCRIPTION

[0025] While various embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be employed.

[0026] Whenever the term “about,” “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “about,” “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0027] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

Overview

[0028] Cell and gene therapy uses living cells to achieve a therapeutic effect. Several technologies exist, for example T-cells used mostly in immuno-oncology, mesenchymal stem cells mostly used for immune-modulation, or induced pluripotent stem cells, used in regenerative medicine or as a source of other cell types.

[0029] For all these technologies, there is a need to process cells ex-vivo, e.g., select cells of interest for a sample (blood, adipose tissue, bone marrow), genetically modify the cells with a viral vector or a protein, differentiate the cells to a target cell type, expand the cells to reach clinically-relevant cell numbers, purify the cells through washing steps and formulate the cells in cryo-preservant or in a solution suitable for infusion into the patient.

[0030] While these processes and their sequence can vary widely, they usually consist of mixing cells with a reagent, leaving the cells in contact with that reagent for a period of time, and then washing away the reagent to replace it with a fresh batch of the same reagent or a new reagent to move to the next step of the process sequence. At a preclinical (and sometimes clinical) level, scientists often carry out these processes manually in standard cell culture vessels (flasks, well plates, bags) and using vast amounts of consumables (pipettes, gloves, etc.). They can take place in biology lab, which contains multiples pieces of equipment that are necessary for the processes (e.g., a laminar flow hood to maintain sterility, an incubator to maintain temperature and humidity conditions, a centrifuge to separate the cells and the reagents).

[0031] For an analytical standpoint, it is desirable to measure certain parameters of the process and gather data on the cells, in order to optimize the process (e.g., choosing the optimal period of time that a given reagent should be in contact with the cells, in order to reduce process time) or to understand the impact of the process on the cells (e.g., impact on the growth rate and viability of the cells). Typically, in research and process development, these measurements are made offline, i.e., a sample of fluid and/or cells is taken and analyzed on a separate device (e.g., microscope, metabolite analyzer, flow cytometer). The data from these measurements being captured in different places, scientists can then have to manually aggregate the data in order to analyze it and correlate it. Finally, these measurements are often made at the end of the process sequence rather than during the sequence, for operators often limit the number of samples taken, because they create a sterility breach risk and manipulation overhead (e.g., sample preparation, fluid top ups).

[0032] Each technology has a wide variety of process sequences and within each process, a wide range of process parameters. However, ultimately, most processes involve mixing the cells with a reagent or a combination of reagents, provide the cells with an environment that promotes their viability (e.g., temperature, CO2 concentration). In some cases, processes involve cells moving, e.g., for cell separation, where cells, attached to magnetic beads via an antibody bound, move towards a magnet, or cell passage or cell transfer, where cells are harvested from a cell culture vessel to another cell culture vessel or system.

[0033] In a process development context, scientists design experiments based on initial knowledge of what could constitute critical process parameters. These process parameters could include cell seeding density, types of reagents, reagent concentration, reagent replacement rate, carbon dioxide (CO2) or oxygen (O2) concentration, pH, or temperature. Based on these parameters, scientists will run several experiments with slightly different parameter spaces (Design of Experiment, DoE) to understand the impact of these parameters on their cells. Each experiment will typically be run in triplicates to obtain statistics on measurements values. Often, the number of experiments that can be carried out simultaneously is limited by the capacity of the research team, the lab space, or the analytical capabilities. This process is highly iterative, with often hundreds of experiments being carried out before a process can move to the clinic and continues for many years in clinical stages to generate further data and optimizations.

[0034] This traditional setup can result in variability in final cell product from manual operations and standard cell culture vessels. Although processes steps are typically well documented, manual operations create variability due to the large number of undocumented degrees of freedom in a given process: pipetting technique, mixing method, speed of execution in the hood, human errors can all be source of variability in the final cell product. Cell culture vessels can also create variability within the product, for example how well spread the cells are within a flask, or how well mixed a bag is. These sources of variability make it difficult to separate the unavoidable variability from the starting cell material from the rest. This results in a poorly characterized process, and a poor understanding of cell behavior. Later, this translates into unwanted variability in the final cell product, which can lead to batch rejection, longer process times, sub-optimal cell potency.

[0035] This traditional setup can result in additional variability in final cell product from lack of in-process measurement, leading to a lack of understanding in key process parameters. Due to the burden of sampling, running the analysis and extracting data, scientists often have a sparse view of their process parameters and cell health. For example, continuous monitoring of basic parameters such as pH and dissolved oxygen are only available on a handful of highly expensive research devices. Another example, cell counting, can be difficult to implement at the research scale, because the sampled cells represent a non-negligible amount compared to the total number of cells in culture. This lack of measurement prevents scientists from gaining a deep understanding of their process parameters and the impact on their cells. This later leads to poorly defined and controlled processes; which in turn leads to non-conform cell product, variability in final cell product, or suboptimal process efficiency.

[0036] This traditional setup can result in long preclinical development cycles. The mixture of limited capacity due to lengthy and numerous manual operations (for processing and analytics), additional variability from operators and cell culture vessels, sterility issues, space limitations, sparse and dispersed analytical measurements, combined with the vast parameter space to explore, leads to process development times that can span years. The pace at which experiments can be carried out becomes proportional to the size of the team and capital available, which is often a limiting factor for biotech companies. Even when many experiments can be carried out, the lack of data available, or that can be exploited and correlated, can contribute to unsolved issues (e.g., high percentage of non-conformity of final product) that requires even longer development times or lead to high cost of goods later on.

[0037] The bioprocessing system described herein can comprise one or more reagent or mixing tanks. The one or more reagent or mixing tanks can be kept at different temperatures. The various temperature zones can correspond to storage requirements of reagents. The system can comprise at least one reagent loading bay, which can be called a main reagent stock. Here, stock reagents can be connected sterilely to the system, via a sterile connector system. Reagents can be loaded via an aseptic click and connect process. Reagent tanks can be in the form of bags, bottles, or vials. Referring to FIG. 1 and FIG. 2, the system can comprise three reagent (or mixing) tanks. Reagent tank 100 can be kept at a temperature of 20°C (ambient temperature). Reagent tank 105 can be kept at a temperature of 4°C. Reagent tank 110 can be kept at a temperature of -20°C. Any particular reagent in the -20°C bay can be reheated to ambient temperature when needed and brought back to -20°C. Any reagent loaded onto the system can be mixed together with another, in a ratio of the user’s choice. In some cases, the bioprocessing system contains 3 reagents, 4 reagents, 5 reagents, 6 reagents, 7 reagents, 8 reagents, 9 reagents, or 10 reagents. In some cases, the bioprocessing system contains at least 3 reagents, 4 reagents, 5 reagents, 6 reagents, 7 reagents, 8 reagents, or 9 reagents. In some cases, the bioprocessing system contains at most 4 reagents, 5 reagents, 6 reagents, 7 reagents, 8 reagents, 9 reagents, or 10 reagents. In some cases, at least one empty container can be connected to the system via tubing and a sterile connector. This empty container can be used as a mixing tank, where several connected reagents can be combined. The empty container can also be used as a holding tank for further heating of reagents originating from the refrigerated or the freezer bay. FIG. 4 shows a picture of three reagent tanks, in accordance with some embodiments.

[0038] Reagents can be fed through a connector box 120 and through a selector valve 125 and optionally through another connector box 140. One or more pumps 130 and 165 can be used to facilitate flow of reagents. The pump 130 or 165 can be an auto-sampler, a peristaltic pump, a syringe pump, or a pressure pump. The goal of this system is to send the correct reagent or reagent mix from a particular stock reagent or stock reagent mix (100, 105, or 110) to a predetermined cell culture vessel 150.

[0039] One or more selector valves 125 can be able to direct fluid from the reagent stock to a predetermined cell culture vessel 150. The valve system can take the form of a manifold fitted with pinch valves, or one or several rotary valves, e.g., n-to-1 valves and 1-to-n valves combinations. The one or more selector valves 125 can be fully automated.

[0040] In some cases, the bioprocessing system contains one or more cell culture vessels 150. In some cases, the cell culture vessels can be a cassette or chip. The cell culture vessels can be in a microfluidic cassette, on which several chambers are parallelized and represent replicates. In some cases, the bioprocessing system contains 9 cell culture vessels, 12 cell culture vessels, 15 cell culture vessels, 25 cell culture vessels, or 30 cell culture vessels. In some cases, the bioprocessing system contains at least 9 cell culture vessels, 12 cell culture vessels, 15 cell culture vessels, or 25 cell culture vessels. In some cases, the bioprocessing system contains at most 12 cell culture vessels, 15 cell culture vessels, 25 cell culture vessels, or 30 cell culture vessels. Each cell culture vessel can come pre-connected with at least one reagent tank, such that the output of the reagent tank is the input of the cell culture vessels. The input of the reagent tank can be fitted with tubing connected to a naturally closed connector, which could be a sterile connector. The fluid output and input of each cell culture vessel can be fitted with tubing and naturally closed connector, which could be a sterile connector. A sampling output can also be fitted on each cell culture vessel, so that an operator can connect via a sterile connector and take a sample of cells and fluid within the cell culture vessel. A cell culture vessel can contain one or more sub-culture vessels. In some cases, a cell culture vessel contains two, three, or four sub-culture vessels. Cells and fluid flow can be evenly distributed across the one or more sub-culture vessels, allowing for the replication of each experiment.

[0041] The bioprocessing system described herein can comprise a gas input 115. A cell culture vessel 150 can fitted with a gas input, and optionally a gas output, each fitted with tubing and connector, which can be a sterile connector. A gas mixture can be flowed into each cell culture vessel through a valve 135 and via a gas line 160. A cell culture vessel can contain a vent fitted with a filter. In some cases, the filter can comprise 0.2pm pores. In some cases, a gas mixture can be flowed into each cell culture vessel via a gas permeable membrane in contact with the fluid in the cell culture vessel. In some cases, the system can accommodate up to 3 compressed gases. The compressed gases can include carbon dioxide (CO2), air or nitrogen. Gases can be mixed together with another, in a ratio of the user’s choice. For example, if CO2 and air are connected, the device can flow the following gas mixtures (all units in volume proportions): 1) 0% CO2, 100% air; 2) 5% CO2, 95% air; 3) 10% CO2, 90% air.

[0042] A cell culture vessel can be connected to its own pH sensor configured to measure pH of the vessel. A cell culture vessel can be connected to its own a dissolved oxygen sensor configured to measure a level of dissolved oxygen in the vessel. A cell culture vessel can be connected to its own temperature sensor configured to measure temperature of the vessel. A sensor can be made from a single-use part which can be attached on the inside of the cell culture vessel. In some cases, a sensor can be made from a reusable part which can move to any cell culture vessel to take a measurement. In some cases, cells and fluids in each cell culture vessel are agitated by a mechanical agitation device, in contact with each cell culture vessel.

[0043] A series of analytical and bioprocessing tools can be located adjacent to the stacks of cell culture vessels. In some cases, the analytical and bioprocessing tools are able to move to (optionally over, underneath or to the side of) any cell culture vessels. Analytical tools can include at least one imaging system 155, which can be a brightfield or holographic microscope, at least one pH sensor reader, at least one dissolved oxygen sensor reader, or at least one temperature sensor. The imaging system can record images of cells in the cell culture vessels and can be linked with an algorithm which detects cells in the cell culture vessels and provides an estimate of cell confluency and cell count. This information is stored on the device, or in the cloud, where it can be accessed by the user remotely. FIG. 5A shows the output of a cell imaging system. FIG. 5B shows the estimated cell count for a cell culture vessel over a period of time. The imaging system can comprise a green or red channel. In some cases, an algorithm is able to measure an estimate of cell viability. The imaging system can detect cells through the entire depth of the vessel or can more simply detect cells at a particular focal depth and perform an estimate of cell confluency and cell count via the algorithm. Bioprocessing tools can use at least one magnetic plate. In some cases, the magnetic plate is an electromagnetic plate.

[0044] The system can contain one or more waste tanks 175. The one or more waste tanks can be fluidically connected to the one or more vessels via selector valve 170 and a connector box 160. The one or more waste tanks can be connected and disconnected from the system via a sterile connector. Each cell culture vessels fluid output line can lead to the one or more waste tanks via a sterile barrier, e.g., an autosampler.

[0045] In some cases, the system is fitted with an inline metabolite analyzer 180. The metabolite analyzer 180 can be connected to each cell culture vessel fluid output via a selector valve 170. The selector valve 170 can be able to direct fluid from a cell culture vessel to the metabolite analyzer 180. The valve system can take the form of a manifold fitted with pinch valves, or one or several rotary valves, e.g., n-to-1 valves and 1-to-n valves combinations. The selector valve 170 can be fully automated.

[0046] In order to provide a sterile environment, the system can be able to self-sterilize. The sterilization concerns any internal tubing and surfaces with which fluid or cells can be in contact with. The system can use steam sterilization. A steam generator 145 can generate steam which can flow through the system at a temperature at or above 121 °C which has a corresponding pressure of 2 bars in absolute value. The system can first create a vacuum to optimize steam distribution and reduce the probability of a trapped air pocket within all tubes and surfaces. The system can generate hydrogen peroxide vapor instead of steam. The ports for the reagents, the cell culture vessels, and the waste can be naturally closed so that contaminants cannot penetrate the system when the ports are unused. These parts of the system would be initially supplied in a sterile state. The system can contain a reusable connector system, where each connector port can be sterilized by the system before a fluidic connection is established between the reagent and the system, or the cell culture vessels and the system or the waste tank and the system. The system can comprise one or more steam drains.

[0047] FIG. 3 shows a picture of a bioprocessing system in accordance with some embodiments. The bioprocessing system can comprise reagent or cell loading bays 1, a cell culture bay 3 comprising one or more microfluidic cassettes 2, and a user interface screen 5. The user interface screen 5 can display real-time process information collected by one or more sensors. A user can use the user interface to pre-program up to one month of running time. The user interface screen 5 can display imaging data, cell count, and/or metabolite concentration time series, which can also be downloaded in CSV, PDF, TIF, or TXT. The system can comprise an imaging system 4 configured to perform real time cell imaging. The system can comprise an inline metabolite analyzer 6. Cell culture vessels can be loaded into cell culture bay 3 using an aseptic click and connect process. In some cases, the cell culture bay 3 does not require a hood. The bioprocessing system may include an inline metabolite analyser 6, which can be used for automated supernatant sampling and analysis. FIG. 7 shows an example of an inline metabolite analyzer utilizing a robot arm for automated supernatant sampling and analysis.

Cell Therapy

[0048] The systems and methods of the present disclosure can be used for research and development for cell therapy applications. Cell therapy can be a treatment approach in which engineered or manipulated cells are administered into or to a subject (e.g., a patient).

[0049] The systems of the present disclosure are designed to improve the cell culture process. In one aspect, the present disclosure provides cell culture vessels comprising a bioprocessing chamber that is capable of performing bioprocessing operations involved in cell culture. The vessel can utilize microfluidics, which can involve manipulating fluids inside channel dimensions of the micrometer range. In some embodiments, the channels described herein (including, for instance, feeding input channels, feeding output channels, input harvest channels, output harvest channels, etc.) can have one or more channel dimensions. The one or more channel dimensions can correspond to one or more of a channel width, a channel length, a channel height, or a channel diameter. The channel dimensions can range from about 1 micrometer to about 10 centimeters. In some cases, the channel dimensions can be less than 1 micrometer. In some cases, the channel dimensions can be greater than 10 centimeters. In some cases, the channels described herein (including, for instance, feeding input channels, feeding output channels, input harvest channels, output harvest channels, etc.) can have a channel volume. The channel volume can range from 10% of the total vessel volume to 90% of the total vessel volume. In some cases, the channel volume can be less than 10% of the total vessel volume. In some cases, the channel volume can be greater than 90% of the total vessel volume.

[0050] Microfluidic cell culture can provide several advantages, including, for instance: (1) better control of process parameters: cells can have equal access to molecules present in the surrounding fluid due to homogenous cell distribution and fluid circulation in microenvironments, which can result in a more homogeneous end product and less process failure (e.g. cell death); (2) better overall cell health: e.g., paracrine effects can be amplified in a small environment (i.e. microscale), which can lead to better cell expansion and phenotype control; (3) a reduction in reactant volume: can be 10-20 fold reduction, due to smaller volumes of fluid used in microfluidic vessels as well as the ability to recirculate unspent reactant (e.g., growth media (fluid) can be re-enriched and recirculated at defined intervals, e.g., due to rapid oxygen or glucose depletion inside the vessel).

Cell Culture Vessels

[0051] In some cases, the bioprocessing system described herein can be used as for research and development. The bioprocessing system can have one or more cell culture vessels, which can each act as a miniature independent bioreactor. The cell culture vessels described herein can result in orders of magnitude improvement in process efficiency. The system may have 3 cell culture vessels to 30 cell culture vessels. The system may have 3 cell culture vessels, 6 cell culture vessels, 12 cell culture vessels, 15 cell culture vessels, 21 cell culture vessels, or 30 cell culture vessels. The system may have at least 3 cell culture vessels, 6 cell culture vessels, 12 cell culture vessels, 15 cell culture vessels, or 21 cell culture vessels. The system may have at most 6 cell culture vessels, 12 cell culture vessels, 15 cell culture vessels, 21 cell culture vessels, or 30 cell culture vessels.

[0052] A cell culture vessel may comprise one or more sub-culture vessels. A cell culture vessel may comprise two, three, or four sub-culture vessels. In some cases, all sub-culture vessels in a cell culture vessel may be replicates. Each cell culture vessel may comprise a bioprocessing chamber.

[0053] A cell culture vessel can be single-use. In some cases, the bioprocessing chamber can comprise a recess with one or more walls that are angled relative to the feeding input channels and/or the feeding output channels. In some non-limiting embodiments, the angle can range from about 45 degrees to about 90 degrees. In some cases, the bioprocessing chamber can have one or more dimensions. The one or more dimensions can comprise, for example, a length, a width, a height, or a depth. The one or more dimensions of the bioprocessing chamber can range from about 1 millimeter to about 60 centimeters. In some cases, the dimensions of the bioprocessing chamber can be less than 1 millimeter. In some cases, the dimensions of the bioprocessing chamber can be greater than 60 centimeters. The bioprocessing chamber can comprise a volume of 10 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, or 0.5 mL. The bioprocessing chamber can comprise a volume of less than 10 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, or 0.5 mL.

[0054] A bioprocessing chamber can hold 1000, 5000, 1 million, 2 million, 5 million, 10 million, 20 million, 40 million, 100 million, 200 million, 500 million, or 800 million cells. A bioprocessing chamber can hold at least 1000, 5000, 1 million, 2 million, 5 million, 10 million, 20 million, 40 million, 100 million, 200 million, 500 million, or 800 million cells. A bioprocessing chamber can hold at most 1000, 5000, 1 million, 2 million, 5 million, 10 million, 20 million, 40 million, 100 million, 200 million, 500 million, or 800 million cells.

[0055] In some cases, the bioprocessing chamber can have a bottom surface, as described elsewhere herein. The bottom surface can be used for cell culturing. Suspension, micro-carrier- or monolayer adherent cell culture processes can be used. The bottom surface can have a surface area ranging from about 1 mm² to about 300 cm². In some cases, the surface area can be less than 1 mm². In some cases, the surface area can be greater than 300 cm². The bottom surface can have a surface area of less than 300 cm², 200 cm², 100 cm², 90 cm², 80 cm², 70 cm², 60 cm², 50 cm², 40 cm², 30 cm², 20 cm², 10 cm², 6 cm², 5 cm², or 1 cm². A sub-culture vessel can hold up to 4 mL of fluid per 12 cm² of surface area.

[0056] In some cases, a length dimension of the bioprocessing chamber can be at least 2x, 3x, 4x, 5x, lOx, 15x, or 20x a width dimension of the bioprocessing chamber. In some cases, the bioprocessing chamber has a height of at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, or 0.5 mm. [0057] The bioprocessing chamber can comprise a cross-sectional shape. The cross-sectional shape can be a circle, an oval, an ellipse, a triangle, a square, a rectangle, or any other polygon having three or more sides. The cross-sectional shape can correspond to a horizontal crosssection and/or a vertical cross-section of the bioprocessing chamber.

[0058] FIG. 6 schematically illustrates a top view of a cell culture vessel 600 comprising four sub-culture vessels 605. A cell culture vessel can comprise a feeding input 610 in a first portion of the cell culture vessel and a feeding output 615 on a second portion of the cell culture vessel. The feeding input 610 can be fluidically connected to one or more sub-culture vessels 605 through one or more feeding input channels 620. The feeding input 610 can be in fluid communication with one or more reagent or mixing tanks. The feeding output 615 can be fluidically connected to one or more sub-culture vessels 605 through one or more feeding output channels 625. The feeding output 615 can be in fluid communication with one or more waste tanks.

[0059] The bioprocessing chamber can connect to a filter (e.g., a filter membrane). The filter can serve as a barrier to prevent cells from exiting the chamber prematurely, hence increasing seeding efficiency. In some cases, the filter can comprise a filter membrane, and the filter membrane can comprise polyethersulfone (PES), e.g., with a pore size structure of about 5 micrometers. In some cases, the filter comprises a pore size of less than 10 pm, less than 7.5 pm, less than 5 pm, or less than 2.5 pm. The shape of the filter can be rectangular or circular. In some cases, the filter can comprise a membrane. The membrane can be permeable or semi- permeable. The membrane can comprise, for example, Polytetrafluoroethylene (PTFE) or expanded polytetrafluoroethylene (ePTFE), polyethersulfone (PES), modified polyethersulfone (mPES), polysulfone (PS), modified polysulfone (mPS), ceramics, polypropylene (PP), cellulose, regenerated cellulose or a cellulose derivative (e.g. cellulose acetate or combinations thereof), polyolefin, polypropylene, polytetrafluoroethylene, polyvinyl chloride, polyester, or any other type of polymer. In some non-limiting embodiments, the membrane can comprise a biomedical polymer, e.g., polyurethane, polyethylene, polypropylene, polyester, poly tetra fluoro-ethylene, polyamides, polycarbonate, or polyethylene-terephthalate.

[0060] The cells described herein can comprise a range of sizes. In some cases, the cells can have a size of at least about 1 micrometer, 5 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, or any size that is between any of the preceding values. In some cases, the cells can have a size that is less than about 1 micrometer. In some cases, the cells can have a size that is at most about 1 micrometer, 900 nanometers, 800 nanometers, 700 nanometers, 600 nanometers, 500 nanometers, 400 nanometers, 300 nanometers, 200 nanometers, 100 nanometers, 90 nanometers, 80 nanometers, 70 nanometers, 60 nanometers, 50 nanometers, 40 nanometers, 30 nanometers, 20 nanometers, 10 nanometers, or less.

[0061] When cells are provided to the vessels described herein, the cells can settle on or come in contact with cyclic olefin copolymer (COC), which can possess glasslike clarity that can exceed thermoplastic substitutes such as polycarbonate. COC can be sterilized using standard methods (e.g., steam, ethylene oxide, gamma irradiation, and hydrogen peroxide) without altering its properties. It can also permit UV transmission, which can be best suited for diagnostic analysis. COC can also have low leachables and extractables, making it best suited for direct drug contact. It can be classified USP Class VI and is ISO 10993 compliant including biocompatibility, USP 661.1 and FDA drug and device master files.

[0062] In some cases, the vessels can comprise a polydimethylsiloxane (PDMS) component, which can form the wall of the bioprocessing chambers, as well as the top layer, which can form part of its ceiling. PDMS can have gas permeability, which can be advantageous for cell growth. PDMS can permit gas equilibration between the bioprocessing chamber and that of the surrounding controlled environment (e.g. incubator), and can withstand autoclave conditions. In some cases, the PDMS component can be replaced with another gas permeable polymer. Cells can settle on the COC portion of the bioprocessing chamber.

[0063] In some cases, the vessels can comprise a plurality of components or layer comprising a plurality of materials. The plurality of materials can comprise different materials. In some cases, the plurality of materials can comprise a cyclic olefin polymer (COP), a cyclic olefin copolymer (COC), or a polydimethylsiloxane (PDMS) material. In some cases, the plurality of materials can comprise a USP Class VI material. In some cases, the plurality of materials can comprise any type of material that is biocompatible and/or biostable. In some cases, the materials for the various components or layers of the vessel can have a high permeability (e.g., liquid or gas permeability) to permit a flow of fluid and/or cells into, out of, or through the vessel (and any components or layers thereof).

[0064] The presently disclosed vessels can also contain a filter, e.g., filter membrane made of polyethersulfone (PES). Contrary to other types of membranes (e.g. PTFE), PES can retain its rigid structure over longer periods of time. The filter can have one or more pores. The pore size can be about 5pm or less, which can be used to retain cells of 10-pm diameter in the bioprocessing chamber.

Materials

[0065] The microfluidic device can be fabricated in optically transparent material or a combination of different types of materials. The bioprocessing chamber that can be used for cell culture can be made of a USP (United States Pharmacopoeia) Class VI Material. Such materials can be transparent so that imaging technology can be coupled. The device can also possess tolerances on the design requirements (e.g. channels) not lower than 5 micrometers in absolute value for the smallest feature and 5% for larger dimensions. This can ensure that fabrication of these devices can be suitable with standard manufacturing processes (e.g. sheet or roll processing). The device can also comprise usable surface culture space (for the individual vessel) that is potentially capable of handling up to at least about 10 million cells, 20 million cells, 30 million cells, 40 million cells, 50 million cells, 60 million cells, 70 million cells, 80 million cells, 90 million cells, 100 million cells, or more.

[0066] The cells can comprise, for example, human cells (e.g., stem cells, bone cells, blood cells, muscle cells, fat cells, skin cells, nerve cells, immune cells (e.g., T-cells) etc.) or nonhuman cells (including, for instance, animal cells, plant cells, bacterial cells, fungal cells, etc.). Advantages

[0067] The systems of the present disclosure can provide a multi-functional design with numerous advantages over other systems. The systems referred to herein can comprise any of the mixing tanks, vessels, pumps, valves, waste tanks, and other devices, hardware, or apparatuses described herein.

[0068] A device or system provided herein can have the ability to run a large number of experiments with different process parameters at the same time. For example, experiments can be performed in 30 cell culture vessels, all of which have different process parameters. Each cell culture vessel can have 4 sub-culture vessels. In this example, the system can run 3 ©different experiments, and each experiment will be replicated four times.

[0069] A device or system provided herein can be fully automated. End-to-end automation can include automated reagent preparation to final formulation. End-to-end automation can include electronic batch records. This can ensure that experiments can be run by moderately skilled personnel and that one person has the capacity to produce several dozens of batches at one time by elimination of numerous manual operations. Manual operations translate into heightened research costs and fundamentally pose the question of scalability due to lack of highly skilled personnel. The systems described herein can automatically prepare reagent mixtures, without the manual intervention by a skilled operator. The systems and methods described herein can use automation for the measurement of all key process parameters and cell health parameters, i.e., temperature, pH, dissolved oxygen, cell morphology and cell count, or a selection of metabolites (e.g., glucose, lactate, glutamine, glutamate). The bioprocessing system described herein may utilize automated seeding, washing, expansion, media exchange, activation, transduction, transfection, differentiation, or formulation, or any combination thereof., The bioprocessing system described herein can utilize a centralized remote control that monitors real-time cell imaging, cell count, pH, and dissolved oxygen of analytes. In some cases, the transition from one process step to another is automated, such that the entire process sequence can also be automated. Therefore, the system can be fully automated to select a reagent, bring the selection of reagent to a predetermined cell culture vessel, and remove the reagent from a cell culture vessel.

[0070] A device or system described herein can have a small footprint as compared to other bioprocessing systems. In some cases, the bioprocessing system described herein can be between 1.5 meters to 2 meters wide. The bioprocessing system can be 1.5 meters wide, 1.6 meters wide, 1.7 meters wide, 1.8 meters wide, 1.9 meters wide, or 2 meters wide. The bioprocessing system can be at most 1.5 meters wide, 1.6 meters wide, 1.7 meters wide, 1.8 meters wide, 1.9 meters wide, or 2 meters wide. In some cases, the bioprocessing system described herein can be between 50 centimeters to 80 centimeters high. The bioprocessing system can be 50 centimeters, 60 centimeters, 70 centimeters, or 80 centimeters high. The bioprocessing system can be at most 50 centimeters, 60 centimeters, 70 centimeters, or 80 centimeters high. In some cases, the bioprocessing system described herein can be between 70 centimeters to 90 centimeters deep. The bioprocessing system can be 70 centimeters, 80 centimeters, or 90 centimeters deep. The bioprocessing system can be at most 70 centimeters, 80 centimeters, or 90 centimeters deep.

[0071] A bioprocessing system provided herein can be closed at all times, i.e., operations can be carried out in a closed environment (no opening of the system at any time). Reagents and cells can never come into contact with the environment. The systems and devices described herein can remain sterile when cell culture vessels are connected or disconnected. The systems and devices described herein can remain sterile while experiments are running.

[0072] A bioprocessing system described herein can comprise integrated in-line analytics. Analytics can be shared across several experiments to reduce the cost per experiment and maximize analytical equipment utilization, such that processes can be monitored and preapproved adjustments to the process can be made in real time and automatically. Analytical inline capabilities can include cell counting, metabolites measurements, etc. In-line analytics can allow deviations in the process to be detected that would not be detected otherwise or that would be detected at a later time. For example, many processes require only one sample a day to be taken and analyzed (with results sometimes taking several hours), which is too infrequent to allow correcting deviation efficiently. In-line analytics can alert operators to a deviation before the sample has been taken and analyzed. This can reduce the need for sampling cells to the strict minimum, i.e., where required analytical processes cannot be integrated or where it would be too onerous to do so.

[0073] A bioprocessing system described herein can limit consumables to the cell culture vessels. All other components of the system can be reusable. This can minimize costs and waste associated with a bioprocessing system.

Cell Processing

[0074] In some cases, systems provided herein can be primed with fluid, e.g., in order to facilitate injection of the growth media. In the presently disclosed vessels, the height of the bioprocessing area can be, e.g., around 3-7mm, which can permit natural "separation" of the cells and occurring bubbles. While the cells settle at the bottom surface, bubbles can be naturally buoyant and thus float towards the top part of the bioprocessing chamber, away from the cells. The environment can be controlled to minimize evaporation and mitigate impacts on the cell growth.

[0075] The vessels provided herein can permit high efficiency cell seeding, minimizing loss. The cells can be spread homogeneously throughout the bioprocessing chamber to enable optimal growth. In some cases, confluent growth can be prematurely reached in some areas and therefore decrease cell culture efficiency.

[0076] The presence of a filter (e.g., filter membrane) can help in blocking cells from prematurely exiting the bioprocessing chamber, ensuring that they stay detained inside the bioprocessing chamber. Mass transport or advection can be a phenomenon due in large part that cells can be relatively large (> 10pm). Their large size can help them sediment into the recess. To help in homogenous distribution, the vessels can be attached to a mechanical agitation device, which can facilitate re-distribution of the seeded cells all throughout the bioprocessing chambers. In some cases, the mechanical agitation device can be used with a single vessel, a plurality of vessels, a portion of the plurality of vessels, or compartments of such vessels. The mechanical agitation device may be a magnetic agitator. Ultrasonic agitation may be used to help in homogenous distribution within cell culture vessels.

[0077] In some cases, the fluid can comprise at least 20,000, 200,000, 350,000, 500,000, 1,000,000, 3,500,000, 10,000,00, 25,000,000, or 50,000,000 cells/mL. In any of the embodiments described herein, the cells can comprise microorganisms or mammalian cells, which can include HEK293 cells, T-cells, Jurkat cells, CHO cells, mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. The bioprocessing chambers can comprise at least 50,000 cells/mL, 100,000 cells/mL, 500,000 cells/mL, 1 million cells/mL, 5 million cells/mL, 10 million cells/mL, 20 million cells/mL, 40 million cells/mL, 60 million cells/mL, or 100 million cells/mL.

[0078] Cells can deplete surrounding media from nutrients in static conditions. The rate of media flow in the vessels can be carefully regulated. In the microscale, numerous parameters can be involved in ensuring cell growth, including temperature gradients, oxygen levels, chemical gradients, cell-to-cell interactions, cell-to-molecule interactions, CO2 level, shear stress, and cell-substrate interactions.

[0079] Nutrient and gas diffusion as well as cell consumption can also be optimized. When cells are not homogeneously distributed during seeding, a consumption rate that follows a Poisson distribution can be expected where there is a higher consumption rate near the position of the feeding input channels (since it can be in first contact with the nutrients). Mechanical agitation can be employed during seeding can be beneficial for perfusion to ensure that nutrients are distributed all throughout the bioprocessing chamber.

[0080] In some embodiments, the methods described herein can further comprise expanding the distributed cells to generate expanded cells. In some cases, the expanding comprises expanding the distributed cells about, or at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 6.5-fold, 7-fold, 8- fold, 9-fold, 10-fold, 11-fold, 12-fold, 25-fold, 50-fold, 100-fold, 150-fold, or 200-fold. In some cases, the expanding occurs over about, or at least 24 hr, 48 h, 72 hr, 96 hr, 120 hr, 144 hr, 168 hr, 192 hr, 216 hr, 240 hr, 264 hr, 288 hr, 312 hr, 336 hr, 360 hr, 720 hr, 1080 hrs, or 1200 hrs.

[0081] Appropriate surface treatment can also be performed inside the bioprocessing chamber of the vessel depending on the experimental conditions. Such surface treatments can allow adherent particles or cells to stick unto the surface such that particle-wall adhesion takes place. Additional coating methods can be used to facilitate cell attachment or detachment on the COC substrate. In some cases, the coating can comprise one or more polymeric surfactants. In some cases, the coating can comprise any type of biocompatible or biostable material that facilitates cell adhesion or growth.

[0082] In some cases, harvesting can comprise harvesting at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells to provide harvested cells. In some cases, the harvesting occurs in 5 min or less, 1 min or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, or 5 seconds or less. In some cases, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, or 94% of the harvested cells can be viable. In some cases, the harvesting of cells from the presently disclosed vessels can result in about, or least, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% cell recovery with about or at least 95%, 94%, 93%, 92%, 91%, or 90% viability.

[0083] In some embodiments, the method can further comprise washing the distributed cells. In some embodiments, the method can further comprise expanding the distributed cells to generate expanded cells. In some cases, the expanding comprises expanding the distributed cells about, or at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 6.5-fold, 7-fold, 8-fold, 9-fold, 10- fold, 11-fold, 12-fold, 25-fold, 50-fold, 100-fold, 150-fold, or 200-fold. In some cases, the expanding occurs over about, or at least 24 hr, 48 h, 72 hr, 96 hr, 120 hr, 144 hr, 168 hr, 192 hr, 216 hr, 240 hr, 264 hr, 288 hr, 312 hr, 336 hr, 360 hr, 720 hr, 1080 hrs, or 1200 hrs.

[0084] In some embodiments, the method can further comprise imaging the distributed cells. In some embodiments, the method can further comprise imaging the expanded cells. In some cases, the system comprises only one imaging system. The imaging system can operate as a brightfield microscope or a holographic microscope. In some cases, the one imaging system is configured to move among the vessels, and the imaging system is configured to capture images of individual vessels. In some cases, the one or more vessels are configured to move to the imaging system, and the imaging system is configured to capture images of individual vessels. [0085] In some embodiments, the system comprises at least one magnetic plate. The magnetic plate can be an electromagnetic plate. In some cases, the one magnetic plate is configured to move among the vessels. In some cases, the one or more vessels are configured to move to the magnetic plate.

[0086] In some embodiments, the method can further comprise using a computer system to predict time to confluence of the expanded cells.

[0087] In some embodiments, the method can further comprise contacting the distributed cells with a reagent after the washing. In some embodiments, the method can further comprise, after the contacting, performing a second wash of the distributed cells. In some embodiments, the method can further comprise, after the second wash of the distributed cells, harvesting the distributed cells.

[0088] The reagents can comprise, for example, balanced salt solutions, buffers, detergents, chelators, or any materials or substances that promote or facilitate cell adhesion.

[0089] In some cases, the cells can be seeded at a flow rate ranging from 0.1 microliters per second (pL/s) to 10 pL/s or more. In some cases, the cells can be seeded at a flow rate of at least about 0.17 pL/s.

[0090] In some cases, the cells can be harvested at a flow rate ranging from about 0.1 microliters per second (pL/s) to about 10 pL/s or more. In some cases, the cells can be harvest at a flow rate ranging from about 0.17 pL/s to about 1.59 pL/s.

Computer Systems

[0091] In an aspect, the present disclosure provides computer systems that are programmed or otherwise configured to implement methods of the disclosure, e.g., any of the subject methods for bioprocessing. FIG. 8 shows a computer system 2001 that is programmed or otherwise configured to implement a method for bioprocessing. The computer system 2001 can be configured to, for example, control a flow of fluid comprising one or more cells into a bioprocessing system. In some cases, the computer system 2001 can be configured to adjust a flow rate or an amount of fluid flow into or through the one or more vessels, based on one or more sensor readings. The computer system can have several functionalities: 1) guiding the user through the set up a Design of Experiment in the system, i.e., by loading the process sequence for each experiment and through system loading (reagents, cell culture vessels);; 2) automated system checks, e.g., testing of valve and pump functionalities, steam flow rate, pressure, temperature, confirmation of fluidic connection with reagents and cell culture vessels, calibration of sensors; 3) providing information to the user about each batch while the batch is run, such as past, current and future sequence steps and timings, sensor measurement values, alerts based on preset parameter values measured by the system, alerts about operational issues (such as reagent shortage or valve failure); 4) visualization and analysis of all sources of data generated by the system, e.g., automated normalization and overlay of time series from several measurement sources, cell image and morphology analysis.

[0092] A computer system can learn and suggest how to improve a research process using analytics and info gained from operations. A computer system may process and gather data on cells in the system during experiments in order to optimize the process (e.g., choosing the optimal period of time that a given reagent should be in contact with the cells, in order to reduce process time) or to understand the impact of the process on the cells (e.g., impact on the growth rate and viability of the cells). A user may wish to vary experiment parameters, and based on the parameters the user wants to vary, a computer system can determine what experiments need to be completed and direct the system to perform the required systems.

[0093] The computer system 2001 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device. The computer system 2001 can include a central processing unit (CPU, also "processor" and "computer processor" herein) 2005, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 2001 also includes memory or memory location 2010 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 2015 (e.g., hard disk), a user interface, a communication interface 2020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 2025, such as cache, other memory, data storage and/or electronic display adapters. The memory 2010, storage unit 2015, interface 2020 and peripheral devices 2025 are in communication with the CPU 2005 through a communication bus (solid lines), such as a motherboard. The storage unit 2015 can be a data storage unit (or data repository) for storing data. The computer system 2001 can be operatively coupled to a computer network ("network") 2030 with the aid of the communication interface 2020. The network 2030 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 2030 in some cases is a telecommunication and/or data network. The network 2030 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 2030, in some cases with the aid of the computer system 2001, can implement a peer-to-peer network, which can enable devices coupled to the computer system 2001 to behave as a client or a server.

[0094] The CPU 2005 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a memory location, such as the memory 2010. The instructions can be directed to the CPU 2005, which can subsequently program or otherwise configure the CPU 2005 to implement methods of the present disclosure. Examples of operations performed by the CPU 2005 can include fetch, decode, execute, and writeback.

[0095] The CPU 2005 can be part of a circuit, such as an integrated circuit. One or more other components of the system 2001 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0096] The storage unit 2015 can store files, such as drivers, libraries and saved programs. The storage unit 2015 can store user data, e.g., user preferences and user programs. The computer system 2001 in some cases can include one or more additional data storage units that are located external to the computer system 2001 (e.g., on a remote server that is in communication with the computer system 2001 through an intranet or the Internet).

[0097] The computer system 2001 can communicate with one or more remote computer systems through the network 2030. For instance, the computer system 2001 can communicate with a remote computer system of a user (e.g., an operator managing or monitoring the bioprocessing). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 2001 via the network 2030.

[0098] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 2001, such as, for example, on the memory 2010 or electronic storage unit 2015. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 2005. In some cases, the code can be retrieved from the storage unit 2015 and stored on the memory 2010 for ready access by the processor 2005. In some situations, the electronic storage unit 2015 can be precluded, and machine-executable instructions are stored on memory 2010.

[0099] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[00100] Aspects of the systems and methods provided herein, such as the computer system 2001, can be embodied in programming. Various aspects of the technology can be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which can provide non-transitory storage at any time for the software programming. All or portions of the software can at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, can enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that can bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also can be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[00101] Hence, a machine readable medium, such as computer-executable code, can take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media including, for example, optical or magnetic disks, or any storage devices in any computer(s) or the like, can be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media can take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media can be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[00102] The computer system 2001 can include or be in communication with an electronic display 2035 that comprises a user interface (LT) 2040 for providing, for example, a portal for an operator to monitor or track one or more steps or operations of the bioprocessing methods and systems described herein. The portal can be provided through an application programming interface (API). A user or entity can also interact with various elements in the portal via the UI. Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.

[00103] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 2005. For example, the algorithm can be configured to adjust a flow rate or an amount of fluid flow into the bioprocessing system, based on one or more sensor readings.

[00104] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the disclosure be limited by the specific examples provided within the specification. While the disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein can be employed in practicing the disclosure. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A bioprocessing system, comprising:

(a) one or more mixing tanks, wherein the one or more reactant tanks comprise reagents;

(b) one or more vessels fluidically connected to the one or more reactant tanks; and

(c) one or more waste tanks fluidically connected to the one or more vessels; wherein at least one of the one or more vessels comprises one or more subvessels, and wherein the dimensions of the one or more sub-vessels are identical.

2. The system of claim 1, wherein a first reactant tank of the one or more reactant tanks is kept at a different temperature than a second reactant tank of the one or more reactant tanks.

3. The system of claim 1 or claim 2, wherein a reactant tank of the one or more reactant tanks is kept at ambient temperature.

4. The system of any preceding claim, wherein a reactant tank of the one or more reactant tanks is kept at a temperature of about 20°C.

5. The system of any one of claims 1 to 3, wherein a reactant tank of the one or more reactant tanks is kept at a temperature of about 4^CC.

6. The system of any one of claims 1 to 4 wherein a reactant tank of the one or more reactant tanks is kept at a temperature of about -20 °C.

7. The system of any preceding claim, wherein the one or more vessels are fluidically connected to the one or more reactant tanks or the one or more waste tanks through a connector system comprising one or more pumps, one or more valves, and tubing.

8. The system of claim 7, further comprising a steam sterilizer.

9. The system of claim 8, wherein the steam sterilizer generates steam that flows through the one or more pumps, the one or more valves, or the tubing at a temperature at or above 121°C at a pressure of 2 bars in absolute value.

10. The system of any preceding claim, further comprising one imaging system.

11. The system of claim 10, wherein the one imaging system is configured to move among the one or more vessels, and wherein the imaging system is configured to capture images of individual vessels of the one or more vessels.

12. The system of claim 10, wherein the one or more vessels are configured to move to the imaging system, and wherein the imaging system is configured to capture images of individual vessels of the one or more vessels.

13. The system of any preceding claim, wherein a vessel of the one or more vessels comprises a pH sensor configured to measure pH of the vessel. The system of any preceding claim, wherein a vessel of the one or more vessels comprises a dissolved oxygen sensor configured to measure a level of dissolved oxygen in the vessel. The system of any preceding claim, wherein a vessel of the one or more vessels comprises a temperature sensor configured to measure a temperature of the vessel. The system of any preceding claim, wherein the one or more sub-vessels each comprise one or more bioprocessing chambers. The system of claim 16, wherein the one or more bioprocessing chambers comprise a volume of less than 10 mL, 7 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL, 0.5 mL, or 0.1 mL. The system of claim 16 or claim 17, wherein the one or more bioprocessing chambers comprise a cell culturing surface of less than 300 cm², 200 cm², 100 cm², 90 cm², 80 cm², 70 cm², 60 cm², 50 cm², 40 cm², 30 cm², 20 cm², 10 cm², 6 cm², 5 cm², or 1 cm². The system of any one of claims 16 to 18, wherein the one or more bioprocessing chambers comprise a biocompatible material. The system of claim 19, wherein the biocompatible material is a U.S. Pharmacopeia Convention (USP) Class VI material. The system of any one of claims 16 to 20, wherein the one or more bioprocessing chambers are sterile. The system of any one of claims 16 to 21, wherein the one or more bioprocessing chambers comprise a plurality of cells. The system of any one of claims 16 to 22, wherein each of the one or more bioprocessing chambers comprise at least 50,000 cells/mL, 100,000 cells/mL, 500,000 cells/mL, 1 million cells/mL, 5 million cells/mL, 10 million cells/mL, 20 million cells/mL, 40 million cells/mL, 60 million cells/mL, or 100 million cells/mL. The system of any one of claims 16 to 23, wherein each of the one or more bioprocessing chambers comprises at least 0.05 million, 0.25 million, 0.5 million, 1 million, 10 million, 50 million, 100 million, 150 million, or 500 million cells. The system of any preceding claim, wherein a vessel of the one or more vessels is configured to have at least one process parameter that differs from another vessel of the one or more vessels. The system of claim 25, wherein the at least one process parameter is temperature. The system of claim 25 or claim 26, wherein the at least one process parameter is pH. The system of any one of claims 25 to 27, wherein the at least one process parameter is dissolved oxygen concentration. The system of any one of claims 25 to 28, wherein the at least one process parameter is reagent concentration. The system of any one of claims 25 to 29, wherein the at least one process parameter is reagent composition. The system of any preceding claim, wherein the at least one of the one or more vessels comprising the one or more sub-vessels comprises cells. The system of claim 31, wherein each of the one or more sub-vessels comprise the same concentration of cells. The system of any preceding claim, further comprising an inline metabolite analyzer fluidically connected to the one or more vessels.