Bioreactor inoculum normalization

Abstract

A method in which a cell health module automates bioreactor start-up and normalizes cell culture inoculum by counting the number of viable cells in a cell sample. A volume of media and a volume of cell sample to receive and combine a desired starting density of cells are determined based on the number of cells in the cell sample, respectively, to result in a predetermined density of cells in the bioreactor. The media and cell sample are combined by feeding a volume of media into the bioreactor and a volume of cell sample into the bioreactor to result in a cell culture.

Description

[Background technology]

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 441,094, filed January 25, 2023, which was filed as a PCT international application on January 23, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (background) Cell line development is the process of culturing cell growth. It allows for the evaluation of differences between individual cells and can be used to confirm the monoclonality of a particular cell line. Cell line development is useful for generating various biological molecules, such as for pharmaceutical development. Cell lines are therefore evaluated based on various criteria, such as protein titer or cell line stability. Cell lines are typically first established by isolating a single viable cell or otherwise establishing the same starting conditions, allowing for equal comparison between cell lines.

[0003] One application of cell line development is the production of therapeutic monoclonal antibodies (mAbs). Producing mAbs requires starting with a single clonal production cell line capable of stably expressing the target mAb. A production cell line for manufacturing needs to be robust, stable, and scalable in large-scale culture in a bioreactor. Some important criteria for a production cell line include monoclonality, high productivity, appropriate post-translational modifications, and excellent quality. Such a production cell line can be generated from a cell line development process that begins with gene transfer, selection, and single-clone isolation for a large number of monoclonal candidate clones. To reduce timelines and costs, a high-throughput workflow is often used to select a small number of good candidate clones. These selected clones are then sent for downstream processing optimization to select the final production cell line. Summary of the Invention [Means for solving the problem]

[0004] (summary) Examples presented herein are directed to a method for automated start-up of a bioreactor, the method including the steps of a cell health module counting the number of viable cells in a cell sample, receiving a desired starting density of cells, determining a volume of media and a volume of cell sample to be combined based on the number of viable cells in the cell sample to provide a predetermined density of cells in the bioreactor, combining the media and cell sample by feeding the volume of media into the bioreactor to provide a cell culture, and feeding the volume of cell sample into the bioreactor.

[0005] In other examples provided herein, the cells are mammalian cells. In yet other examples provided herein, the predetermined number of cells is a predetermined number of viable cells. In yet other examples provided herein, the cell sample is in a source plate. In other examples provided herein, the number of cells is a first number of cells, and the method further includes counting a second number of cells in the bioreactor.

[0006] In other examples provided herein, counting the second number of cells is performed immediately after delivering the volume of the cell sample to the bioreactor and determining the final density of the cells. In further examples provided herein, the method includes determining that the second number of cells in the bioreactor is below a predetermined threshold, calculating a second volume of the cell sample to be injected into the bioreactor to result in the predetermined density of cells, and injecting the second volume of the cell sample into the bioreactor. In yet further examples provided herein, the method includes determining that the second number of cells in the bioreactor is above a predetermined threshold, calculating a second volume of medium to be injected into the bioreactor to result in the predetermined density of cells, and injecting the second volume of medium into the bioreactor.

[0007] In other examples provided herein, counting the second number of cells is performed after a predetermined period of time following dispensing the volume of the cell sample into the bioreactor and determining proliferation by the cells in the bioreactor. In further examples provided herein, the method includes comparing the first number of cells and the second number of cells over time. In yet further examples provided herein, the predetermined period of time is one day or more.

[0008] In other examples provided herein, the cell health module counting the number of viable cells in the cell sample includes counting the number of viable cells in a plurality of cell samples. In yet other examples provided herein, combining the medium and the cell sample by feeding each into the bioreactor includes feeding each of a volume of medium and a volume of cell sample into each well of a plurality of wells in the bioreactor. In yet other examples provided herein, the predetermined density of cells is the same for each well of the plurality of wells in the bioreactor.

[0009] In further examples provided herein, the volume of the cell sample for each well of the plurality of wells is determined from the same cell sample, such that each well of the plurality of wells contains the same type of cell. In yet further examples provided herein, the volume of the cell sample for at least one well of the plurality of wells is determined from a different cell sample than the volume of the cell sample for the remainder of the plurality of wells, such that at least one well of the plurality of wells contains a different type of cell than the remainder of the plurality of wells. In yet further examples provided herein, dispensing the volume of medium into each well of the plurality of wells is performed using a first tip, and dispensing the volume of the cell sample into a first well of the plurality of wells is performed using a first tip.

[0010] In other further examples provided herein, the plurality of wells in the bioreactor comprises a plurality of wells in a series of bioreactors. In yet other further examples provided herein, the method includes culturing a cell sample in each well of the plurality of wells, evaluating cell growth in each well of the plurality of wells in the bioreactor, and identifying one or more wells as containing a cell line with greater productivity compared to other wells of the plurality of wells. In yet further examples provided herein, injecting the volume of medium into each well of the plurality of wells in the bioreactor is performed using a first pipette tip, and injecting the volume of the cell sample into a first well of the plurality of wells in the bioreactor is performed using the first pipette tip. In other examples provided herein, the cell sample is maintained.

[0011] Another example provided herein is directed to a system for automating the inoculation of a bioreactor with a predetermined number of cells, the system including: a source plate configured to hold a cell sample; a cell health evaluator configured to count the number of cells in the cell sample; a bioreactor including a number of wells; and a processor in communication with a memory, the memory storing instructions that, when executed by the processor, cause the system to receive the number of cells in the cell sample, determine a volume of medium and a volume of the cell sample to be combined based on the number of cells in the cell sample, provide cells at a predetermined density, operate a fluid transfer device, deposit the volume of medium into each well of the number of wells in the bioreactor, and deposit the volume of the cell sample into each well of the number of wells in the bioreactor.

[0012] In other examples provided herein, the fluid transfer device is a pipette. In yet other examples provided herein, a first tip for the pipette is used to inject a volume of medium into each well of the plurality of wells and to inject a volume of a cell sample into a first well of the plurality of wells. In yet other examples provided herein, the system includes a receiving zone for the pipette tip.

[0013] In other examples provided herein, the receiving zone is configured to receive several tips, the number of tips equaling the number of wells. In yet other examples provided herein, the source plate is further configured to maintain the cell sample at a predetermined temperature. In further examples provided herein, the predetermined temperature is within the range of 2-8 degrees Celsius.

[0014] Another example presented herein is directed to a method for optimizing pipette tip usage, the method including pipetting a medium into several sample wells using a first pipette tip and pipetting several samples into several sample wells, the number of samples equaling the number of wells, and the first sample being pipetted using the first pipette tip.

[0015] Yet another embodiment presented herein relates to a system for optimizing pipette tip usage, the system including a media source, a sample source, a number of sample wells, a pipette with a number of mandrels equal to the number of sample wells, and a number of pipette tips, one pipette tip associated with each mandrel of the number of mandrels, the pipette filling each sample well of the number of sample wells from the media source using a first pipette tip of the number of pipette tips and adding a sample from the sample source to each of the number of sample wells, the first sample in the first of the pipette wells being added using one pipette tip.

[0016] Various additional aspects of the invention will be set forth in the description that follows. Aspects of the invention can relate to individual features and combinations of features. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the broad inventive concept on which the embodiments disclosed herein are based. [Brief explanation of the drawings]

[0017] The accompanying drawings, which are incorporated in and constitute a part of the description, illustrate several aspects of the present disclosure. A brief description of the drawings follows:

[0018] [Figure 1] FIG. 1 is an exemplary cell line development (CLD) system.

[0019] [Figure 2] FIG. 2 is a component diagram of the exemplary CLD workstation of FIG.

[0020] [Figure 3] FIG. 3 is a deck component layout of the exemplary CLD workstation of FIG.

[0021] [Figure 4] FIG. 4 is a component diagram of the bioreactor of the exemplary CLD workstation of FIG.

[0022] [Figure 5] FIG. 5 is a component diagram of the gantry system of the exemplary CLD workstation of FIG.

[0023] [Figure 6] FIG. 6 is a component diagram of the liquid handler of the gantry system of FIG.

[0024] [Figure 7] FIG. 7 is a process diagram of an exemplary CLD process.

[0025] [Figure 8] FIG. 8 is a flow chart of an exemplary method for implementing automated bioreactor start-up for a CLD process.

[0026] [Figure 9] FIG. 9 is a flow chart of a method for inoculating a bioreactor vessel or plate. DETAILED DESCRIPTION OF THE INVENTION

[0027] (Detailed explanation) Reference will now be made in detail to the exemplary aspects of the present disclosure, which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

[0028] Cell line development (CLD) is the process of serially propagating a cell or cells with distinct and reproducible characteristics that define a cell line. Effective CLD requires high precision in establishing the initial cell or cells from which the cell line originates, so that the characteristics of the entire cell line can be effectively traced back to the cell of origin. This process can be referred to as "seeding" the cell line. As discussed herein, seeding refers to preparing one or more vessels with a precise number of initial viable cells for cell line development, allowing for accurate determination of the strength of the cell line's serial propagation over time during the development process.

[0029] In some cases, the initial cell population may be divided, and individual cells or subpopulations from the initial population may be passaged separately to assess variation within the population of the initial population. In such cases, it may be important to start with a precise amount of cells, particularly with each division of the initial population, to prevent variations in the starting amount of cells from distorting the analysis of differences in growth rates between the divided cell lines. This process may be referred to as "normalizing" the cell line inoculum.

[0030] Currently, no technology exists to enable automated normalization of input cell lines and ensure that all bioreactors or vessels receive the same initial viable cell density. Normalization is currently an offline, manual process with associated human error. As discussed herein, automated inoculation refers to the use of a workstation or platform to perform a CLD process based on initially programmed or preprogrammed instructions, without intervention by an operator or other user. Aspects of the present disclosure may particularly focus on the inoculation and normalization steps within the CLD process.

[0031] 1 , an exemplary automated cell line development (CLD) system 100 is shown. The CLD system may include an input 102, a CLD workstation 104, a computing device 106, and an output 108. The CLD system 100 may be used to grow and develop cell lines based on an input sample, such as the input 102. The CLD system 100 may also provide control of various steps in the CLD process and analysis of the samples and cell lines throughout the process.

[0032] Input 102 provides a starting point for one or more cell lines to be developed using CLD system 100. Input 102 may be an input plate of various cell lines to be developed. The initial cell lines may originate from a cell bank or a prior CLD process.

[0033] The CLD workstation 104 provides an integrated and automated platform for seeding and culturing cell lines. The CLD workstation 104 accepts input 102 and enables analysis of the accepted cell line and combination of input samples and other components for the CLD process. The CLD workstation 104 is discussed in more detail below with respect to FIG. 2.

[0034] The computing device 106 receives data from the CLD workstation 104 and may also receive additional data from an operator or other user. The computing device 106 may be configured to provide commands and operations that control the CLD workstation 104. The computing device 106 may process data received from the CLD workstation 104 or other sources and, in response, generate further commands for the CLD workstation 104 or output, such as output 108. In embodiments, the computing device 106 may be integrated with the workstation 104. In other embodiments, the computing device 106 may be physically separate from the workstation 104 and communicate with the workstation 104 through a local or wireless connection or over a network.

[0035] Computing device 106 includes at least a processor and memory and may be any number of known computing devices or may be a specialized computing device. A computing device is a physical, tangible device that processes data. Exemplary types of computing devices include personal computers, stand-alone server computers, blade server computers, mainframe computers, handheld computers, smartphones, special-purpose computing devices, and other types of devices that process data.

[0036] A computing device generally includes at least one central processing unit ("CPU"), a system memory, and a system bus coupling the system memory to the CPU. The system memory includes random access memory ("RAM") and read-only memory ("ROM"). A basic input / output system containing basic routines that help to transfer information between elements within the device, such as during start-up, is stored in the ROM. The device also includes a mass storage device. The mass storage device is capable of storing software instructions and data.

[0037] A mass storage device and its associated computer-readable data storage medium provide non-volatile, non-transitory storage for a device. While the description of a computer-readable data storage medium contained herein refers to a mass storage device such as a hard disk or CD-ROM drive, it should be understood by those skilled in the art that a computer-readable data storage medium can be any available non-transitory physical device or article of manufacture from which a device can read data and / or instructions.

[0038] Computer-readable data storage media include volatile and non-volatile, removable and non-removable media implemented in any method or technology for storage of information, such as computer-readable software instructions, data structures, program modules, or other data. Exemplary types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technology, CD-ROM, digital versatile disks ("DVDs"), other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store desired information and that can be accessed by a device. In some embodiments, computer-readable data storage media include non-transitory media.

[0039] The computing device may also include an input / output controller for receiving and processing input from a number of other devices, including a keyboard, a mouse, a touch-sensitive user interface display screen, or another type of input device, and similarly, the input / output controller may provide output to a touch-sensitive user interface display screen, a printer, or other type of output device.

[0040] In an embodiment, computing device 106 may be fully integrated within CLD workstation 104 and operated by buttons, keys, or one or more touchscreens on CLD workstation 104. In an example, computing device 106 may be computer software loaded onto any number of common or custom combinations of processors and memory. Computing device 106 may enable analysis of data generated by CLD workstation 104 and may further provide accessible readout of and analysis of the data.

[0041] The output 108 may include the physical cell lines and cell line products produced using the CLD system 100. The output 108 may include data or data analysis related to the developed cell lines, such as the number of cells, the cell health of the cells, the growth rate, the mass or volume of the product, etc.

[0042] Referring now to FIG. 2, a component diagram of the exemplary CLD workstation 104 of FIG. 1 is shown. The CLD workstation 104 may include a deck 110 and a gantry system 112. The gantry system 112 may include a chassis 114, one or more grippers 116, and a liquid handler 118. The CLD workstation 104 provides an integrated and automated platform for seeding and culturing cell lines. The CLD workstation 104 accepts cell line input and enables analysis of the received cell line. The CLD workstation 104 enables automated combination of input samples with other components for the CLD process.

[0043] The CLD workstation 104 may provide liquid and component handling equipment integrated within a modular platform. The modular design and integration with scalable operating software provides a platform for configuring interchangeable accessories, integrating peripheral process devices, and automating laboratory workflows. The CLD workstation 104 may be configured with equipment for pipetting or transferring liquid samples from input sources to other components within the CLD workflow in an automated manner. Automating such sample preparation processes improves liquid volume accuracy and precision by reducing the variability inherent in manual pipetting techniques, which are subject to operator-to-operator variation.

[0044] The deck 110 provides a platform base for the CLD workstations 104. The deck 110 may be arranged in a grid to aid in navigation of the gantry system 112. The deck 110 may include location identification indicators, such as pre-drilled holes in the deck, that may be used to position components on the deck 110, or in embodiments, at off-deck locations. The configuration of the deck 110 may be controlled by a computer program or device, such as the computing device 106 of FIG. 1. The deck 110 and the configuration of the deck 110 are discussed in more detail below in connection with FIG. 3.

[0045] The gantry system 112 provides an automated transport system to position components on the deck 110 and facilitate executing automated protocols, such as the CLD process, within the workstation 104. The chassis 114 provides a frame for the gantry system 112 and, in embodiments, provides a base platform for the entire workstation 104, which may also support the deck 110. The chassis 114 supports the gripper 116 and other arms of the gantry system 112.

[0046] Gripper 116 provides for the movement of labware and microplates from one deck location to another, including to peripheral process devices, e.g., heating / cooling or shaking devices, as well as to off-deck instruments using a shuttle transport system.

[0047] The liquid handler 118 provides interchangeable heads that can accept a variety of laboratory manipulation tools and, through the use of the gantry system 112, can provide automated operation of such tools. The liquid handler 118 may generally be an automated liquid-handling pipette. In embodiments, the liquid handler 118 may be a single- or multi-channel pipette. The liquid handler 118 may be configured to perform specific liquid handling procedures. For example, the liquid handler 118 may be configured to aspirate from a well at a location by first touching the top of the sample and then gradually moving downward as the sample is removed. This may avoid excess cells accumulating on the outside of the pipette tip, as occurs when the tip is immersed all the way to the bottom. The liquid handler 118 may also be configured to store or receive vessel geometry parameters and provide effective suction for a particular vessel.

[0048] Depending on the head and desired liquid handling procedure, different tools and / or tip types may be associated with the liquid handler 118. Replaceable heads disposed on the liquid handler 118 may be used to aspirate and dispense liquids, for example, using disposable tips. The liquid handler 118 may be configured to hold tip interfaces for fixed or disposable tips and to perform both liquid level sensing and non-liquid level sensing operations.

[0049] The CLD workstation 104 may incorporate other elements and features not directly related to the CLD process. For example, some embodiments may incorporate a deck observation system or one or more tools to assist in the organization and alignment of labware for an automated CLD workflow, as needed.

[0050] Referring now to FIG. 3 , a deck component layout of the deck 110 of the exemplary CLD workstation 104 of FIG. 2 is shown. The deck 110 may include an input receiving area 122, one or more bioreactors 124, a media source 126, a tip source 128, and a cell health module 130. The layout of the components on the deck 110 can be important for the effective execution of a CLD process, especially if the process is automated. Those skilled in the art will understand that numerous layouts are possible and suitable for a CLD process. The layout presented in FIG. 3 is presented as a non-limiting example of one possible layout and demonstrates components and considerations for a CLD process layout. The deck component layout may be varied according to the particular procedure being performed and the tools being used. For example, the tip source 128 may be omitted when disposable tips are not required or used.

[0051] Input receiving area 122 is an area for receiving an initial cell line sample, such as input 102 of FIG. 1. The cell sample or other input may be a single sample or multiple samples. The samples may be introduced onto the plate prior to initiating the automated CLD process by workstation 104. Multiple cell line samples may be arrayed on one or more input plates, and workstation 104 may be configured to inoculate cell lines derived from more than one sample based on the array of cell line samples on input receiving area 122.

[0052] The bioreactor 124 is a receiving zone for a vessel or vessels to receive an initial cell line sample and media and provide growth conditions for the inoculated cell culture. The bioreactor 124 is configured to maintain the conditions necessary for cell growth during the growth phase of the CLD process. In embodiments, the bioreactor 124 may refer to a vessel assembly to be installed within a bioreactor positioned outside the workstation 104. For example, the bioreactor 124 may include a 96-well plate, with a distinct cell line inoculated into each of the 96 wells. In embodiments, the bioreactor may be a microbioreactor or microreactor. The bioreactor 124 will be discussed in further detail in connection with FIG. 4.

[0053] The media source 126 may provide a known location for the workstation 104 to retrieve media when performing the CLD process. The media provides nutrients necessary for cell line development.

[0054] The tip source 128 may provide a known location for the workstation 104 to retrieve clean, uncontaminated tips throughout the execution of the CLD process. For example, clean tips may be used to inoculate individual cell lines to avoid contamination or carryover from the inoculation of one cell line to the inoculation of the next. In embodiments, a single tip may be used to deliver media to each vessel or well in the bioreactor 124 before each cell line is inoculated. In embodiments, the same tip used to deliver media may also be used to inoculate cell lines before being discarded. The same tip may generally be used to inoculate a single vessel or well. The same tip may generally be used to inoculate a first vessel following the dispensing of media into all vessels to be inoculated.

[0055] Cell health module 130 may provide instruments and known locations for determining the total number of cells and / or the number of viable cells in a sample. For example, cell health module 130 may contain instruments for measuring electrical impedance and / or light scattering from cells in the analyzed sample. Cell health module 130 may include programming for determining the total number of cells and / or the number of viable cells in a sample. Cell health module 130 may be in communication with computing device 106, which may store appropriate instructions and / or programming for cell health module 130. Cell health module 130 may also be configured to determine other parameters associated with the evaluated cell population, such as the average cell diameter of the evaluated cell population.

[0056] The deck 110 may further include other components and zones, as needed, for the effective execution of the CLD process by the workstation 104. For example, the deck 110 may further include a waste zone in which a waste container or tip waste container may be arranged.

[0057] Referring now to Figure 4, a components diagram of the exemplary bioreactor 124 of the exemplary CLD workstation 104 of Figure 2 is shown. The bioreactor 124 contains a housing 132, a culture chamber 134, and, in some embodiments, a control panel 136.

[0058] The housing 132 defines a culture chamber 134. The bioreactor 124 may measure parameters such as biomass, pH, dissolved oxygen (DO), and fluorescence online during cultivation inside the chamber 134. The control panel 136 may be configured to allow a user to control the agitation rate, temperature, gas concentration, gas flow rate, and humidity inside the culture chamber 134. Alternatively, or in addition, the bioreactor 124 may be communicatively coupled to a separate computing device, such as the computing device 106 of FIG. 1, that may enable such control. In embodiments, closed-loop control of pH and dissolved oxygen may be used, and the gas flow rate may be automatically adjusted according to observed changes in pH and / or dissolved oxygen.

[0059] Referring now to Figure 5, there is shown a component diagram of the gantry system 112 of the exemplary CLD workstation 104 of Figure 2. The gripper 116 and liquid handler 118 are shown in further detail.

[0060] The gripper 116 enables movement of labware around the deck 110. The gripper 116 may be one of multiple grippers 116 incorporated into the gantry system 112. The gripper 116 is configured for 360° rotation and may include offset fingers. The gripper 116 enables loading and unloading of labware and movement of plates and microplates from one deck location to another, including movement to peripheral process devices such as heating / cooling and shaking devices. The gripper 116 may also support user interaction with off-deck instruments using a shuttle transport system or the like.

[0061] Labware movement may be controlled programmatically, such as through communication to a computing device, such as computing device 106 of Figure 1. The user interface may allow instrument deck layouts and labware types to be defined, and automated sample preparation methods to be imported and exported.

[0062] The liquid handler 118 provides liquid transfer between components within the workstation 104. The liquid handler 118 may be configured as a pipette. Liquid transfer may be implemented using air or liquid displacement. In an embodiment, when aspirating a sample, a hydraulic piston in the head of the liquid handler mechanically draws liquid into a pipette tip, which is immersed in the sample. Air in the pipette tip is displaced by liquid entering the tip. In another embodiment, a syringe pump, connected to a disposable or fixed tip via hydraulic tubing lines, mechanically moves system fluid, thereby displacing air in the tip with liquid entering the tip. When dispensing liquid into a destination labware, the piston or syringe pump movement is reversed and liquid is removed from the pipette tip.

[0063] Referring now to FIG. 6, a component schematic of the liquid handler 118 of the gantry system 112 of FIG. 5 is shown. The liquid handler 118 may include one or more mandrels 138 and one or more tips 140. In an example, the liquid handler 118 may be configured as an 8×12 or 16×24 pipette dispensing array, for example, so that pipette dispensing operations can be completed with up to 96 or 384 samples at a time. The liquid handler 118 may be configured with one or more independent pipette dispensing probes. The probes may be configured to extend and retract to support liquid transfer to and from labware with different well spacings and orifice sizes. In an embodiment, one or more pipette dispensing probes may be arranged in a linear plane.

[0064] Each mandrel 138 is an elongated hollow structure that serves to couple a tip 140 to the liquid handler 118. The mandrels 138 may generally be metal. The tips 140 engage with the mandrels 138 to form a seal. The tips 140 provide high precision liquid transfer into and out of the container. In embodiments, the tips 140 are disposable. Disposable pipette tips may generally be preferred for transferring liquid from a source container, such as a tube or microplate, to a destination container.

[0065] 7, a process diagram of an exemplary CLD process 200 is shown. Process 200 may involve an input plate 202, an automated transfer system 204, a cell health module 206, a computing device 208, a media reservoir 210, and one or more bioreactors 212. Process 200 may be performed using a CLD workstation, such as CLD workstation 104 in FIGS. 1 and 2.

[0066] An input plate 202, which may represent a sample input such as input 102 in FIG. 1, may be introduced into the workstation prior to initiating the CLD process 200. The input plate 202 may contain one or more initial cell line samples. The input plate 202 may be, for example, a microtiter plate having any number of wells. In one example, the input plate 202 may be a 1-96 well microtiter plate. The input plate 202 is the sample source and may contain one or more cell lines of interest in different wells. The cell lines in the input plate 202 may undergo titer comparison. In embodiments, the input plate 202 may be maintained by the system in a temperature-controlled manner to ensure that cell health is not reduced.

[0067] Each of these cell lines may be expected to have variable cell densities at this stage in the cell line development process. The cell lines in input plate 202 may be transferred, such as by an automated transfer system 204, to cell health module 206, which may assess the initial cell density within each sample cell line. Automated transfer system 204, which may represent a liquid transfer system such as liquid handler 118 of FIGS. 2 and 5, provides for the movement of liquid to different containers and components as required by CLD process 200. Automated transfer system 204 may be operated by or receive instructions from computing device 208. Cell health module 206, which may represent a cell health module such as cell health module 130 of FIG. 3, provides the instrumentation necessary to determine cell density and viability within cell samples and cell line cultures.

[0068] The initial cell line sample from the input plate 202 may be transferred to the cell health module 206 and evaluated for cell density. In embodiments, the cell line sample may be evaluated specifically for viable cell density. The determined cell density of the cell line sample may be communicated to the computing device 208.

[0069] Computing device 208, which may represent a computing device such as computing device 106 of FIG. 1, may store instructions for execution of CLD process 200 and communicate the instructions to components to enable execution of CLD process 200.

[0070] The computing device 208 may receive a target cell culture density for inoculating the bioreactor 212. The computing device 208 determines the respective volumes of media and each cell line sample to be combined to result in a cell culture with the target cell culture density. In embodiments, multiple cell cultures may be inoculated from a single sample cell line. Each cell culture may thus be inoculated with the same volume of media and each of the sample cell lines. In embodiments, cell cultures may be inoculated from multiple sample cell lines. Each cell culture may thus be inoculated with a different volume of media and each of the corresponding sample cell lines, since each cell line of the multiple cell lines will generally have a different cell density.

[0071] The computing device 208 may provide control of system components, such as the automated transfer system 204, according to stored instructions and calculated values. Once the cell density of the initial cell line sample in the input plate 202 is received from the cell health module 206, the computing device 208 calculates a volume of media and a volume of one or more of the sample cell lines from the input plate 202. The computing device 208 operates or provides instructions to the automated transfer system 204 to deliver the calculated volumes of media and one or more of the initial sample cell lines to the bioreactor 212.

[0072] Media reservoir 210, which may represent a media source such as media source 126 in Figure 3, provides a source of media to feed bioreactor 212 and prepare cell cultures for cell line development. Automated transfer system 204 may be programmed or operated to move a calculated volume of media from media reservoir 210 to bioreactor 212.

[0073] Bioreactor 212, which may represent a bioreactor such as bioreactor 124 of FIGS. 3 and 4, provides an environment configured to be conducive to cell growth and cell line development. Bioreactor 212 receives calculated volumes of media and initial cell line sample, which may be combined in a vessel or plate within bioreactor 212 or combined externally to bioreactor 212 and then placed inside the bioreactor. Bioreactor 212 may represent integrated or separate components that together maintain a desired CLD environment. Parameters for the CLD environment may be stored or determined by computing device 208. Bioreactor 212 may be operated by or receive instructions from computing device 208 to establish and maintain a desired CLD environment, e.g., thermal cycling, shaking, humidity, etc.

[0074] Referring now to FIG. 8, a flowchart of an exemplary method 300 for implementing automated bioreactor start-up for a CLD process is shown. Method 300 may be a fully automated method executed by an automated workstation, such as workstation 104 of FIGS. 1 and 2. Instructions for implementing method 300 may be stored and communicated by a computing device, such as computing device 106 of FIG. 1 or computing device 208 of FIG. 7. The computing device may be in wired or wireless communication with the automated workstation to receive data from and provide instructions to components of the workstation. The computing device may also receive initial or target parameters from a user prior to initiation of method 300. The initial or target parameters may be determined internally by the computing device according to previously established parameters or stored instructions.

[0075] At 302, a sample source plate may be cooled. The source plate may correspond to input 102 of FIG. 1 and / or input plate 202 of FIG. 7. Cooling the source plate may reduce cellular activity in the sample cell line and improve the accuracy of cell counts and viable cell counts. In embodiments, step 302 may be omitted and the sample cell line may be evaluated for cell density without cooling. In embodiments, the input plate may be pre-cooled prior to receipt.

[0076] At 304, the cell density of the sample cell lines is determined. The number of cells in each sample cell line may be counted. Only the number of viable cells in each sample cell line may be counted. The counting may be performed by a cell health module, such as cell health module 130 of FIG. 3 or cell health module 206 of FIG. 7. The counting may be accomplished by placing or transferring a full or partial sample of each sample line into the cell health module. The transfer may be accomplished by an automated transfer system, such as gantry system 112 of FIGS. 2, 5, and 6 or automated transfer system 204 of FIG. 7.

[0077] In embodiments, a single sample cell line may be used. In other embodiments, multiple sample cell lines may be used and each may be counted individually. In cases involving multiple sample cell lines, each sample may be identified based on its location on the input plate or its orientation within the automated workstation.

[0078] A verification that all initial sample cell lines have been counted is performed at 306. If samples remain to be counted, additional samples are counted at 304. If all initial sample cell lines have been counted, calculations proceed at 306. In embodiments, the system may proceed with calculations for samples already counted while continuing to count additional samples.

[0079] At 308, the volumes of media and cell line sample to be combined to result in a cell culture are calculated. The calculated volume is determined according to the cell count or cell density measurement performed at 304. The calculated volume may also take into account the density of viable cells, the ratio of viable cells to total cell count, a predetermined target cell density in the cell culture, the properties of the media to be used in the cell culture, etc.

[0080] The volume is calculated so that when the volume of media and the volume of the initial cell line sample are combined to result in cell cultures, each cell culture will have the same cell density, e.g., a predetermined target cell density, regardless of variations in the cell density of the initial sample cell line. The target cell density is generally a number of cells per unit volume, e.g., 2 x 10 6 It may be set in terms of cells / mL. In some cases, it may be desirable to have a default target cell density, such as 1 cell per cell culture. The calculation may be performed by a computing device, such as computing device 106 of FIG. 1 or computing device 208 of FIG. 7.

[0081] At 310, the bioreactor vessel or plate is inoculated with the cell culture. The calculated volumes of the media and each of the one or more initial cell line samples are combined in a vessel configured to be placed in the culture chamber of the bioreactor. This method of inoculating a bioreactor vessel or plate is described in further detail in connection with FIG. 9. The inoculation may proceed automatically in response to the calculation of the media and cell sample volumes.

[0082] Following seeding, each cell culture may be measured for cell density prior to being placed into a bioreactor or prior to starting the bioreactor and providing growth conditions to the cells. Measuring cell density following seeding, but prior to cell growth, verifies compliance with a uniform cell density or target cell density for each cell culture prior to starting the culture. If a particular well or vessel is found to have a cell density lower than the target cell density, an additional volume of the associated cell line sample may be calculated and added to bring the cell density up to the target cell density. If a particular well or vessel is found to have a cell density higher than the target cell density, an additional volume of media may be calculated and added to bring the cell density down to the target cell density.

[0083] At 312, the cell culture is shaken. In embodiments, the cell culture may be shaken for about 2 hours. Shaking the cell culture is one example of conditions established to promote cell growth, but is not limiting of the factors and conditions that may be established during the growth phase of the CLD process.

[0084] At 314, cell density within the cultured cell lines is evaluated. The cultured cell lines may be measured, for example, using the same cell health module used in 304. Individual cultured cell lines may be counted for overall cell density and / or viable cell density. The cultured cell lines may be compared based on measured characteristics. For example, the final cell densities between two or more cultured cell lines may be compared to determine the cell line with the highest or fastest growth rate. The cultured cell lines may also be evaluated and compared based on other factors and characteristics, including, but not limited to, production of proteins and other compounds.

[0085] 9, a flow chart of a method 400 for seeding a bioreactor vessel or location is shown. Method 400 may be performed by an automated liquid transfer system, such as the gantry system 112 of FIGS. 2, 5 and 6 or the automated transfer system 204 of FIG. 7.

[0086] At 402, a first tip is loaded onto a pipette or other liquid transfer device. The tip provides a clean and precise user interaction point with the fluid to be transferred. In embodiments, method 400 may be performed using sterile tips. In embodiments, the first tip may refer to a first set of tips.

[0087] At 404, a first tip is used to load and dispense media into a cell culture vessel or plate. The volume of the loaded and dispensed media may be pre-calculated according to the initial cell line sample cell density and the target cell density for the cell culture.

[0088] At 406, a verification is performed to assess whether all culture vessels or wells have been supplied with media. For example, if a culture plate contains multiple wells, each well may need to be supplied with a volume of media. If wells still remain that need to be supplied with media, loading and dispensing of the calculated volume of media continues as in 404. If all wells have been supplied with media, loading and dispensing of sample may proceed.

[0089] At 408, a first sample is dispensed into the culture vessel or well that has been loaded and supplied with medium. The volume of the loaded and dispensed first sample may be pre-calculated according to the initial cell line sample cell density and the target cell density for the cell culture. The first sample may refer to a first set of samples.

[0090] At 410, the first tip is discarded. Once a tip has been used to transfer a sample, it may be understood that the tip is contaminated and unsuitable for use to transfer any additional samples.

[0091] At 412, an additional tip is loaded. The additional tip may refer to an additional set of tips. At 414, an additional sample is loaded and dispensed. The additional sample may refer to an additional set of samples. At 416, the additional tip is discarded.

[0092] At 418, an assessment is made of whether all samples have been dispensed. In embodiments, the assessment may be made by determining whether all wells have been provided with sample. In other embodiments, the assessment may be made by determining whether a volume has been drawn and dispensed from each initial cell line sample. If a determination is made that additional samples remain to be dispensed, additional tips may be loaded as in 412. If a determination is made that all samples have been dispensed, the seeding process may end at 420.

[0093] Illustrative examples of the systems and methods described herein are provided below. Embodiments of the systems or methods described herein may include any one or more of the following notes, and any combination thereof:

[0094] Appendix 1. A method for automated start-up of a bioreactor, the method including: a cell health module counting the number of viable cells in a cell sample; receiving a desired starting density of cells; determining a volume of media and a volume of cell sample to be combined based on the number of viable cells in the cell sample to bring cells of the predetermined density into the bioreactor; combining the media and cell sample by feeding the volume of media into the bioreactor to bring a cell culture; and feeding the volume of cell sample into the bioreactor.

[0095] Appendix 2. The method of Appendix 1, wherein the cell is a mammalian cell.

[0096] Appendix 3. The method of Appendix 1 or 2, wherein the predetermined number of cells is a predetermined number of viable cells.

[0097] Clause 4. The method of any of the preceding clauses, wherein the cell sample is in a source plate.

[0098] Clause 5. The method of any of the above clauses, wherein the number of cells is a first number of cells, and the method further comprises counting a second number of cells in the bioreactor.

[0099] Clause 6. The method of clause 5, wherein counting the second number of cells is performed immediately after delivering the cell sample volume to the bioreactor and determining the final density of the cells.

[0100] Clause 7. The method of Clause 6, further comprising determining that a second number of cells in the bioreactor is below a predetermined threshold, calculating a second volume of the cell sample to be injected into the bioreactor to result in a predetermined density of cells, and injecting the second volume of the cell sample into the bioreactor.

[0101] Clause 8. The method of Clause 6, further comprising determining that a second number of cells in the bioreactor is above a predetermined threshold, calculating a second volume of medium to be infused into the bioreactor to result in a predetermined density of cells, and injecting the second volume of medium into the bioreactor.

[0102] Appendix 9. The method of Appendix 5 or 6, wherein counting the second number of cells is performed after a predetermined period of time following dispensing the cell sample volume into a bioreactor and determining proliferation by the cells in the bioreactor.

[0103] Clause 10. The method of Clause 9, further comprising comparing the first number of cells and the second number of cells over time.

[0104] Clause 11. The method of clause 9 or clause 10, wherein the predetermined time period is one day or more.

[0105] Clause 12. The method of any of the above clauses, wherein the cell health module counting the number of viable cells in a cell sample includes counting the number of viable cells in a plurality of cell samples.

[0106] Clause 13. The method of any of the above clauses, wherein combining the medium and the cell sample by feeding each into the bioreactor includes feeding each of the volume of medium and the volume of the cell sample into each well of a plurality of wells in the bioreactor.

[0107] Clause 14. The method of clause 13, wherein the predetermined density of cells is the same for each well of a plurality of wells in the bioreactor.

[0108] Clause 15. The method of clause 13 or 14, wherein the volume of the cell sample per well of the plurality of wells is determined from the same cell sample, such that each well of the plurality of wells contains the same type of cell.

[0109] Appendix 16. The method of any of Appendixes 13-15, wherein the volume of the cell sample for at least one well of the plurality of wells is determined from a cell sample that is different from the volume of the cell sample for the remainder of the plurality of wells, such that the at least one well of the plurality of wells contains a different type of cell than the remainder of the plurality of wells.

[0110] Appendix 17. The method of any of Appendixes 13-16, wherein dispensing the volume of medium into each well of the plurality of wells is performed using a first tip, and dispensing the volume of cell sample into a first well of the plurality of wells is performed using a first tip.

[0111] Appendix 18. The method of any one of Appendixes 13-17, wherein the plurality of wells in the bioreactor comprises a plurality of wells in a series of bioreactors.

[0112] Appendix 19. The method of any of appendices 13-18, further comprising culturing a cell sample in each well of the plurality of wells; assessing cell growth in each well of the plurality of wells in the bioreactor; and identifying one or more wells as containing a cell line with greater productivity compared to other wells of the plurality of wells.

[0113] Appendix 20. The method of any of Appendixes 13-19, wherein injecting the volume of medium into each well of the plurality of wells in the bioreactor is performed using a first pipette tip, and injecting the volume of cell sample into a first well of the plurality of wells in the bioreactor is performed using a first pipette tip.

[0114] Clause 22. The method of any of the above clauses, wherein the cell sample is maintained.

[0115] Appendix 23. A system for automating the inoculation of a bioreactor with a predetermined number of cells, the system including: a source plate configured to hold a cell sample; a cell health evaluator configured to count the number of cells in the cell sample; a bioreactor including a number of wells; and a processor in communication with a memory, the memory storing instructions that, when executed by the processor, cause the system to receive the number of cells in the cell sample; determine a volume of medium and a volume of the cell sample to be combined based on the number of cells in the cell sample; result in cells of a predetermined density; operate a fluid transfer device; deposit the volume of medium into each well of the number of wells in the bioreactor; and deposit the volume of the cell sample into each well of the number of wells in the bioreactor.

[0116] Clause 24. The system of clause 23, wherein the fluid transfer device is a pipette.

[0117] 25. The system of claim 23 or 24, wherein a first tip for the pipette is used to inject a volume of medium into each well of the plurality of wells and a volume of a cell sample into a first well of the plurality of wells.

[0118] Item 26. The system of any one of Items 23-25, further comprising a receiving zone for a pipette tip.

[0119] Addendum 27. A system according to any one of Addendums 23 to 26, wherein the receiving zone is configured to receive several tips, the number of tips being equal to the number of wells.

[0120] Addendum 28. The system of any of Addendums 23-27, wherein the source plate is further configured to maintain the cell sample at a predetermined temperature.

[0121] Clause 29. The system of clause 28, wherein the predetermined temperature is within the range of 2 to 8 degrees Celsius.

[0122] Clause 30. A method for optimizing pipette tip usage, the method comprising: pipetting a medium into several sample wells using a first pipette tip; and pipetting several samples into several sample wells, the number of samples equaling the number of wells, and the first sample being pipetted using the first pipette tip.

[0123] Addendum 31. A system for optimizing pipette tip usage, the system including: a media source; a sample source; a number of sample wells; a pipette with a number of mandrels equal to the number of sample wells; and a number of pipette tips, one pipette tip associated with each mandrel of the number of mandrels, wherein the pipette fills each sample well of the number of sample wells from the media source using a first pipette tip of the number of pipette tips and adds a sample from the sample source to each of the number of sample wells, wherein a first sample in a first of the pipette wells is added using one pipette tip.

[0124] While preferred aspects and implementations of the present disclosure have been described, modifications and equivalents of the disclosed concepts may readily occur to those skilled in the art, however, such modifications and equivalents are intended to be included within the scope of the claims appended hereto.

Claims

1. 1. A method for automated start-up of a bioreactor, the method comprising: a cell health module counting the number of viable cells in the cell sample; receiving a desired starting density of cells; determining a volume of medium and a volume of the cell sample to be combined based on the number of viable cells in the cell sample to provide a predetermined density of cells in a bioreactor; combining the medium and cell sample by delivering a volume of the medium to the bioreactor to provide a cell culture; providing the volume of the cell sample into the bioreactor; A method comprising:

2. The method of claim 1 , wherein the cell is a mammalian cell.

3. 3. The method of claim 1 or 2, wherein the predetermined number of cells is a predetermined number of viable cells.

4. 4. The method of claim 1, wherein the number of cells is a first number of cells, and the method further comprises counting a second number of cells in the bioreactor.

5. 5. The method of claim 4, wherein counting the second number of cells is performed immediately after delivering the volume of the cell sample to the bioreactor and determining a final density of cells.

6. determining that a second number of the cells in the bioreactor is below a predetermined threshold; calculating a second volume of the cell sample to be injected into the bioreactor to result in the predetermined density of cells; injecting the second volume of the cell sample into the bioreactor; The method of claim 5 further comprising:

7. determining that a second number of the cells in the bioreactor is above a predetermined threshold; calculating a second volume of media to be injected into the bioreactor to result in the predetermined density of cells; injecting a second volume of the medium into the bioreactor; The method of claim 5 further comprising:

8. 6. The method of claim 4 or 5, wherein counting the second number of cells is performed a predetermined period of time following providing the volume of the cell sample into the bioreactor and determining proliferation by the cells in the bioreactor.

9. 9. The method of claim 8, further comprising comparing the first number of cells and the second number of cells over time.

10. 10. The method of claim 1, wherein the cell health module counting the number of viable cells in the cell sample comprises counting the number of viable cells in a plurality of cell samples.

11. The method of any one of claims 1 to 10, wherein the predetermined density of cells is the same for each well of a plurality of wells in the bioreactor.

12. The method of any one of claims 1 to 11, wherein the volume of the cell sample for each well of a plurality of wells is determined from the same cell sample, such that each well of the plurality of wells contains the same type of cell.

13. dispensing the volume of medium into each well of the plurality of wells is performed using a first tip; The method of any of claims 1 to 12, wherein delivering the volume of the cell sample to a first well of the plurality of wells is performed using the first tip.

14. culturing the cell sample in each well of a plurality of wells; assessing cell growth in each well of the plurality of wells in the bioreactor; and identifying one or more wells as containing a cell line with greater productivity compared to other wells of said plurality of wells; The method of any one of claims 1 to 13, further comprising:

15. The method according to any one of claims 1 to 14, wherein the cell sample is maintained.

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