EP4655383A1

EP4655383A1 - Normalization of seeding of bioreactors

Info

Publication number: EP4655383A1
Application number: EP24708295.1A
Authority: EP
Inventors: Abraham J. OLSON; Alexis MESA; Yingjian YOU
Original assignee: Beckman Coulter Inc
Current assignee: Beckman Coulter Inc
Priority date: 2023-01-25
Filing date: 2024-01-23
Publication date: 2025-12-03
Also published as: JP2026503534A; KR20250135878A; CN120500526A; WO2024158817A1

Abstract

A method of automating startup of a bioreactor and normalizing seeding of cell cultures by counting a number of viable cells in a cell sample by a cell health module. A desired starting density of cells is received and a volume of media and a volume of the cell sample are each determined, based on the number of cells in the cell sample, to be combined to yield a predetermined density of cells in a bioreactor. The media and cell sample are combined to yield a cell culture by supplying the volume of media to the bioreactor and supplying the volume of the cell sample to the bioreactor.

Description

NORMALIZATION OF SEEDING OF BIOREACTORS

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

BACKGROUND

[0002] Cell line development is a process of cultivating cell growth. Cell line development enables evaluation of differences between individual cells and can be used to confirm monoclonality of a particular cell line. Cell line development is useful for generating various biological molecules, such as for the development of pharmaceuticals. Cell lines are therefore subject to evaluation based on various criteria, such as protein titer or cell line stability . Cell lines are ty pically established by first isolating a single viable cell or otherwise established identical starting conditions to enable equal comparison for each cell line.

[0003] One application of cell line development is the manufacture of therapeutic monoclonal antibodies (mAb). Manufacturing mAbs requires starting from a single clonal production cell line that can stably express a target mAb of interest. A production cell line for manufacturing needs to be robust, stable and can scale-up in large scale culture in bioreactors. Some critical criteria of a production cell line include monoclonality⁷, high productivity⁷, proper post translational modification and good quality. Such a production cell line can be generated from a cell line development process that starts from transfection, selection and single clonal isolation for a large number of monoclonal candidate clones. To reduce timeline and cost, a high throughput workflow is often used for screening for a few good candidate clones. Those selected clones are then sent for dow nstream processing optimization to choose a final production cell line.

SUMMARY

[0004] Examples presented herein are directed to a method of automated startup of a bioreactor. The method includes steps of counting, by a cell health module, a number of viable cells in a cell sample; receiving a desired starting density of cells; determining a volume of media and a volume of the cell sample, based on the number of viable cells in the cell sample, to be combined to yield a predetermined density⁷ of cells in a bioreactor; combining media and cell sample to yield a cell culture by supplying the volume of media to the bioreactor; and supplying the volume of the cell sample to the bioreactor.

[0005] In other examples presented herein, the cells are mammalian cells. In still other examples presented herein, the predetermined number of cells is a predetermined number of viable cells. In yet other examples presented herein, the cell sample is in a source plate. In other examples presented 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 presented herein, counting the second number of cells is performed immediately after supplying the volume of the cell sample to the bioreactor to determine an ending density of cells. In further examples presented herein, the method includes determining the second number of cells in the bioreactor is below a predetermined threshold; calculating a second volume of the cell sample the inject into the bioreactor to yield the predetermined density of cells; and injecting the second volume of the cell sample into the bioreactor. In still further examples presented herein, the method includes determining the second number of cells in the bioreactor is above a predetermined threshold; calculating a second volume of media the inject into the bioreactor to yield the predetermined density⁷ of cells; and injecting the second volume of media into the bioreactor.

[0007] In other examples presented herein, counting the second number of cells is performed after a predetermined time period following supplying the volume of the cell sample into the bioreactor to determine growth by the cells in the bioreactor. In further examples presented herein, the method includes comparing the first number of cells and the second number of cells over time. In still further examples presented herein, the predetermined time period is one or more days.

[0008] In other examples presented herein, counting, by a cell health module, the number of viable cells in the cell sample comprises counting a number of viable cells in a plurality of cell samples. In still other examples presented herein, combining media and the cell sample by supplying each to a bioreactor comprises supplying each of the volume of media and the volume of the cell sample to each well of a plurality of wells in the bioreactor. In yet other examples presented herein, the predetermined density⁷ of cells is the same for each well of the plurality of wells in the bioreactor.

[0009] In further examples presented herein, the volume of the cell sample for each well of the plurality of wells is taken from a same cell sample such that each w ell of the plurality of wells contains a same kind of cells. In still further examples presented herein, the volume of the cell sample for at least one well of the plurality of wells is taken from a different cell sample than the volume of the cell sample for a remainder of the plurality of wells such that the at least one well of the plurality of wells contains a different kind of cells than the remainder of the plurality of wells. In yet further examples presented herein, supplying the volume of media to each well of the plurality of wells is performed using a first tip; and supplying a volume of the cell sample to a first well of the plurality of wells is performed using the first tip.

[0010] In other further examples presented herein, the plurality of w ells in the bioreactor comprise a plurality of wells among a series of bioreactors. In yet other further examples presented herein, the method includes culturing the 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 as compared to other wells of the plurality' of wells. In still further examples presented herein, injecting the volume of media into each well of a plurality of wells in a bioreactor is performed using a first pipette tip; and injecting the volume of the cell sample into a first w ell of the plurality of w ells in the bioreactor is performed using the first pipette tip. In other examples presented herein, the cell sample is maintained.

[0011] Other examples presented herein are directed to a system for automating seeding a bioreactor with a predetermined number of cells The system includes a source plate configured to hold a cell sample; a cell health evaluator configured to count a number of cells in the cell sample; the bioreactor including a number of w ells; and a processor in communication with a memory, the memory storing instructions which, when executed by the processor, cause the system to: receive the number of cells in the cell sample; determine a volume of media and a volume of the cell sample, based on the number of cells in the cell sample, to be combined to yield a predetermined density of cells; operate a fluid transfer device to: deposit the volume of media in 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 presented herein, the fluid transfer device is a pipette. In still other examples presented herein, a first tip for the pipette is used to inject the volume of media into each well of the plurality of wells and to inj ect the volume of the cell sample into a first well of the plurality of wells. In yet other examples presented herein, the system includes a receiving zone for pipette tips. [0013] In other examples presented herein, the receiving zone is configured to receive a number of tips, wherein the number of tips is equal to the number of wells. In yet other examples presented herein, the source plate is further configured to maintain the cell sample at a predetermined temperature. In further examples presented herein, the predetermined temperature is within a range of 2 to 8 degrees Celsius.

[0014] Other examples presented herein are directed to a method for optimizing pipette tip usage. The method includes pipetting a media into a number of sample wells using a first pipette tip; and pipetting a number of samples in the number of sample wells, the number of samples equal to the number of wells, wherein the first sample is pipetted using the first pipette tip.

[0015] Still other examples presented herein are direct to a system for optimizing pipette tip usage. The system includes a media source; a sample source; a number of sample wells; a pipettor with a number of mandrels equal to the number of sample wells; and a number of pipette tips such that one pipette tip is associated with each mandrel of the number of mandrels; wherein the pipettor fills each sample well of the number of sample wells from the media source with 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 with a first sample in a first of the pipette wells being added using the first pipette tip.

[0016] A variety of additional inventive aspects will be set forth in the description that follows. The inventive aspects can relate to individual features and to combinations of features. It is to be understood that both the forgoing general description and the following detailed description are exemplar}' and explanatory only and are not restrictive of the broad inventive concepts upon which the embodiments disclosed herein are based.

BRIEF DESCRIPTION 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 is as follows:

[0018] FIG. I is an example cell line development (CLD) system.

[0019] FIG. 2 is a component diagram of the example CED workstation of FIG. 1.

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

1.

[0021] FIG. 4 is a component diagram of the bioreactor of the example CLD workstation of FIG. 1. [0022] FIG. 5 is a component diagram of the gantry' system of the example CLD workstation of FIG. 1.

[0023] FIG. 6 is a component diagram of the liquid handler of the gantry system of FIG. 5.

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

[0025] FIG. 8 is a flowchart of an example method of performing an automated bioreactor startup for a CLD process.

[0026] FIG. 9 is a flowchart of a method of inoculating a bioreactor container or plate.

DETAILED DESCRIPTION

[0027] Reference will now be made in detail to exemplary aspects of the present disclosure that 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 a process of propagating a cell or cells with distinct and reproducible characteristics that define the cell line. Effective CLD requires precision in establishing the initial cell or cells to initiate the cell line, so that characteristics of the cell line overall can be effectively traced back to a progenitor. This process may be referred to as “seeding” the cell line. As discussed herein, seeding refers to preparing one or more containers with a precise number of initial viable cells for cell line development to enable accurate determinations of the strength of a cell line’s propagation over time during the development process.

[0029] In some cases, an initial group of cells may be divided and individual cells or small groups from the initial group propagated separately to evaluate variations among the population of the initial group. In such cases it may be particularly important to begin with a precise quantity of cells for each division of the initial population, to prevent variations in the starting quantity of cells from distorting analysis of differences in growth rate among the divided cell lines. This process may be referred to as “normalizing” the seeding of the cell line.

[0030] There is presently no technology to enable automated normalization of the input cell lines to ensure that all bioreactors or all containers are receiving the same initial viable cell density. Normalization is currently an off-line, manual process fraught with associated human error. As discussed herein, automated seeding refers to 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 provide particular emphasis on the steps of seeding and normalizing within the CLD process.

[0031] Referring now to FIG. 1 , an example 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. CLD system 100 may be used to grow and develop cell lines based on input samples, such as input 102. CLD system 100 may also provide for control of various steps in a 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 developed using the CLD system 100. Input 102 may be an input plate of various cell lines to be developed. Initial cell lines may come from a cell bank or an earlier CLD process.

[0033] CLD workstation 104 provides an integrated and automated platform for seeding and culturing cell lines. CLD workstation 104 receives input 102 and enables analysis of received cell lines and combination of input samples with other components for a CLD process. CLD workstation 104 is discussed is greater detail below with respect to FIG. 2.

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

[0035] Computing device 106 includes at least a processor and a 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. Example types of computing devices include personal computers, standalone server computers, blade server computers, mainframe computers, handheld computers, smart phones, special purpose computing devices, and other types of devices that process data. [0036] Computing devices generally include at least one central processing unit ("CPU"), a system memory, and a system bus that couples the system memory to the CPU. The system memory includes a random access memory ('‘RAM”) and a read-only memory (“ROM”). A basic input/output system containing the basic routines that help to transfer information between elements within the device, such as during startup, is stored in the ROM. The device further includes a mass storage device. The mass storage device is able to store software instructions and data.

[0037] The mass storage device and its associated computer-readable data storage media provide non-volatile, non-transitory storage for the device. Although the description of computer-readable data storage media contained herein refers to a mass storage device, such as a hard disk or CD-ROM drive, it should be appreciated by those skilled in the art that computer-readable data storage media can be any available non- transitory', physical device or article of manufacture from which the 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. Example ty pes 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-ROMs, digital versatile discs (“DVDs”), other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can be accessed by the device. In some embodiments, the computer-readable data storage media includes non-transitory’ media.

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

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

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

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

[0043] CLD workstation 104 may provide for liquid- and component-handling instruments to be integrated in a module-based platform. A modular design and integration with extensible operating software, provides a platform for configuring interchangeable accessories and for integrating peripheral process devices to automate laboratory⁷ workflows. CLD workstation 104 may be configured with instruments to pipette, or transfer liquid samples, from an input source to other components in a CLD workflow in an automated fashion. Automating such sample preparation processes improves liquid quantity⁷ accuracy and precision by reducing the variability inherent to manual pipetting techniques, which are subjected to operator-to-operator differences.

[0044] Deck 110 provides a platform base for CLD w orkstation 104. Deck 110 may be arranged in a grid to assist in navigation of gantry system 112. Deck 110 may include locating indicators, such as predrilled holes in the deck that may be used to position components on deck 110 or, in embodiments, an off-deck position. Configuration of deck 110 may be controlled by a computer program or device, such as computing device 106 of FIG. 1. Deck 110 and the configuration of deck 110 is discussed in greater detail below in relation to FIG. 3.

[0045] Gantry system 112 provides an automated transfer system to facilitate positioning components on deck 110 and executing automated protocols, such as a CLD process, within workstation 104. Chassis 114 provides a frame for gantry⁷ system 112 and, in embodiments, provides a base platform for workstation 104 overall and may support deck 110 as well. Chassis 114 supports grippers 116 and other arms of gantry system 112.

[0046] Grippers 116 provide for movement of labware and microplates from one deck position to another, including movement to peripheral process devices, e.g., heating/cooling or shaking devices, as well as movement to off-deck instruments by means of shuttle transport systems.

[0047] Liquid handler 118 provides an interchangeable head which can receive various lab manipulation tools and provide for automated operation so such tools through use of gantry system 112. Liquid handler 118 may generally be an automated liquid handling pipettor. In embodiments, liquid handler 118 may be a single or multichannel pipettor. Liquid handler 118 may be configured to perform a specific liquid-handling procedure. For example, liquid handler 118 may be configured to aspirate from wells of a place by first touching the top of the sample and then gradually move downward as sample is removed. This may avoid accumulating excess cells on the outside of the pipette tip, as occurs when a tip is dipped all the way to the bottom. Liquid handler 118 may be configured to store or receive geometry parameters of a container to provide for effective aspiration for a particular container.

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

[0049] 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 framing and arranging lab ware as needed for an automated CLD workflow.

[0050] Referring now to FIG. 3, a deck component layout of the deck 110 example CLD workstation 104 of FIG. 2 is shown. The deck 110 may include an input receiving region 122, one or more bioreactors 124, a media source 126, a tip source 128, and a cell health module 130. The layout of components on deck 110 may be important for effective execution of a CLD process, especially in cases where the process is automated. Those of skill 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 to demonstrate components and considerations for CLD process layouts. The deck component layout may be varied according to the particular procedure conducted and tools used. For example, when disposable tips are not required or used, tip source 128 may be excluded.

[0051] Input receiving region 122 is a region for receiving beginning cell line samples, such as input 102 of FIG. 1. Cell samples or other input may be a single sample or a plurality of samples. Samples may be introduced on plates prior to initiating an automated CLD process by workstation 104. Multiple cell line samples may be arranged on one or more input plates, and workstation 104 may be configured to seed cells lines originating from more than one sample based on the arrangement of the cell line samples on the input receiving region 122.

[0052] Bioreactors 124 is a receiving zone for a container or plurality of containers for receiving initial cell line samples and media and providing growth conditions for the seeded cell cultures. Bioreactors 124 are configured to maintain conditions necessary for cell growth during a growth phase of the CLD process. In embodiments, bioreactors 124 may refer to a container assembly to be placed in a bioreactor positioned outside of workstation 104. For example, bioreactors 124 may include a 96-well plate, with a separate cell line seeded into each of the 96 wells. In embodiments, the bioreactor may be a micro bioreactor or a microreactor. Bioreactors 124 are discussed in more detail in relation to FIG. 4.

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

[0054] Tip source 128 may provide a known location for workstation 104 to retrieve clean, uncontaminated tips throughout execution of a CLD process. For example, a clean tip may be used for seeding individual cell lines to avoid contamination or carryover from the seeding of one cell line to the next. In embodiments, a single tip may be used to supply media to each container or well in bioreactor 124 before each cell line is seeded. In embodiments, a same tip as used for supplying the media may also be used to seed a cell line before being discarded. The same tip may generally be used to seed a single container or well. The same tip may be generally be used to seed a first container following dispensing of media to all containers to be seeded.

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

[0056] Deck 110 may further include other components and zones as necessary⁷ for effective execution of a CLD process by workstation 104. For example, deck 110 may further include a waste zone where a waste container or a tip discard container may be arranged.

[0057] Referring now to FIG. 4, a component diagram of example bioreactor 124 of the example CLD workstation 104 of FIG. 2 is shown. Bioreactor 124 contains a housing 132, a cultivation chamber 134, and, in some embodiments, a control panel 136.

[0058] Housing 132 defines cultivation chamber 134. Bioreactor 124 may measure parameters such as biomass, pH, dissolved oxygen (DO), and fluorescence online while running a cultivation inside the cultivation chamber 134. Control panel 136 may be configured to allow a user to control shaking speed, temperature, gas concentration, gas flow rate, and humidity inside the cultivation chamber 134. Alternatively or additionally, bioreactor 124 may be communicatively coupled to a separate computing device, such as computing device 106 of FIG. 1, that may allow for such control. In embodiments, a 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 FIG. 5, a component diagram of gantry system 1 12 of the example CLD workstation 104 of FIG. 2 is shown. The gripper 116 and liquid handler 118 are shown in greater detail.

[0060] Gripper 116 enables movement of lab ware around deck 110. Gripper 116 may be one of multiple grippers 116 incorporated into gantry system 112. Gripper 116 may be configured for 360° rotation and include offset fingers. Gripper 116 enables stacking and unstacking of labware and movement of plates and microplates from one deck position to another, including movement to peripheral process devices such as heating/cooling and shaking devices. Gripper 116 may further support interaction with off-deck instruments, such as by means of a shuttle transport system. [0061] Labware movement actions may be controlled by programming, such as through communications to a computing device, such as computing device 106 of FIG. 1. A user interface may enable instrument deck layouts and labware types to be defined and automated sample preparation methods to be imported and exported.

[0062] Liquid handler 118 provides for liquid transport among the components within workstation 104. Liquid handler 118 may be configured as a pipette. Liquid transport may be implemented by means of air or liquid displacement. In examples, when aspirating a sample, a hydraulic piston in the head of the liquid handler mechanically pulls liquid into the pipette tip, which is immersed in the sample. The air in the pipette tip is displaced by the liquid entering the tip. In other examples, a syringe pump connected to the disposable or fixed tip via a hydraulic tubing line mechanically moves system fluid, thereby displacing the air in the tip with the liquid entering the tip. In dispensing the liquid to the destination labware, the piston or syringe pump movement is reversed, and the liquid is dispelled from the pipette tip.

[0063] Referring now to FIG. 6, a component diagram of liquid handler 118 of gantry system 112 of FIG. 5 is shown. Liquid handler 118 may include one or more mandrels 138 and one or more tips 140. In examples, liquid handler 118 may be configured as an 8 x 12 or 16 x 24 pipetting array, for instance such that pipetting action may be completed with up to 96 or 384 samples at one time. Liquid handler 118 may be configured with one or more independent pipetting probes. Probes may be configured to be expanded and contracted to support liquid transport to and from labware of differing well spacing and opening size. In embodiments, the one or more pipetting probes may be arranged in a linear plane.

[0064] Mandrels 138 are each an elongated hollow structure that serves to couple tips 140 to liquid handler 1 18. Mandrels 138 may generally be metallic. Tips 140 engage with mandrels 138 and form a seal. Tips 140 provide for precision movement of liquids out of and into containers. In embodiments, tips 140 are disposable. Disposable pipette tips may generally be preferred to transfer liquid from a source container, such as a tube or microplate, to a destination container.

[0065] Referring now' to FIG. 7, a process diagram of an example CLD process 200 is shown. The 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. The process 200 may be performed using a CLD workstation such as CLD workstation 104 in FIGS. 1 and 2. [0066] Input plate 202, which may represent a sample input such as input 102 of FIG. 1. may be introduced to a workstation prior to initiating the CLD process 200. Input plate 202 may contain one or more initial cell line samples. Input plate 202 may be, for example, a microtiter plate having any number of wells. In one example, input plate 202 may be a 1-96 well microtiter plate. Input plate 202 may be a sample source and contain one or more cell lines of interest in different wells. Cell lines in input plate 202 may be subject to titer comparison. In embodiments, input plate 202 may be held by the system in a temperature-controlled manner to ensure cell health does not decrease.

[0067] It may be expected for each of these cell lines at this stage in the cell line development process to have variable cell densities. Cell lines in input plate 202 may be transferred, such as by automated transfer system 204, to a cell health module 206 to evaluate initial cell density in 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 movement of liquids to the different containers and components as required by the 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 instrumentation necessary to determine cell density⁷ and viability⁷ in cell samples and cell line cultures.

[0068] Initial cell line samples from input plate 202 may be transferred to cell health module 206 and evaluated for cell density. In embodiments, the cell line samples may be evaluated specifically for viable cell density. The determined cell densities of the cell line samples may be communicated to 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 the execution of CLD process 200 and communicate instructions to components to enable execution of CLD process 200.

[0070] Computing device 208 may receive a target cell culture density⁷ for seeding bioreactors 212. Computing device 208 determines a volume of each of media and each cell line sample to be combined to yield cell cultures with the target cell culture density. In embodiments, multiple cell cultures may be seeded from a single sample cell line. Each cell culture may accordingly be seeded with the same volume of each of media and the sample cell line. In embodiments, cell cultures may be seeded from multiple sample cell lines. Each cell culture may accordingly be seeded with a different volume of each of media and the corresponding sample cell line, as each cell line of the multiple cell lines will generally have a different cell density.

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

[0072] Media reservoir 210, which may represent a media source such as media source 126 of FIG. 3, provides a source of media for supplying to bioreactors 212 to prepare a cell culture for cell line development. Automated transfer system 204 may be programmed or operated to move calculated volumes of media from media reservoir 210 to bioreactors 212.

[0073] Bioreactors 212. which may represent bioreactors such as bioreactors 124 of FIGS. 3 and 4, provide an environment configured to promote cell growth and cell line development. Bioreactors 212 receive calculated volumes of media and initial cell lines samples, which may be combined in a container or plate within bioreactors 212 or combined external to bioreactors 212 and then placed inside the bioreactors. Bioreactors 212 may represent integrated or separate components that together maintain a desired CLD environment. Parameters for the CLD environment may be stored by or determined by computing device 208. Bioreactors 212 may be operated by or receive instructions from computing device 208 to establish and maintain the desired CLD environment, e.g., thermocycling, shaking, humidity, etc.

[0074] Referring now to FIG. 8, a flowchart of an example method 300 of performing an automated bioreactor startup 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 performing 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 wared or wireless communication with the automated workstation, and receive data from and provide instructions to components of the workstation. The computing device may further receive initial or target parameters from a user prior to initiation of method 300. Initial or target parameters may be determined internally by the computing device according to previously established parameters or stored instructions.

[0075] At 302, the 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 can reduce cell activity⁷ in the sample cell lines, improving the accuracy of cell counting and viable cell counting. In embodiments, step 302 may be excluded, and the sample cell lines may be evaluated for cell density without being cooled. In embodiments, an input plate may be received precooled.

[0076] At 304, cell density of sample cell lines is determined. A number of cells in each sample cell line may be counted. A number of only the viable cells in each sample cell line may be counted. Counting may⁷ be performed by a cell health module, such as cell health module 130 of FIG. 3 or cell health module 206 of FIG. 7. Counting may be accomplished by placing or transferring full or partial samples of each sample line into the cell health module. Transfer may be accomplished by an automated transfer system, such as gantry system 112 of FIGS. 2, 5, and 6 or automated transfers 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 counted individually. In cases involving multiple sample cell lines, each sample by be identified based on its location on an input plate or its orientation within the automated workstation.

[0078] At 306, verification that all initial sample cell lines have been counted is performed. 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 that have already been counted in parallel with continuing to count additional samples.

[0079] At 308, volumes of media and cell line samples to be combined to yield a cell culture are calculated. Calculated volumes are determined according to the cell count or cell density measurement take at 304. Calculated volumes may also take into account density of viable cells, ratio of viable cells to total cell count, a predetermined target cell density⁷ in cell cultures, characteristics of a media to be used in the cell cultures, etc.

[0080] Volumes are calculated such that when the volume of the media and the volume of the initial cell line sample are combined to yield a cell culture, each cell culture has the same cell density⁷, e.g., the predetermined target cell density, regardless of variations in cell density of the initial sample cell lines. A target cell density may generally be set in terms of cells per unit volume, e.g., 2 x 10⁶ cells/mL. In some cases, it may be desirable to have default target cell density, such as one cell per cell culture. Calculations 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, a bioreactor container or plate is inoculated with the cell culture. Each of the calculated volumes for media and each of one or more initial cell line samples are combined in a container configured to be placed in a cultivation chamber of the bioreactor. The method of inoculating the bioreactor container or plate is described in more detail in relation to FIG. 9. Inoculation may proceed automatically in response to calculation of volumes of media and cell sample.

[0082] Following inoculation, each cell culture may be measured for cell density prior to being placed in the bioreactor or prior to initiating the bioreactor to provide growth conditions to the cells. Measuring cell density following inoculation but prior to cell growth verifies uniform cell density⁷ or compliance with the target cell density for each cell culture prior to initiating cultivation. If a particular well or container 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 container is found to have a cell density⁷ 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 two hours. Shaking the cell culture is one example of conditions established to promote cell growth, but is not limiting on the factors and conditions that may be set during a growth phase of a CLD process.

[0084] At 314, the cell density in the cultured cell lines is evaluated. Cultured cell lines may be measured, for instance using a same cell health module as used at 304. Individual cultured cell lines may be counted for overall cell density⁷ and/or viable cell density. Cultured cell lines may be compared based on measured characteristics. For example, final cell density between two or more cultured cell lines may be compared to determine a cell line with the highest or fastest grow th rate. Cultured cell lines may be evaluated and compared based on other factors and characteristics, including but not limited to production of proteins and other compounds.

[0085] Referring now to FIG. 9, a flowchart of a method 400 of inoculating a bioreactor container or place is shown. Method 400 may be performed by an automated liquid transfer system, such gantry system 112 of FIGS. 2, 5 and 6, or 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 point of interaction with fluids to be transferred. In embodiments, method 400 may be performed with sterile tips. In embodiments, a first tip may refer to a first set of tips.

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

[0088] At 406, a verification is performed to assess whether all cultivation containers or wells have been supplied with media. For example, if the cultivation plate contains a plurality⁷ of wells, each well may need to be supplied with a volume of media. If wells remain that still need to be supplied with media, loading and dispensing of calculated volumes of media continues as at 404. If all wells have been supplied with media, loading and dispensing of samples may proceed.

[0089] At 408, a first sample is loaded and dispensed into a cultivation container or well that has already been supplied with media. The volume of the first sample loaded and dispensed may be precalculated according to an initial cell line sample cell density and a target cell density for a cell culture. A first sample may refer to a first set of samples. [0090] At 410, the first tip is discarded. Once a tip is used to transfer a sample, that tip may be understood to be contaminated and unsuitable for use to transfer any additional samples.

[0091] At 412, an additional tip is loaded. An additional tip may refer to an additional set of tips. At 414, an additional sample is loaded and dispensed. An additional sample may refer to an additional set of samples. At 416, the additional tip is discarded. [0092] At 418, an evaluation of whether all samples have been dispensed is performed. In embodiments, the evaluation may be performed by determining whether all wells have been supplied with a sample. In other embodiments, the evaluation may be performed by determining whether a volume have been drawn and dispensed from each initial cell line sample. If the determination is made that additional samples remain to be dispensed, additional tips may be loaded as at 412. If the determination is made that all samples have been dispensed, the inoculation process may end as at 420. [0093] Illustrative examples of the systems and methods described herein are provided below. An embodiment of the system or method described herein may include any one or more, and any combination of, the clauses described below.

[0094] Clause 1. A method of automated startup of a bioreactor, the method including: counting, by a cell health module, a number of viable cells in a cell sample; receiving a desired starting density of cells; determining a volume of media and a volume of the cell sample, based on the number of viable cells in the cell sample, to be combined to yield a predetermined density of cells in a bioreactor; combining media and cell sample to yield a cell culture by supplying the volume of media to the bioreactor; and supplying the volume of the cell sample to the bioreactor.

[0095] Clause 2. The method of clause 1, wherein the cells are mammalian cells.

[0096] Clause 3. The method of clause 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 above 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 includes: 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 supplying the volume of the cell sample to the bioreactor to determine an ending density of cells.

[0100] Clause 7. The method of clause 6, further including: determining the second number of cells in the bioreactor is below a predetermined threshold; calculating a second volume of the cell sample the inject into the bioreactor to yield the 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 including: determining the second number of cells in the bioreactor is above a predetermined threshold; calculating a second volume of media the inj ect into the bioreactor to yield the predetermined density’ of cells; and injecting the second volume of media into the bioreactor.

[0102] Clause 9. The method of clause 5 or 6, wherein counting the second number of cells is performed after a predetermined time period following supplying the volume of the cell sample into the bioreactor to determine growth by the cells in the bioreactor.

[0103] Clause 10. The method of clause 9, further including: comparing the first number of cells and the second number of cells over time. [0104] Clause 11. The method of clause 9 or 10, wherein the predetermined time period is one or more days.

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

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

[0108] Clause 15. The method of clause 13 or 14, wherein the volume of the cell sample for each well of the plurality' of wells is taken from a same cell sample such that each well of the plurality' of wells contains a same kind of cells.

[0109] Clause 16. The method of any of clauses 13-15, wherein the volume of the cell sample for at least one well of the plurality of wells is taken from a different cell sample than the volume of the cell sample for a remainder of the plurality of wells such that the at least one well of the plurality' of wells contains a different kind of cells than the remainder of the plurality of wells.

[0110] Clause 17. The method of any of clauses 13-16, wherein supplying the volume of media to each well of the plurality of wells is performed using a first tip; and supplying a volume of the cell sample to a first well of the plurality of wells is performed using the first tip.

[OHl] Clause 18. The method of any of clauses 13-17, wherein the plurality of wells in the bioreactor include a plurality of wells among a series of bioreactors.

[0112] Clause 19. The method of any of clauses 13-18, further including: culturing the 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 as compared to other wells of the plurality of wells.

[0113] Clause 20. The method of any of clauses 13-19, wherein injectingthe volume of media into each well of a plurality of wells in a 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. [0114] Clause 22. The method of any of the above clauses, wherein the cell sample is maintained.

[0115] Clause 23. A system for automating seeding 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 a number of cells in the cell sample; the bioreactor including a number of wells; and a processor in communication with a memory, the memory storing instructions which, when executed by the processor, cause the system to: receive the number of cells in the cell sample; determine a volume of media and a volume of the cell sample, based on the number of cells in the cell sample, to be combined to yield a predetermined density of cells; operate a fluid transfer device to: deposit the volume of media in 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 pipete.

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

[0118] Clause 26. The system of any of clauses 23-25, further including a receiving zone for pipete tips.

[0119] Clause 27. The system of any of clauses 23-26, wherein the receiving zone is configured to receive a number of tips, wherein the number of tips is equal to the number of wells.

[0120] Clause 28. The system of any of clauses 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 a range of 2 to 8 degrees Celsius.

[0122] Clause 30. A method for optimizing pipete tip usage, the method including: pipeting a media into a number of sample wells using a first pipete tip; and pipeting a number of samples in the number of sample wells, the number of samples equal to the number of wells, wherein the first sample is pipeted using the first pipete tip.

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

[0124] Having described the preferred aspects and implementations of the present disclosure, modifications and equivalents of the disclosed concepts may readily occur to one skilled in the art. However, it is intended that such modifications and equivalents be included within the scope of the claims which are appended hereto.

Claims

What is claimed is:

1. A method of automated startup of a bioreactor, the method comprising: counting, by a cell health module, a number of viable cells in a cell sample; receiving a desired starting density of cells; determining a volume of media and a volume of the cell sample, based on the number of viable cells in the cell sample, to be combined to yield a predetermined density of cells in a bioreactor; combining media and cell sample to yield a cell culture by supplying the volume of media to the bioreactor; and supplying the volume of the cell sample to the bioreactor.

2. The method of claim 1, wherein the cells are mammalian cells.

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

4. The method of any of claims 1-3, 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. The method of claim 4, wherein counting the second number of cells is performed immediately after supplying the volume of the cell sample to the bioreactor to determine an ending density of cells.

6. The method of claim 5, further comprising: determining the second number of cells in the bioreactor is below a predetermined threshold; calculating a second volume of the cell sample the inject into the bioreactor to yield the predetermined density of cells; and injecting the second volume of the cell sample into the bioreactor.

7. The method of claim 5, further comprising: determining the second number of cells in the bioreactor is above a predetermined threshold; calculating a second volume of media the inject into the bioreactor to yield the predetermined density of cells; and injecting the second volume of media into the bioreactor.

8. The method of claim 4 or 5, wherein counting the second number of cells is performed after a predetermined time period following supplying the volume of the cell sample into the bioreactor to determine growth by the cells in the bioreactor.

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

10. The method of any of claims 1-9, wherein counting, by a cell health module, the number of viable cells in the cell sample comprises counting a number of viable cells in a plurality of cell samples.

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

12. The method of any of claims 1-11, wherein the volume of the cell sample for each well of the plurality of wells is taken from a same cell sample such that each well of the plurality of wells contains a same kind of cells.

13. The method of any of claims 1-12, wherein supplying the volume of media to each well of the plurality' of wells is performed using a first tip; and supplying a volume of the cell sample to a first well of the plurality of wells is performed using the first tip.

14. The method of any of claims 1-13, further comprising: culturing the cell sample in each w ell of the plurality' of wells; evaluating cell grow th 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 as compared to other wells of the plurality of wells.

15. The method of any of claims 1-14. wherein the cell sample is maintained.