Normalization of bioreactor vaccination

Abstract

A method for automated start-up of a bioreactor and standardized inoculation of a cell culture by counting the number of viable cells in a cell sample by a cell health module. A desired starting cell density is received, and a volume of culture medium and a volume of the cell sample to be combined in the bioreactor to produce a predetermined cell density are respectively determined based on the number of cells in the cell sample. The culture medium and the cell sample are combined by supplying the volume of culture medium to the bioreactor and supplying the volume of cell sample to the bioreactor to produce a cell culture.

Description

Technical Field

[0001] This application was filed as a PCT International Application on January 23, 2024, 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 incorporated herein by reference in its entirety. Background Art

[0002] Cell line development is the process of growing cells in culture. Cell line development enables the evaluation of differences between individual cells and can be used to confirm the monoclonality of a particular cell line. Cell line development can be used to generate a variety of biomolecules, such as for the development of pharmaceuticals. Therefore, cell lines are evaluated based on a variety of criteria, such as protein titer or cell line stability. Cell lines are typically established by first isolating single living cells or otherwise establishing identical starting conditions to enable equal comparison of each cell line.

[0003] One application of cell line development is the production of therapeutic monoclonal antibodies (mAbs). Production of mAbs begins with a single, clonal producer cell line that stably expresses the target mAb of interest. The producer cell line used for production needs to be robust, stable, and scalable for large-scale cultivation in bioreactors. Key criteria for a producer cell line include monoclonality, high productivity, appropriate post-translational modifications, and good quality. Such a producer cell line can be generated from a cell line development process that begins with transfection, selection, and isolation of individual clones from a large number of monoclonal candidate clones. To shorten timelines and reduce costs, high-throughput workflows are often used to screen a small number of good candidate clones. These selected clones are then sent for downstream processing optimization to select the final producer cell line. Summary of the Invention

[0004] Examples presented herein relate to a method for automating the startup of a bioreactor. The method comprises the following steps: counting the number of viable cells in a cell sample using a cell health module; receiving a desired starting cell density; determining, based on the number of viable cells in the cell sample, a volume of culture medium and a volume of the cell sample to be combined in the bioreactor to produce a predetermined cell density; and combining the culture medium and the cell sample to produce a cell culture by supplying the volume of culture medium and 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 still other examples presented herein, the cell sample is in a source plate. In still other examples presented herein, the number of cells is a first number of cells, and the method further comprises counting a second number of cells in the bioreactor.

[0006] In some 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 the final cell density. In other examples presented 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 injected into the bioreactor to produce a predetermined cell density; and injecting the second volume of the cell sample into the bioreactor. In some further examples presented herein, the method includes determining that the second number of cells in the bioreactor is above a predetermined threshold; calculating a second volume of culture medium injected into the bioreactor to produce a predetermined cell density; and injecting the second volume of culture medium into the bioreactor.

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

[0008] In some other examples presented herein, counting the number of viable cells in the cell sample by the cell health module includes counting the number of viable cells in a plurality of cell samples. In some other examples presented herein, combining the culture medium and the cell sample by supplying the culture medium and the cell sample to the bioreactor includes supplying each of the volume of culture medium and the volume of the cell sample to each of the plurality of wells in the bioreactor. In some further examples presented herein, the predetermined cell density of each of the plurality of wells in the bioreactor is the same.

[0009] In other examples presented herein, the volume of cell sample in each of the plurality of wells is taken from the same cell sample, such that each of the plurality of wells contains the same type of cells. In still other examples presented herein, the volume of cell sample in at least one of the plurality of wells is taken from a different cell sample than the volume of cell sample in the remaining wells in the plurality of wells, such that at least one of the plurality of wells contains a different type of cells than the remaining wells in the plurality of wells. In still other examples presented herein, supplying the volume of culture medium to each of the plurality of wells is performed using a first pipette tip; and supplying the volume of cell sample to the first well in the plurality of wells is performed using a first pipette tip.

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

[0011] Other examples presented herein relate to a system for automatically inoculating 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 the number of cells in the cell sample; a bioreactor comprising a plurality 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, based on the number of cells in the cell sample, a volume of culture medium and a volume of the cell sample to be combined to produce a predetermined cell density; operate a fluid transfer device to: deposit the volume of culture medium in each of the plurality of wells in the bioreactor; and deposit the volume of the cell sample in each of the plurality 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 of the pipette is used to inject the volume of culture medium into each of the plurality of wells and to inject the volume of cell sample into a first well of the plurality of wells. In still other examples presented herein, the system includes a receiving area for a pipette tip.

[0013] In some other examples presented herein, the receiving area is configured to receive a plurality of pipette tips, wherein the number of pipette tips is equal to the number of wells. In some other examples presented herein, the source plate is further configured to maintain the cell sample at a predetermined temperature. In some other examples presented herein, the predetermined temperature is in the range of 2 degrees Celsius to 8 degrees Celsius.

[0014] Other examples presented herein relate to methods for optimizing pipette tip usage. The methods include pipetting culture medium into a plurality of sample wells using a first pipette tip; and pipetting a plurality of samples into the plurality of sample wells, the number of samples being equal to the number of wells, wherein the first sample is pipetted using the first pipette tip.

[0015] Still other examples presented herein relate to a system for optimizing pipette tip usage. The system includes: a culture medium source; a sample source; a plurality of sample wells; a pipette having a plurality of mandrels equal to the number of sample wells; and a plurality of pipette tips, such that each mandrel in the plurality of mandrels is associated with a pipette tip; wherein the pipette fills each of the plurality of sample wells with a first pipette tip from the plurality of pipette tips from the culture medium source, and adds a sample from the sample source to each of the plurality of sample wells, wherein a first sample in the first pipette well is added using the first pipette tip.

[0016] In the following description, various additional inventive aspects will be described. These inventive aspects may relate to individual features or to combinations of features. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and illustrative and are not intended to limit the broad inventive concepts on which the embodiments disclosed herein are based. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0019] Figure 2 yes Figure 1 Component diagram of an exemplary CLD workstation.

[0020] Figure 3 yes Figure 1 Platform component layout of an exemplary CLD workstation.

[0021] Figure 4 yes Figure 1 Diagram of the bioreactor components of an exemplary CLD workstation.

[0022] Figure 5 yes Figure 1 Component diagram of an exemplary CLD workstation's gantry system.

[0023] Figure 6 yes Figure 5 Diagram of the components of the gantry system for the liquid handler.

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

[0025] Figure 8 is a flow chart of an exemplary method for performing automated bioreactor startup for a CLD process.

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

[0027] 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 numerals will be used throughout the drawings to refer to the same or like parts.

[0028] Cell line development (CLD) is the process of propagating one or more cells with unique and reproducible characteristics that define a cell line. Effective CLD requires the precise establishment of one or more initial cells to initiate a cell line so that the overall characteristics of the cell line can be effectively traced back to the progenitor cells. This process can be referred to as "seeding" the cell line. As discussed herein, seeding refers to the preparation of one or more containers for cell line development with a precise number of initial viable cells, enabling the robustness of cell line proliferation to be accurately determined over time during the development process.

[0029] In some cases, the initial cell group can be separated and the individual cells or groups from the initial group can be propagated separately to evaluate the variation between the populations of the initial group. In such cases, it may be particularly important to start with an accurate amount of cells for each separation of the initial population to prevent variations in the initial amount of cells from interfering with the analysis of growth rate differences between the cell lines separated. This process can be referred to as "standardizing" the inoculation of the cell line.

[0030] Currently, there is no technology that can automatically standardize input cell lines to ensure that all bioreactors or all containers receive the same initial viable cell density. Standardization 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 the CLD process based on initial programmed or pre-programmed instructions without the need for operator or other user intervention. Some aspects of the present disclosure may particularly emphasize the inoculation and standardization steps in the CLD process.

[0031] Now refer to Figure 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 culture and develop cell lines based on input samples (e.g., input 102). The CLD system 100 may also provide control over various steps in the CLD process and analysis of samples and cell lines throughout the process.

[0032] Input 102 provides a starting point for developing one or more cell lines using the CLD system 100. Input 102 can be an input palette of multiple cell lines to be developed. The initial cell line can come from a cell bank or an earlier CLD process.

[0033] The CLD workstation 104 provides an integrated and automated platform for seeding and culturing cell lines. The CLD workstation 104 receives input 102 and is capable of analyzing the received cell line and combining the input sample with other components for the CLD process. Figure 2 The CLD workstation 104 is discussed in more detail.

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

[0035] Computing device 106 includes at least a processor and memory and can be any number of known computing devices or can be a special-purpose computing device. A computing device is a physical, tangible device that processes data. Some 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 typically includes at least one central processing unit (CPU), system memory, and a system bus that connects the system memory to the CPU. System memory includes random access memory (RAM) and read-only memory (ROM). ROM stores a basic input / output system (BIOS), containing the basic routines that help transfer information between elements within the device, such as during startup. The device also includes a mass storage device. The mass storage device is capable of storing software instructions and data.

[0037] Mass storage devices and their associated computer-readable data storage media provide non-volatile, non-transitory storage for devices. Although the descriptions of computer-readable data storage media contained herein refer to mass storage devices, such as hard disks or CD-ROM drives, those skilled in the art will understand that a computer-readable data storage medium can be any available non-transitory physical device or article from which the device can read data and / or instructions.

[0038] Computer-readable data storage media include volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storing information (e.g., computer-readable software instructions, data structures, program modules, or other data). Some 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 the desired information and can be accessed by a computing device. In some embodiments, the computer-readable data storage medium includes non-transitory media.

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

[0040] In some embodiments, the computing device 106 is fully integrated within the CLD workstation 104 and can be operated via buttons, keys, or one or more touch screens on the CLD workstation 104. In some examples, the computing device 106 can be computer software loaded onto any number of general-purpose or custom combinations of processors and memory. The computing device 106 can enable analysis of data generated by the CLD workstation 104 and can also provide accessible readouts of the data and analysis of the data.

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

[0042] Now refer to Figure 2 , showing Figure 1 FIG2 is a component diagram of an exemplary CLD workstation 104. The CLD workstation 104 may include a platform 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 receives cell line input and is capable of analyzing the received cell line. The CLD workstation 104 is capable of automatically combining the input sample with other components used in the CLD process.

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

[0044] The platform 110 provides a platform base for the CLD workstation 104. The platforms 110 may be arranged in a grid to assist in guiding the gantry system 112. The platforms 110 may include positioning indicators, such as pre-drilled holes in the platform, which may be used to position components on the platform 110 or, in some embodiments, off-platform. The configuration of the platform 110 may be controlled by a computer program or device (e.g., Figure 1 The following is about Figure 3 The platform 110 and the configuration of the platform 110 are discussed in greater detail.

[0045] The gantry system 112 provides an automated transfer system to facilitate positioning components on the platform 110 and executing automated solutions, such as CLD processes, within the workstation 104. The chassis 114 provides a framework for the gantry system 112 and, in some embodiments, a base platform for the workstation 104 as a whole, and may also support the platform 110. The chassis 114 supports the gripper 116 and other arms of the gantry system 112.

[0046] The gripper 116 provides for movement of labware and microplates from one platform location to another, including movement to peripheral handling devices such as heating / cooling or shaking devices, and movement to off-platform instruments via a shuttle transport system.

[0047] Liquid handler 118 provides interchangeable head, and it can receive multiple laboratory operation tools and provides the automation operation of such tool by using gantry system 112.Liquid handler 118 can be usually automated liquid handling pipette.In some embodiments, liquid handler 118 can be single channel or multi-channel pipette.Liquid handler 118 can be configured to carry out specific liquid handling program.For example, liquid handler 118 can be configured to aspirate from the hole of one position by first contacting the top of sample and then gradually moving downward as sample is moved out.This can avoid accumulating excessive cells on the outside of pipette tip, as occurs when tip is immersed in bottom all the time.Liquid handler 118 can be configured to store or receive the geometric parameters of container to provide effective aspiration to specific container.

[0048] Different tools and / or tip types can be associated with the liquid handler 118, depending on the head and the desired liquid handling procedure. Interchangeable heads mounted on the liquid handler 118 can be used, for example, to aspirate and dispense liquids using disposable tips. The liquid handler 118 can be configured to accommodate a tip interface for fixed or disposable tips, and to perform both level sensing and non-level sensing operations.

[0049] The CLD workstation 104 may include other elements and features not directly related to the CLD process. For example, some embodiments may include a platform viewing system or one or more tools to help build and arrange labware as needed for an automated CLD workflow.

[0050] Now refer to Figure 3 , showing Figure 21 shows a layout of platform components of the platform 110 of an exemplary CLD workstation 104. The platform 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 platform 110 may be important for the efficient execution of a CLD process, particularly where the process is automated. Those skilled in the art will appreciate that many layouts are possible and suitable for a CLD process. Figure 3 The layout presented in FIG. 1 is presented as a non-limiting example of one possible layout to illustrate components and considerations for a CLD process layout. The platform component layout may vary depending on the specific procedure being performed and the tools being used. For example, the tip source 128 may not be included when disposable tips are not needed or used.

[0051] The input receiving area 122 is used to receive a starting cell line sample (e.g. Figure 1 The workstation 104 may be configured to receive a cell line from a single plate or multiple plates (input 102). The cell sample or other input may be a single sample or multiple samples. The sample may be introduced to the plate before the automated CLD process is initiated at the workstation 104. Multiple cell line samples may be arranged on one or more input plates, and the workstation 104 may be configured to seed cell lines derived from more than one sample based on the arrangement of the cell line samples on the input receiving area 122.

[0052] The bioreactor 124 is a receiving area of one or more containers for receiving the initial cell line sample and culture medium and providing 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 some embodiments, the bioreactor 124 may refer to a container component placed in a bioreactor located external to the workstation 104. For example, the bioreactor 124 may comprise a 96-well plate, wherein each of the 96 wells is inoculated with a separate cell line. In some embodiments, the bioreactor may be a microbioreactor or a microreactor. Figure 4 Bioreactor 124 is discussed in more detail.

[0053] The culture medium source 126 can provide a known location for the workstation 104 to obtain culture medium when performing the CLD process. The culture medium provides the nutrients required for cell line development.

[0054] The tip source 128 provides the workstation 104 with a known location for obtaining clean, uncontaminated tips throughout the execution of the CLD process. For example, a clean tip can be used to inoculate a single cell line to avoid contamination or carryover from one cell line inoculation to the next. In some embodiments, a single tip can be used to supply culture medium to each vessel or well in the bioreactor 124 before inoculating each cell line. In some embodiments, the same tip used to supply culture medium can also be used to inoculate the cell line before being discarded. The same tip can generally be used to inoculate a single vessel or well. After dispensing culture medium to all vessels to be inoculated, the same tip can generally be used to inoculate the first vessel.

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

[0056] The platform 110 may also include other components and areas required to effectively perform the CLD process by the workstation 104. For example, the platform 110 may also include a waste area where a waste container or a tip disposal container may be located.

[0057] Reference Figure 4 , showing Figure 2 Component diagram of an exemplary bioreactor 124 of an exemplary CLD workstation 104. The bioreactor 124 includes 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 can measure parameters such as biomass, pH, dissolved oxygen (DO), and fluorescence online while the culture is running in the culture chamber 134. The control panel 136 can be configured to allow the user to control the shaking speed, temperature, gas concentration, gas flow rate, and humidity within the culture chamber 134. Alternatively or additionally, the bioreactor 124 can be connected to a separate computing device (e.g., a computer) that can allow such control. Figure 1 In some embodiments, closed-loop control of pH and dissolved oxygen can be used, and the gas flow rate can be automatically adjusted based on observed changes in pH and / or dissolved oxygen.

[0059] Reference Figure 5 , showing Figure 2 FIG. 1 is a component diagram of the gantry system 112 of an exemplary CLD workstation 104. The gripper 116 and liquid handler 118 are shown in greater detail.

[0060] The gripper 116 is capable of moving laboratory equipment on the platform 110. The gripper 116 can be one of multiple grippers 116 incorporated into the gantry system 112. The gripper 116 can be configured for 360° rotation and include an offset finger. The gripper 116 can stack and unstack laboratory equipment and move plates and microplates from one platform location to another, including to peripheral processing devices such as heating / cooling and shaking devices. The gripper 116 can also support interaction with off-platform instruments (e.g., via a shuttle transport system).

[0061] The movement of the laboratory equipment can be programmed, for example, by communicating with a computing device (e.g. Figure 1 The user interface may enable definition of instrument platform layouts and labware types, and import and export of automated sample preparation methods.

[0062] The liquid handler 118 provides liquid transport between components within the workstation 104. The liquid handler 118 can be configured as a pipette. Liquid transport can be achieved by air or liquid displacement. In some instances, when aspirating a sample, a hydraulic piston in the head of the liquid handler mechanically pulls the liquid into a pipette tip immersed in the sample. The air in the pipette tip is replaced by the liquid entering the tip. In other instances, a syringe pump connected to a disposable or fixed tip via a hydraulic line mechanically moves the system fluid, thereby replacing the air in the tip with the liquid entering the tip. When the liquid is dispensed into the target laboratory appliance, the piston or syringe pump reverses motion and the liquid is discharged from the pipette tip.

[0063] Reference Figure 6 , showing Figure 5Component diagram of the liquid handler 118 of the gantry system 112. The liquid handler 118 may include one or more mandrels 138 and one or more tips 140. In some examples, the liquid handler 118 may be configured as an 8×12 or 16×24 pipetting array, for example, so that pipetting actions can be performed on up to 96 or 384 samples at a time. The liquid handler 118 may be configured with one or more independent pipetting probes. The probes may be configured to extend and retract to support the delivery of liquids to and from laboratory equipment with different well spacings and opening sizes. In some embodiments, one or more pipetting probes may be arranged in a linear plane.

[0064] Each mandrel 138 is an elongated, hollow structure used to connect a pipette tip 140 to the liquid handler 118. The mandrel 138 can typically be metal. The pipette tip 140 engages the mandrel 138 and forms a seal. The pipette tip 140 provides precise movement of liquids into and out of a container. In some embodiments, the pipette tip 140 is disposable. Disposable pipette tips are generally preferred for transferring liquids from a source container (e.g., a tube or microplate) to a destination container.

[0065] Reference Figure 7 , shows a process diagram of an exemplary CLD process 200. The process 200 may involve an input plate 202, an automated transfer system 204, a cell health module 206, a computing device 208, a culture medium reservoir 210, and one or more bioreactors 212. The process 200 may be performed using a CLD workstation (e.g., Figure 1 and Figure 2 The CLD workstation 104 is used.

[0066] Input plate 202 (which may represent a sample input, e.g. Figure 1 Input plate 202 (input 102) can be introduced into the workstation prior to initiating the CLD process 200. Input plate 202 can contain one or more initial cell line samples. Input plate 202 can be, for example, a microtiter plate having any number of wells. In one example, input plate 202 can be a 1- to 96-well microtiter plate. Input plate 202 can be the sample source and contain one or more cell lines of interest in different wells. The cell lines in input plate 202 can be compared for titer. In some embodiments, input plate 202 can be maintained by the system in a temperature-controlled manner to ensure that cell health is not compromised.

[0067] At this stage in the cell line development process, it is expected that each of these cell lines will have varying cell densities. The cell lines in the input plate 202 can be transferred to the cell health module 206 (e.g., via an automated transfer system 204) to assess the initial cell density of each sample cell line. The automated transfer system 204 (which can represent a liquid transfer system, e.g., Figure 2 and Figure 5 The liquid handler 118 of the CLD process 200 provides for the movement of liquids to various containers and components as needed by the CLD process 200. The automated transfer system 204 may be operated by or receive instructions from a computing device 208. The cell health module 206 (which may represent a cell health module, such as Figure 3 The Cell Health Module 130) provides the instrumentation necessary to determine cell density and viability in cell samples and cell line cultures.

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

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

[0070] The computing device 208 can receive the target cell culture density for inoculating the bioreactor 212. The computing device 208 determines the volume of each culture medium and each cell line sample to be combined to produce a cell culture having the target cell culture density. In some embodiments, multiple cell cultures can be inoculated from a single sample cell line. Therefore, each cell culture can be inoculated with the same volume of each culture medium and sample cell line. In some embodiments, cell cultures can be inoculated from multiple sample cell lines. Each cell culture can be inoculated with different volumes of each culture medium and corresponding sample cell line, respectively, because each cell line in the multiple cell lines typically has a different cell density.

[0071] The computing device 208 can provide control of system components, such as the automated transfer system 204, based on stored instructions and calculated values. Upon receiving the cell density of the initial cell line sample in the input plate 202 from the cell health module 206, the computing device 208 calculates the volume of one or more sample cell lines and the volume of culture medium from the input plate 202. The computing device 208 operates the automated transfer system 204 or provides instructions to the automated transfer system 204 to supply the calculated volume of culture medium and the one or more initial sample cell lines to the bioreactor 212.

[0072] Culture medium reservoir 210 (which may represent a culture medium source, e.g. Figure 3 The culture medium source 126 provides a source of culture medium for supply to the bioreactor 212 to prepare cell cultures for cell line development. The automated transfer system 204 can be programmed or operated to move a calculated volume of culture medium from the culture medium reservoir 210 to the bioreactor 212.

[0073] Bioreactor 212 (which may represent a bioreactor, e.g. Figure 3 and Figure 4 The bioreactor 212 (of the embodiment of the present invention) provides an environment configured to promote cell growth and cell line development. The bioreactor 212 receives calculated volumes of culture medium and an initial cell line sample, which can be combined in a container or plate within the bioreactor 212 or combined outside the bioreactor 212 and then placed within the bioreactor. The bioreactor 212 can represent an integrated or separate component that together maintain a desired CLD environment. Parameters for the CLD environment can be stored by or determined by the computing device 208. The bioreactor 212 can be operated by the computing device 208 or receive instructions from the computing device 208 to establish and maintain the desired CLD environment, such as thermal cycling, shaking, humidity, etc.

[0074] Reference Figure 8 , a flow chart showing an exemplary method 300 for automated bioreactor startup of a CLD process is shown. The method 300 may be performed by an automated workstation (e.g., Figure 1 and Figure 2 The instructions for performing the method 300 may be provided by a computing device (e.g., a workstation 104). Figure 1 computing device 106 or Figure 7 The computing device 208 may store and transmit the data to the automated workstation. The computing device may communicate with the automated workstation via wired or wireless communication, receiving data from components of the workstation and providing instructions to components of the workstation. The computing device may also receive initial or target parameters from a user prior to initiating method 300. The initial or target parameters may be internally determined by the computing device based on previously established parameters or stored instructions.

[0075] At 302, a sample source plate may be cooled. The source plate may correspond to Figure 1 Input 102 and / or Figure 7 The input plate 202 is cooled. Cooling the source plate can reduce cell viability in the sample cell line, improving the accuracy of cell counts and viable cell counts. In some embodiments, step 302 may not be included, and the cell density of the sample cell line may be assessed without cooling. In some embodiments, the input plate may be received pre-cooled.

[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 count may be performed by a cell health module (e.g., Figure 3 Cell Health Module 130 or Figure 7 The counting can be done by placing or transferring all or part of each sample into the cell health module. The transfer can be done by an automated transfer system (e.g. Figure 2 、 Figure 5 and Figure 6 gantry system 112 or Figure 7 This is accomplished by an automated transfer system 204).

[0077] In some embodiments, a single sample cell line can be used. In other embodiments, multiple sample cell lines can be used and each counted separately. In cases where multiple sample cell lines are involved, each sample can be identified based on its position on the input plate or its orientation within the automated workstation.

[0078] 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, a calculation is performed at 306. In some embodiments, the system can perform calculations on already counted samples in parallel while continuing to count additional samples.

[0079] At 308, the volumes of culture medium and cell line sample to be combined to produce the cell culture are calculated. The calculated volume is determined based on 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, characteristics of the culture medium to be used in the cell culture, etc.

[0080] The volumes are calculated so that when the volume of culture medium and the volume of the initial cell line sample are combined to produce the cell cultures, each cell culture has 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 can typically be calculated in terms of cells per unit volume, e.g., 2 × 10 6 In some cases, it may be desirable to have a default target cell density, such as one cell per cell culture. The calculation may be performed by a computing device (e.g. Figure 1 computing device 106 or Figure 7 The calculation device 208 is performed.

[0081] At 310, a bioreactor container or plate is inoculated with a cell culture. Each of the calculated volumes of culture medium and each of the one or more initial cell line samples are combined in a container configured to be placed in a culture chamber of a bioreactor. Figure 9 Methods for inoculating a bioreactor vessel or plate are described in more detail. Inoculation can be automated in response to calculations of culture medium and cell sample volumes.

[0082] After inoculation, before placement in a bioreactor or before starting the bioreactor to provide growth conditions for the cells, the cell density of each cell culture can be measured. Measuring cell density after inoculation but before cell growth verifies uniform cell density or compliance with a target cell density for each cell culture before initiating culture. If a particular well or container is found to have a cell density below the target cell density, an additional volume of the relevant cell line sample can be calculated and added to increase the cell density to the target cell density. If a particular well or container is found to have a cell density above the target cell density, an additional volume of culture medium can be calculated and added to reduce the cell density to the target cell density.

[0083] At 312, the cell culture is shaken. In some 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 limited to factors and conditions that may be set during the growth phase of a CLD process.

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

[0085] Reference Figure 9 , a flow chart of a method 400 for inoculating a bioreactor vessel or plate is shown. The method 400 can be performed by an automated liquid transfer system (e.g., Figure 2 、 Figure 5 and Figure 6 gantry system 112 or Figure 7 The automated transfer system 204) is used.

[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 the fluid to be transferred. In some embodiments, method 400 can be performed using sterile tips. In some embodiments, the first tip can refer to a first set of tips.

[0087] At 404, a first pipette tip is used to load and dispense culture medium into a cell culture vessel or plate. The volume of culture medium to be loaded and dispensed can be pre-calculated based on 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 containers or wells have been supplied with culture medium. For example, if a culture plate comprises multiple wells, each well may need to be supplied with a certain volume of culture medium. If wells still require culture medium, the calculated volume of culture medium is loaded and dispensed as described at 404. If all wells have been supplied with culture medium, sample loading and dispensing may proceed.

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

[0090] The first tip is discarded at 410. Once a tip is used to transfer a sample, the tip may be considered contaminated and unsuitable for use in transferring any additional samples.

[0091] At 412, additional tips are loaded. Additional tips may refer to additional sets of tips. At 414, additional samples are loaded and dispensed. Additional samples may refer to additional sets of samples. At 416, the additional tips are discarded.

[0092] At 418, an assessment is made as to whether all samples have been dispensed. In some embodiments, this assessment can be made by determining whether all wells have been supplied with samples. In other embodiments, this assessment can be made by determining whether a certain volume has been drawn and dispensed from each initial cell line sample. If it is determined that additional samples remain to be dispensed, additional tips can be loaded as per 412. If it is determined that all samples have been dispensed, the inoculation process can be terminated as per 420.

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

[0094] Item 1. A method for automating the start-up of a bioreactor, the method comprising: counting the number of viable cells in a cell sample by a cell health module; receiving a desired starting cell density; determining a volume of culture medium and a volume of cell sample to be combined in a bioreactor to produce a predetermined cell density based on the number of viable cells in the cell sample; and combining the culture medium and cell sample to produce a cell culture by supplying the volume of culture medium to the bioreactor and supplying the volume of cell sample to the bioreactor.

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

[0096] Clause 3. The method of Clause 1 or 2, wherein the predetermined number of cells is a predetermined number of living 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 preceding clause, 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 supplying the volume of cell sample to the bioreactor to determine a final cell density.

[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 produce a predetermined cell density; 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 culture medium to be injected into the bioreactor to produce a predetermined cell density; and injecting the second volume of culture medium 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 period of time after supplying the volume of the cell sample to the bioreactor to determine growth of 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 10, wherein the predetermined time period is one or more days.

[0105] Clause 12. The method of any one of the preceding clauses, wherein counting the number of viable cells in the cell sample by the cell health module comprises 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 culture medium and the cell sample by supplying the culture medium and the cell sample to a bioreactor comprises supplying each of the volume of culture medium and the volume of cell sample to each well of a plurality of wells in the bioreactor.

[0107] Clause 14. The method of Clause 13, wherein the predetermined cell density is the same in each of the plurality of wells in the bioreactor.

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

[0109] Item 16. The method of any one of Items 13 to 15, wherein the volume of cell sample in at least one of the plurality of wells is taken from a different cell sample than the volume of cell samples in the remaining wells of the plurality of wells, so that at least one of the plurality of wells contains cells of a different type than the remaining wells of the plurality of wells.

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

[0111] Clause 18. The method of any one of Clauses 13 to 17, wherein the plurality of wells in the bioreactor comprises a plurality of wells in a series of bioreactors.

[0112] Clause 19. The method of any one of clauses 13 to 18, further comprising: 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 a higher productivity compared to other wells of the plurality of wells.

[0113] Clause 20. The method of any one of clauses 13 to 19, wherein injecting the volume of culture medium into each of a plurality of wells in a bioreactor is performed using a first pipette tip; and injecting the volume of cell sample into a first well of a plurality of wells in the bioreactor is performed using the first pipette tip.

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

[0115] Item 23. A system for automatically inoculating a bioreactor with a predetermined number of cells, the system comprising: 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 comprising a plurality 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, based on the number of cells in the cell sample, a volume of culture medium and a volume of the cell sample to be combined to produce a predetermined cell density; operate a fluid transfer device to: deposit the volume of culture medium in each of the plurality of wells in the bioreactor; and deposit the volume of the cell sample in each of the plurality of wells in the bioreactor.

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

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

[0118] Clause 26. The system of any one of clauses 23 to 25, further comprising a receiving area for a pipette tip.

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

[0120] Clause 28. The system of any one of clauses 23 to 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 in the range of 2 to 8 degrees Celsius.

[0122] Item 30. A method for optimizing pipette tip usage, the method comprising: pipetting culture medium into a plurality of sample wells using a first pipette tip; and pipetting a plurality of samples into a plurality of sample wells, the number of samples being equal to the number of wells, wherein a first sample is pipetted using the first tip.

[0123] Item 31. A system for optimizing the use of pipette tips, the system comprising: a culture medium source; a sample source; a plurality of sample wells; a pipette having a plurality of core shafts equal to the number of sample wells; and a plurality of pipette tips, such that each core shaft in the plurality of core shafts is associated with a pipette tip; wherein the pipette fills each of the plurality of sample wells with a first pipette tip from the culture medium source and adds a sample from the sample source to each of the plurality of sample wells, wherein a first sample in a first pipette well is added using the first pipette tip.

[0124] Having described preferred aspects and implementations of the present disclosure, 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. A method for automatically starting a bioreactor, the method comprising: Count the number of live cells in the cell sample using the cell health module; Receiving the desired starting cell density; determining, based on the number of viable cells in the cell sample, a volume of culture medium and a volume of the cell sample to be combined in a bioreactor to produce a predetermined cell density; The culture medium and the cell sample are combined to produce a cell culture by supplying the volume of culture medium to the bioreactor and supplying the volume of the cell sample to the bioreactor.

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

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

4. The method of any one of claims 1 to 3, wherein the number of cells is a first number of cells, and the method further comprises: A second number of cells in the bioreactor is counted.

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

6. The method of claim 5, 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 produce the predetermined cell density; as well as The second volume of the cell sample is injected into the bioreactor.

7. The method of claim 5, further comprising: determining that a second number of cells in the bioreactor is above a predetermined threshold; calculating a second volume of culture medium to be injected into the bioreactor to produce the predetermined cell density; as well as The second volume of culture medium is injected into the bioreactor.

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

9. The method of claim 8, further comprising: The first number of cells and the second number of cells are compared over time. 10 . The method of claim 1 , wherein counting the number of viable cells in the cell sample by the cell health module 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 cell density is the same in each of the plurality of wells in the bioreactor.

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

13. The method of any one of claims 1 to 12, wherein supplying the volume of culture medium to each well of the plurality of wells is performed using a first pipette tip; and Supplying the cell sample volume to a first well of the plurality of wells is performed using the first pipette tip.

14. The method of any one of claims 1 to 13, further comprising: 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; as well as One or more wells are identified as containing a cell line having a higher productivity compared to other wells in the plurality of wells.

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

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