Automated system for clone selection based on antibody production and measurement of cell health parameters

Abstract

Systems and method for and automated laboratory instrument for measuring protein concentration and cell health of a biological sample. The automated laboratory instrument includes a housing containing a biological sample container for receiving at least one biological sample, a pipettor to aspirate and dispense the biological sample, a titer instrument to receive the biological sample and measure protein concentration of the biological sample, a cell health instrument to receive the biological sample and measure cell health of the biological sample and processing circuitry having a memory for storing instructions. When executed by the processing circuitry, the instructions cause the processing circuitry to aspirate the biological sample from the container into the pipettor, dispense the biological sample into the titer and cell health instruments, measure protein concentration in the biological sample with the titer instrument, and measure cell health in the biological sample with the cell health instrument.

Description

AUTOMATED SYSTEM FOR CLONE SELECTION BASED ON ANTIBODYPRODUCTION AND MEASUREMENT OF CELL HEALTH PARAMETERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is being filed on October 11, 2024, as a PCT International application and claims the benefit of and priority to U.S. Application No. 63 / 612,256, filed on December 19, 2023, entitled AUTOMATED SYSTEM FOR CLONE SELECTION BASED ON ANTIBODY PRODUCTION AND MEASUREMENT OF CELL HEALTH PARAMETERS, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Cell culturing campaigns are conducted to cultivate and maintain cells outside their natural environment for a variety7of scientific, medical, and industrial purposes. In research, cell culturing allows scientists to study cellular behavior, explore disease mechanisms, and test potential treatments. It serves as a crucial tool in drug development, enabling the screening of compounds and the production of therapeutic proteins. Biotechnological applications involve the large-scale production of cells for the manufacturing of vaccines, antibodies, and other bio-based products. Additionally, cell culturing is integral to regenerative medicine, where it plays a role in growing tissues and organs for transplantation. The controlled environment of a cell culture enables researchers to manipulate and observe cells under specific conditions, providing valuable insights into fundamental biological processes and facilitating advancements across various fields.SUMMARY

[0003] Examples presented herein relate to an automated laboratory instrument for measuring protein concentration and cell health of a biological sample. The automated laboratory instrument includes a housing containing a biological sample container for receiving at least one biological sample, a pipettor configured to aspirate and dispense the biological sample, a titer instrument configured to receive the biological sample and measure protein concentration of the biological sample, a cell health instrument configured to receive the biological sample and measure parameters of cell health of the biological sample and processing circuitry having a memory for storing instructions.When executed by the processing circuitry, the instructions cause the processing circuitry to aspirate the biological sample from the container into the pipettor, dispense the biological sample into the titer and cell health instruments, measure protein concentration in the biological sample with the titer instrument, and measure cell health in the biological sample with the cell health instrument.

[0004] Other examples presented herein relate to a method of clone selection and / or automated cell line development using an automated laboratory instrument. The method includes obtaining, from a titer instrument of the automated laboratory instrument, protein concentration in a sample of a plurality of biological samples where the protein can be, among other things, Immunoglobulin G (IgG); obtaining, from a cell health instrument of the automated laboratory instrument, measurements of parameters that describe cell health in the sample; and repeating, by a processing circuitry of the automated laboratory instrument, the steps of obtaining protein concentration and measure of cell health parameters for each sample of the plurality of biological samples at a predetermined interval(s) throughout a cell culture campaign.

[0005] 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 exemplary 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

[0006] 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:

[0007] FIG. 1 is an isometric view of an example of an automated cell culturing system that automates one or more aspects of a cell culturing campaign.

[0008] FIG. 2 is another isometric view of the cell culturing system having the top cover removed from the housing.

[0009] FIG. 3 is a top view of the work platform supported inside the housing.

[0010] FIG. 4 is an isometric view of the temperature controlled plate storage.

[0011] FIG. 5 is another isometric view of the temperature controlled plate storage.

[0012] FIG. 6 schematically illustrates an example of the titer instrument supported on the work platform.

[0013] FIG. 7 is an isometric view of an example titer module of the cell culturing system of FIG. 1.

[0014] FIG. 8 is an exploded view of the titer container assembly of titer module of FIG. 7.

[0015] FIG. 9 is a graph of an example of five different lots of fluorescent beads at different concentrations.

[0016] FIG. 10 is an isometric view of cell health module.

[0017] FIG. 1 1 is an isometric view of an example of a bioreactor.

[0018] FIG. 12 is a top view of the interior of the cultivation chamber of bioreactor.

[0019] FIG. 13 is a cutaway side view of a well of bioreactor plate.

[0020] FIG. 14 shows an example of a counterbalanced shaker table.

[0021] FIG. 15 shows another example of the counterbalanced shaker table.

[0022] FIG. 16 shows another example of the counterbalanced shaker table.

[0023] FIG. 17 shows another example of the counterbalanced shaker table.

[0024] FIG. 18 shows another example of the counterbalanced shaker table.

[0025] FIG. 19 is a flowchart of an example method for automated monitoring of a cell culturing campaign.

[0026] FIG. 20 is a flowchart of an example method for automated cell line development using an automated laboratory instrument.

[0027] FIG. 21 is a flowchart of an example method for operating an automated laboratory instrument for cell line development.

[0028] FIG. 22 shows an example view of a user interface for the automated cell culturing system.

[0029] FIG. 23 shows another example view of the user interface for the automated cell culturing system.

[0030] FIG. 24 shows another example view of the user interface for the automated cell culturing system.

[0031] FIG. 25 shows another example view of the user interface for the automated cell culturing system.

[0032] FIG. 26 shows another example view of the user interface for the automated cell culturing system.

[0033] FIG. 27 shows another example view of the user interface for the automated cell culturing system.

[0034] FIG. 28 shows another example view of the user interface for the automated cell culturing system.

[0035] FIG. 29 shows another example view of the user interface for the automated cell culturing system.

[0036] FIG. 30 shows an example view of a user interface for generating a new protocol.

[0037] FIG. 31 shows another example view of a user interface for generating a new protocol.

[0038] FIG. 32 shows another example view of the user interface for generating a new protocol.

[0039] FIG. 33 shows another example view of the user interface for generating a new protocol.

[0040] FIG. 34 shows another example view of the user interface for generating a new protocol.

[0041] FIG. 35 shows another example view of the user interface for generating a new protocol.

[0042] FIG. 36 shows another example view of the user interface for generating a new protocol.

[0043] FIG. 37 schematically illustrates an example of a controller of the cell culturing system.DETAILED DESCRIPTION

[0044] Disclosed herein is an automated liquid-handler housing titer and cell health measuring modules which provides an efficient, stable platform for time intensive cell culturing campaigns. In embodiments, platforms according to the present disclosure further incorporate a bioreactor or other amplification module, and other modules to support the cell culturing campaign.

[0045] Cell culturing is used in numerous research and production environments, such as the development and manufacture of biopharmaceuticals. Selecting a desirable cell line for production of a particular protein involves consideration of a number of factors, including per-cell productivity and cell density' at the production scale. For example, in some cases a highest producing cell line at the per-cell level may be increasing its population too slowly to match the volumetric productivity of a cell line that is reproducing more rapidly but producing the protein at a lower per-cell rate or ahigh-producing cell line may lose production significantly or entirely as its population expands. Because of this, cell lines cannot be effectively selected for large scale production based on protein output alone. In many cases, monoclonality is verified to ensure consistent production as population increases and multiple cloning rounds may be required to verify the consistent production.

[0046] 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.

[0047] FIG. 1 is an isometric view of an example of an automated cell culturing system 100 that automates one or more aspects of a cell culturing campaign. For example, cell samples may be analyzed to determine the cell health of a plurality of cell samples. For a single cell sample, the cell culturing system 100 may measure cell count, cell viability, antibody concentration (e.g., protein titer), and other cell characteristics with minimal interaction from a user of the system. Samples may be biological or control samples. Control samples may include, as some non-hmiting examples, biological standards, reference beads that fluoresce, etc.

[0048] The cell culturing system 100 may incorporate or include connectivity to automated bioreactors and other systems and devices. The cell culturing system 100 automates sample preparation, and minimizes sample volume requirements for measuring cell and other sample characteristics. The cell culturing system 100 provides remote access to data including the measured cell and other sample characteristics, supports multiple users at once, and is compatible with various information technology (IT) structures.

[0049] As shown in FIG. 1, the cell culturing system 100 includes a housing 102 that supports a work platform 200. The housing 102 includes a top cover 104 that can support a dispensing system 106 that will be described in more detail with reference to FIGS. 2 and 3. In embodiments, top cover 104 includes indicator 108 and air filtration system 110. Housing 102 further includes a door 116 for access to work platform 200 and sealing of the housing 102 during a cell culturing campaign for cleanliness and integrity. In embodiments, housing 102 is mounted on a cart 118, which may provide one or more of storage and mobility for the cell culturing system 100. Cell culturing system 100 is configured with one or more instruments to perform analysis in support of a cellculturing campaign. Instruments include titer instrument 400, cell health instrument 500, and bioreactor 600.

[0050] In embodiments, cell culturing system 100 further includes a controller 300 storing instructions for various operations of the cell culturing system 100. A single controller 300 may be configured to operate the various components of cell culturing system 100, or individual components may be associated with dedicated controllers. In some cases, the controller 300 may be arranged separately from cell culturing system 100 and remotely connected or controller 300 may be an integrated component of cell culturing system 100. Controller may include various input devices 352, including a code scanner 354. In examples, code scanner 354 may be a optical scanner, such as a barcode scanner or a QR code scanner.

[0051] FIG. 2 is another isometric view of the cell culturing system 100 having the top cover 104 removed from the housing 102, and thereby exposing the dispensing system 106. The dispensing system 106 includes a gripper 112 and a liquid handling pod 114 that is movably mounted within the housing 102 in the space and a gantry above the work platform 200. In some embodiments, liquid handling pod 1 14 includes multiple probes 120 which either move together as a unit for the purposes of aspirating and dispensing groups of adjacent samples from the containers, or may move independently for the purposes of aspirating and dispensing from a variety of non-adjacent locations. In some implementations, probe tips 122 are fixed, such that they are permanent until replacement during a service process, while in other implementations, probe tips 122 are disposable and may be replaced after each use. Embodiments with multiple probes 120 may include a combination of fixed and disposable tips.

[0052] The liquid handling pod 114 is mounted for movement along three mutually perpendicular axes (e.g., X-axis, Y-axis, and Z-axis of a three-dimensional cartesian coordinate system). The three-dimensional movement allows a distal end of the liquid handling pod 114, through which liquid aspiration and dispensing occurs, to access any container held on the work platform 200 inside the housing 102. A proximal end of the liquid handling pod 1 14 is fluidly connected to a bi-directional pump 124 having a movable actuator 126 that controls the mode of the pump. For example, a first mode can include aspirating and a second mode can include dispensing, and the movable actuator controls switching between the first and second modes, as well as a rate of liquid that is aspirated and dispensed under the first and second modes. As an example, the bidirectional pump can include a syringe pump. Movement of the liquid handling pod 114and the movable actuator is controlled by one or more step motors that operate under the control of a controller 300 that is programmable.

[0053] A tip wash station 128 cleans the probes 120 on the liquid handling pod 114. It contains a number of wells, with a portion of the wells containing tip wash reagent, and another portion containing DI water. The tip washing station cleans the fixed probes by first lowering the probes into the wells containing tip wash reagent, followed by rinsing on the water side of the device. To mitigate contamination, the water wells may be flushed with tip wash reagent daily. All wells may share a common drain. The tip wash station may be fixedly or movably mounted to the deck. Pressure for tip wash station 128 may be provided by pumps 130. Waste containers 132 and supply containers 134 are also associated with tip wash station 128.

[0054] FIG. 3 is a top view of the work platform 200 supported inside the housing 102. Referring now to FIGS. 2 and 3, the work platform 200 supports one or more tube racks that hold a plurality of containers containing samples and various types of reagents. In some embodiments, other forms of sample and / or reagent containers are used, such as multi-well plates. In some instances, at least some of the containers are empty. As shown in FIG. 3, the housing 102 is dimensioned to have a width W, a depth D, and a height H.

[0055] As shown in FIG. 3, the work platform 200 supports a sample receiving zone 202 where user may input a sample receptacle. Also provided are a disposal chute 204, tip storage 206. and temperature controlled plate storage 208. Work platform 200 supports one or more instruments to perform analysis in support of a cell culturing campaign. Instruments include titer instrument 400, cell health instrument 500, and bioreactor 600.

[0056] In some cases, uncontrolled condensation from the plate cooler will potentially cause water to pool up and damage equipment in the instrument, or short circuit electronics. FIGS. 4 and 5 are isometric view s of the temperature controlled plate storage 208. The feature places an plate receptacle 250 atop a temperature regulator 252 that collects any condensation formed on the cooled surfaces of the tub or labware placed inside the plate receptacle 250. In embodiments, plate receptacle 250 is formed of aluminum. Temperature regulator 252 may be a thermoelectric cooler (“TEC”). The condensation will pool together in a trough 256 along the perimeter of the plate receptacle 250. From there, it will move through a channel 258 in the side of the plate receptacle 250 and collect in a reservoir 260 sitting outside the plate receptacle 250. The reservoir 260 may also be aluminum and sits in the flow of the exhaust 262 emitted by thetemperature regulator 252. The exhaust 262 heats the reservoir, assisting in evaporating the collected condensation. A plastic insulator 264 may placed between the reservoir 260 and the plate receptacle 250 to limit heat transfer from the reservoir 260 to the plate receptacle 250.

[0057] FIG. 6 schematically illustrates an example of the titer instrument 400 supported on the work platform 200. The titer instrument 400 includes optical components that are used to measure a concentration of an antibody or another protein present in a cell sample. For example, the titer instrument 400 measures a concentration of Immunoglobulin G (IgG) present in a sample of cells.

[0058] More specifically, the titer instrument 400 calculates a fluorescence polarization measurement for measuring the concentration of protein in the sample of cells. Fluorescence polarization includes mixing the sample of cells with a fluorescence polarization assay, and subsequently measuring fluorescence polarization for determining the concentration of the protein in the sample of cells. While the titer instrument 400 is described herein with reference to examples measuring IgG concentration, the titer instrument 400 and the measurement techniques described herein can be used to measure the concentrations of additional types of proteins and antibodies.

[0059] In embodiments, the fluorescence polarization assay may be provided as a titer plate pre-coated with fluorescently labelled probes. The probes may be reconstituted in the different plates using, for example, fresh cell culture media or phosphate-buffered saline (PBS). Following this, a standard or test sample is added to the plate. After a short incubation period, the plate is measured using a plate reader with FP, as discussed in greater detail below with respect to the various components of the titer instrument 400. Depending on the plate reader and workflow, in embodiments results can be rapidly obtained from crude samples, e.g., in less than 15-minutes total assay time. In some examples, Valita®Titer or Valita® Aggregation may be used as the fluorescence polarization assay.

[0060] As shown in FIG. 6. the titer instrument 400 includes a light source 424 that emits light 436 toward a titer container 410 holding a solution of cells mixed with the fluorescence polarization assay. Titer container 410 is a sample receptacle for titer instrument 400. The fluorescence polarization assay binds with antibodies of interest (e.g., IgG) produced by the cells. As discussed above, the dispensing system 106 of the cell culturing system 100 is automated to mix the fluorescence polarization assay together with the sample of cells in the titer container 410, such that the user of the systemdoes not need to manually mix the solution in the titer container 410. In some examples, the titer container 410 is a cuvette, tube, and the like. Examples discussed herein focus on titer containers configured as an open-topped cuvette, but other configurations are contemplated.

[0061] The light source 424 emits the light 436 without polarization such that the light 436 is unpolarized light. In some examples, the light source 424 is a light-emitting diode (LED). In some examples, the titer instrument 400 includes a focusing lens 426 and a spectral filter 428 that respectively focus and filter the light 436 emitted from the light source 424.

[0062] The light 436 passes through a first polarizing filter 444 that polarizes the light 436 in a first direction. In some examples, the first direction is in a linear direction. For example, for purposes of discussion of the example shown in FIG. 6, the first direction is vertically linear. In alternative examples, the first direction is horizontally linear. Additional polarization directions are possible.

[0063] The light 436, once polarized in the first direction, is absorbed by the solution of cells mixed with the fluorescence polarization assay in the titer container 410. This causes the solution in the titer container 410 to emit polarized fluorescence light 438.

[0064] The polarized fluorescence light 438 can pass through alens 430 for focusing the polarized fluorescence light 438 after emission from the solution in the titer container 410. Afterwards, the polarized fluorescence light 438 alternately passes through a second polarizing filter 446 and a third polarizing filter 448. The second and third polarizing filters 446, 448 are mounted to a structure 450 controlled by a controller to alternate placement of the second and third polarizing filters 446, 448 into the path of the polarized fluorescence light 438.

[0065] The second polarizing filter 446 is polarized in a direction parallel to the direction of the first polarizing filter 444 (i.e., in the first direction). The second polarizing filter 446 restricts passage of the polarized fluorescence light 438 to the first direction.

[0066] The third polarizing filter 448 is polarized in a second direction perpendicular to the first direction of the first polarizing filter 444. The third polarizing filter 448 restricts passage of the polarized fluorescence light 438 to the second direction.

[0067] Thereafter, the polarized fluorescence light 438 passes through a spectral filter 432 before detection by a detector 434. In some examples, the detector 434 a photomultiplier tube (PMT). The detector 434 converts the polarized fluorescence light438 into voltage values for input into a calculation to determine a measure of fluorescence polarization (FP).

[0068] The measured FP is correlated with the concentration of antibody in the sample of cells. For example, a higher detected FP correlates to a higher concentration of antibody or other protein, and a lower detected FP correlates to a lower concentration of antibody or other protein. This is because a higher concentration of antibody will have increased binding with the fluorescence polarization assay such that the voltage of parallel fluorescence is larger, and the voltage of perpendicular fluorescence is smaller, which results in a larger detected FP.

[0069] A photodiode 440 on the far side (opposite of light source 424) of the titer container 410 is used to monitor the direct output light 442 of the LED. This direct output light 442 is somewhat altered by whether the titer container 410 is empty, filled with water, filled with sample, etc. Output of the photodiode 440 may be used to monitor the intensity of the direct output light 442 available from the light source 424. The measured intensity of direct output light 442 can be used to diagnose loss of light, such as from actual decrease of the light source itself or from contamination by absorbing material in the path of the light. Sources of contamination of absorbing material include, for example, sample material that adheres to the surfaces of the titer container. For example, if the measured intensity of output light 442 drops below a predetermined threshold or minimum, or if a decreasing trend in intensity is observed, the system may provide an indication to an operator that the light source should be checked.

[0070] Photodiode 440 enables detection of the presence or absence of liquid in titer container 410. Using photodiode 440 in this way, the system determines whether the cuvette has successfully performed its intended drain sequence and / or wash sequence after a measurement and is fully empty and in a suitable condition to receive the next sample. This washing sequence and arrangement of the titer module are described in more detail in US Provisional Application No. 63 / 509,243.

[0071] Referring now collectively to FIGS. 7 and 8, the titer container assembly 402 of the titer instrument 400 will be discussed in more detail. FIG. 7 is an isometric view of an example titer instrument 400 of the cell culturing system 100. Titer instrument 400 includes a titer reagent storage 460, deck locations 462 for receiving labware, a disposal receptacle 464, titer container assembly 402, and a tip guide 422 to assist in providing samples into the internal titer container assembly 402. FIG. 8 is an exploded view of thetiter container assembly 402 of titer instrument 400. Titer container assembly 402 includes a two-part body 404, 406, and a top retainer 408.

[0072] Body 404 supports a titer container 410 and includes an upper seal 412, a lower seal 414, and an insert 416. Titer container 410 is held mechanically in place by the supporting structure of body 404 and insert 416. Titer container 410 is sealed at the top and bottom, by upper seal 412 and lower seal 414, each sealing against upper or lower surfaces of titer container 410. Upper seal 412 and lower seal 414 may be formed by any appropriate sealing means and, in some embodiments, may each be an O-ring. In some embodiments, upper seal 412 and lower seal 414 may be formed from materials with different properties and / or have dimensions different from each other.

[0073] In addition to support, body 404. 406 may provide thermal control for samples loaded into titer container 410. Thermal control of samples aid in providing constant temperature for measurements taken by titer instrument 400. Together, body 404, 406 and titer container 410 provide a thermally controlled sample receptacle. Titer measurements are sensitive to temperature and consistency in sample temperature improves the accuracy of readings determined according to standard curves, which may be temperature specific. In some embodiments, body 404, 406 is made of aluminum. Body 406 secures and retains titer container 410 within the support of body 404. Body 404, 406 includes optical input aperture and output aperture to permit the beam of the optical interrogation system to pass through a sample loaded into titer container 410. In embodiments, body 406 further includes a thermoelectric cooler (TEC) assembly 418. In some cases, the TEC assembly is a printed circuit board assembly.

[0074] A thermal pad 419 may be included to enhance the thermal interface between the TEC module and the target surface it is cooling or heating. By filling microscopic gaps and irregularities, the thermal pad ensures improved thermal conductivity and minimizes thermal resistance. Typically composed of thermally conductive materials like silicone-based compounds, the pad facilitates efficient heat conduction between the TEC and the surface, allowing the TEC to effectively transfer heat. Some thermal pads also provide insulation on one side to prevent electrical contact between the TEC and the surfaces it interfaces with, avoiding potential short circuits.

[0075] A TEC heat sink 420 is included to manage the heat generated by a thermoelectric cooler (TEC) during its operation. Typically constructed from thermally conductive materials like aluminum or copper, features such as fins may be included to increase surface area, promoting efficient heat dissipation through convection. A thermalinterface material, such as thermal paste or a thermal pad, enhances the thermal contact between the TEC module and the heat sink, reducing thermal resistance. In some cases, TEC heat sink 420 also incorporates a fan to further enhance heat dissipation through forced convection.

[0076] In embodiments, titer container 410 is a hollow, open-topped and open- bottomed container for liquid. In some embodiments, titer container 410 has a fourwalled construction, and may have, for example, a square or rectangular cross-section. Titer container 410 may be formed of fused silica or other optically compatible material, such as quartz. Titer container 410 may have internal dimensions configured based on desired sample volumes to use and the resulting fill level in titer container 410 when the sample is loaded, to ensure adequate sample presence for effective analysis. In some embodiments, the internal dimensions of the titer container may be 3 mm x 3mm. In some embodiments, the internal dimensions may be larger or smaller, or the two dimensions may differ from one another, e.g., a rectangular container.

[0077] Titer container 410 is oriented vertically in operation with an upper opening, lying inside of upper seal 412. available to receive sample liquid dispensed by the pipette of a liquid handler, such as liquid handling pod 114 of FIG. 2.

[0078] Upper seal 412 mates against a fill port to prevent any liquid from spilling over the top edge of titer container 410 and contaminating the exterior optical surfaces of titer container 410. Liquid may be either dispensed into the titer container, such as a sample, or pushed into the titer container during flushing and / or cleaning.

[0079] Lower seal 414 mates against a drain outlet port that has a access port. The access port may extend through insert 416. The dimensions of the access port may be kept relatively small, e.g., on the scale of about 0.7 mm, such as specifying dimensions of about 0.5-0.9 mm or 0.3-1.1 mm, etc. In some embodiments, samples may be loaded through the top of titer container 410 with other fluid exchanges associated with titer container 410 generally performed using the access port in the lower portion of titer container 410. Using a single access port for both draining the titer container and filling of flushing and cleaning agents provide manufacturing advantages by limiting the number of holes drilled or otherwise formed during the construction of the titer container. Further, use of a combined access port may simplify the assembly and arrangement of the components of the titer instrument 400 and the dispensing system 106.

[0080] In some embodiments, the titer container may be configured with a drain port and a separate fill port. For example, a fill port may be provided towards the top of oneor more sidewall of titer container 410. In another example, a fill port is provided from the interior of top retainer 408. Top retainer 408 secures upper seal 412. Top retainer may include tip guide 422, which may assist in guiding liquid handling pod 114 to access titer container 410, such as for sample loading. In embodiments, top retainer 408 may be removable to provide access to titer container 410, such as for maintenance.

[0081] A liquid handling system associated with titer container 410, in embodiments, includes a bi-directional pump and a multiport valve. The liquid handling system may be fluidly in communication with the interior of titer container 410 via an access port integrated with titer container 410 and insert 416.

[0082] The access port of titer container 410 is attached via tubing to bi-directional pump, which can pull fluid out of the cuvette through the access port and subsequently dispense the discarded fluid to a waste container. The bi-directional pump can further provide a variety of flushing and / or cleaning liquids from bottled sources into the titer container through the same access port. As discussed above, titer container 410 is integrated with titer instrument 400. which may include an optical titer analysis system. A sample loaded into titer container 410 may be subjected to an optical titer analysis by the components of titer instrument 400.

[0083] The titer analysis system further may include a processing circuitry' in communication with a memory, which may be a non-lransitory memory. The processing circuitry and memory form part of a controller, which may be a dedicated controller for the titer analysis system or may form part of a controller responsible for multiple modules of the cell culturing system. The memory' stores instructions which, when executed by the processing circuitry, cause the processing circuitry to perform one or more of the methods disclosed herein.

[0084] In embodiments, controls are provided to ensure that titer instrument 400 is functioning properly. These controls ensure usable test data is acquired from titer analysis. In embodiments, users can test for and ensure quality' by running a titer measurement test using a titer quality probe alone. Expected values to be detected from the probe alone may be stored within the system and presented to a user or otherwise made available to the user. The detected values are compared to known values and any variation will indicate the quality of the measurement.

[0085] Other controls to ensure proper functioning by titer instrument 400 include use of a fluorescent polarization (FP) reference standard to determine the function andaccuracy of the titer module. FP reference dyes are dependent on dilution, pH, and dye purity, and have to the potential to stain the optical cuvette or tubing.

[0086] As is disclosed herein, fluorescent dyes immobilized on a substrate provide a stable FP reference standard suitable to measure titer module function and accuracy. The substrate may be a solid, for example a thin rod, or commercially available fluorescent beads. Various substrates were determined to satisfy the requirements for a FP standard by remaining stable, providing a consistent FP measurement despite different dilutions, and being easily removed from the measurement cuvette. In embodiments, one of a plurality of fluorescent beads or non-bead solid standards is selected as a standard for a particular experimental run based on the fluorescent wavelength required. FIG. 9 is a graph of an example of five different lots of fluorescent beads at five different concentrations. The graph of FIG. 9 is starting at a nominal concentration of one million beads per milliliter. As can be seen in FIG. 9, the fluorescent polarization (mP) is the same across the bead lots and dilution.

[0087] In some cases, a NIST (National Institute of Standards and Technology ) traceable fluorescence brightness standard is used as a reference material in fluorescence measurements to ensure accuracy and traceability to a recognized standard. NIST traceability means that the measurement or calibration of an instrument is linked to national or international measurement standards maintained by NIST. In the context of fluorescence, brightness standards are often used to calibrate fluorescence instruments, such as fluorometers or microplate readers, by providing a known reference for fluorescence intensity. These standards typically consist of fluorophores with well- defined and stable fluorescence properties. One example of a NIST traceable fluorescence brightness standard is fluorescein sodium salt.

[0088] As is disclosed herein, a diluted solution of fluorescein sodium salt, for example diluted to 0.43pM in water, is used to adjust the optical excitation intensity' on the instruments, e.g., by vary ing the electrical current in the LED light source, so that they operate in an optimal range for signal to noise ratio. As is further disclosed herein, the diluted solution of fluorescein sodium salt is used as a sample when performing “G- factor” measurement procedure. The G-factor, as discussed herein, refers to a correction factor calculated to compensate for optical effects of the hardware, such as small differences in the detection paths (parallel and perpendicular).

[0089] Quality of the titer measurement can be further controlled by testing three types of particles, for example when maintenance is performed by service ormanufacturing engineers. As with the titer quality probe test above, each of these particles has known values for each test, and the output is compared against the known values to indicate the quality of the workflow.

[0090] A first type of bead is embedded with fluorescent molecules of a known fluorescence. This bead is tested to measure the known fluorescence of the particle and ensures an absence of a target protein, e.g., IgG, in manufacturing. A second type of particle is fluorescein sodium salts, which are tested to measure the system’s brightness and low polarization quality. The third type of particle is scatter beads, which are used for measuring stray light control of the module.

[0091] In fluorescence measurements for biological applications, such as in titer instrument 400 which measures fluorescence polarization, light from the excitation beam directed towards the detection site is typically orders of magnitude more intense than the fluorescence produced. Because of this, the excitation light w avelength needs to be filtered out by spectral filters with high extinction ratios, typically 105 or higher. This is well understood by those in the art and many spectral filters specifically for fluorescence applications are available.

[0092] The fluorescence polarization (FP) measurement is particularly sensitive to remaining excitation light as it is strongly polarized and can introduce large errors in FP measurements. For titer application, the detected excitation light may be no more than 0.1% of the fluorescence and still yield an error <0.5mP (mP = milliPolarization unit). Effective high rejection of the excitation wavelength is especially important when measuring samples that contain particles as they scatter light in all directions including towards the detection optics, such as when measuring samples with cell culture. Rejection of the excitation light can be negatively affected by, for example, fabrication errors of the filters, poor handling (e.g., scratches, finger prints) or incorrect mounting.

[0093] In order to ensure that the excitation light is adequately rejected, filtering of the excitation light is performed is verified. Using a sample of plain polystyrene microbeads diluted to 0.1% solid was found, as is disclosed herein, to scatter light towards the detector about 15 times more than a CHO cell culture sample at 20 M / mL concentration. Excitation light rejection is performed by evaluating an of detected signal attributable to the diluted polystyrene microbeads. In embodiments, the instrument is tested during manufacturing and is failed if the measured signal is too high (e.g., indicating excess excitation light detectable).

[0094] FIG. 10 is an isometric view of cell health instrument 500. Cell health instrument 500 includes a cell health instrument 502, sample inlet 504, a tip wash station 506, a temperature controlled plate storage 508, a reagent storage 510, and tip storage 512.

[0095] Cell health instrument 500 may provide instruments and a known location for measuring one or more parameters of cell health. Examples of cell health parameters which may be evaluated in various embodiments include cell growth rate, cell density, viable cell density, antibody titer, and product quality. In embodiments, antibody titer is performed by titer instrument 400 with one or more parameters of cell growth rate, cell density, viable cell density, and product quality performed by cell health instrument 500. Together, the measured parameters of cell health are evaluated by a user or the system to form a picture of the overall health and production capacity' of a particular cell line. For example, a cell line with high protein production but low- growth rate and / or low viable cell density may be less desirable than a cell line with lower per-cell protein production but high growth rate and viable cell density.

[0096] Cell growth rate is assessed, for example, by counting cell numbers over a defined period, using methods such as hemocytometer counting or automated cell counters. Cell density, representing the number of cells per unit volume, can be measured using similar techniques. Viable cell density, indicating the number of live cells in a culture, is determined by employing vital dyes, e.g.. Trypan Blue, or flow cytometry. An automated cell counter streamlines the process of cell counting using imaging technology7. Operation begins with a prepared cell suspension, which may include a viability stain, being loaded onto the automated cell counter. The instrument captures high-resolution images of the cells using, for example, microscopy or flow cytometry, and these images are subjected to image processing algorithms which distinguish between viable and non-viable cells based on staining characteristics, while identity ing individual cells and determining their parameters such as size. The automated cell counter may then calculate total and viable cell counts, cell size, and other relevant metrics. Results are displayed on the instrument's screen or can be exported for further analysis. In embodiment, additional features, e.g., data storage, are included, allowing users to track cell growth and compare results across experiments.

[0097] Other vital aspects of cell health, such as metabolic activity, cell viability', cell cycle distribution, apoptosis, and morphological characteristics, are assessed usingvarious assays and microscopy methods. Additionally, the release of signaling molecules can be evaluated through cytokine / chemokine assays.

[0098] In embodiments, cell health instrument 500 is configured to determine a total number of cells and / or a number of viable cells in a sample. For example, cell health instrument 500 may contain instruments for the measurement of electrical impedance and / or light scatter from cells in an analyzed sample. Cell health instrument 500 may include programming to determine a total number of cells and / or a number of viable cells in a sample. Cell health instrument 500 may be in communication w ith computing device 300, and computing device 300 may store appropriate instructions and / or programming for cell health instrument 500. Cell health instrument 500 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.

[0099] Cell health instrument 500 may provide instrumentation for cell counting. Cell health instrument 500 may be configured to count all cells in a particle sample or portion, or to count specifically all live cells the sample or portion. Counting may be accomplished by placing or transferring full or partial samples of each sample line into the cell health instrument.

[0100] In embodiments, cell health instrument 500 is configured to automate a Try pan Blue dye exclusion method. A sample is mixed with a Trypan Blue suspension allowed to incubate for a short period. During this time, viable cells will exclude the dye, while non-viable cells will take up the dye and appear blue. Cell health instrument 500 employs advanced imaging technology7to analyze the size and staining patterns of cells, providing accurate cell counts and viability assessments.

[0101] Integration of cell health instrument 500 enables cell culturing system 100 to provide cell viability and density measurements for samples taken during a cell cultivation campaign. In embodiments where a bioreactor or other amplification module is also incorporated or communicatively linked with the cell culturing system 100, samples may be taken directly from the bioreactor. Dispensing system 106 aspirates samples from the bioreactor module and dispenses them into cell health instrument 500 at a sample inlet 504 using fixed pipette tips. Cell health instrument 500 may also be accessed with disposable tips, such as during seeding and normalization.

[0102] Reagent storage 510 may include one or more reagents for performing cell health analysis. In examples, reagent storage 510 includes receiving area for distilled water, cleaning agents, cell health agents (e.g., Trypan Blue), a cell health dilutant, and / orisopropyl alcohol. Trypan blue is suitable cell viability assays to distinguish between live and dead cells. Live cells have intact cell membranes and exclude the dye, while dead cells with compromised membranes take up the dye and appear blue under a microscope.

[0103] In embodiments, cell health instrument 500 is further configured to evaluate a product qualify produced by a particular cell line. For example, an assay for clonal stability may be integrated with the cell health instrument 500 or provided by another instrument within the cell culturing system 100. Assessment of clone stability demonstrates a cell line is unlikely to change during production in a manner deleterious to product qualify, and helps guard against product loss. A clonal stability assay may evaluate one or more of genetic stability, transcriptomic stability, and proteomic stability of a cell line, as each may contribute to production instability over time. While a complete omics (genomics, proteomics, metabolomics, etc.) analysis of cell lines may represent an ideal for gaining deep cellular insights to understand phenotypic and clone stability, such robust analysis may be to time and cost intensive for effective evaluation in the course of a cell -culturing and clone selection campaign.

[0104] In examples, the clonal stability assay may be a ChemStress® assay to provide a phenotypic profile of a cell line. In this example, a challenge plate, e.g., a 96- well plate, is prepared with each well is coated with a chemical compound, selected based on its published ability to simulate specific conditions, or effect specific cellular pathways. Incubating cells on the plate induces a characteristic set of changes to cell function that together provide a phenotypic profile of the cell line. Cellular responses (e.g. cell viability, productivity, cellular metabolism, host cell protein production etc.) to the various conditions produced in the challenge plate. In embodiments, cells may be incubated in the challenge plate for a predetermined period of time, e.g., 3 days.

[0105] In embodiments, cell health parameters, e.g., protein titer, cell viability, etc., may be read directly from the challenge plate following incubation. For example, fluorescence intensity for cell viability and fluorescence polarization for cell productivity (protein titer) can be analyzed directly on the challenge plate. In embodiments, readings may be provided to a processing circuit or application configured to determine a quantitative biological classification of clone stability with, for example, a respective clone ranking based on the readings.

[0106] In embodiments, output from the processing circuit or application includes a metric, referred to herein as an index value, identifying stable clones that remain unperturbed across many components of cell function with a phenotypic characterizationusing a panel of multiple chemical stressors. Advanced analytics and multivariate statistics applied to the rich data output generated with the challenge plate enables the system to calculate the index value. This is a summary statistic describing the variation between different challenge profdes for cell lines and an offset from a baseline. The larger this number, the greater the deviation from the baseline, the more dissimilar the fingerprints over the generations, the more unstable the clone. In embodiments, readings are taken from the challenge plate at multiple time points to provide data on cell line behavior over time.

[0107] As with titer instrument 400, the accuracy of cell health instrument 500 must be assessed to ensure that analysis quality is tightly controlled and flawed test data is not recorded. In embodiments, samples are diluted 1:4 in cell health instrument 500. A focus control bead is used in an automated procedure to optimize the focal plane by adjusting the instrument's focus settings. Images of the focus control beads are then analyzed by the system's software. The cell health instrument’s autofocus algorithm may be calibrated or adjusted using the information obtained from the focus control beads. This ensures that the counter will consistently and accurately focus on cells during subsequent analyses. Quality control metrics may include measures of bead concentration, accuracy in focus optimization, and precision in counting.

[0108] In another instance, control beads are used for generating the concentration control curve. In this example, three different concentration beads (4 mill / mL. 2 mill / mL, 10 mill / mL) are used for concentration control, and a slope is created and the recorded values are compared to known values. For example, a series of beads, which may be provided with the instrument or otherwise be associated with the instrument, are sampled by the use. These may include 2M. 4M, and 10 M concentration control beads which ensure the system is provided correct concentrarions. Another tested bead type provide assurance that cell size is accurately reported. For the system to pass QC the values measured must be within a predetermined range of expected values.

[0109] FIG. 11 is an isometric view of an example of a bioreactor 600. In embodiments, bioreactor 600 may be a microbioreactor. As shown in FIG. 11, the bioreactor 600 includes a housing 602 that defines a cultivation chamber 604. The bioreactor 600 measures parameters such as biomass, pH, dissolved oxygen (DO), and fluorescence online while running a cultivation inside the cultivation chamber 604. Additionally, the bioreactor 600 may include a touchscreen display 306. as an example input device, that allows a user to control the shaking speed, temperature, gasconcentration, gas flow rate, and humidity inside the cultivation chamber 604. Alternatively or additionally, the bioreactor 600 may be communicatively coupled to a separate computing device that may allow for such control. While shown as a separate module in the example of FIG. 11, in embodiments, microbioreactor 600 is integrated with cell culturing system 100 and may be situated on w ork platform 200 of FIG. 3

[0110] In some aspects, the bioreactor 600 can share similar components, features, and functionalities with the microreactors described in U.S. Patent No. 8,268,632. titled Method and Device for Recording Process Parameters of Reaction Fluids in Several Agitated Microreactors, issued on September 18, 2012, U.S. Patent No. 8,828,337, titled Microreactor, issued on September 9, 2014, U.S. Patent No. 8,932,544, titled Microreactor Array, Device Comprising a Microreactor Array, and Method for Using a Microreactor Array, issued on January 13, 2015, and U.S. Patent No. 10,421,071, titled Microreactor System, issued on September 24, 2019, the entireties of which are hereby incorporated by reference.[OHl] FIG. 12 is a top view of the interior of the cultivation chamber 604 of bioreactor 600. Within cultivation chamber 604 are bioreactor plates 608. A door 610 is provided to seal the incubation chamber for temperature and gassing control during cultivation cycles. Door 610 opens during experiments to provide the liquid handling probes access to the cultivations for analysis. Cultivation chamber 604 holds the bioreactor plates and provides shaking, gassing, and temperature control for ideal cultivation conditions. The bioreactor 600 provides temperature control as well as N2, 02, and CO2 gassing to the bioreactor plates.

[0112] FIG. 13 is a cutaw ay side view of a well 612 of bioreactor plate 608. Each well 612 includes a well wall 614, a well bottom 616, and a sterile barrier 618. A fiber optic cable 620 emits an excitation light beam 622 into the well 612 and functions as an optode to monitor conditions in the well 612. In embodiments, bioreactor plate 608 further includes a shaker 624. Displacement position 626 of the well is shown for reference. Also shown for reference is schematic system 628. The bioreactor measures pH, dissolved oxygen (DO), and biomass of the samples in the wells using optodes. Each optode is read through a fiber bundle underneath the plates using a designated filter.

[0113] In practice, each lot of bioreactor plates will have variables in their sensitivity for pH detection. If these sensitivities are not assessed and accounted for, flawed test data could result. Accordingly, as is disclosed herein, each lot of bioreactor plates is tested for calibration data prior to being made available to end users. Tests assess the optodesresponse to changes in pH. The test protocols may be selected according to guidance by an optode manufacturer.

[0114] The calibration data, and the coefficients resulting from it, are stored in correlation with the lot of plates and may be accessed via a code reader, such as code reader 354 as seen in FIG. 1. The code may, for example, appear on the packaging of each lot of plates.

[0115] Optimization of a fill level of the bioreactor wells 612 may, for example, prohibit spilling or sloshing, while still ensuring enough sample depth to allow proper measurement. As is disclosed herein, fill level of wells 612 may be optimized, for example, at considering one or more of a max to avoid spilling, liquid touching vents, or contamination (e.g., 8mL); a min to allow proper reading for optodes and sufficient depth (e.g., 3mL). In this example, and optimal level of 3mL-5mL may be determined. Dispensing system 106 may then be configured, such as by instructions stored on controller 300, with a range of 5mL-8mL allowable. This accounts for machine errors outside that range and prevents operations that would exceed 8mL.

[0116] In embodiments, the bioreactor includes shaking. Shaking may be orbital, e g., in a 3 mm diameter orbit. The bioreactor shaking range, in embodiments, is 600-800 RPM which may provide for optimal gas diffusion rates within the wells and to avoid excessive vibration from resonance frequencies in the system. During shaking, conical guides may be included above a seal on the bioreactor plates to direct pipette tips through a slit in the seal and into the well openings in the gassing chip.

[0117] In embodiments, a shaker table using eccentric pulleys to oscillate the table of the bioreactor is used, which sen es the purposes of “shaking'’ it and the contents in its wells. The shaking causes angular momentum variations within the body of the instrument. If the angular momentum is not counterbalanced, it can cause the instrument to vibrate or to move in unwanted ways, which can damage equipment. As is disclosed herein, a series of counterbalances attached to the pulleys of the devices can counteract the shaking of the table. FIGS. 12-18 show examples of a counterbalanced shaker table 700. In this example, three sets of counterbalances 702. respectively, counteract angular momentum from the eccentric pulley 704 and the table, along with torque from the table.

[0118] FIG. 19 is a flowchart of an example method 800 for automated monitoring of a cell culturing campaign. Method 800 may be executed by a processing circuitry associated with cell culturing system 100. such as controller 300 of FIG. 1.

[0119] At operation 802, a biological sample is aspirated from the container into the pipettor. In embodiments, the container may be a sample container or a well of a bioreactor, for example to evaluate a current cultivation state of a cell line being cultivated in the bioreactor. In some cases the bioreactor may be arranged outside of the analysis system performing method 800. The biological sample may contain a cell population in aqueous solution. The sample may not be associated with a bioreactor or cell cultivation campaign, but may otherwise be desired to be analyzed by both titration and cell health.

[0120] In embodiments, the pipettor may be liquid handling pod 114 of FIG. 1.

[0121] At operation 804, the biological sample is dispensed into the titer and cell health instruments. In embodiments, the biological sample aspirated into the pipettor includes a first and second volume of the biological sample. For example, a first volume may be aspirated first and dispensed into the titer instrument, a second may then be aspirated and dispensed into the cell health instrument. In another example, each of the first and second volume are aspirated together by the pipettor. In some cases, the first and second volume are aspired together by the pipettor and separated by an air gap.

[0122] At operation 806, protein concentration in the biological sample is measured with the titer instrument. At operation 808, cell health in the biological sample is measured with the cell health instrument. The cell health instrument may be one or both of a cell viability measuring instrument and a cell density measuring instrument. In some cases, the cell health instrument is cell health instrument 500 of FIG. 1. In embodiments, protein concentration and cell health measurements, operations 806 and 808 respectively, are capable of being performed in parallel or sequentially.

[0123] In some cases, the at least one biological sample further comprises a at least two biological samples. In such cases, the protein concentration and cell health measurements between the at least two biological sample within the at least two biological samples may be compared. Following the comparison, biological samples from the at least two biological samples may be selected to transfer to a container based on the comparison of the protein concentration and cell health measurements.

[0124] For example, following a cell culture in the bioreactor, a user may desire to transfer one or more culture to another plate for use in off-instrument analytical processing. The desired transfer volume may be a sample or a harvest. Harvest implies the culture is over and a significant volume will be transferred. Sample implies the culturewill continue. For example, a sample be taken for metabolite measurements from an off deck instrument.

[0125] A user may select wells for transfer, e.g., all of the wells, based on measurements taken of the wells and the user’s knowledge of the reaction. The instrument may be configured to then transfer the volume after the user has determined that the instrument is set up for the transfer. The system notifies the user when the transfer is finished, then the user can remove the plate.

[0126] A harvest workflow happens at end of the reaction process. In embodiments, only the best results are harvested, based on interpretation of the data. The remainder may be left behind in the well and discarded. In examples, the volume is transferred 120uL at a time. Following the analysis, a user may be presented with a file of the data, such a .csv file.

[0127] FIG. 20 is a flowchart of an example method 900 for automated cell line development using an automated laboratory instrument. At operation 902, a cell line is cultured in a bioreactor. At operation 904, a protein concentration is obtained from a titer instrument of the automated laboratory instrument for a sample of a plurality of biological samples. At operation 906, cell health data is obtained from a cell health instrument of the automated laboratory instrument for the sample of a plurality' of biological samples. At operation 908, the steps of obtaining protein concentration and cell health are repeated for each sample of the plurality of biological samples at a predetermined interval throughout a cell culture campaign. In embodiments, the predetermined interval is adjusted based on one or more of the protein concentration or the cell health measurement. The predetermined interval may in some cases be adjusted based on a user input.

[0128] FIG. 21 is a flowchart of an example method 1000 for operating an automated laboratory instrument for cell line development. At operation 1002, at least one sample of a cell line to be developed is received. At operation 1004, a protein concentration in the at least one sample is obtained from a titer instrument. At operation 1006, cell health in the at least one sample is obtained from a cell health instrument.

[0129] At operation 1008, a determination is made whether predetermined interval has elapsed. If the interval has elapsed, the system returns to operation 1004 and repeats the steps of obtaining protein concentration (operation 1004) and cell health (operation 1006) for each sample of the plurality of biological samples. If the predetermined interval has not elapsed, the system waits, at operation 1010, for the interval to elapse. Together,operations 1004 -1008 represent a protocol is executed at regular intervals to monitor the development of the at least one cell line. The interval may be determined based on a cell culture campaign as preselected by a user and, in embodiments, may be adjusted based on a parameter measured by the bioreactor (e.g., pH, dissolved O2, etc.) or based on parameters measured by the titer and cell health instruments.

[0130] FIGS. 22-29 are an example user interface 1100 for the automated cell culturing system. User interface 1100 may be generated by a controller or processing circuit associated with the cell culturing system, such as controller 300 of FIG. 1. User interface 1100 presents data obtained from various instruments of the cell culturing system for review and study by a user. User interface 1100 further provides various controls for setting and adjusting automated operations of the cell cultivation system, and preforming various other control operations of the cell cultivation system.

[0131] FIG. 22 shows a “new experiment” view of user interface 1100a. This view depicts a layout of one or more cell culture plates, each cell culture plate having one or more wells. Each of the cell culture plates and cell culture wells is configured to be named by a user and receive operational commands from the user to be executed by the automated laboratory instrument. FIG. 23 is a view of the user interface 1100b showing active areas in a layout of a worksurface of the cell culturing system, such as work platform 200 of FIGS. 1 and 3. User interface 1100b solicits automated or manual input of parameters for culturing one or more plates. Automated input may be performed, for example, by scanning a code, such as a QR or barcode associated with each well or plate. FIG. 24 is a view of user interface 1100c showing an indication of the state of plates in the bioreactor. In this example, proper orientation, e.g., not being tilted, and securement, e.g., plates affixed with clips, of the plates is shown.

[0132] FIG. 25 is a view of user interface HOOd showing a calendar display depicting scheduled protocols. The calendar display is further configured to display the scheduled protocols associated with a user selected one of the one or more cell culture plates or one or more wells. A selected plate and well is visible in the left side of interface 1 lOOd and several scheduled actions appear in the right side.

[0133] Below the selected plate and / or well is a set of last measured values for one or more parameters of the well. Measured parameters include parameters measured by the bioreactor, such as pH, dissolved oxygen, volume, and biomass. Together these parameters depicts a display of current bioreactor conditions. The bioreactor conditionsmay be displayed per well of the one or more wells, such as for a user selected well of the one or more wells.

[0134] Above the selected plate and / or well is a schedule showing a next planned interaction by the cell culturing system with the plate or well. In embodiments, automated actions include feeding each well of the one or more wells and obtaining, for each well of the one or more wells, at least one of protein concentration from the titer instrument and cell health from the cell health instrument. Other automated actions may include measuring of other parameters of the culture in each well, drawing samples, etc. In embodiments, interface 1100 further includes an indication of performance of automated actions at times of the automated actions, e.g., a banner at the top of the screen identifying the action occurring. In embodiments the calendar display further depicts events requiring user input.

[0135] FIG. 26 is a view of a user interface HOOe showing user interaction to schedule anew scheduled action. In this example, a user is scheduling an additional feed for a target well or plate. FIG. 27 is a view of example user interface 11 OOf showing user operations for harvesting an output plate or selecting samples for a sample plate. One or more wells may be selected, based on measured values and / or user knowledge of the culture experiment. Once selected, transfer of culture from each of the one or more selected wells to an output plate may be automated by the cell culture system. In embodiments, harvesting the output plate comprises receiving the selection of one or more wells to be transferred to the output plate.

[0136] FIG. 28 is an example view of a user interface 1100g showing user operations to end cultivation in a select well. In embodiments, current measured values for the selected well may be presented to aid the user in making the determination whether to end cultivation in the well. Cultivation in a well may be ended due to bad data, possible contamination (e.g., indicated by a spike in biomass), or a bad clone. FIG. 29 is an example view of a user interface 1 lOOh showing available protocols for selection and / or, in embodiments, editing. Selection of a protocol may generate a view of each protocol parameter and schedule element for review.

[0137] FIGS. 30 - 36 show a series of example views of user interface 1200 for generating a new protocol. FIG. 30 is a view of user interface 1200a for entering a name and or description for the protocol, or otherwise identifying anew protocol. FIG. 31 is a view of an example user interface 1200b for defining a plate setup for the new protocol. Various parameters may be defined for each plate and / or each well, such as volume andmedia present. FIG. 32 is a view of an example user interface 1200c for defining a setpoint parameters for the protocol. Various environmental conditions may be defined for the protocol, for example, temperature, shaking parameters such as speed and direction, pH and dissolved oxygen, including target values and acceptable ranges.

[0138] FIG. 33 is a view of an example user interface 1200d for defining which reagents will be available and / or used throughout the protocol. Reagents for various environmental control features may be provided, such as pH control, as well as feed solutions and other additives. FIG. 34 is a view of an example user interface 1200e for defining an automated feed schedule to be carried out throughout the cell culturing campaign of the protocol. FIGS. 35 and 36 views of example user interfaces 1200f and 12006 for defining an automated testing schedule to evaluate the cell culture campaign. Testing for protein titer and cell health may be defined by a user using user interfaces 1200f and 1200g. In embodiments, execution of protocols defined using user interfaces 1200f and 1200g may embody method 800 of FIG. 19.

[0139] FIG. 37 schematically illustrates an example of the controller 300 of the cell culturing system 100 that can be used to implement aspects described herein, such as controller 300 of FIG. 1 . As shown in FIG. 37, the controller 300 includes one or more processing devices 302, a memory' storage device 304, and a system bus 306 that couples the memory storage device 304 to the one or more processing devices 302. The one or more processing devices 302 can include central processing units (CPU). In some instances, the one or more processing devices 302 are part of a processing circuitry having a memory' for storing instructions which, when executed by the processing circuitry, cause the processing circuitry' to perform the various aspects, features, and functionalities described herein.

[0140] As shown in FIG. 37, the memory storage device 304 can include a randomaccess memory (“RAM”) 308 and a read-only memory' (“ROM”) 310. Basic input and output logic having basic routines that help to transfer information between elements within the controller 300, such as during startup, can be stored in the ROM 310.

[0141] The controller 300 can also include a mass storage device 312 that can include an operating system 314 and store software instructions and data 316. The mass storage device 312 is connected to the processing device 302 through the system bus 306. The mass storage device 312 and associated computer-readable data storage media provide non-volatile, non-transitory storage for the controller 300.

[0142] Although the description of computer-readable data storage media contained herein refers to the mass storage device 312, 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 controller 300 can read data and / or instructions. The computer-readable storage media can be comprised of entirely non-transitory media. The mass storage device 312 is an example of a computer-readable storage device.

[0143] 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 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, or any other medium which can be used to store information, and which can be accessed by the device.

[0144] The controller 300 can operate in a networked environment using logical connections to the other devices through the network 320. The controller 300 connects to the network 320 through a network interface unit 318 connected to the system bus 306. The network interface unit 318 can also connect to additional types of communications networks and devices, including through Bluetooth, Wi-Fi, and cellular telecommunications networks including 4G and 5G networks. The network interface unit 318 can connect the controller 300 to additional networks, systems, and devices. The controller 300 also includes an input / output unit 322 for receiving and processing inputs and outputs from peripheral devices.

[0145] The mass storage device 312 and the RAM 908 can store software instructions and data. The software instructions can include an operating system 314 suitable for controlling the operation of the cell culturing system 100. The mass storage device 312 and / or the RAM 908 can also store the software instructions and data 316, which when executed by the processing device 302, provide the functionality of the cell culturing system 100 discussed herein.

[0146] 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.

[0147] Clause 1. A method of operating an automated laboratory instrument for cell line development, the method comprising: receiving at least one sample of a cell line tobe developed; and executing a protocol at regular intervals to monitor the development of the at least one cell line, the protocol including: obtaining, from a titer instrument, protein concentration in the at least one sample; obtaining, from a cell health instrument, cell health in the at least one sample; and repeating the steps of obtaining protein concentration and cell health for each sample of the plurality of biological samples at a predetermined interval throughout a cell culture campaign.

[0148] Clause 2. A system for cell line development, the system comprising: an automated laboratory instrument including a titer instrument and a cell health instrument; processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: obtain data from the titer instrument and the cell health instrument; generate a user interface including: a layout of one or more cell culture plates, each cell culture plate having one or more wells, wherein each of the cell culture plates and cell culture wells is configured to be named by a user and receive operational commands from the user to be executed by the automated laboratory instrument; a calendar display depicting scheduled protocols, configured to display the scheduled protocols associated with a user selected one of the one or more cell culture place or one or more wells; and an indication of performance of automated actions at times of the automated actions.

[0149] Clause 3. The system of clause 2, wherein the automated actions include feeding each well of the one or more wells and obtaining, for each well of the one or more wells, at least one of protein concentration from the titer instrument and cell health from the cell health instrument.

[0150] Clause 4. The system of clause 2, wherein the user interface further comprises user operations for harvesting an output plate.

[0151] Clause 5. The system of clause 4, wherein harvesting the output plate comprises receiving the selection of one or more wells to be transferred to the output plate.

[0152] Clause 6. The system of clause 2, wherein the calendar display further depicts events requiring user input.

[0153] Clause 7. An automated laboratory instrument for measuring protein concentration and cell health of a biological sample, the automated laboratory instrument comprising: a housing containing: a biological sample container for receiving a biological sample; an pipettor configured to aspirate and dispense the biological sample; a titer instrument configured to receive the biological sample and measure proteinconcentration of the biological sample; a cell health instrument configured to receive the biological sample and measure cell health of the biological sample; a population development module configured to culture at least one cell population; processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: aspirate a portion of the biological sample from the container into the pipettor; dispense the portion of the biological sample into the titer and cell health instruments; measure at least one of pH, biomass, volume, and dissolved oxygen with the population development module; measure protein concentration in the biological sample with the titer instrument; and measure cell health in the biological sample with the cell health instrument.

[0154] Clause 8. The instrument of clause 7, wherein the portion of the biological sample aspirated into the pipettor includes a first and second volume of the biological sample.

[0155] Clause 9. The instrument of clause 8, wherein the first and second volumes of the biological sample are spaced apart from one another in the pipettor.

[0156] Clause 10. The instrument of clause 9, wherein an air gap separates the spaced apart first and second volumes of the biological sample.

[0157] Clause 11. The instrument of clause 8, wherein the first volume of the biological sample is dispensed into the titer instrument.

[0158] Clause 12. The instrument of clause 8, wherein the second volume of the biological sample is dispensed into the cell health instrument.

[0159] Clause 13. The instrument of clause 7, wherein the protein concentration and cell health measurements are capable of being performed in parallel or sequentially.

[0160] Clause 14. The instrument of clause 7, wherein the biological sample contains a cell population in aqueous solution.

[0161] Clause 15. The instrument of clause 7, wherein the biological sample in a container resides in the population development module.

[0162] Clause 16. The instrument of clause 15, wherein the population development module is a bioreactor.

[0163] Clause 17. The instrument of clause 7, wherein the instructions further cause the processing circuitry to: perform a plurality of protein concentration and cell health measurements throughout a cell culture campaign.

[0164] Clause 18. The instrument of clause 17, wherein the instructions further cause the processing circuitry to: perform a plurality of protein concentration and cell health measurements throughout a cell culture campaign as preselected by a user.

[0165] Clause 19. The instrument of clause 17, wherein the instructions further cause the processing circuitry to: perform a plurality of protein concentration and cell health measurements throughout a cell culture campaign based on a parameter measured by the bioreactor.

[0166] Clause 20. The instrument of clause 17, wherein the instructions further cause the processing circuitry' to: perform a plurality' of protein concentration and cell health measurements throughout a cell culture campaign based on parameters measured by the titer and cell health instruments.

[0167] Clause 21. The instrument of clause 7, wherein the cell health instrument is a cell viability measuring instrument.

[0168] Clause 22. The instrument of clause 7, wherein the cell health instrument is a cell density measuring instrument.

[0169] Clause 23. A method of cell line development using an automated laboratory instrument, the method comprising: culturing a plurality of biological samples in a bioreactor; obtaining, from a titer instrument of the automated laboratory' instrument, protein concentration in a sample of the plurality of biological samples from the bioreactor; obtaining, from a cell health instrument of the automated laboratory instrument, cell health in the sample of the plurality biological samples; and repeating the steps of obtaining protein concentration and cell health for each sample of the plurality of biological samples at a predetermined interval throughout a cell culture campaign.

[0170] Clause 24. The method of clause 23, further comprising obtaining, from the bioreactor, a measurement of one or more of pH, dissolved oxygen, volume, and biomass.

[0171] Clause 25. The method of clause 23, further comprising adjusting the predetermined interval based on one or more measurements by the bioreactor.

[0172] Clause 26. The method of clause 23, further comprising adjusting the predetermined interval based on one or more the protein concentration or the cell health measurement.

[0173] Clause 27. The method of clause 23, further comprising adjusting the predetermined interval based a user input.

[0174] Clause 28. The method of clause 23, further comprising comparing the protein concentration and cell health between each biological sample of the plurality- of biological samples.

[0175] Clause 29. The method of clause 28, further comprising, based on the comparison of protein concentration and cell health, selecting at least one biological sample to transfer to an output plate.

[0176] Clause 30. A method of operating an automated laboratory instrument for cell line development, the method comprising: receiving a container with at least one sample of a cell line to be developed in a bioreactor; and executing a protocol at regular intervals to develop the at least one cell line, the protocol including: obtain, from a titer instrument, protein concentration in the at least one sample; obtain, from a cell health instrument, cell health in the at least one sample; repeat the steps of obtaining protein concentration and cell health for each sample of the plurality- of biological samples at a predetermined interval throughout a cell culture campaign.

[0177] Clause 31. A system for cell line development, the system comprising: an automated laboratory instrument including a titer instrument and a cell health instrument; processing circuitry having a memory^ for storing instructions which, when executed by the processing circuitry-, cause the processing circuitry- to: obtain data from the titer instrument and the cell health instrument; generate a user interface including: a layout of one or more cell culture plates, each cell culture plate having one or more wells, wherein each of the cell culture plates and cell culture wells is configured to be named by a user and receive operational commands from the user to be executed by the automated laboratory instrument; a layout of a bioreactor in the automated laboratory- instrument, wherein the one or more cell culture plates are configured to be loaded into the bioreactor and shown in the layout of the bioreactor; a calendar display depicting scheduled protocols, configured to displays the scheduled protocols associated with a user selected one of the one or more cell culture place or one or more wells; and an indication performance of automated actions at the time of the automated actions.

[0178] Clause 32. The system of clause 31, wherein the automated actions include feeding each well of the one or more wells and obtaining, for each well of the one or more wells, at least one of pH, protein concentration from the titer instrument, and cell health from the cell health instrument.

[0179] Clause 33. The system of clause 31, wherein the user interface further comprises user operations for harvesting an output plate.

[0180] Clause 34. The system of clause 33, wherein harvesting the output plate comprises receiving the selection of one or more wells to be transferred to the output plate.

[0181] Clause 35. The system of clause 31, wherein the calendar display further depicts events requiring user input.

[0182] Clause 36. The system of clause 31, wherein the user interface further includes a display of bioreactor conditions.

[0183] Clause 37. The system of clause 36, wherein the bioreactor conditions include one or more of pH, biomass, dissolved oxygen, and volume.

[0184] Clause 38. The system of clause 36, wherein the bioreactor conditions are displayed per well of the one or more wells.

[0185] Clause 39. The system of clause 36, wherein the bioreactor conditions are displayed for a user selected well of the one or more wells.

[0186] Clause 40. A method of automated cell line development using an automated laboratory instrument, the method comprising obtaining, from a titer instrument of the automated laboratory instrument, protein concentration in a sample of a plurality of biological samples; obtaining, from a cell health instrument of the automated laboratory instrument, cell health in the sample of the plurality biological samples; and repeating, by a processing circuitry of the automated laboratory instrument, the steps of obtaining protein concentration and cell health for each sample of the plurality of biological samples at a predetermined interval throughout a cell culture campaign.

[0187] Clause 41. The method of clause 40, further comprising adjusting the predetermined interval based on one or more of the protein concentration or the cell health measurement.

[0188] Clause 42. The method of clause 40, further comprising adjusting the predetermined interval based on a user input.

[0189] 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. An automated laboratory instrument for measuring protein concentration and cell health of a biological sample, the automated laboratory instrument comprising: a housing containing: a biological sample container for receiving at least one biological sample; a pipettor configured to aspirate and dispense the biological sample; a titer instrument configured to receive the biological sample and measure protein concentration of the biological sample; a cell health instrument configured to receive the biological sample and measure cell health of the biological sample; processing circuitry having a memory for storing instructions which, when executed by the processing circuitry, cause the processing circuitry to: aspirate the biological sample from the container into the pipettor; dispense the biological sample into the titer and cell health instruments; measure protein concentration in the biological sample with the titer instrument; and measure one or more parameters of cell health in the biological sample with the cell health instrument.

2. The instrument of claim 1 , wherein biological sample aspirated into the pipettor includes a first and second volume of the biological sample.

3. The instrument of claim 2. wherein the first volume of the biological sample is dispensed into the titer instrument.

4. The instrument of claim 2, wherein the second volume of the biological sample is dispensed into the cell health instrument.

5. The instrument of claim 2, wherein the first and second volumes of the biological sample are spaced apart from one another in the pipettor, and wherein an air gap separates the spaced apart first and second volumes of the biological sample.

6. The instrument of claim 1, wherein the protein concentration and one or more parameters of cell health measurements are capable of being performed in parallel or sequentially.

7. The instrument of claim 1, wherein the biological sample contains a cell population in aqueous solution.

8. The instrument of claim 1, wherein the cell health instrument is a cell viability measuring instrument.

9. The instrument of claim 1, wherein the cell health instrument is a cell density measuring instrument.

10. The instrument of claim 1 , wherein the at least one biological sample further comprises at least two biological samples.1 1. The instrument of claim 10, wherein the processing circuitry is further configured to compare the protein concentration and cell health measurements between the at least two biological sample within the at least two biological samples.

12. The instrument of claim 1 1 , wherein the processing circuitry is further configured to select biological samples from the at least two biological samples to transfer to a container based on the comparison of the protein concentration and cell health measurements.

13. The instrument of claim 1, further comprising a population development module configured to culture at least one cell population.

14. The instrument of claim 13, wherein the population development module is a bioreactor and the biological sample in a container resides in the population development module.

15. The instrument of claim 1, wherein the instructions further cause the processing circuitry to: perform a plurality of protein concentration and cell health measurements throughout a cell culture campaign.

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