Fluorescence polarization analysis using a temperature controlled sample whose dilution may be adjusted

Abstract

Systems and method for titration analysis to indicate a protein concentration present in an experimental sample. The titration analysis system includes a titration analysis instrument with a light source, a thermally controlled sample receptacle, and a light detector configured to detect light from the light source after the light has passed through the thermally controlled sample receptacle. A non-transitory memory stores a reusable titration curve and a processing circuit is configured to receive a signal from the light detector, determine a fluorescence polarization value based on the signal, retrieve the reusable titration curve from the non-transitory memory as a reference curve, and fit the fluorescence polarization value to the reusable titration curve to indicate the protein concentration present in the experimental sample.

Description

FLUORESCENCE POLARIZATION ANALYSIS USING A TEMPERATURE CONTROLLED SAMPLE WHOSE DILUTION MAY BE ADJUSTEDCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is being filed on January 8, 2025 as a PCT International application and claims the benefit of and priority to U.S. Provisional Application No. 63 / 622,277, filed on January 18, 2024, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Protein titer determination is used in biologies research and development, representing the concentration of a specific protein within a sample. Protein analytics devices for measuring protein concentration utilize laser light and, in some cases, fluorescently labeled antibodies to analyze and sort cells based on their specific surface or intracellular protein markers. The protein titer refers to the concentration or abundance of a particular protein within a cell population. Protein titer plays a role in optimizing bioprocesses early in development, assessing cell culture efficiency, and predicting overall yield. It is used in quality control during manufacturing, ensuring product consistency and meeting specified concentration requirements. Monitoring protein titer over time aids in understanding cell culture growth and determining optimal harvesting times. Additionally, it plays a role in evaluating downstream purification processes, ensuring efficient removal of impurities and verifying target protein concentration. Accurate protein titer measurements are central for analytical method development, using techniques like flow cytometry, ELISA, HPLC, and mass spectrometry. These measurements are used to ensure regulatory compliance, providing thorough characterization data during the approval process. Ultimately, protein titer helps achieve the quality, consistency, and efficiency necessary for successful biologies production.SUMMARY

[0003] Generating a standard curve for protein titer traditionally presents challenges in biologies research and development. One frequent issue is variability in assay conditions (e.g., temperature, pH and other environmental factors), which can lead to inaccuracies in the standard curve. Factors such as pipetting errors, temperature fluctuations, and reagent inconsistencies may contribute to imprecise measurements.Additionally, protein stability and solubility can impact the reliability of the standard curve, especially when dealing with complex biological samples.

[0004] Because of these challenges, standard curves must traditionally be painstakingly regenerated for each run or each new sample. Disclosed herein is a reusable standard curve which eliminates the need for frequently regenerating standard curves. In embodiments, because of the temperature, pH, and environmental controls provided, a “default” standard curve is ensured to be appropriate for analysis of samples across a broad timescale. In addition, techniques are disclosed herein for comparing samples to one another based on their fitted location on the standard curve.

[0005] Examples presented herein relate to a titration analysis system to indicate a protein concentration present in an experimental sample. The titration analysis system includes a titration analysis instrument with a thermally controlled sample receptacle, a light source to direct light toward a sample in the thermally controlled sample receptacle, and a light detector configured to detect light emitted from the sample in the thermally controlled sample receptacle, a non-transitory memory storing a reusable titration curve, and a processing circuit. The processing circuit is configured to receive a signal from the light detector, determine a fluorescence polarization value based on the signal, retrieve the reusable titration curve from the non-transitory memory as a reference curve, and fit the fluorescence polarization value to the reusable titration curve to indicate the protein concentration present in the experimental sample.

[0006] In other aspects presented herein, the reusable titration curve embodies a protein concentration associated with a fluorescence polarization output. In yet other aspects presented herein, the thermally controlled sample receptacle regulates a temperature of a sample received in the sample receptacle.

[0007] In still other aspects presented herein, the reusable titration curve is a default curve.

[0008] In other aspects presented herein, the processing circuitry is further configured to combine the sample with a pH stable buffer and a probe. In further aspects presented herein, the processing circuitry is further configured to determine the sample is a high range sample. In yet further aspects presented herein, the processing circuitry is further configured to calculate a dilution of the sample with the pH stable buffer to yield an in-range sample. In still further aspects presented herein, the processing circuitry is further configured to calculate a multiplier, using the dilution, to apply to the reusable standard curve. In further aspects presented herein, the pH stable buffer comprises aMOPS((3-(N-morpholino)propanesulfonic acid))-based buffer. In still further aspects presented herein, the pH stable buffer is stored in an evaporation limiting container.

[0009] Other examples presented herein relate to a method of indicating relative protein concentrations present in a plurality of samples having an unknown protein concentration using a reusable titration curve. The method includes interrogating a plurality of samples of unknown protein concentrations with light from a light source, receiving a signal from a light detector detecting light fluoresced by the plurality of samples, determining a fluorescence polarization value of the plurality of samples having an unknown protein concentration, retrieving the reusable titration curve from the non- transitory memory as a reference curve for determining a protein concentration present in a plurality of experimental samples over a period of time and fitting the fluorescence polarization value to the reusable titration curve to indicate the protein concentrations present in the plurality of samples. In some aspects presented herein, the method further includes comparing the protein concentrations in the plurality of samples.

[0010] In other aspects presented herein, the method further includes harvesting a selection of the plurality of samples based on the comparison between protein concentrations. In still other aspects presented herein, running the plurality of samples of unknown concentrations comprises using a pH stable buffer. In further aspects presented herein, the method further includes storing the pH stable buffer in an evaporation limiting container.

[0011] In other aspects presented herein, running the plurality of samples of unknown protein concentrations further comprises maintaining each sample environmentally constant. In further aspects presented herein, maintaining each sample environmentally constant includes one or more of receiving each sample in a thermally controlled sample receptacle, suspending and / or diluting each sample with a pH stable buffer, and providing a controlled voltage to a photomultiplier tube during sample interrogation.

[0012] In other aspects presented herein, the method further includes combining the sample with a pH stable buffer and a probe. In further aspects presented herein, the method further includes determining the sample is a high range sample. In other further aspects presented herein, the method further includes calculating, in response to determining the sample is the high range sample, a dilution of the sample with the pH stable buffer to yield an in-range sample, and calculating a multiple, using the dilution, to apply to the reusable standard curve.

[0013] 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

[0014] 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:

[0015] FIG. 1 is an isometric view of an example of a sample analysis system that analyzes characteristics of samples.

[0016] FIG. 2 is another isometric view of the sample analysis system of FIG. 1 having a top cover removed from a housing of the sample analysis system.

[0017] FIG. 3 is a top view of a work platform supported inside the housing of the sample analysis system of FIG. 1.

[0018] FIG. 4 schematically illustrates an example of a titer module supported on the work platform of FIG. 3.

[0019] FIG. 5 is an isometric view of an example titer container assembly of the titer module of FIG. 4.

[0020] FIG. 6 is an exploded view of the titer container assembly of FIG. 5.

[0021] FIG. 7 is a flowchart of an example workflow for indicating a protein concentration present in an experimental sample.

[0022] FIG. 8 is a flowchart of an example workflow for generating and using a reusable titration curve is shown.

[0023] Referring now to FIG. 9, a flowchart of an example method 1000 of expanding a dynamic range of a reusable standard curve is shown.

[0024] FIG. 10 schematically illustrates an example of the controller of the sample analysis system that can be used to implement aspects described herein.

[0025] FIG. 11 is an example of a graphical user interface depicting a reusable standard curve.

[0026] FIG. 12 is an example of a graphical user interface depicting data entry and calculations for the reusable standard curve of FIG. 11.

[0027] FIG. 13 is an example of a graphical user interface depicting fitting a measurement of an unknow sample to the reusable standard curve of FIG. 11.DETAILED DESCRIPTION

[0028] 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.

[0029] FIG. 1 is an isometric view of an example of an automated sample analysis system 100 that analyzes samples for various properties. For example, cell samples may be analyzed to determine the cell health of a plurality of cell samples. For a single cell sample, the sample analysis 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-limiting examples, biological standards, reference beads that fluoresce, etc.

[0030] The sample analysis system 100 may include connectivity to automated bioreactors and other systems and devices. The sample analysis system 100 automates sample preparation and minimizes sample volume requirements for measuring cell and other sample characteristics. The sample analysis 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.

[0031] As shown in FIG. 1, the sample analysis system 100 includes a housing 102 that supports a work platform 300. 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, sample analysis system 100 further includes a controller (not pictured) storing instructions for various operations of the sample analysis system 100. A single controller may be configured to operate the various components of sample analysis system 100, or individual components may be associated with dedicated controllers. In some cases, the controller may be arranged separately from sample analysis system 100 and remotely connected.

[0032] FIG. 2 is another isometric view of the sample analysis system 100 having the top cover 104 removed from the housing 102, and thereby exposing the dispensing system 106. FIG. 3 is a top view of the work platform 300 supported inside the housing102. Referring now to FIGS. 2 and 3, the work platform 300 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.

[0033] The dispensing system 106 includes a liquid handler 108 that is movably mounted within the housing 102 in the space above the work platform 300. In some embodiments, liquid handler 108 includes multiple probes 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, the probe tips are fixed, such that they are permanent until replacement during a service process, while in other implementations, probe tips are disposable and may be replaced after each use. Embodiments with multiple probes may include a combination of fixed and disposable tips.

[0034] The liquid handler 108 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 handler 108, through which liquid aspiration and dispensing occurs, to access any container held on the work platform 300 inside the housing 102.

[0035] A proximal end of the liquid handler 108 is fluidly connected to a bidirectional pump having a movable actuator 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 bi-directional pump can include a syringe pump. Movement of the liquid handler 108 and the movable actuator is controlled by one or more step motors that operate under the control of a controller 900 that is programmable.

[0036] As shown in FIG. 2, the housing 102 is dimensioned to have a width W, a depth D, and a height H. As an illustrative example, the width W is about 24 inches to about 36 inches, the depth D is about 24 inches to about 36 inches, and the height H is about 24 inches to about 36 inches. In some examples, the housing 102 is dimensioned to have a cube shape.

[0037] As shown in FIG. 3, the work platform 300 supports a sample transfer module 302, one or more mixing plates 304, a cell health module 306, one or more tipracks 308, one or more titer plates 310, a tip waste bin 312, a diluent reservoir 314, sample inputs 316 such as tube trays and well plates, and a titer module 400. The cell health module 306 measures cell health, including cell count and cell viability. The titer module 400 measures, for example, protein titer.

[0038] FIG. 4 schematically illustrates an example of the titer module 400 supported on the work platform 300. As discussed herein, titer module 400 is implemented as a module that measure fluorescence polarization, which is used to perform protein analytics by comparing a fluorescence polarization value to a titration curve. The titer module 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 module 400 measures a concentration of Immunoglobulin G (IgG) present in a sample of cells.

[0039] More specifically, the titer module 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 module 400 is described herein with reference to examples measuring IgG concentration, the titer module 400 and the measurement techniques described herein can be used to measure the concentrations of additional types of proteins and antibodies.

[0040] As shown in FIG. 4, the titer module 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 module 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 sample analysis 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 system does 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.

[0041] 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-emiting diode (LED). In some examples, the titer module 400 includes a focusing lens 426 and aspectral filter 428 that respectively focus and filter the light 436 emitted from the light source 424.

[0042] 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. 4, the first direction is vertically linear. In alternative examples, the first direction is horizontally linear. Additional polarization directions are possible.

[0043] 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.

[0044] The polarized fluorescence light 438 can pass through a lens 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.

[0045] 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.

[0046] 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.

[0047] 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 light 438 into voltage values for input into a calculation to determine a measure of fluorescence polarization (FP).

[0048] 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. When the probe molecule is unbound, it disrupts the polarization of the light which causes a difference between parallel and perpendicularfluorescence to be smaller and leading to a lower fluorescence polarization. In contrast, when the probe molecule is bound to the larger antibody, the polarization is maintained, leading to a larger difference between parallel and perpendicular fluorescence and therefore to a higher fluorescence polarization. As the concentration of antibody increases, more and more of the probe molecules become bounded, increasing the sample fluorescence polarization.

[0049] Measurement output is determined by a ratio of a bound probe, referring to a population of the probe attached to antibody or other protein to be measured, to an unbound probe, referring to a population of free probes present. Measurement is based on this ratio, rather than total brightness or emission. The total amount of probe per milliliter determines brightness and an excitability of the probe is affected by pH and temperature. In embodiments, about 10: 1 dynamic range for the standard curve is expected. Introducing dilutions, as discussed in further detail below increases the dynamic range.

[0050] 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 intensify 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 intensify is observed, the system may provide an indication to an operator that the light source should be checked.

[0051] 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.

[0052] Referring now collectively to FIGS. 5 and 6, the titer container assembly 402 of the titer module 400 will be discussed in more detail. FIG. 5 is an isometric view ofan example titer container assembly 402 of the sample analysis system 100. FIG. 6 is an exploded view of the titer container assembly 402 of FIG. 5. Titer container assembly 402 includes a two-part body 404, 406, and a top retainer 408.

[0053] 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.

[0054] 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 module 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.

[0055] 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.

[0056] 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 toincrease surface area, promoting efficient heat dissipation through convection. A thermal interface 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.

[0057] 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.

[0058] 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 handler 108 of FIG. 2.

[0059] 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.

[0060] 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 module 400 and the liquid handling system 500.

[0061] 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 one or more sidewall of titer container 410. In another example, a fill port is provided from the interior of top retainer 408.

[0062] Top retainer 408 secures upper seal 412. Top retainer may include tip guide 422, which may assist in guiding liquid handler 108 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.

[0063] 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.

[0064] 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 module 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 module 400.

[0065] The titer analysis system further may include a processing circuitry in communication with a memory, which may be a non-transitory 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 sample analysis 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.

[0066] Many methods of quantitative analysis, such a protein concentration titer, rely on running a known sample, generally referred to as a standard, at known concentrations and recording the results. These results are used to create a reference for determining concentration of experimental samples, often referred to as a standard curve. This does not have to be a curve. It could be a matrix, or any other type of lookup table. A single curve or multiple joined curves are often used because they can be represented mathematically, such that any point along the x-axis is a defined value in the y-axis.Many standard curves consist of running a plurality of samples, plotting them on two or more axes and then performing a fit. Linear fitting is common, as well as polynomial fits, but any type of fit could be used. In the case of protein titer measurements, it is common practice to run a standard immediately before and sometimes after every instance of running unknown samples. This helps to minimize any environmental effects, like temperature, humidity, reagent decay, and other factors which could affect the results. This is problematic, because it requires constantly running known samples, which themselves can be tricky to work with and store. It also requires a significant additional time investment for each run of an unknown sample, in order to get a result.

[0067] Systems and methods disclosed herein are directed to creating a standard curve or a plurality of standard curves which can be stored on the instrument and associated with any unknown sample to determine the protein concentration. As disclosed herein and described in further detail below, standard curves can be created by running a plurality of known samples at known concentrations and stored in the system to be recalled for future use.

[0068] The system and reagents are configured to produce results stable enough over time such that stored standard curves are suitable to be used at a time which is not concurrent with the time the sample is run. In other words, a standard curve is created and stored for use in an hour, a day, a week, or any other time in the future without the need to run a new standard curve.

[0069] In embodiments, the system is configured with a method of choosing a standard curve to assign to any unknown sample. This may be done at the time the sample is defined or it could be done at a later point in time to re-calculate a result. In embodiments, the system is configured to work such that a single, or multiple, reusable standard curves are created at a known concentration, but then future samples are able to be run at different concentrations while still being referenced back to the stored standard curve. Further, by enabling the system to dilute the samples, the system is then able to use the reference curve to calculate concentrations that are higher or lower than the original curve created.

[0070] For example, an example standard curve is created by running known samples from lOOug / mL up to l,000ug / mL. The instrument may then perform the calculations to determine the curve definition. An experimental sample is then submitted, which may be identified as the same protein as the known samples used to generate the standard curve, but with a concentration somewhere around 6,000ug / mL. The systemautomatically calculates and performs a dilution (1: 10 in this example) such that the curve is valid from l,000ug / mL - 10,000ug / mL. When the experimental sample is run on the system, the system compares the result to the standard curve and determines the unknown sample had a concentration of 4,387ug / mL.

[0071] In embodiments, an instrument is shipped to an end user with a generic standard curve that the end user can run an unknown sample against and receive a result. While in some cases the result received may not represent a precise measurement, it will yield a repeatable result for a given protein. In embodiments, the system is further configured so that an end user is able to run known concentrations of an identified specific protein to be measured, which will improve the result accuracy. The more specific the protein used for the standard curve, the more accurate the expected results may be. For users with interests related to relative results between samples of the same type, ultimate accuracy may not matter, and a simple generic curve is adequate. However, the system is configured such that users with interest in more particular determinations can refine the results.

[0072] In embodiments, the system is configured to create the standard curve automatically from a single sample of target protein. A user may be prompted to tell the system what the concentration of the sample is and / or confirm the curve is good after creation. The system may be configured to calculate statistics and provide automated guidance on a usable range for the curve. In embodiments, the system is further configured to assist a user through software to determine a best range for running a particular experimental sample. For example, a user may run two samples. A first sample to determine a usable range for a standard curve to a be applied and a second sample to generate a result.

[0073] In some cases, the system provides feedback after running a sample to indicate if the range of the standard curve was appropriate for the sample, e.g., if the sample was in the middle, or functional range, of the standard curve or if it was near an edge of the curve where accuracy is reduced. The system may be further configured to provide guidance to the user regarding a predicted optimal range for the sample based on the result to assist in selection of an effective range for subsequent runs of the sample or related samples.

[0074] Referring now to FIG. 7, a flowchart of an example method 700 to indicate a protein concentration present in an experimental sample. To indicate, as discussed in the method 700, refers to a comparison between values rather than to an absolutemeasurement. In embodiments, the method 700 may be executed by a processing circuitry, such as a processing circuitry configured to operate the overall sample analysis system or a dedicated processing circuitry to execute a titer analysis. In embodiments, a memory associated with the processing circuitry includes a reusable titration curve.

[0075] At operation 701, a sample is loaded into a titration analysis system. In embodiments, the system may be configured to automatically draw and load a sample or the sample may be loaded by a user.

[0076] At operation 702, the sample may be combined with a pH stable buffer and a probe. In embodiments, when a sample is prepared for a protein concentration measurement, the sample is combined with the pH stable buffer, such as a MOPS buffer, and a dried down probe molecule. This helps to stabilize the sample pH and ensuring consistency in measurements over time. Addition of the pH stable buffer also contributes to rehydrating the probe molecule, which is maintained in a dried down state. The probe is maintained is this state, dry and cold, in order to extend its life in storage.

[0077] Dilution may be performed, for example, to prepare a high concentration sample to produce a result that can be effectively fit to a lower concentration reusable standard curve. In other words, the system also supports an optional additional dilution to extend the overall measurement range of the system. In embodiments, the additional dilution a high concentration sample, which may also be referred to herein as a high range sample due to appearing in the upper range of the reusable standard curve, is performed as an additional step of adding a calculated or predetermined volume of the pH stable buffer to the sample prior to preparing the sample for measurement, e.g., combining with the pH stable buffer and the probe. In such examples, the method includes two dilutions. A first dilution to bring the concentration of the sample in-range on the reusable standard curve and a second dilution to rehydrate the probe and prepare the sample for measurement.

[0078] In embodiments, the system may automatically perform the dilution based on a user entered concentration of a sample or a determination of the concentration of the sample based a prior run. For example, a first run produces a result that is fit to the reusable standard curve and is found to he at an edge of the curve and, in response, the system performs a calculation to dilute the sample based on that fit. In embodiments, the system performs the dilution calculation, automatically or in response to a user request, and provides the calculation result to the user for further action. In embodiments, the dilution may be performed externally to the titer module.

[0079] Use of a pH stable buffer contributes to the stability of the environment within the titer module and the predictability of titer results to support use of the reusable standard curve. Using the pH stable buffer limits the variability in pH between samples and runs of samples. The sample analysis system may be configured to accommodate the pH stable buffer, such by providing an evaporation limiting container for storage of the buffer.

[0080] In embodiments, the pH stable buffer comprises a MOPS-based buffer. “MOPS” refers to 3-(N-morpholino)propanesulfonic acid, a buffer often employed in electrophoresis and nucleic acid gel electrophoresis. A MOPS-based buffer is a solution made by combining MOPS in water to a specific concentration, used to maintain a stable pH in experiments.

[0081] At operation 704, one or more samples of unknown protein concentrations are excited with light from a light source. Samples are loaded into a thermally controlled sample receptacle and interrogated using fluorescent polarization, as discussed in greater detail above with respect to FIG. 4.

[0082] The thermally controlled sample receptacle regulates a temperature of a sample received in the sample receptacle and reports a measured temperature. Use of the thermally controlled sample receptacle limits the variability in temperature between samples and sample runs. In embodiments, temperature control of the sample receptacle may be achieved using a thermoelectric cooler (TEC) and feedback, such as closed loop feedback, from one or more thermistors located on the TEC assembly. The measured temperature is used for calculating temperature compensation to support adaption for minor changes in temperature, e.g., between the conditions for the generation of a reusable standard curve and the later conditions for a sample measurement. In some cases, the TEC assembly is a printed circuit board assembly.

[0083] At operation 706, a signal is received from a light detector. In embodiments, the light detector generates the signal in response to fluorescence generated by the sample as the light passes through the thermally controlled sample receptacle and excites the sample. At operation 708, a fluorescence polarization value is determined based on the signal. More specifically, a titer module, such as titer module 400 discussed in more detail above with reference to FIGS. 4-6, calculates a fluorescence polarization measurement for measuring the concentration of protein in the sample of cells. In some cases, the system measures the fluorescence intensity through parallel and perpendicularpolarizers., and the determined value is a ratio between the parallel and perpendicular polarizations.

[0084] At operation 710, a reusable titration curve is retrieved from the non- transitory memory as a reference curve. The reusable titration curve embodies a protein concentration associated with a fluorescence polarization output. In embodiments, the reusable titration curve may be loaded onto the instrument prior to any sample runs or may be generated based on a sample of known concentration run on the instrument. The reusable titration curve may be used as a reference curve for determining a protein concentration present in one or more experimental samples over a period of time.

[0085] The reusable titration curve may take the form of a matrix, a lookup table, a single curve, or a plurality of joined curves. In some cases, the reusable titration curve is plotted on two or more axes. In embodiments, the reusable titration curve is used a period of time after it was generated. The period of time may be one or more days, one or more weeks, one or more months, etc. In embodiments, one or more experimental samples are run without a time association to a known sample.

[0086] In embodiments, the reusable titration curve is one of a plurality of reusable titration curves. Curves among the plurality of reusable titration curves may vary by concentration and / or protein of interest. Each of the reusable titration curves may be created based on a unique known concentration. The reusable titration curve may be selected based on being a default curve or, in some cases, the reference curve is selected based on a dilution of the experimental sample.

[0087] At operation 712, the fluorescence polarization value is fit to the reusable titration curve to indicate the protein concentration present in the experimental sample. Linear and polynomial fitting are two example techniques used in curve fitting. Linear fitting involves fitting data to a straight line and is simple and easily interpretable but may not capture more complex relationships. Polynomial fitting extends the concept to include higher-degree polynomials. While polynomial fitting can capture more intricate patterns but may require regularization techniques to mitigate overfitting. Other fitting techniques include nonlinear regression, where data is fit to a nonlinear equation, and spline fitting, which uses piecewise-defined functions to interpolate or smooth the data. Additionally, advanced machine learning methods like support vector machines and neural networks may be advantageously applied to complex and high-dimensional datasets, offering more flexibility in capturing nonlinear relationships.

[0088] At operation 714, in examples where a plurality of samples are analyzed, the protein concentrations in the additional samples are run. At operation 716, the concentrations of the plurality of samples are compared. Comparison among the samples analyzed provides an indication of relative protein concentration across the plurality of samples. In cases where a plurality of samples are interrogated and analyzed, interrogation may be performed in parallel for each sample or sequentially, or in some combination of parallel and ordered analysis. Each sample may be interrogated by a common light source or with a separate light source for each or some of the samples.

[0089] In embodiments, a selection of the plurality of samples is harvested based on the comparison between protein concentrations. As discussed herein, harvesting refers to removing selected samples and placing them in an output plate or container for further processing.

[0090] Running the plurality of samples of unknown protein concentrations may further include measures to maintain each sample environmentally constant, such as use of a pH stable buffer and the thermally controlled sample receptacle. For example, maintaining each sample environmentally constant includes one or more of receiving each sample in a thermally controlled sample receptacle, suspending and / or diluting each sample with a pH stable buffer, and maintaining a constant voltage to a photomultiplier tube during sample interrogation. As changes in the voltage of the photomultiplier tube may influence the starting point of a curve and therefore cause data to be offset, maintaining the constant voltage contributes to the stable environment of the instrument and the reusability of the standard curve.

[0091] Referring now to FIG. 8, a flowchart of an example method 800 of generating and using a reusable titration curve is shown. In embodiments, the method 800 may be executed by a processing circuitry, such as a processing circuitry configured to operate the overall sample analysis system or a dedicated processing circuitry to execute a titer analysis.

[0092] The reusable standard curve establishes a quantitative relationship that can be used to determine the concentration of an unknown protein sample based on its measured response. In embodiments, the method 800 may be performed and the reusable titration curve may be loaded onto the instrument prior to any sample runs or the method 800 may be performed by a user of the titration analysis system and the curve may be generated based on a sample of known concentration run on the instrument. The reusable titration curve may be used as a reference curve for determining a protein concentrationpresent in one or more experimental samples over a period of time. The reusable titration curve may take the form of a matrix, a lookup table, a single curve, or a plurality of j oined curves. In embodiments, the reusable titration curve is used a period of time after it was generated. The period of time may be one or more days, one or more weeks, one or more months, etc. In embodiments, one or more experimental samples are run without a time association to a known sample. In embodiments, the reusable titration curve is one of a plurality of reusable titration curves. Curves among the plurality of reusable titration curves may vary by concentration and / or protein of interest. Each of the reusable titration curves may be created based on a unique known concentration.

[0093] In embodiments, it is preferable for a user to generate a standard curve appropriate for their analysis by using a known sample of the particular target protein or target subclass. In some cases, a default is provided, however, better results may generally be achieved through use a reusable standard curve based on the particular target protein or protein subclass, rather than a generic curve or a curve based a different protein. The system may store multiple curves, based on any number of target proteins. In embodiments, a user may select the curve to be used, either directly or by indicating a target protein to measured.

[0094] At operation 802, a plurality of known samples at known concentrations are run, including performing fluorescence polarization measurements per the method 700 in FIG. 7 on a series of samples with known protein concentration. Running each sample includes loading the sample into a titer container, interrogation by fluorescence polarization, and measuring a resulting fluorescence polarization associated with the sample. In embodiments, running the plurality of known samples at known concentrations comprises using a pH stable buffer. In embodiments, operation 802 may be preceded by running a sample with probe to determine appropriate gain settings for the photomultiplier tube to use for the standard curve to be generated. In examples, the known concentrations are selected from a range including 100 mg / L to 1200 mg / L.

[0095] Known concentrations may be prepared, for example, by precise combinations of media with the high concentration input protein of interest. In embodiments, the system may prepare the series of samples automatically and provide those sample to the titer module for analysis.

[0096] In some cases, an experimental sample of unknown concentration may be run with the plurality of known samples at known concentrations. In embodiments, running the plurality of known samples and the experimental sample further comprisesmaintaining each sample environmentally constant. Maintaining each sample environmentally constant may include receiving each sample in a thermally controlled sample receptacle, suspending and / or diluting each sample in a pH stable buffer, and maintaining a constant voltage to a photomultiplier tube during sample interrogation.

[0097] At operation 804, a curve definition of the reusable titration curve is determined using data from running the plurality of known samples at the known concentrations. The curve definition may be, for example, linear relationship depending on the assay characteristics. In one example, the titer module calculates regression coefficients using a 4th order polynomial fit with protein concentration on X-axis and fluorescence polarization on the Y-axis. This fit may be preferred in some cases because it is easy to implement in software and succeeds without a lot of careful handling.

[0098] In addition to the curve definition, e.g., the regression coefficients, the output of the standard curve procedure may include quality criteria to help determine if the resulting standard curve is suitable for use. For example, a check on the range of fluorescence polarization values measured during the procedure (delta shift) may be performed. Based on the predictable performance of the titer probe, it may be known, in one example, that there is typically lOOmP difference between zero protein and saturation. The delta shift criteria may then be used to ensure that the procedure did reach saturation.

[0099] In embodiments, the delta shift is further applied to help the user determine if the curve fails to reach saturation, limiting the utility of this particular curve. For example, if the protein of interest has a low affinity for the probe molecule, it may saturate well beyond the typical range of the standard curve procedure. This results in a low-resolution curve that does not effectively differentiate between protein concentrations, e.g., low precision. In some embodiments, the user may elect to modify the sample preparation for the protein of interest to increase the volume of specimen, thereby increasing the slope of the curve, and in effect the resolution, and optimizing the functional range of the standard curve. In such an example, where the user decides to modify the specimen volume for the protein of interest, a new standard curve procedure is run with the modified sample preparation. Any samples of unknow n concentration that are to be analyzed with this standard curve are prepared with a matching specimen volume and sample preparation.

[0100] In addition, a check on the repeatability of replicates may be performed. For example, the series of samples may be prepared with three replicates per knownconcentration. If, for example, one measurement is an extreme outlier compared to the others at a given concentration, that sample is suspect and may have produced bad regression coefficients. This check may be performed to confirm that the samples that should be similar were indeed similar.

[0101] A third check performed in some embodiments is on the protein functional range. For example, some criteria may be set on how steep the curve needs to be (mP / protein) to be able to differentiate one value from another. Then, using this resolution criteria, a range of protein concentrations that are able to be measured is determined. This range is configured to be sufficiently large to ensure that samples throughout a cell culturing sequence, for example, generally land in the functional range of the standard curve.

[0102] In embodiments, the protein functional range assists a user in determining if the curve saturates too early, limiting the utility of this particular curve. For example, if the protein of interest has a high affinity for the probe molecule, it may saturate as early as 600mg / L. Not only does this make it more difficult to measure a sample of unknown concentration, it also limits the maximum concentration the user may measure with the system. In some embodiments, the user may elect to modify the sample preparation for the protein of interest to decrease the volume of specimen, thereby delaying saturation to a higher protein concentration and extending the functional range of the standard curve. In such an example, where the user decides to modify the specimen volume for the protein of interest, a new standard curve procedure is run with the modified sample preparation. Any samples of unknown concentration that are to be analyzed with this standard curve are prepared with a matching specimen volume and sample preparation.

[0103] At operation 806, the reusable titration curve is stored in the non-transitory memory. In embodiments, a plurality of reusable titration curves may be generated, each according to the method 800. Each of the reusable titration curves may be created using a unique known concentration.

[0104] At operation 808, the reusable titration curve is retrieved from the non- transitory memory as a reference curve for determining a protein concentration present in a plurality of experimental samples over a period of time. The curve is used as a reference to interpolate or extrapolate the concentration of unknown samples based on their corresponding assay responses.

[0105] For example, following a run of an experimental sample, a sample curve of the sample is plotted. The sample curve may then be fit to the reusable titration curve.Fitting the curve to the reusable titration curve may be one of a linear fitting and a polynomial fitting. An output is generated indicating a fit between the sample curve and the reusable titration curve. The output may indicate a deviation between the fit and a reference fit, e.g., the data fits the curve near an edge and outside the functional range of the curve. In some cases, a dilution for the sample is calculated based on the deviation between the fit and the reference fit. The sample may then be diluted, automatically or in response to a user input, using the dilution calculated. In some cases, the output includes an optimal dilution range for a subsequent run of the sample.

[0106] In embodiments, the plurality of known samples are prepared at known concentrations as a series of dilutions. Running a plurality7of known samples at known concentrations may include at least a first run and a second run of each known concentration. A curve definition of the reusable titration curve may be determined using data from each of the first run and the second run to determine a first curve and a second curve and by comparing the first curve and the second curve for consistency. In examples, running a plurality of known samples at known concentrations includes a third run of each known concentration, determining a curve definition of the reusable titration curve using data the third run to determine a third curve, and comparing the third curve to each of the first curve and the second curve for consistency.

[0107] In embodiments, the reusable titration curve is associated with analysis of a sample of known concentration run at a time independent of the sample of unknown concentration. The sample of known concentration is run at least one day before the sample of unknown concentration, at least one week before the sample of unknown concentration, at least one month before the sample of unknown concentration, etc.

[0108] Referring now to FIG. 9, a flowchart of an example method 1000 of expanding a dynamic range of a reusable standard curve is shown. In embodiments, the method 1000 may be executed by a processing circuitry, such as a processing circuitry configured to operate the overall sample analysis system or a dedicated processing circuitry to execute a titer analysis.

[0109] At operation 1002, a fluorescence polarization value is determined for an unknown sample. As discussed in greater detail above, the unknown sample may be combined with a pH stable buffer and a probe and excited with a light source. Fluorescence generated by the unknown sample and probe response to the excitation is detected through both parallel and perpendicular polarizers., and the determined value is a ratio between the parallel and perpendicular polarizations.

[0110] At operation 1004, a determination is made whether the sample is a high range reading or an in-range reading. If the determination is made at operation 1004 that the sample yields a high range reading, the system may proceed to calculate a dilution for the sample, at operation 1006. For example, if the determine value is fit to the reusable standard curve and found to be outside of the reliable range of the curve, the system may automatically determine a dilution of the sample and determine a second fluorescence polarization value using the diluted sample. In embodiments, a user may view the high range reading and manually adjust dilution and / or system settings to obtain the second fluorescence polarization value using a dilute sample At operation 1008, a multiplier for the reusable standard curve is calculated. The multiplier is determined using the dilution calculated at operation 1006. By applying a multiplier to the reusable standard curve, the dynamic range of the reusable standard curve can be expanded to give usable results based on the diluted sample.

[0111] If the determination is made at operation 1004 that the sample yields an inrange reading, the system may proceed to fit the measurement to the reusable standard curve, at operation 1010.

[0112] FIG. 10 schematically illustrates an example of the controller 900 of the sample analysis system 100 that can be used to implement aspects described herein, including features of the titer module 400 and liquid handling system 500. As shown in FIG. 10, the controller 900 includes one or more processing devices 902, a memory storage device 904, and a system bus 906 that couples the memory storage device 904 to the one or more processing devices 902. The one or more processing devices 902 can include central processing units (CPU). In some instances, the one or more processing devices 902 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.

[0113] As shown in FIG. 10, the memory storage device 904 can include a randomaccess memory ("RAM") 908 and a read-only memory (“ROM”) 910. Basic input and output logic having basic routines that help to transfer information between elements within the controller 900, such as during startup, can be stored in the ROM 910.

[0114] The controller 900 can also include a mass storage device 912 that can include an operating system 914 and store software instructions and data 916. The mass storage device 912 is connected to the processing device 902 through the system bus 906.The mass storage device 912 and associated computer-readable data storage media provide non-volatile, non-transitory storage for the controller 900.

[0115] Although the description of computer-readable data storage media contained herein refers to the mass storage device 912, 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 900 can read data and / or instructions. The computer-readable storage media can be comprised of entirely non-transitory media. The mass storage device 912 is an example of a computer-readable storage device.

[0116] 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.

[0117] The controller 900 can operate in a networked environment using logical connections to the other devices through the network 920. The controller 900 connects to the network 920 through a network interface unit 918 connected to the system bus 906. The network interface unit 918 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 918 can connect the controller 900 to additional networks, systems, and devices. The controller 900 also includes an input / output unit 922 for receiving and processing inputs and outputs from peripheral devices.

[0118] The mass storage device 912 and the RAM 908 can store software instructions and data. The software instructions can include an operating system 914 suitable for controlling the operation of the sample analysis system 100. The mass storage device 912 and / or the RAM 908 can also store the software instructions and data 916, which when executed by the processing device 902, provide the functionality of the sample analysis system 100 discussed herein.

[0119] FIG. 11 is an example of a graphical user interface 1100 depicting a reusable standard curve 1102. Reusable standard curve 1102 is plotted on an x-axis 1104 of fluorescence polarization, measured in units of mP, and a y-axis 1106 of proteinconcentration. Data points 1108 are determined by running samples of known protein concentration and measuring their fluorescence polarization output. Reusable standard curve 1102 may be generated and used using the method 800 of FIG. 8 as described above. The reusable standard curve 1102 is shown plotted into two distinct regions 1110 and 1112. A first, in-range region 1110 shows a usable range of the curve 1102. A second, high range region 1112 is delineated to depict to a user where the curve 1102 may become unreliable and assist the user is making dilution and other adjustment decisions. In embodiments, datapoints 1114 are included to demonstrate the fit of reusable standard curve 1102. For example, control samples are run in duplicate or triplicate, etc. during generation of the standard curve to enable the identification of outlier readings. By depicting each datapoint 1114, a user is able to see where points overlap versus where points are spread, indicating the fit may rely on an average of somewhat distributed measured values.

[0120] In addition to the curve plot itself, a curve summary 1116 presents parameters of the curve to assist a user in effectively applying it. Some example parameters shown in this example are a protein range 1118, a R2value 1120, and a delta shift 1122. Protein range 1118 may assist a user in determining when a particular sample is out of range or a high range sample. R2value 1120 provides a metric of how well the reusable standard curve 1102 fits the data, e.g., datapoints 1114. Delta shift 1122 is discussed is greater detail above and provides a user with a metric useful, for example, in evaluating the saturation of a curve. Curve equation 1124 mathematically defined reusable standard curve 1102.

[0121] FIG. 12 is an example of a graphical user interface 1200 depicting data entry and calculations for the reusable standard curve 1102 of FIG. 11. A standard data summary 1202 provides additional detail of the data values used to plot datapoint 1114 and underlying the reusable standard curve 1102. Background data 1204 is also provided, showing, by example, an origination date and approval of the reusable standard curve 1102.

[0122] FIG. 13 is an example of a graphical user interface 1300 depicting fitting a measurement 1302 of an unknown sample to the reusable standard curve 1102 of FIG. 11. Measurement 1302 of the unknown sample is shown graphically fit 1304 to the reusable standard curve 1102, allowing a user to readily see whether the measurement 1302 is in-range or high range. A numerical summary 1306 is the protein concentration determination based on the reusable standard curve 1102 is also provided. The numericalsummary 1306 may include snapshot metrics, such as temperature at the time of reading, to aide a user is determining suitability of results. As, for example, the temperature at the time of generating the reusable standard curve is also recorded, the two values can be compared to evaluate the fit of the unknown sample to the curve. In embodiments, graphical user interface 1300 may incorporate input from other modules, such a cell counter or cell health module, to provide further protein concentration metrics, such as protein per cell.

[0123] 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.

[0124] Clause 1. A titration analysis system to indicate a protein concentration present in an experimental sample, the titration analysis system including a titration analysis instrument including: a thermally controlled sample receptacle; a light source to direct light toward a sample in the thermally controlled sample receptacle; and a light detector configured to detect light t emitted from the sample in the thermally controlled sample receptacle; a non-transitory memory storing a reusable titration curve; and a processing circuit configured to: receive a signal from the light detector; determine a fluorescence polarization value based on the signal; retrieve the reusable titration curve from the non-transitory memory as a reference curve; and fit the fluorescence polarization value to the reusable titration curve to indicate the protein concentration present in the experimental sample.

[0125] Clause 2. The titration analysis system of clause 1, wherein the reusable titration curve embodies a protein concentration associated with a fluorescence polarization output.

[0126] Clause 3. The titration analysis system of any one of clauses 1 or 2, wherein the thermally controlled sample receptacle regulates a temperature of the sample received in the sample receptacle.

[0127] Clause 4. The titration analysis system of any one of clauses 1-3, wherein the reusable titration curve is a default curve.

[0128] Clause 5. The titration analysis system of any one of clauses 1-4, wherein the processing circuitry' is further configured to combine the sample with a pH stable buffer and a probe.

[0129] Clause 6. The titration analysis system of clause 5, wherein the processing circuitry is further configured to determine the sample is a high range sample.

[0130] Clause 7. The titration analysis system of clause 6, wherein the processing circuitry is further configured to calculate a dilution of the sample with the pH stable buffer to yield an in-range sample.

[0131] Clause 8. The titration analysis system of clause 7, wherein the processing circuitry is further configured to calculate a multiplier, using the dilution, to apply to the reusable standard curve.

[0132] Clause 9. The titration analysis system of clause 5, wherein the pH stable buffer includes a MOPs-based buffer.

[0133] Clause 10. The titration analysis system of clause 9, wherein the pH stable buffer is stored in an evaporation limiting container.

[0134] Clause 11. A method of indicating relative protein concentrations present in a plurality of samples having an unknown protein concentration using a reusable titration curve, the method including interrogating a plurality of samples of unknown protein concentrations with light from a light source; receiving a signal from a light detector detecting light fluoresced by the plurality of samples; determining a fluorescence polarization value of the plurality of samples having an unknown protein concentration; retrieving the reusable titration curve from the non-transitory memory as a reference curve for determining a protein concentration present in a plurality of experimental samples over a period of time; and fitting the fluorescence polanzation value to the reusable titration curve to indicate the protein concentrations present in the plurality of samples.

[0135] Clause 12. The method of clause 11, further including harvesting a selection of the plurality of samples based on the comparison between protein concentrations.

[0136] Clause 13. The method of any one of clauses 11 or 12, wherein running the plurality of samples of unknown concentrations includes using a pH stable buffer.

[0137] Clause 14. The method of clause 13, further including storing the pH stable buffer in an evaporation limiting container.

[0138] Clause 15. The method of any one of clauses 11-13, wherein running the plurality of samples of unknown protein concentrations further includes maintaining each sample environmentally constant.

[0139] Clause 16. The method of clause 15, wherein maintaining each sample environmentally constant includes one or more of receiving each sample in a thermally controlled sample receptacle, suspending and / or diluting each sample with a pH stablebuffer, and maintaining a constant voltage to a photomultiplier tube during sample interrogation.

[0140] Clause 17. The method of any one of clauses 11-16, further including comparing the protein concentrations in the plurality of samples.

[0141] Clause 18. The method of any one of clauses 11-17, further including combining the sample with a pH stable buffer and a probe.

[0142] Clause 19. The method of clause 18, further including determining the sample is a high range sample.

[0143] Clause 20. The method of clause 19, further including calculating, in response to determining the sample is the high range sample, a dilution of the sample with the pH stable buffer to yield an in-range sample; and calculating a multiple, using the dilution, to apply to the reusable standard curve.

[0144] The various embodiments described above are provided by way of illustration only and should not be construed to be limiting in any way. Various modifications can be made to the embodiments described above without departing from the true spirit and scope of the disclosure.

Claims

WHAT IS CLAIMED IS:

1. A titration analysis system to indicate a protein concentration present in an experimental sample, the titration analysis system comprising: a titration analysis instrument including: a thermally controlled sample receptacle; a light source to direct light toward a sample in the thermally controlled sample receptacle; and a light detector configured to detect light t emitted from the sample in the thermally controlled sample receptacle; a non-transitory memory storing a reusable titration curve; and a processing circuit configured to: receive a signal from the light detector; determine a fluorescence polarization value based on the signal; retrieve the reusable titration curve from the non-transitory memory as a reference curve; and fit the fluorescence polarization value to the reusable titration curve to indicate the protein concentration present in the experimental sample.

2. The titration analysis system of claim 1, wherein the reusable titration curve embodies a protein concentration associated with a fluorescence polarization output.

3. The titration analysis system of any one of claims 1 or 2, wherein the thermally controlled sample receptacle regulates a temperature of the sample received in the sample receptacle.

4. The titration analysis system of any one of claims 1-3, wherein the processing circuitry is further configured to combine the sample with a pH stable buffer and a probe.

5. The titration analysis system of claim 4, wherein the processing circuitry is further configured to determine the sample is a high range sample and to calculate a dilution of the sample with the pH stable buffer to yield an in-range sample.

6. The titration analysis system of claim 5, wherein the processing circuitry is further configured to calculate a multiplier, using the dilution, to apply to the reusable standard curve.

7. The titration analysis system of claim 4, wherein the pH stable buffer comprises a MOPs-based buffer.

8. The titration analysis system of claim 7, wherein the pH stable buffer is stored in an evaporation limiting container.

9. A method of indicating relative protein concentrations present in a plurality of samples having an unknown protein concentration using a reusable titration curve, the method comprising: interrogating a plurality of samples of unknown protein concentrations with light from a light source; receiving a signal from a light detector detecting light fluoresced by the plurality of samples; determining a fluorescence polarization value of the plurality of samples having an unknown protein concentration; retrieving the reusable titration curve from the non-transitory memory as a reference curve for determining a protein concentration present in a plurality of experimental samples over a period of time; and fitting the fluorescence polarization value to the reusable titration curve to indicate the protein concentrations present in the plurality of samples.

10. The method of claim 9, further comprising harvesting a selection of the plurality of samples based on the comparison between protein concentrations.

11. The method of any one of claims 9 or 10, wherein running the plurality of samples of unknown concentrations comprises using a pH stable buffer.

12. The method of claim 11, further comprising storing the pH stable buffer in an evaporation limiting container.

13. The method of any one of claims 9-12, wherein running the plurality of samples of unknown protein concentrations further comprises maintaining each sample environmentally constant.

14. The method of claim 13, wherein maintaining each sample environmentally constant includes one or more of receiving each sample in a thermally controlled sample receptacle, suspending and / or diluting each sample with a pH stable buffer, and maintaining a constant voltage to a photomultiplier tube during sample interrogation.

15. The method of any one of claims 9-14, further comprising: combining the sample with a pH stable buffer and a probe; determining the sample is a high range sample; calculating, in response to determining the sample is the high range sample, a dilution of the sample with the pH stable buffer to yield an in-range sample; and calculating a multiple, using the dilution, to apply to the reusable standard curve.

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