Systems and methods for improved tracking, fractionation, collection, and identification of analytes in a sample

Abstract

A method comprising introducing, at a first time, a sample containing a plurality of analytes and a plurality of isoelectric point (pI) markers in a conductive medium into a capillary, each pI marker from the plurality of pI markers having a known isoelectric point from a plurality of isoelectric points, the plurality of pI markers including a subset of pI markers, each pI marker from the subset of pI markers being configured to be excited by light of a wavelength within a known excitation range of wavelengths to emit a non-native fluorescence signal at a wavelength within a range of emission wavelengths; separating, at a second time after the first time, the plurality of pI markers and at least a subset of the plurality of analytes, each pI marker and each analyte being focused according to their isoelectric points, by applying a voltage across the capillary; eluting a portion of the sample that includes at least one of an analyte from the plurality of analytes or a pI marker from the subset of pI markers into a well at a third time after the second time; probing the portion of the sample including the at least one of the analyte or the pI marker from the subset of pI markers with light within the known excitation range of wavelengths to cause the pI marker to emit the non-native fluorescence signal; and detecting the non-native fluorescence signal indicating a presence of the pI marker in the portion of the sample.

Description

SYSTEMS AND METHODS FOR IMPROVED TRACKING, FRACTIONATION, COLLECTION, AND IDENTIFICATION OF ANALYTES IN A SAMPLERELATED APPLICATIONS

[1001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 640,162 filed April 29, 2024, which is incorporated herein by reference in its entirety for all purposes.BACKGROUND

[1002] The embodiments described herein relate generally to systems and methods for tracking, separating, analyzing, and eluting fractions and locating or identification of separated fractions from sample mixtures.

[1003] In known capillary electrophoresis techniques, a sample is separated and then individual fractions of the sample are eluted into individual wells of a sample collection plate. One known technique to identify fractions within the sample collection plate involves placing the sample collection plate under a fluorescence or UV reader to measure the native fluorescence or UV absorbance of the collected material directly. This technique, however, may only be suitable for identify ing wells having a high concentration of an analyte (e.g., the main peak) and may not have sufficient sensitivity to identify wells containing fractions with smaller analyte concentrations.

[1004] Determination of the location of a particular analyte using known techniques may call for testing the contents of each w ell using a method or modality orthogonal to the modality- used for detection of analytes during separation. This testing of contents of each well, however, can take costly additional time, be inefficient, and could lead to significant sample loss. There is a need for quick, efficient, and accurate methods and systems to detect a location of fractions containing analytes before and / or after elution and fraction collection.Brief Description of the Drawings

[1005] FIG. 1 is a schematic illustration of a fractionation system, a system configured to perform capillary electrophoresis, fractionate, and / or locate analytes separated by capillary electrophoresis, according to an embodiment.

[1006] FIG. 2 is a flowchart of a method of using a fractionation system, according to one implementation.

[1007] FIG. 3 is a flowchart of a method of using a fractionation system, according to one implementation.

[1008] FIGS. 4A is an example plot showing results from separation and isoelectric focusing of a sample.

[1009] FIG. 4B represents an elution of a series of fractions of the sample of FIG. 4A into a series of wells in a sample plate.

[1010] FIG. 4C illustrates experimental results from analysis of contents in a subset of wells shown in FIG. 4B.Detailed Description

[1011] Techniques such as Isoelectric focusing (IEF) can be a powerful approach to separating analytes in a sample, for example, charge variants of protein molecules such as monoclonal antibodies (mAbs) or other biological molecules, with good resolution and sensitivity. Therapeutic monoclonal antibodies (mAbs) make up a large portion of the rapidly growing drug market. Ensuring safety and efficacy through comprehensive understanding of these products’ critical quality attributes (CQAs), including charge heterogeneity, is a regulator}7requirement. Various charge isoforms of mAbs can result from cell culture or production processes, potentially affecting the mAb structure and function. Imaged capillary isoelectric focusing (icIEF) is a method that can be used for charge profiling. Ion-exchange chromatography (IEC) has also been a major tool for fractionation combined with characterization. IEC, however, is not compatible with certain types of molecules, hydrophobic antibody drug conjugates (ADCs) for example, and icIEF typically provides higher separation resolution. Moreover, an individual charge variant obtained from IEC fractionation may not be comparable to the variant peak in the icIEF profile. Therefore, there is an unmet need for IEF- based fractionation of charge variants for characterization.

[1012] IEF can be performed with the sample mixed with ampholytes sandwiched between an acid and a base reservoir. Under an electric field, each charged component of the sample migrates to a position along a pH gradient formed by the ampholytes where the pH is the sameas that component's isoelectric point (pl). Capillary isoelectric focusing (cIEF) is a variant of this approach where IEF is performed in a sample held in a lumen of a capillary.

[1013] In case of cIEF, due to the miniatured fluidic path and insignificant Joule heating involved, larger magnitude electric fields can be applied for the separation of components in a sample held in the lumen of a capillary, resulting in fast separation and better resolution of separation of the analytes in the sample. Whole column (icIEF) is a method that can used as described in U.S. Patent 10,794,860 entitled, “Systems and methods for capillary electrophoresis, isoelectric point, and molecular weight analysis,” the disclosure of which is incorporated herein by reference in its entirety. icIEF further improves the speed, resolution, and precision of the assay due to the fact that no sample mobilization is needed for the detection and a shorter capillary can be used for the separation. Because of its superior performance, icIEF can be widely used as a standardized analytical tool in several industries including the pharmaceutical industry for the characterization and quality control of therapeutic proteins including mAbs. antibody drug conjugates (ADCs) and other biological molecules. cIEF systems can also be used for fractionation and sample collection, such as described in U.S. Patent No. 11,420,202, entitled “Systems and Methods for Fractionation and Collection of Analytes in a Sample,” the disclosure of which is incorporated herein by reference in its entirety.

[1014] While icIEF is a powerful method yielding rich information about charge variants of proteins, sometimes additional information may be desired to fully characterize and identify a molecule, for example, to identify and / or isolate unknown impurities that may arise from formulation or bioprocessing stages of generating the molecule. It may be desirable to not only separate the charge variants but also to isolate them for further analysis using methods such as Mass Spectrometry (MS) or other biological assays.

[1015] Methods explored to utilize additional processing in conjunction with separation techniques like icIEF can be categorized into two groups: fraction collection and hyphenated cIEF-MS. Fraction collection methods allow individual fractions of charge variants in a sample to be collected and further processed according to desired needs. Hyphenated cIEF-MS methods interface directly from the capillary of the cIEF system into the ionization source of a MS system. While the hyphenated cIEF-MS method circumvents the efforts needed for fraction collection, it has some limitations: (1) the fractions cannot be analyzed by downstream analysis method other than MS; (2) performance of the MS will be compromised if the cIEF run needsUREA or any other additives that are unfriendly to MS systems; (3) peptide mapping is not possible on such hyphenated platforms. On the other hand, multiple fraction collection devices have been developed and commercialized but with limited success either because of the poor performance (e.g., poor resolution, insufficient sensitivity, low yield, etc.) or because they are difficult to operate (e.g., complicated device set up, lack of robustness, etc.).

[1016] Fraction collection methods can include isolation and / or collection of fractions of the sample using multi-well (e.g., 96-well) plates. Fractions are typically isolated and / or collected following a separation process. One known technique to identity7fractions within the sample collection plate involves placing the sample collection plate under a fluorescence reader to detect analytes via native fluorescence. This technique, however, may only be suitable for identifying wells having a high concentration of an analyte (e.g., the main peak) and may not have sufficient sensitivity7to identity’ wells containing fractions with smaller analyte concentrations. For example, w ells containing sample fractions with low7analyte concentration may exhibit significant non-sample emissions (e.g.. non-sample UV noise). Determination of the location of a particular analyte may call for testing the contents of each well using a method or modality7orthogonal to the modality7used for detection of analytes during separation and / or a repetition of cIEF analysis to identify which analytes have been captured and / or eliminated. This testing of contents of each well, however, can take costly additional time, be inefficient, and could lead to significant sample loss. There is a need for quick, efficient, and accurate methods and systems to detect a location of fractions containing analytes before and / or after elution and fraction collection. Even in implementations of hyphenated cIEF-MS methods there exists a need for tracking and locating isolated charge variants in a sample to increase accuracy of separation. Embodiments disclosed herein provide methods for tracking and locating isolated charge variants in a sample following separation and isolation.

[1017] Embodiments disclosed herein provide a novel cIEF fractionation solution, which involves cIEF separation and collection of charge variants with ability to locate, identify, and isolate charge variants prior to and / or following elution and collection of one or more fractions. Some embodiments described enable Maurice™ icIEF-based peak identification followed by downstream determination of location of individual fractions, identification of analytes in a fraction, and / or characterization of a fraction, such as native analysis of collected charge variants using ZipChip (CE-ESI) due to the broad sample matrix compatibility, easy sample prep, and fast mass spectrometry analysis time.

[1018] Embodiments described herein include apparatus, methods, and systems for performing fractionation and location of a fraction of analytes in a sample using a suitable separation technique (e.g., capillary isoelectric focusing) such that there is streamlined, semiautomatic, separation, visualization, detection, fractionation of analytes in a sample into fraction collection wells and / or determination of location of the analytes before or after elution of fractions. The fractionation and / or fraction identification can be performed such that the collected analytes can be further processed using any suitable technique without any restriction as in the case of the hyphenated cIEF-MS methods.Definitions

[1019] As used in this specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherw ise. Thus, for example, the term “a member” is intended to mean a single member or a combination of members, “a material” is intended to mean one or more materials, or a combination thereof.

[1020] As used herein, the terms “about” and “approximately” mean plus or minus 10% of the value stated and all values in between. For example, about 0.5 would include 0.45 and 0.55, about 10 would include 9 to 11, about 1000 would include 900 to 1100. The term “substantially” when used in connection with presence or absence of a signal or substance (e.g., substantially no native fluorescence) is intended to convey that the presence or absence of the signal or substance meets the criterion to not impact or impede the intended action that requires the presence or absence. The term “negligible” when used in connection with presence or absence of a signal or substance (e.g., negligible native fluorescence) is intended to convey that there is no amount of signal or substance that is beyond a criterion that may impact or impede the intended action (e.g., detection of a native fluorescence of an analyte) that calls for the negligible amount of signal or substance. For example, a signal having a strength less than 3 times the noise floor may be considered negligible in some contexts. In other contexts, a signal having an area of less than 1% of a signal of interest may be considered negligible.

[1021] As used herein the term “module” refers to any assembly and / or set of operatively- coupled electrical components that can include, for example, a memory, a processor, electrical traces, optical connectors, software (executed in hardware), and / or the like. For example, a module executed in the processor can be any combination of hardware-based module (e.g., a field-programmable gate array (FPGA), an application specific integrated circuit (ASIC), adigital signal processor (DSP)) and / or software-based module (e.g., a module of computer code stored in memory’ and / or executed at the processor) capable of performing one or more specific functions associated with that module.

[1022] As used herein, the terms “analyte” and / or “target analyte” refer to any molecule or compound to be separated and / or detected with the methods, apparatus and systems provided herein. Suitable analytes include, but are not limited to. small chemical molecules such as. for example, environmental molecules, clinical molecules, chemicals, pollutants, and / or biomolecules. More specifically, such chemical molecules can include, but are not limited to pesticides, insecticides, toxins, therapeutic and / or abused drugs, antibiotics, organic materials, hormones, antibodies, antibody fragments, antibody-molecule conjugates (e.g.. antibody-drug conjugates), antigens, cellular membrane antigen, proteins (e.g., enzymes, immunoglobulins, and / or glycoproteins), nucleic acids (e.g., DNA and / or RNA), lipids, lectins, carbohydrates, whole cells (e.g., prokaryotic cells such as pathogenic bacteria and / or eukaryotic cells such as mammalian tumor cells), viruses, spores, polysaccharides, glycoproteins, metabolites, cofactors, nucleotides, polynucleotides (comprising ribonucleic acid and / or deoxyribonucleic acid), transition state analogs, inhibitors, receptors, receptor ligands (e.g., neural receptors or their ligands, hormonal receptors or their ligands, nutrient receptors or their ligands, and / or cell surface receptors or their ligands), receptor-ligand complexes, nutrients, electrolytes, growth factors and other biomolecules and / or non-biomolecules, as well as fragments and combinations thereof. In some embodiments, the analyte is a protein or a protein complex, and the sample is a cellular lysate or a purified protein. Other suitable analytes can include aggregates, agglomerates, floc, and / or dispersed phase droplets or particles of colloids and / or emulsions.

[1023] As used herein, the term “sample” refers to a composition that contains an analyte or analytes to be detected. A sample, in some embodiments, is heterogeneous, containing a variety of components (e.g., different proteins) or homogenous, containing one component (e.g., a population of one protein). In some instances, a sample can be naturally occurring, a biological material, and / or a manufactured material. Furthermore, a sample can be in a native (e.g., a cell suspension) or denatured form (e.g., a lysate). In some instances, a sample can be a single cell (or contents of a single cell, e.g., as a cellular lysate from the single cell, or a purified protein) or multiple cells (or contents of multiple cells, e.g.. as a cellular lysate from the multiple cells, or a purified protein from the multiple cells), a blood sample, a tissue sample,a skin sample, a urine sample, a water sample, and / or a soil sample. In some instances, a sample can be from a living organism, such as a eukaryote, prokaryote, mammal, human, yeast, and / or bacterium or the sample can be from a virus.

[1024] In some embodiments, the sample is a heterogeneous biological sample or derived from a heterogeneous biological sample, for example a tissue lysate, a cellular lysate or a mixture of biomolecules such as proteins (e.g., a purified protein). In a further embodiment, a protein within the cellular lysate is the analyte to be detected by the methods and systems described herein. In a further embodiment, the apparatus, systems, and methods provided herein provide for the detection of a particular form of a protein, for example, a phosphorylated protein. The cellular lysate, for example, can be the lysate of one cell or a mixture of cells. Moreover, the cellular lysate can include a single cell type, or multiple cell types. The cell ty pe, in some embodiments, includes a stem cell or a cancer cell, or a population of stem cells, or a population of cancer cells. In some embodiments, a sample comprises one or more stem cells (e.g., any cell that has the ability’ to divide for indefinite time periods and to give rise to specialized cells) and / or stem cell lysates. Suitable examples of stem cells can include but are not limited to embryonic stem cells (e.g., human embryonic stem cells (hES)), and non- embryonic stems cells (e.g., mesenchymal, hematopoietic, induced pluripotent stem cells (iPS cells), or adult stem cells (MSC)).

[1025] In some instances, prior to detecting and / or fractionating an analyte in a sample with the apparatus and systems provided herein, processing may be performed on the sample. For example, a sample can be subj ected to a lysing step, denaturation step, heating step, purification step (e.g., protein purification), precipitation step, immunoprecipitation step, column chromatography step, centrifugation, etc. In some embodiments, a sample is subjected to a denaturation step prior detecting and / or separating a target analyte in a sample with the methods, apparatus, and systems described herein. The processing step on the sample, in some embodiments, is performed in one of the apparatus or systems described herein. In another embodiment, the processing step is performed prior to introducing the sample into one of the apparatus or systems set forth herein.

[1026] As used herein, the term isoelectric point (pl) marker refers to a component having a known isoelectric point that is configured to be added to a sample. Markers can also be referred to as standards and / or a marker can be a standard that is readily detectable, for example because of a label, dye, or other detectable characteristic (e.g., native fluorescence). In someinstances, a marker is different from the analyte but behaves in a way similar to or the same as the analyte, enabling relevant comparative measurements. In some embodiments, a marker that is suitable for use can be any of those described in U.S. Patent Application Publication No. 2007 / 0062813 entitled, “Electrophoresis Standards, Methods and Kits,” filed on September 20, 2006, the disclosure of which is incorporated herein by reference in its entirety. For example, a pl marker can be a stable, salt-free, purified protein with a known pl point. A set of pl markers (e.g.. a lyophilized mixture of stable, salt-free, purified proteins) can form a “ladder” when separated.

[1027] In some implementations, pl markers can be categorized as a separation marker and / or a fractionation marker. A separation marker can be a pl marker that is used for indicating a location of a known isoelectric point along a pH gradient resulting from applying voltage for example along the length of a capillary in an implementation of capillary' isoelectric focusing (cIEF). Separation markers can be visualized and / or detected using a first detection modality'. In some implementations, the first detection modality’ can be the same methods used for visualizing the separated analytes themselves, for example, native fluorescence emission or UV absorbance generating a record of a separation intensity' plot (e.g., FIG. 4A). A separation marker can be a pl marker having native fluorescence and or UV absorbance in a predetermined range of w avelengths designated for analyte identification. A native fluorescence or UV absorbance signal associated with a separation marker at one or more known pl can thus occur and be detected and recorded alongside native fluorescence or UV absorbance signal associated with a plurality' of analytes in the sample each analyte separated at its pl. In some instances, it may thus be important for the separation markers to be selected such that the pl associated w ith each separation marker is distinct and far removed from a pl associated with an analyte included in the sample so that there is no interference between the signal from the separation marker and the signal from the analyte with a near adjacent pl. In some implementations, separation markers can be used to align separation intensity' plots obtained from multiple across multiple samples.

[1028] A fractionation marker can be a pl marker that is used for indicating a location of a fraction having a known isoelectric point, the fraction obtained from a pH gradient resulting from applying voltage for example along the length of a capillary in an implementation of capillary isoelectric focusing (cIEF). In some implementations, the fractionation markers can have pl that are substantially different than pl associated with the analytes included in thesample. In some implementations, the fractionation markers can have pl that are substantially overlapping and / or proximal to the pl associated with the analytes in the sample. Fractionation markers can be visualized and / or detected using a second detection modality different than and / or orthogonal to the first detection modality used for detection of separation markers and / or analytes included in the sample. Similarly stated, fractionation markers can be visualized and / or detected using a different mechanism and / or property for visualizing the separated analytes themselves. For example, in an instance in which the separated analytes are detected and / or visualized using native fluorescence emission or UV absorbance, the second modality can be non-native fluorescent emission (e.g., emissions of a fluorescent dye or tag) that is excited by light of a particular wavelength. Fractionation markers can be configured to be excited by light of a wavelength within a known excitation range of wavelengths to emit a nonnative fluorescence signal at a wavelength within a range of emission wavelengths. In some instances, a fractionation marker can be a pl marker with a label added to the pl marker, for example, a fluorescent label that is excitable by the light of the wavelength within the known excitation range of wavelengths. The label can be configured to emit the non-native fluorescence signal at the wavelength within the range of emission wavelengths. In some instances, the fractionation marker can have negligible native fluorescence in the predetermined range of w avelengths designated for analyte identification so that there is little to no interference between native fluorescence from a pl marker and native fluorescence from an analyte. The wavelength or range of wavelengths for excitation of one or more fractionation markers can be the same or can be different as desired and / or based on application / selection. The wavelength or range of wavelengths for emission of signal upon excitation from of one or more fractionation markers can be the same or can be different as desired and / or based on application / selection.

[1029] In the implementations in which the pl markers have pls that are substantially different than pl associated with the analytes included in the sample, there is less concern of interference between signals from pl markers and signals from analytes. In some such implementations, it might be acceptable to have cross-talk betw een signals from fractionation markers (or separation markers, as discussed above) and the analytes as long as the separation makers can be adequately identified and detected in the separation intensity7plot (e.g., of the capillary prior to elution) and the fractionation markers can be adequately detected and identified in a collection plate (e.g., after elution) or at the tip of a capillary during the elution of one or more fractions following separation. In the implementations in which thefractionation markers have pl that are substantially overlapping and / or proximal to the pl associated with the analytes in the sample, however, there is heightened concern over potential leak or interference between signals from pl markers and signals from analytes. In some such implementations, interference can be reduced by increasing separation and reducing cross-talk between signals from pl markers and signals from analytes, between the detection channels and / or detection modalities.

[1030] In some embodiments, pl markers added to a sample can include a first subset of pl markers that serve as fractionation markers, which can be fluorescently labeled and a second subset of pl markers different than the first subset that serve as separation markers, which can be natively fluorescent. Fluorescently labeled pl markers and natively fluorescent pl markers are commercially available. In some embodiments, one or more pl markers can serve as both separation and fractionation markers.Overview of a Fractionation Process

[1031] Embodiments described include systems and methods to perform separation, detection, and / or fractionation of one or more analytes in a sample (e.g., based on molecular weight and / or isoelectric point). The sample can be prepared in a conductive medium and loaded into capillary that is in turn loaded into a fractionation system (also referred to herein as "the system”).

[1032] The embodiments of fractionation systems described herein can be used to separate analytes, detect and / or visualize separated analytes, and selectively fractionate one or more analytes in a sample, based on the separation and / or visualization, using a single system. Embodiments described herein can use microfluidic separation techniques, thereby enabling the analysis of very’ small volume samples.

[1033] In some instances, multiple analytes can be separated, detected, and / or fractionated from a sample loaded in a single capillary' by the system using apparatus and / or methods provided herein. For example, in some instances, a user can load a capillary cartridge into the system and can initiate and / or otherwise provide instructions to the system to cause the system to at least semi -automatically separate analytes (e.g., proteins) within the sample by isoelectric point.Separation

[1034] Analytes and / or markers described above, can be separated using a fractionation system by taking advantage of any suitable mobility parameter such as charge, molecular weight, electrophoretic mobility (e.g., influenced by molecular weight, characteristic length, area, or volume, oligonucleotide length, or other suitable characteristic), and / or the like. The sample can be loaded into a capillary and the capillary' can be positioned in the system such that a first end of the capillary is coupled to a first running buffer having a first pH and a second end of the capillary can be ionically coupled to a second running buffer having a second pH, such that a pH gradient is formed along the length of the capillary via the lumen of the capillary. The analytes can be separated (e.g., according to their isoelectric points) by applying a voltage across the first running buffer and the second running buffer For example, in some embodiments a voltage can be applied between the first running buffer having the first pH that is ionically coupled to a first end of the capillary, and a second running buffer having the second pH that is ionically7coupled to a second end of the capillary. The applying of the voltage across the ends of the capillary can induce separation of analytes along a fluid path in the capillary lumen comprising the sample, based on a mobility parameter such as an isoelectric point and / or the like.

[1035] In some embodiments, the capillary' can include a separation matrix, which can be added in an automated fashion. Capillary electrophoresis through a separation matrix using the system can be analogous to separation in a polymeric gel. such as a polyacrylamide gel or an agarose gel, where molecules are separated on the basis of the mobility parameter of the molecules in the sample, by providing a porous passageway of fluid path through which the molecules can travel.Visualization and Elution

[1036] In some embodiments, the separated analytes and / or separation markers can be visualized and their relative localization along the fluid path can be determined. The apparatus can then be manipulated to elute one or more analytes individually based on the visualization and / or relative localization of the separated analytes. That is, following visualization one or more of the separated analytes can be made to migrate towards a distal end of the capillary', and eluted out of the capillary to be collected in a collection well. The separated analytes can be mobilized towards the distal end of the capillary using any suitable technique including pressure ejection, voltage induced mobilization towards a distal end of the capillary, elutionusing chemical mobilizers, etc. In some instances, chemicals with different negative ions, for example, acetate and phosphate, can be used as chemical mobilizers.

[1037] In some implementations, the migration of separated bands of analytes and serial elution of isolated bands of one or more analytes can be conducted while still maintaining separation of the analytes by providing appropriate counterbalancing force to prevent mixing of the separated analytes. For example, a counter balancing force of negative pressure (via vacuum source) can be used to counter the effects of gravity on a vertically oriented capillary during elution of separated analytes.

[1038] In some implementations, the visualization and / or detection of analytes can be conducted in a real-time or semi-real-time manner such that the separated analytes and their relative localization (e.g., localization of a peak concentration of each separated analyte in the fluid path) can be monitored as the separated analytes are made to migrate towards the distal end of the capillary. In some implementations, the apparatus can be manipulated such that a movement of a sample plate including a plurality of collection wells can be coordinated based on the relative localization of analytes and / or separation markers (e.g., relative location of peak concentrations of each separated analyte) and / or a rate of migration of each analyte (e.g., rate of migration of a peak concentration of each analyte). For example, the sample plate can include multiple collection wells and the capillary can be moved between collection wells eluting a fraction of the sample (e g., one or more separated analytes) into each collection well. In some embodiments, each collection well can contain a chemical mobilizer (e.g., the same or different chemical mobilizers), and the capillary can be moved from collection well to collection well. While in a collection well a voltage can be applied until a fraction of the sample is eluted. Once the fraction of the sample is eluted (e.g., as determined by continuous monitoring of the capillary), the capillary can be moved to another collection well, where a subsequent fraction of the sample can be eluted. In addition, or as an alternative to chemical mobilization, a fraction of the sample can be eluted into each collection well by applying a pressure to the capillary. Using known techniques, however, separation markers may interfere with visualization of nearby analytes, while analytes further from separation markers may be challenging to detect or resolve along the fluid path during separation and / or focusing, particularly for analytes having a relatively low concentration. In some embodiments, therefore markers can be added to the sample to bracket analytes of interest. In addition or alternatively, fractionation markers that do not interfere with analyte visualization (e g., by having negligiblenative fluorescence within a detection band associated with analytes, by not having a fluorescent label or a fluorescent label that has a similar excitation wavelength to analytes, etc.) can be added to the sample.A Fractionation System

[1039] FIG. 1 is a schematic illustration of a portion of a fractionation system 100 (also referred to herein as “the system”) configured to perform separation, detection, fractionation and / or determination of location of fractions including one or more analytes in a sample (e g., separation based on molecular weight and / or isoelectric point) according to an embodiment.

[1040] Embodiments of the fractionation system 100 described herein can be used to facilitate separation of one or more analytes in a single system, visualization and / or detection of analytes within a sample before, during, and / or after the separation, fractionation of one or more separated analytes based on the separation and visualization and detection, and determination of location of fractions with one or more analytes. Embodiments described herein can provide the functionality of pipettes and microfluidic paths, thereby enabling the separation, analysis, and / or fractionation of very small volume samples. Such apparatus and / or systems can include any suitable device, mechanism, assembly, subassembly, electronic device, actuator, and / or the like that can enable the apparatus and / or system to, for example, separate, visualize, detect, fractionate any suitable target analytes, and / or locate fractions collected that include one or more analytes.

[1041] The system 100 includes a housing 101, a probe system 102, a cartridge retainer 103 configured to receive and / or secure a capillary' cartridge 104, a sample plate assembly 107, and an electronic system 108. While not shown in FIG. 1, the electronic system 108 can include a processor, a memory, a communicator, and / or a power source. The electronic system 108 can be configured to permit communications with external compute devices using any suitable mode of communication, for example, to receive / transmit data and / or instructions. In some embodiments, the system 100 can be configured such that the electronic system 108 includes any suitable system or assembly with a power source, a processor, and a memory that can be configured and / or otherwise programmed to perform one or more processes (e.g., hardware module and / or software module stored in the memory and executed in the processor) associated with performing at least a semi-automatic electrophoretic separation. Similarly, the system 100 can include any suitable fluid flow system or assembly that defines one or more fluid flowpaths configured to receive a fluid such as, for example, a sample, one or more reagents, and / or the like, which can flow through the system 100 as described in further detail herein with reference to specific embodiments.

[1042] The housing 101 of the system 100 can be any suitable shape, size, or configuration and can be arranged to at least partially enclose or at least partially house any suitable component of the system 100. For example, the housing 101 can at least partially enclose the probe system 102, the sample plate assembly 107, the capillary cartridge retainer 103, and the electronic system 108. Although not shown in FIG. 1, in some embodiments, the housing 101 can be configured to form one or more portions, chambers, inner volumes, etc. that are configured to allow at least some of the components of the system 100 to be disposed therein. In some embodiments, the housing 100 can include a door configured to provide access to the inner volume defined thereby. For example, a user can open the door of the housing 100 to position a capillary cartridge 104 within the capillary cartridge retainer 103, as described in further detail herein. In some embodiments, at least a portion of the housing 101 can be light tight such that no substantial quantify of light leaks through the housing into a chamber defined by the housing. In some embodiments, the housing 101 can define at least one climate- controlled chamber. Similarly stated, the system 100 can be operable to maintain a chamber of the housing at a constant and / or preset temperature, humidity, and / or other environmental parameter (e.g., illumination, etc.).

[1043] The probe system 102 of the system 100 can be fixedly disposed within the housing 101. In some embodiments, the probe system 102 can be disposed in a predetermined and fixed position relative to the cartridge retainer 103 and / or one or more components in association with the cartridge retainer 103 or the cartridge 104 (e.g., a viewing window (not shown) defined on the cartridge, the capillary 106, etc.,). For example, the probe system 102 can be arranged within the housing 101 such that predetermined portions of the probe system 102 are aligned with and / or otherwise disposed in a desired position relative to predetermined portions of the cartridge retainer 103. In some embodiments, the probe system 102 and / or cartridge retainer 103 can include any suitable adjustment mechanism or the like to ensure a desired alignment between the probe system 102 and the cartridge retainer 103.

[1044] The probe system 102 can include any suitable device, mechanism, and / or assembly that is configured to capture and / or detect digital or analog data (e.g., images) of, for example, an analyte and / or marker and / or to detect a signal emitted by the analyte and / or marker (e.g.,in a sample held in the capillary 106) such that any suitable analyses may be performed using the signal (e.g., analyses conducted by the electronic system 108). In some embodiments, the probe system 102 can include one or more emitters and one or more detectors (not shown in FIG. 1).

[1045] The emitters included in the probe system 102 can be any suitable device, member, mechanism, assembly, and / or the like that is configured to release energy (e.g., heat, photons, radiation, etc.). For example, in some embodiments, the emitters can include LEDs, arrays of LEDs, a deuterium lamp, a laser, an incandescent light source, a fluorescent light source, or any other suitable light source. The emitter, in some embodiments, can be optically coupled to the cartridge retainer 103, the cartridge 104, and / or the capillary 106 via one or more lenses, mirrors, prisms, fiber optics, and / or the like. The emitter(s) can be powered and / or excited to emit light at, for example, a predetermined wavelength and / or range of wavelengths. In some embodiments, the probe system 102 can include one or more mirrors, lenses, filters and / or the like configured to direct, focus, and / or convert the wavelength of photons emitted by the emitter(s). For example, the probe system 102 can include any suitable lens and / or filter (e.g., a TAMRA filter) that is associated with chemiluminescence, fluorescence (e.g., native fluorescence, fluorescence of label moieties, etc.), absorbance, and / or the like. In some embodiments, the probe system 102 can include a sequence of emitters, for example, a grid array of fiber optic outputs, LEDs, or the like (e.g., a column of light). In some embodiments, the probe system 102 can be configured such that the one or more emitters can be used to convey energy (e.g., excitation energy7) to a sample via one or more apertures, filters, blockers, reflectors and / or refractors, etc. The energy7conveyed can be configured to interact with at least a portion of a sample contained within the capillary 106 of the cartridge 104 when the cartridge 104 is retained by the cartridge retainer 103.

[1046] The detectors included in the probe system 102 can be any device that can receive or acquire a signal emitted or associated with a portion of a sample in the capillary 106 and convey data or information associated with the acquired signal to a processor (e.g., a processor included in the electronic system 108). In some embodiments, the detectors can be configured to receive signals in one form that can be transduced to another form or to data that can be transmitted to the processor. For example, the detectors can include any suitable digital or analog detectors that can capture a signal in the form of light emitted by a portion of the sample and transduce the captured signal into data (e.g., digital data conveying information related tointensity, wavelength, quality, duration of emission, etc.) that can be used to perform suitable analyses of the portion of the sample from which the signal was received. As an example, in some embodiments, one or more detectors can be and / or can include a photodiode, an array of photodiodes, a photomultiplier tube (PMT), a charged coupled device (CCD) array, and / or the like. The detectors can be used to capture an image and / or signal associated with the analyte and / or marker within a sample. In some embodiments, the detectors can be operable to capture images and / or signals emitted from the analyte and / or marker periodically and / or continuously. In some embodiments, the detectors can be operable to monitor the analyte and / or marker, in real time or substantially in real time, which can allow a user to rapidly determine whether an analyte is present in the sample, a rate of migration of a portion of the sample (e.g., during elution), an amount or activity of the analyte, a molecular weight of the analyte, and / or the like.

[1047] In some embodiments, a detector can be used during a fractionation to detect and / or image, substantially in real time, a flow of a sample through the capillary 106 of the cartridge 104 (in this instance, configured for use in fractionation based on isoelectric focusing and associated analyses) when the cartridge 104 is retained by the cartridge retainer 103. In some embodiments, a detector can be used during and / or after isoelectric focusing. For example, the detector can be used to detect the separation of analytes substantially in real time as analytes separate and focus and / or after analytes have been focused and, optionally, migrated towards an end of the capillars’ to be fractionated out in isolation. Similarly stated, the detector can detect a signal (e.g., fluorescence, absorbance, etc.) associated with one or more analytes included in a sample that has been separated and / or is in the process of being eluted from within the capillary 106 of the cartridge 104 (in this instance, configured for use in isoelectric focusing) when the cartridge 104 is retained by the cartridge retainer 103. As describe previously, the probe system 102 can be operably coupled to any suitable electrical or electronic circuit included in the electronic system 108 and / or associated with a remote device. The probe system 102 can be configured to send and / or receive signals from a processor and / or the like (e.g., the probe system 102 can send one or more signals to the processor or the like in the electronic system 108 to cause data associated with the captured images and / or detected signals to be stored, for example, in a memory or database). The probe system 102 can include a single detector or multiple detectors (e.g., more than two) configured to detect a portion of energy (e.g., light of specified wavelength range) produced by the emitters and / or interacted by a portion of the sample (e.g., fluorescence from a separated analyte in a sample held in capillary 106).

[1048] As described above, the system 100 is configured to receive a capillary cartridge 104 (also referred to herein as ‘"cartridge”). In some embodiments, the system 100 is configured to receive a cartridge 104 including one or more capillaries 106 and to expose at least a portion of the cartridge 104 to negative pressure differential (e.g., produced by a vacuum source) operable to draw a volume of fluid (e.g., one or more reagents, samples, buffers, washes, detectors, analytes, ampholytes, and / or the like) from one or more wells or trays included in the apparatus and / or system into the capillary(ies) 106 of the cartridge 104.

[1049] In some embodiments, the system 100 is configured to receive the cartridge 104 including capillaries 106 and to expose at least a portion of the cartridge 104 to a positive pressure differential (e.g., produced by a pressure source) operable to inject or eject a volume of fluid (e.g., one or more reagents, samples, buffers, washes, detectors, analytes, ampholytes, and / or the like) from the capillary 106 to one or more wells or trays.

[1050] In some embodiments, the system 100 is configured to receive the cartridge 104 including one or more capillaries 106 and to expose at least a portion of the cartridge 104 to a source of applied voltage or a source of electric current that can be operable to draw a volume of fluid via electrokinetic injection (e.g., one or more reagents, samples, buffers, washes, detectors, analytes, ampholytes, and / or the like) from one or more wells or trays into the capillary 106. In some embodiments, the system 100 can be configured to expose at least a portion of the cartridge 104 to a source of applied voltage or a source of electric current that can be operable to inject a volume of fluid via electrokinetic injection (e.g., one or more reagents, samples, buffers, washes, detectors, analytes, ampholytes, and / or the like) from the capillary 106 to one or more wells or trays. Electrokinetic injection can be injection of a substance (inj ectate) by applying a voltage or a current via a substance in a capillar}' 106. An amount of the substance injected into a well or vial can depend on a mobility of components included in the substance, a diameter of the capillary 106, the applied electric field and / or the injection time (e.g., time of application of the voltage).

[1051] The cartridge 104 can include at least a body portion that is fixedly coupled to at least one capillary' 106. The cartridge 104 can be any suitable shape, size, or configuration.

[1052] The capillary cartridge retainer 103 (also referred to herein as “cartridge retainer”) is fixedly disposed within the housing 101. For example, in some embodiments, the cartridge retainer 103 can be coupled to a frame or the like that maintains the cartridge retainer 103 in asubstantially fixed position within the housing 101. In some embodiments, the cartridge retainer 103 can also be coupled to and / or otherwise disposed in a fixed position relative to the analysis system 102, as described in further detail herein.

[1053] The cartridge retainer 103 can be any suitable shape, size, or configuration. For example, the cartridge retainer 103 can include, for example, a set of sidewalls that define an inner volume configured to receive at least a portion of a capillary cartridge 104 (also referred to herein as ‘‘cartridge”). More particularly, the cartridge retainer 103 can have or define a substantially C-shaped cross-section with at least one side of the cartridge retainer 103 being substantially open. A user can insert the cartridge 104 through the substantially open side of the cartridge retainer 103 to position at least a portion of the cartridge 104 within the inner volume. In some embodiments, the cartridge retainer 103 can include a latch mechanism suitable to form a friction fit, a snap fit, a threaded coupling, and / or the like with at least a portion of the cartridge 104 to couple the cartridge 104 to the cartridge retainer 103. In other words, the cartridge retainer 103 at least temporarily couples to the cartridge 104 when the portion of the cartridge 104 is inserted into the inner volume to maintain the cartridge 104 in a substantially fixed position relative to the cartridge retainer 103.

[1054] The cartridge retainer 103 can be configured to receive the cartridge 104 in a predetermined orientation (e.g., only one orientation or way). Although not shown in FIG. 1, the cartridge retainer 103 can include any suitable alignment feature or sensor configured to engage and / or sense a portion of the cartridge 104 as the cartridge 104 is positioned within the cartridge retainer 103. More particularly, the cartridge retainer 103 can include, for example, any number of features (e.g., protrusions, openings, grooves, etc.), assemblies, mechanisms, sensors, and / or the like, each of which engage and / or sense a portion of the cartridge 104 to ensure the cartridge 104 is retained within the cartridge retainer 103 at a desired position and / or in a desired orientation.

[1055] The cartridge 104 is configured to receive and house a capillary 106 in a specified orientation such that when the cartridge 104 is engaged with the cartridge retainer 103 the capillary 106 can be engaged with one or more components of the system 100 to enable forming a fluid path that can be used to manipulate a sample (e.g., draw the sample, separate analytes in the sample, analyze one or more constituents of the sample, fractionate one or more analytes, etc.)

[1056] The capillary 106 is configured to be placed in fluid communication with one or more fluid reservoirs (e.g., disposed in the cartridge body and / or disposed in or defined by a reagent tray or the like). In some embodiments, the one or more fluid reservoirs can be wells or the like containing a fluid with constituents having any of the chemistries described above. In some embodiments, the one or more fluid reservoirs can be wells (e.g., sample collection wells) having solutions for separation and / or fractionation of analytes including running buffers (e.g.. acid or base solutions), chemical mobilizers. etc. as described herein.

[1057] The capillary 106 of the cartridge 104 defines a lumen that receives at least a portion of a sample, solution, reagent, analyte, and / or any other suitable fluid or gel. In some embodiments, the capillary 106 can include a separation matrix configured to support generation of a pH gradient and / or separation of analytes (e.g., via isoelectric focusing). In some embodiments, the capillary 106 of a cartridge 104 can be an elongate member having a rounded or circular cross-sectional shape or a polygonal cross-sectional shape (e.g., trapezoidal, rectangular, square, pentagonal, octagonal, etc.). In some embodiments, the shape and / or size of the lumen defined by the capillary 106 can be based at least in part on the sample, the sample volume, and / or the type of analysis (e.g., with an inner diameter of about 10 micrometers or “microns” (pm) to about 1000 pm). For example, a capillary' 106 having a relatively small inner diameter can be associated with and / or otherwise used for relatively low sample volumes, which can be suitable for expensive samples or reagents. Conversely’, a capillary 106 defining a relatively larger inner diameter can be associated with and / or otherwise used for relatively high sample volumes, which in some instances, can result in improved signal detection or the like. In other embodiments, the inner diameter can be based at least in part on the analysis to be performed (e.g., molecular weight-based separation, isoelectric focusing, etc.). In some embodiments, capillaries with multiple segments having different inner diameters (including transition segments with variable inner diameter) can be used, as described herein.

[1058] The capillary 106 can be any suitable shape, size, volume, or configuration and can be formed from any suitable material (e.g., glass, plastic, silicon, fused silica, gel, metal, carbon nanotubes, PYREX™ (amorphous glass), and / or the like) that allow s a liquid and / or dissolved molecules to flow through the lumen. The capillary 106 can have any suitable length and any suitable inner diameter and a suitable outer diameter. For example, in some embodiments, the capillary 106 can have a length of approximately 50 to 120 mm (e.g., a length as small as 5mm,10mm, 20mm, 30mm, 40mm, 50mm, 60mm 70mm. 80mm, 90mm, 100mm, 110mm. 120mm, or any suitable length therebetween). In some embodiments, the capillary 106 can have a length of approximately 100 mm to 1000 mm (e.g., a length as small as 100mm, 200mm, 300mm, 400mm, 500mm, 600mm 700mm, 800mm, 900mm, 1000mm, 1100mm, or any suitable length therebetween). In some embodiments, the capillary' 106 can have a single constant inner diameter and / or outer diameter. For example, in some embodiments the capillary can have an inner diameter of approximately 320 to 530 pm (e.g., an inner diameter of 320pm, 420pm, 530pm, or any suitable inner diameter therebetween.

[1059] In some embodiments, the length of the capillary can be based at least in part on factors such as sample size or volume and the extent of sample separation when resolving the analyte or analytes of interest (e.g., between about 2 centimeters (cm) and about 20 cm), where a longer capillary' can result in increased separation of samples, which in turn, can improve resolution of complex mixtures and / or mixtures having a low abundance of analytes. In some embodiments, the capillar}' 106 can have any suitable diameter which can be based at least in part on factors such as sample size or volume, an extent of sample separation when resolving the analyte or analytes of interest, a speed of separation, a speed of migration for elution or fractionation of analytes in a sample (e.g., between Imm / min and 5mm / min), where a capillary with a larger diameter can result in larger volumes and / or increased speed of migration of separated analytes in the samples, which in turn, can improve or increase a fractionated amount of a separated analyte, and / or a speed of fractionation of the analytes from a complex mixtures and / or mixtures in the sample. The diameter of the capillary can be based at least in part on factors such as sample size or volume, the extent of sample separation desired for an analyte or analytes of interest, and / or a speed of fractionation.

[1060] In some embodiments, the capillary 106 can include two or more portions each portion associated with a specified length and location and each portion having a specified variable inner diameter and / or outer diameter, as described in further detail in following sections. In some embodiments, the capillary 106 can be made as a unibody element. In some embodiments, the capillary' 106 can be made by joining two or more portions together.

[1061] In some embodiments, the cartridge retainer 103 can include and / or otherwise can be coupled to any suitable assembly, mechanism, device, and / or the like configured to engage the cartridge 104 to control, for example, a flow of fluid through at least a portion of the cartridge 104. For example, in some embodiments, the cartridge retainer 103 can include and / orcan be coupled to a vacuum source or assembly (not shown in FIG. 1). The vacuum source or assembly can be configured to be brought in fluidic connection with the cartridge 104 via suitable connection (e.g., tubing, port, and / or the like) and the vacuum source or assembly can be configured to produce a negative pressure within a volume of the cartridge 104 such as, for example, within a lumen of a capillary7106 or a portion of a fluidic path defined in the cartridge and / or the like. Said in another way, the vacuum source can be fluidically coupled to a portion of the cartridge retainer 103 via a port or a tubing or the like that places the vacuum source in fluid communication with the lumen of the capillary 106 in the cartridge 104 when the cartridge 104 is retained by the cartridge retainer 103.

[1062] In some instances, the vacuum source can be a pressure source capable of applying positive and / or negative pressure differential. For example, the pressure source can apply a negative pressure differential to draw a sample or substance from a sample well into the capillary 106. As another example, the pressure source can apply a positive pressure differential to inj ect or elute a portion of the sample or substance (e.g. , a separated analyte or set of analytes) from the capillary 106 and into a collection well. In some embodiments, the pressure source can be activated and / or controlled (e.g., by a manual switch or controlled and / or by an electrical switch or controller included in an electrical circuit and controlled by a processor) to produce and / or modulate a negative pressure of a desired magnitude within the cartridge 104. as described in detail below with reference to specific embodiments. In some embodiments, the cartridge retainer 103 can include a device or mechanism configured to engage the cartridge 104 to selectively limit a bulk flow of fluid through a portion of the cartridge 104. For example, the cartridge retainer 103 can include an actuator or the like that is selectively placed in contact with a pinch valve or the like included in the cartridge 104 to limit and / or substantially prevent a bulk flow of fluid through, for example, a lumen of a capillary7included in the capillary cartridge 104.

[1063] In some embodiments, the cartridge 104 can include one or more running buffer reservoirs 105 that can be configured to hold one or more running buffers that can be used to probe, separate, analyze, and / or fractionate one or more analytes in a sample held in the capillary 106. As an example, a first running buffer held in the running buffer reservoir 105 can include a buffer having a first pH and / or having a specified concentration of desired ions. For example, in some embodiments, the running buffer reservoir 105 can hold a specified quantity of an acid with a desired pH. The capillary 106 can be positioned such that a lumen ofthe capillary 106 at a first end or a proximal end of the capillary 106 is fluidically and / or ionically coupled with the running buffer held in the running buffer reservoir 105. The acid and / or its pH may be selected such that one or more analytes included in a sample, that is drawn into the capillary 106, can be separated using techniques like isoelectric focusing by taking advantage of the pH of the running buffer. That is, in some embodiments, the sample in the capillary can be ionically coupled via a first end (e.g., a proximal end) to a first running buffer in the running buffer reservoir and having a first pH. The sample in the capillary can be ionically coupled via a second end (e g., a distal end) to a second running buffer in a well and having a second pH different than the first pH. The well holding the second running buffer can, for example, be in the sample plate held by the sample plate assembly 107, and the distal end of the capillar}’ may be configured to be dipped into the well by the system 100. The first running buffer at the first pH and the second running buffer at the second pH can induce a pH gradient to be established along the capillary lumen and through the sample. The pH gradient can cause one or more analytes in the sample to separate and / or migrate along the gradient to be positioned and / or accumulated at their respective equilibrium pH based on the ionic balance of the respective analytes.

[1064] Following separation and / or focusing of the one or more analytes in the sample along the pH gradient the one or more analytes can be mobilized and eluted using any suitable process including hydrodynamic mobilization and / or chemical mobilization. Hydrodynamic mobilization can refer to a mobilization of analytes due to movement of fluids (e.g., due to a flow driven by gravitational forces acting on fluids in a vertically oriented capillary, a flow driven by a pressure differential, and / or the like). In some instances, the application of electric field can be continued during hydrodynamic mobilization, which can help to maintain separation between the analytes. Chemical mobilization can refer to a mobilization of analytes by a process in which an anolyte (electrolyte on the anode side of the capillar}’ 106) or a catholyte (electrolyte on the cathode side of the capillary) can be replaced by another electrolyte with a high ionic strength and / or a different pH compared to the running buffers associated with the anode side or cathode side of the capillary 106. The electrolyte with the high ionic strength and / or different pH can be introduced via a chemical mobilizer (also referred to herein as an elution buffer) in conjunction with an applied electric field between a first running buffer and the chemical mobilizer via the capillary 106.

[1065] A chemical mobilizer can be a buffer that provides ions that can migrate into the capillary 106 and disrupt the pH gradient in the capillary 106 to again impart a charge to the separated analytes that may have been rendered neutralized and stable. When a one end (e.g., a distal end) of the capillary 106 is disposed in a chemical mobilizer the electrolyte can be introduced into the capillary' 106 and can disrupt the pH gradient. Ampholytic analytes in the now-disrupted pH gradient can therefore experience a force causing them to migrate and / or be eluted. In some embodiments, the cartridge 104 can be configured such that the first running buffer in the running buffer reservoir 105 can be used to elute and / or fractionate one or more analytes in a sample held in the capillary' 106 in conjunction with a chemical mobilizer. As an example, the first running buffer held in the running buffer reserv oir 105 can include a buffer having a first pH and / or having a specified concentration of desired ions. For example, in some embodiments, the running buffer reservoir 105 can hold a specified quantity of an acid with a desired pH. A collection w ell can include a chemical mobilizer with a buffer having a third pH and / or having a third specified concentration of a desired set of ions, for example a specified quantity of base with a desired pH.

[1066] In some implementations, a single chemical mobilizer can be chosen to elute separated analytes in a capillary' 106. One or more wells holding the chemical mobilizer can, for example, be in the sample plate held by the sample plate assembly 107, and the distal end of the capillary may be configured to be disposed into the well holding the chemical mobilizer by the system 100. The chemical mobilizer can induce elution of the separated analytes along the pH gradient generated during the separation phase (e.g., established using the second running buffer) such that analytes focused in a specified portion of the pH gradient can be collected in the collection well. In some implementations, multiple wells can contain the chemical mobilizer such that the distal end of the capillary 106 can be moved from collection well to collection well, and a specified portion of the separated one or more analytes can be collected in each (or at least a subset) of the wells. Movement of the capillary 106 from collection well to collection well can be coordinated (e.g.. with real-time imaging of the column) such that separated analytes with slightly different pl values can be tracked and / or selectively eluted and collected as fractions in separate collection well via chemical mobilization.

[1067] Some of the properties of a chemical mobilizer / elution buffer to be considered when choosing chemical mobilizers for mobilizing analytes include: (1 ) compatibility with massspectrometry (e.g., a volatility of chemical mobilizers can help remove the chemical mobilizer components during electrospray during a mass spectroscopy analysis following fractionation and collection), (2) a pH that permits stable storage of the analytes (e.g., proteins) that may remain in the chemical mobilier after elution (e.g., a pH range of 4 to 9, in some implementations, a pH range of 5 to 8), (3) sufficient buffer capacity to support the current during chemical mobilization. In some implementations, a sufficient buffer capacity can be achieved by increasing a concentration of ions in the chemical mobilizer / elution buffer. Some example chemical mobilizers include NaCl, Acetic Acid, Acetate salt, Formic Acid, Phosphate Salt, Phosphoric Acid, Ammonium Acetate, and Formate. Ammonium Acetate is an example chemical mobilizer that can have a pH 6.7 and can be used to mobilize all analytes (e.g., proteins) in a sample. Chemical mobilizer can be chosen at a suitable concentration based on desired mobilization and / or elution parameters (e.g., speed, precision, etc.) As an example, NaCl can be chosen as a chemical mobilizer at a concentration of about 10 to 200 mM, acetic acid can be used at a concentrations from about 20mM to 200mM, acetate salt (e.g., ammonium) can be used at a concentration from about ImM to 20mM, formic acid can be used at a concentration from about 0.01% to 1%, phosphate salt can be used at a concentration from about 0.5mM to 20mM, formate can be used at a concentration from about 0.5mM to 20mM.

[1068] The cartridge retainer 103 can also include any number of electrical contacts (not shown in FIG. 1) or the like configured to electrically couple a portion the cartridge 104 to an electrical or electronic assembly included in the system 100. For example, in some embodiments, the cartridge retainer 103, the cartridge 104, and / or the capillary 106 can include one or more electrically conductive contact members, clips, surfaces, etc. that are placed in contact with an associated electrically conductive contact member, clip, surface, etc. of the cartridge 104 when the cartridge 104 is retained therein. In some embodiments, at least a portion the cartridge 104 can be formed from an electrically conductive material such as stainless steel, electrically conductive plastic or the like. Thus, when the cartridge 104 is retained in a desired position within the cartridge retainer 103 the one or more electrically conductive members or the like of the cartridge retainer 103 contact one or more predetermined electrically conductive portions of the cartridge 104, which in turn, places the cartridge 104 in electrical or electronic communication with the electrical and / or electronic assembly included in the system 100. By way of example, the cartridge retainer 103 can electrically connect a conductive capillary of the cartridge 104 and / or a conductive fluid within a lumen of a capillaryto the electrical and / or electronic assembly of the system 100. as described in further detail herein.

[1069] In some embodiments, at least a part of the cartridge 104 and / or the capillary 106 can be electrically conductive. The electrically conductive part of the cartridge 104 and / or the capillary 106 can be formed from any suitable electrically conductive material. For example, the electrically conductive part can be formed from a metal including copper, platinum, stainless steel, electrically conductive microplate plastic, carbon-infused plastic, an electrically conductive polymer, and / or any other suitable material. As an example, in some embodiments, the cartridge 104 can include a capillary 106 with a metal tip at the distal end of the capillary that is electrically conductive. When the capillary is dipped into a solution (e.g., in a collection well or a reagent well in a sample plate) the metal tip can be disposed in the solution and can be used to provide an electrical connectivity between an external source of electrical power and the solution and / or the lumen of the capillary. The metal tip can be used to apply a voltage through the solution that is occupying the lumen of the capillary’ and that optionally contains a pH gradient between ionically coupled running buffers (e.g., disposed on opposite sides of the capillary). Said in another way, the metal tip can be used to provide a voltage across the length of the capillary' 106 and between solutions and / or buffers that are coupled to the proximal and distal ends of the capillary 106, respectively, such that analytes in a sample held in the lumen of the capillary 106 can be separated and / or fractionated by applying a voltage.

[1070] In some embodiments, the cartridge 104 can include one or more electrical leads or points of electrical connectivity that are configured such that the first running buffer and / or the second running buffer that is ionically coupled to the sample in the capillary can each be also electrically coupled to a power source (e.g., a power source included in the electronic system 108). In some embodiments, as described in further detail in the following sections, the first running buffer can be electrically coupled to a first electrical lead electrically coupled to the running buffer reservoir and a voltage source, and the second running buffer (e.g., in a well in a sample plate held by the sample plate assembly 107) can be electrically coupled to a second lead positioned at the distal tip on the capillary 106 holding the sample based on the distal end of the capillary' being dipped into the second running buffer. The second lead positioned at the distal tip of the capillary 106 can be configured to be electrically coupled to the voltage source such that the system 100 can provide apply a specified voltage across the first lead and the second lead, thereby applying the voltage between the first running buffer and the secondrunning buffer via the sample held in the lumen of the capillary 106. In some instances, the voltage applied can be selected to induce isoelectric focusing of the components included in the sample such that one or more analytes and / or markers included in the sample can separate and / or migrate to their respective isoelectric points (pl) (i.e., the pH at which each analyte has no net charge and therefore is at equilibrium with no more net movement along the pH gradient) along the length of the capillary based on their respective ionic balance with respect to the pH gradient generated between the first running buffer and the second running buffer.

[1071] As described previously, the capillary' 106 can be of any suitable shape, size, or configuration and can be arranged to be received by the cartridge 104 and to have a lumen that can be made continuous with a fluidic path that is defined in the cartridge 104 (e.g., a lumen that is configured to be arranged in fluidic connection with atubing that is coupled to a vacuum source, to a first running buffer in a running buffer reservoir, to a sample w ell in a sample well plate, to a second running buffer in a w ell included in a sample well plate, etc.

[1072] Some embodiments described herein relate to capillary-containing cartridges suitable for use with capillary' electrophoresis instruments, such as Maurice™ by ProteinSimple®. U.S Patent No. 10,794,860, issued on October 6, 2020 and entitled ‘‘Systems and Method for Capillary Electrophoresis, Isoelectric Point, and Molecular Weigh Analysis." the entire disclosure of which is hereby incorporated by reference, includes additional disclosure of a suitable capillary electrophoresis instrument and cartridges suitable for capillary electrophoresis. Embodiments described herein generally relate to cartridges that include a capillary 106 that can serve or that includes portions that can serve to transfer solutions, hold a sample including analytes and / or markers, support a separation of one or more analytes / markers in the sample, and elute one or more separated analytes via fractionation. The capillary 106 can be configured to transfer a buffer or maintain fluidic contact w ith one or more buffer reservoirs such that the capillary 106 can be to be in contact with a buffer reservoir(s). The capillary 106 can be configured to transfer a sample and / or receive the sample into a lumen of the capillary' 106 such that the contents in a lumen of the capillary can be subjected to an applied voltage to form a pH gradient.

[1073] In some embodiments, the cartridge 104 and the capillary 106 can be configured such that the capillary 106 is in a substantially vertical orientation with respect to gravity with the distal end protruding away' from the cartridge 104 such that a portion of the distal end can be removably disposed in any suitable well, vial, reservoir, and / or the like that can beintroduced at the correct position with reference to the capillary 106 and / or the cartridge 104. In some embodiments, the protruding end (e.g.. a distal end) of the capillary 106 can be configured to be disposed in sample reservoir in a sample well plate and / or a buffer reservoir in collection wells (e.g., in a sample well plate).

[1074] In some embodiments, the proximal end of the capillary’ 106 can be configured to be coupled to a suitable mechanism that can be used to load a sample into the lumen of the capillary 106. For example, during a loading step suction applied through a sheath interface or a tubing coupled to the proximal end of the capillary 106 via the vacuum source can provide a negative pressure to draw a sample or buffer from a sample or buffer reservoir and bring the sample or buffer into the lumen of the capillary 106.

[1075] The capillary 106 can be configured for separation of analytes included in the sample, for example via isoelectric focusing when an electric potential (i.e., voltage) is applied across the proximal and distal ends of the capillary 106. The electric potential can be applied across the lumen of a substantially vertically oriented capillary 106 via a first or top running buffer reservoir and a second or bottom running buffer reservoir. In some embodiments, the capillary 106 can be configured such that one end of the capillary 106 (e.g., a proximal end) can be disposed in a first running buffer reservoir included in the cartridge 104. The other end protruding from the cartridge 104 can be configured to be disposed in a second running buffer reservoir in a sample well included in a sample well plate. Said in another way, the top running buffer reservoir can be disposed in the cartridge 104, and a top end of the capillary 106 can be disposed in the top running buffer reserv oir. A bottom of the capillary 106 can be disposed in a bottom running buffer reservoir, which can be disposed in a sample plate that is a portion of or accessed by the system 100. Similarly stated, the capillary 106 can extend from the cartridge and be '‘dipped” into a bottom running buffer reservoir.

[1076] In some embodiments, one or both ends of the capillary’ 106 can be configured to include a portion of a porous membrane (not shown in FIG. 1) having a predefined pore size and covering an access to the lumen of the capillary 106. Some embodiments can include membranes on both ends of the capillary used to separate the analytes. Some embodiments can include a membrane on just one end of the capillary 106. For example, the proximal (e.g., top) end of the capillary' 106 that is disposed in a first running buffer held in a first running buffer reservoir can include a porous membrane that reduces movement of the first running buffer into the capillary’ 106, for example due to a gravitational and / or capillary’ forces. The porousmembrane(s) can serve as a hydrodynamic barrier preventing the first running buffer (e.g., acid) and / or the second running buffer (e.g., base) from entering the capillary’ during separation. The ions such as H+and OH' can, however, freely transport through the membrane(s) under electric field allowing the separation (e.g., via isoelectric focusing process) to occur. In addition to the membranes, some embodiments can include one or more valves located at or close to one or more ends of the capillary 106 (e g., a loading end of the capillary used to load sample, an elution end of the capillary used to elute fractions). The valve at a loading end / elution end can be configured to be opened during sample loading and / or fraction elution and to be closed during the separation phase to minimize hydrodynamic flow during separation. In some embodiments, the porous membrane may be configured to be removed during elution (e.g., using a suitable actuation).

[1077] In some embodiments, a porous membrane at a distal (e.g., bottom) portion of the capillary, through which sample is injected and / or fractions eluted, can have a predefined selectivity to reduce infiltration of the first running buffer into the lumen of the capillary 106 while allowing analytes and / or protons and / or hydroxyl ions to pass therethrough under an applied electric field. In some embodiments, the porous membrane can be in the form of a tubing coupled to one end (e.g., the proximal end) of the capillary’, and also referred to as "membrane tubing". Such a membrane tubing defines a lumen through which fluid can flow hydrodynamically when a pressure difference exists between the two ends of the membrane tubing. In contrast, the porous wall that encloses the lumen can permit transport of ions (e.g., by electrophoresis or diffusion) while substantially restricting hydrodynamic flow. In some embodiments, the membrane tubing can be configured to connect a vacuum source to the capillary 106 (e.g.. at the proximal end of the capillary 106) and can be in contact with and / or submerged in the running buffer reservoir. The porous membrane or membrane tubing can have a molecular weight cut-off (MWCO) of approximately 10 kDa to 500 kDa in some embodiments. Some such embodiments, the porous membrane can be configured to be used with a cartridge 104 with a capillary’ 106 that has a single inner diameter of about 320 - 530 pm with a length of about 60 - 120 mm. Some such embodiments can be configured to increase a sample loading capacity while at the same time maintaining an advantage of real time monitoring of progress of separation of analytes, for example via isoelectric focusing. In some embodiments, the porous membrane can be selected to enable effective mobilization speed during chemical mobilization and elution of fractions of the sample. The porous membrane canbe selected to reduce a disruption of a pH gradient generated during separation at the stage of mobilization and elution of fractions.

[1078] In some embodiments, the selection of the porous membrane can be based on a size location, and / or orientation of the running buffer reservoir (e.g., 500pl - 4000pl), a volume associated with the membrane tubing, and / or an inner and / or outer diameter of the capillary 106. For example, the volume associated with the membrane tubing can be a portion of tubing of approximately 300 pm to 700 urn inner diameter, and a length of approximately 0.5mm to 2mm. In some instances, the porous membrane, the running buffer reservoir (and the cartridge associated with a running buffer reservoir), and the capillary can be selected to suitably match each other to achieve a desired separation, mobilization, and / or elution of analytes (e.g., a target efficiency of separation, a target speed of mobilization, etc ).

[1079] A size, location, and / or orientation of the running buffer reserv oir can affect a hydrodynamic flow associated with contents of the lumen of the capillary 106, as does the porous membrane (e.g., pore size of the membrane). For example, a volume and / or size of the running buffer reservoir that may be located and / or oriented at a vertically elevated position with respect to a capillary 106 can impart a hydrodynamic flow based on gravitational forces acting on the running buffer released from the running buffer reservoir. The pore size of the porous membrane can not only affect the degree of hydrodynamic flow but also the efficiency of ion-exchange (i.e., level of electric current under applied voltage). In some embodiments, the running buffer reservoir can be configured to hold a first running buffer which for example can be an acid. The running buffer reservoir can thus serve as a supplier of protons during the separation and mobilization process. The larger the size or volume of the sample to be separated, which can depend inner diameter of the capillary 106, the larger may be the need for protons. Thus, the inner diameter of the capillary' 106 can affect the sample size, which can determine a size of running buffer reservoir that may be used to supply a suitable number of protons that can be supplied.

[1080] When a sample is introduced into the cartridge 104, the sample can distribute in both the lumen of the capillary 106 as well as the volume associated with the membrane tubing. As described previously, the lumen of the capillary’ 106 can include a separation matrix. Under applied voltage, if the separation matrix includes no spacers, which are electrolyte solution added to a capillary to electrophoretically block specific segments of the capillary from being used to focus analytes (e.g., Iminodiacetic acid (IDA) and / or Arginine), the length of the pHgradient can extend over the entire length of the capillary and the length of the portion of tubing that includes the volume associated with the membrane tubing. The inner diameter and length of the membrane tubing can thus affect the distribution of the pH gradient, subsequently, the quality of separation as well as the quality of mobilization. Therefore, the volume associated with the membrane tubing can be an important factor in the uality of separation and / or mobilization. Additionally, the inner diameter of the porous membrane tubing can be selected to match the outer diameter of the capillary 106 such that the junction between the membrane tubing and the capillary 106 is sealed and does not become a defect area that may encourage potentially detrimental effects (e.g., bubble formation).

[1081] In some embodiments, the capillary 106 can include one or more valves located at any suitable position along the length of the capillary 106 and operational to open and / or close fluid flow through the capillary 106 at any desired time. For example, the capillary' 106 can include a valve located at or near a distal end such that the valve can be opened during loading the capillary 106 with a sample and the valve can be closed following loading and while separating the analytes (e.g., via isoelectric focusing) to reduce or minimize hydrodynamic flow (e.g., flow caused by gravity due to the vertical orientation of the capillary 106) during separation. In some embodiments, the capillary' 106 and / or the cartridge 104 can be configured to include one or more adaptations to permit an application of electric potential across the lumen of the vertically oriented capillary 106 via the first or top running buffer reservoir and the second or bottom running buffer reservoir, as described in further detail herein. For example, in some embodiments, as described previously, the capillary' 106 and / or the cartridge 104 can include one or more electrical contacts that can be used to connect to a source of electrical power. For example, the capillary 106 and / or the cartridge 104 can include one or more electrical contacts disposed at or near the proximal and and / or the distal end of the capillary 106.

[1082] The sample plate assembly 107 of the system 100 can be any suitable shape, size, or configuration and can be arranged to receive, house, and / or store at least a portion of a sample plate or a reagent tray (not shown in FIG. 1). For example, the reagent tray or sample plate can hold and / or otherwise define a set of vials, wells, well plates, microwell plates, troughs, and / or the like (any of which may be generically described as a “well” or “microwell”). The wells and / or microwells can be any suitable size and can be disposed along and / or otherwise defined by a surface of the reagent tray in any suitable arrangement. Althoughspecific examples of reagent trays are described herein, the sample plate assembly 107 can be configured to receive and / or include any suitable reagent tray of similar size and / or shape that can define any number and / or any arrangement of wells and / or microwells. The wells and / or microwells included in or defined by the reagent tray can contain and / or receive any suitable volume of a solution, fluid, gel, lysate, buffer, sample, analyte, ampholyte, agent, reagent, protein, matrix, and / or the like. In some embodiments, the wells and / or microwells can receive a vial or the like containing a volume of any suitable fluid. In some embodiments, the sample plate assembly 107 and / or a portion of the sample plate assembly 107 is electrically conductive and electrically coupled to the electrical and / or electronic assembly 108 included in the system 100. In such embodiments, the sample plate and / or a portion of the sample plate can also be electrically conductive and can facilitate an electrical connection to a fluid disposed within the sample plate through the coupling of the sample plate to the sample plate assembly 107.

[1083] At least a portion of the sample plate assembly 107 is movably disposed within and / or movably coupled to the housing 101. In some embodiments, the sample plate assembly 107 can be movably disposed in the housing 101 such that the system 100 can automatically or semi-automatically manipulate a sample plate with respect to the cartridge retainer 103. The sample plate assembly 107 can be configured to move relative to the cartridge retainer 103 to place the capillary 106 of the cartridge 104 in fluid communication with a reagent or sample or buffer volume in one or more vials or wells in a sample plate manipulated by the sample plate assembly 107. In some embodiments, the system 100 can be configured such that the sample plate assembly 107 can be moved relative to the cartridge retainer 103 such that the distal end of the capillary 106 can be sequentially disposed in a set of vials or wells that include a sample, a running buffer, an elution reagent, a chemical mobilizer, and or the like.

[1084] For example, the sample plate assembly 107 can be movably coupled to one or more tracks, racks, lead screws, slides, pistons, and / or the like that can be operable to move the sample plate assembly 107 relative to the housing 101. The sample plate assembly 107 (or at least a sample vial or at least a reagent tray included therein) can be moved in a direction closer to or further from the cartridge retainer 103, as indicated by the arrow AA in FIG. 1. In other words, the sample plate assembly 107 can be moved in a direction parallel to an axis defined by the capillary 106 of the cartridge 104 when the cartridge 104 is retained by the cartridge retainer 103. In addition, the sample plate assembly 107 can be moved in one or more directions along a plane normal to the cartridge retainer 103, as indicated by the arrow BB. That is to say,the sample plate assembly 107 (or the sample plate included therein or at least a vial or well from the sample plate) can be moved along a plane normal to the axis defined by the capillary106 of the cartridge 104 when the cartridge 104 is retained by the cartridge retainer 103. Said another way, the sample plate assembly 107 (or the sample plate included therein or at least a vial from the sample plate) can be moved within the housing 101 in the X-direction (e.g., left or right), the Y-direction (e.g., up or down), and the Z-direction (e.g., front or back) relative to the cartridge retainer 103.

[1085] In this manner, the sample plate assembly 107 is configured to move at least the sample plate or at least a vial or well from the sample plate (not shown in FIG. 1) relative to the cartridge 104 retained by the cartridge retainer 103 to dispose at least a distal end portion of the capillary 106 of the cartridge 104 in the wells, microwells, vials, and / or the like of the sample plate. Moreover, the sample plate assembly 107 can move at least the vial well or sample plate through any suitable number of positions relative to the cartridge 104 and / or cartridge retainer 103 to place the capillary 106 in any of the wells, microwells, and / or vials included in the sample plate, or any suitable combination thereof. The sample plate assembly107 can be moved to draw solutions / sample into the capillary 106 and / or to elute fractions from the capillary 106 and into collection wells, using any suitable method including pressure-based injection, pressure-based elution, chemical injection, chemical mobilization, electrochemical injection, electrochemical mobilization, etc.

[1086] For example, with the capillary7106 in fluid communication with the vacuum source (as described above), a negative pressure can be produced within the capillary7106 that is operable to draw a volume of fluid, such as those described above, from any7suitable well or wells of the reagent tray and into the capillary' 106. In some instances, an electric field can be applied across the lumen of the capillary 106, which can apply an electrokinetic force is operable to draw charged moi eties into the capillary 106 via electrokinetic injection, from any suitable well or wells of the reagent tray and into the capillary 106. In some embodiments, a well, microwell, vial, etc., can be fluidically coupled to a positive pressure source via a pressure conduit (not shown in FIG. 1) inserted into the well, microw ell, vial etc. ith the capillary7106. The sample plate assembly 107, the capillary7106, and / or a portion of the cartridge 104 can be operable to seal the well, microwell, vial, etc. against the cartridge 104 such that a positive pressure can urge fluid from the well, microwell, vial etc. into the capillary 106.

[1087] In some embodiments, the system 100 can be configured to manipulate the sample plate assembly 107 such that a portion of the distal end of the capillary 106 can be disposed in a sample reservoir in a sample plate at a first time and the suction or electrokinetic force is applied to draw the sample, and then the capillary 106 can be disposed in a in a second running buffer reservoir in a sample well plate. In some instances, the sample plate assembly 107 can be configured to be manipulated such that the proximal end of the capillary distal end capillary 106 can be configured to be disposed in sample reservoir in a sample well plate and / or a buffer reservoir in a sample well plate. Suction applied through a sheath interface of the capillary 106 or a tubing coupled to the proximal end of the capillary 106 via the vacuum source can draw sample / buffer from such reserv oirs and bring the sample / buffer into the lumen of the capillary 106. Then the system 100 can be manipulated to perform a separation of analytes in the sample (e.g., via isoelectric focusing by applying a voltage across the proximal and distal ends of the capillary while top and bottom running buffers induce a pH gradient across the capillary). The separated analytes can be analyzed to determine a degree of separation that can be measured between each separated analyte. The analysis can be conducted using the probe system 102 and / or electronic system 108.

[1088] The separated analytes can be mobilized to be eluted using any suitable mechanism or driving force. For example, the separated analytes can be mobilized to migrate towards the distal end of the capillary 106 at a specified rate using an applied positive pressure at the proximal end of the capillary 106. Based on the measured separation between analytes an expected rate or duration of elution of each analyte can be calculated and the sample plate assembly 107 can be manipulated to introduce isolated collection vials or collection wells in the sample plate to receive each eluted analyte or fraction as a product of fractionation. In some instances, the analytes separated in the capillary 106 can be simultaneously or near- simultaneously detected and / or visualized during the elution phase to monitor a degree of separation and / or a relative location of each analyte (e.g., peak of each distribution corresponding to each analyte)

[1089] As another example, the separated analytes can be induced to migrate towards the distal end of the capillary7106 by manipulating the sample plate assembly 107 such that a set of collection wells or a collection vials having chemical mobilizers are sequentially introduced for the distal end to be disposed in. The set of collection well with chemical mobilizers may be configured such that each collection well includes a chemical mobilizer (e g., the same ordifferent chemical mobilizers) configured to disrupt the pH gradient formed during separation. The chemical mobilizers can be carefully selected to induce chemical mobilization to draw out and elute the most distal separated analyte or analytes. The chemical mobilizers can be used to carefully draw the desired portion of the separated sample while not substantially drawing the next subsequent separated analyte that is undesired to be mixed with the collected fraction of analyte or analytes. For example, each collection well can contain a buffer configured to incrementally decrease the pH gradient across the capillary such that analyte(s) migrate towards the sample plate and / or into the collection well(s).

[1090] In some instances, one or more of the separated analytes can be electrochemically mobilized and / or induced to migrate towards the distal end of the capillary 106 to be eluted. The elution can be monitored, and / or controlled as desired. For example, in some embodiments a detector disposed to detect a distal end portion of the capillary 106 can be operable to detect markers as they approach and / or exit the capillary. Any suitable driving force can be used including pressure-based elution, chemical mobilization, electrochemical mobilization, etc. For ease of discussion, isoelectric point (pl) markers are generally discussed herein, but it should be understood that whenever pl markers are discussed, similar techniques can be applied using molecular weight or other suitable markers when, for example, separation modalities other than isoelectric focusing are used.

[1091] A sequence of collection wells can be included in a sample plate such that the distal portion of the capillary 106 can be sequentially disposed in each successive collection well. The sample plate assembly 107 and or the capillary' 106 can be manipulated such that the distal end of the capillary 106 is disposed in a sequence of collection well in a sequential manner while eluting separated analytes in successive collection well of the sample plate.

[1092] In some instances, the system 100 can be configured such that the elution can be monitored, and / or controlled by using detection of one or more pl markers mixed in with the sample. For example, separation markers and / or fractionation markers with known pl can be visualized at the distal end of the capillary 106 and a rate of mobilization and / or a rate of elution can be controlled by modifying an elution rate, an elution volume, an elution duration, etc. based on the visualization, detection, and / or determination of a location of the one or more pl markers. Detection at the distal end of capillary 106 is typically distinct from detection using probe system 102 as discussed above. In this way, the sample can be probed while in the main body of the capillary 106 using the probe system 102 to detect, for example, separationprogress, analytes of interest, etc., while elution can be monitored at the distal end of the capillary 106 using, in some instances, different detection techniques. For example, the probe system 102 can be operable to detect separation markers, while at the distal end of the capillary detection can be operable to detect fractionation markers. Similarly stated, an analyte or a separation marker can be detected and the location of the focused analyte or focused separation marker can be determined using the probe system 102 and a first modality such as a detection of a native fluorescence or UV absorbance in a predetermined range of wavelengths (e.g., within UV range, in some embodiments, a suitable predetermined wavelength range for naive fluorescence is 320nm - 450 nm or 330 nm to 420 nm; in some embodiments a suitable predetermined wavelength for absorbance is 330 nm to 420 nm) designated for analyte identification. A fractionation marker can be detected using a second detection modality that can, under some implementations, be an orthogonal detection modality to decrease cross talk with the first modality. For example, the second modality can be excitation by light of a wavelength within a known excitation range of wavelengths to emit a non-native fluorescence signal at a wavelength within a range of emission wavelengths.

[1093] In some embodiments, in addition to or as an alternate to detecting elution at the distal end of capillary 106, the system 100 and / or an external instrument can be configured determine location of one or more analytes after elution by detecting of one or more isoelectric point (pl) markers mixed in with the sample by probing the sample plate 107. For example, separation markers and / or fractionation markers can be visualized after being eluted into wells in the sample plate 107 using a top-down detector or similar technique. For example, fractionation markers can be detected to infer and determine the location of the focused analytes or focused separation markers using a second modality excitation by light of a wavelength within a known excitation range of wavelengths to emit a non-native fluorescence signal at a wavelength within a range of emission wavelengths. Optical detection of fractionation markers in the fractions collected in the numerous wells can be efficient and effective for the low concentration of fractions in the collected fractions while not impacting the sample collected in the fractions.

[1094] In some instances, a user can load a capillary' cartridge 104 into the system 100 and can initiate and / or otherwise provide instructions to the system 100 to cause the system 100 to at least semi-automatically separate analytes within the sample by isoelectric point. In some instances, the system 100 draws a sample (e.g., including any suitable agent, reagent, protein,analyte, buffer, lysate, etc.) into a capillary, separates and / or focuses analytes in the sample within the capillar}’, and detects the presence or the absence of a target analyte and / or detects the location of analytes within the sample (e.g., analytes that have migrated to different positions along the capillary associated with their isoelectric points) using a first modality (e.g., native fluorescence or UV absorption) . The system 100 can identify locations associated with a target analyte within the sample using separation markers, which are pl markers configured to have native fluorescence or UV absorbance in the same predetermined range of wavelengths designated for analyte identification. The system 100 can selectively elute at least some constituents of the sample within the capillary’ (e.g., one or more separated analytes) in a serial manner such that the separated analytes can be collected in collection wells and be used for further processing. The system 100 can detect the location(s) and / or identify fractions of sample associated with the analytes and / or manage elution using one or more fractionation markers with a using a second modality7(e.g., non-native fluorescence from a protein tagged with a fluorescent label).

[1095] In use, for example, the system 100 can be set, programed, and / or otherwise placed in a configuration to perform fractionation of a sample, which can include, for example, preparing samples and / or reagents as w ell as preparing the cartridge 104. A user can then insert the cartridge 104 into the cartridge retainer 103 in a single, predetermined orientation, as described above. The cartridge retainer 103, in turn, at least temporarily couples to the cartridge 104 to retain the cartridge 104 in a substantially fixed (e.g., vertically oriented) position. The cartridge retainer 103 can couple to the cartridge 104 such that the proximal end of the capillary’ 106 is disposed in a first running buffer (e.g., acid of a desired acidic pH) held in a running buffer reservoir 105. The proximal portion of the contents of the lumen of the capillary 106 can be electrically and ionically coupled to the first running buffer via electrical contacts and / or fluidic connection. The sample plate assembly 107 is moved relative to the capillary 106 such that a distal end portion of the capillary' 106 is disposed in a first vial or w ell containing a sample solution. A suitable driving force, for example, a pump operable as a vacuum / pressure source, is used to draw and load the sample into the capillary 106. The drawing and loading the sample can be accompanied by suitably operating one or more values that may be associated with loading the sample. For example, a valve in the distal end of the capillary’ 106 can be opened to allow the sample to be loaded while a valve in the proximal end can be closed to reduce movement of first running buffer into the capillary 106. The sample plate assembly may then be operated to remove the first vial or well and introduce a second w ell or vial that caninclude a second running buffer in which the distal end of the capillary' 106 is disposed. The distal portion of the contents of the lumen of the capillary 106 can be electrically and ionically coupled to the second running buffer via electrical contacts and / or fluidic connection. The electronic system 108 can provide instructions to apply and / or apply an electric field between the first running buffer and the second running buffer via the contents of the lumen of the capillary 106. The applied electric field can induce separation of one or more analytes in the sample via isoelectric focusing. The analytes can be charge variants each associated with a different charge such that each separated analyte migrates and localizes at a point that corresponds to its isoelectric point (of neutralized charges) along the length of the capillary 106. The first running buffer and the second running buffer can be selected such that they define a pH range that encompasses the isoelectric points (pl) of the analytes or fractions that are desired to be separated and fractionated from the sample.

[1096] Following separation, the probe system 102 can be used to analyze the separated analytes using any suitable mechanism under a first modality (e.g., fluorescence emission analysis, optical density analysis, UV absorbance, etc.) The arrangement of the cartridge retainer 103 and the probe system 102 within the housing 101 is such that predetermined portions of the probe system 102 (e.g., an emitter and a detector) are aligned with predetermined portions of the cartridge retainer 103 (e.g., one or more openings or viewing windows or the like). Thus, by aligning the probe system 102 with the cartridge retainer 103 and with the cartridge retainer 103 retaining the cartridge 104 in a predetermined, fixed position, the emitter and the detector can be aligned with, for example, a portion of the capillary 106 of the cartridge 104. Therefore, energy and / or light emitted by the first emitter can be directed to a predetermined portion or length of the capillary 106. The detector can capture the signal emitted by the separated analytes and analyze the captured signal to determine a degree of separation, location of separation (e.g., relative location of peak quantity or a defined portion of a quantity) of a separated analyte along the length of the capillary' 106, and / or an identity' of the separated analyte(s).

[1097] In some instances, a full-column detection portion of a detector can detect ‘‘fullcolumn” images or signals and / or otherwise can perform “full-column” detection of the sample within the capillary 106. Similarly stated, the detector can include an imaging device included in the probe system 102 can be operable to capture more than a single point along the capillary 106. For example, the imaging device can be operable to capture and / or detect a sufficientlength of the capillary 106 to visualize separation and / or focusing of analytes during the separation and / or mobilization process (e.g., a length of about 1 cm, about 3 cm, about 5 cm, about 10 cm, about 20 cm, about 50 cm, or any other suitable length of the capillary 106). In addition, or alternatively, the imaging device can be operable to capture and / or detect native fluorescence, and absorbance of analytes within the capillary' 106. For example, the probe system 102 can include a fdter wheel associated with the detector and / or emitter such that the filter wheel can be rotated to change the optical signal presented to the sample and / or received from the sample while analytes are being separated, focused, and / or mobilized within the capillary 106. Thus, during a single run, a sample can be characterized for native fluorescence, absorbance, and / or any other suitable optical characteristic along the full-column while the analytes separate, focused, and / or are mobilized to be eluted in fractions.

[1098] The sample plate 107 can be operated, in conjunction with the analysis by the probe system 102 to dispose the distal end of the capillary 106 in successive collection wells including running buffers and / or chemical mobilizers that can be used to draw and elute a fraction of the separated analytes by applying a suitable driving force such as positive pressure using a pump , or an applied electric field of a specified magnitude, and / or a chemical mobilizer with a desired pH. In some instances, elution can be monitored in progress using a detector configured to probe a distal portion of the capillary 106. The distal portion of the capillary 106 can be probed using a different modality than used for full-column imaging discussed above (e.g., non-native fluorescence from a protein tagged with a fluorescent label) to visualize bands of focused analytes and / or pl markers mixed in the sample to determine a location of a particular focused analyte and / or pl marker (e.g., separation marker). The visualization and / or detection at the viewing window and analysis of relative location of peaks of separated analytes can be used to inform elution of fractions (e.g., rate of elution, duration of elution of each fraction, volume of each fraction, and the like).The sample plate 107 can be moved at a specified rate based on areal-time peak analysis (analysis of relative location of peaks of separated analytes), or an analysis of relative distribution of quantities of separated fractions of the analytes. The fractions once collected can then be used for any further downstream processing as desired.Determining location of analytes or fractions using pl markers

[1099] FIG. 2 is a flowchart illustrating an exemplary method 200 of using one or more pl markers (e.g., separation markers and / or fractionation markers) to monitor and determine thelocation of separated analytes before or after elution into fractions, according to an embodiment.

[1100] The method 200 includes, at 271, introducing a sample containing a plurality of analytes and a plurality of isoelectric point (pl) markers in a conductive medium into a capillary, each pl marker from the plurality of pl markers having a known isoelectric point from a plurality of isoelectric points, the plurality of pl markers including a subset of pl markers each pl marker from the subset of pl markers having negligible native fluorescence in a predetermined range of wavelengths designated for analyte identification. This subset of pl markers can be fractionation markers as described herein. Each pl marker from the subset of pl markers can be configured to be excited by light of a wavelength wi thin a known excitation range of wavelengths to emit a non-native fluorescence signal at a wavelength within a range of emission wavelengths. Prior to introducing the sample the plurality of pl markers can be dissolved or mixed in with the plurality' of analytes in the sample in any suitable manner.[HOI] In some implementations, each pl marker from the subset of pl markers (e.g., each fractionation marker) is tagged with a fluorescent label. The fluorescent label can be configured to be excited by light within the known excitation range to cause the subset of pl markers to emit the non-native fluorescence signal within a range of emission wavelengths. The known excitation range of wavelengths and / or the range of emission wavelengths can be any suitable range. The subset of pl markers can be selected, for example, as having pls that correspond to and / or are configured to bracket pls of an analyte, analytes of interest, and / or to produce a ladder suitable for identifying boundaries of particular pl ranges.

[1102] In some implementations, the subset of pl markers (e.g., each fractionation marker) can be a first subset and the plurality of pl markers can also include a second subset of pl markers (e.g., separation markers). Each pl marker from the second subset of pl markers can have an isoelectric point different than the isoelectric point(s) associated with analytes. Markers from the second subset (e.g., separation markers) can be configured to fluoresce (e.g., natively and / or non-natively) in the predetermined range of wavelengths designated for analyte identification. In some implementations, the second subset of pl markers (separation markers) can be different than the first subset of pl markers (fractionation markers). In some implementations, the second subset of pl markers (separation markers) can be the same as the first subset of pl markers (fractionation markers). In some implementations, the second subset of pl markers (separation markers) can be overlapping with the first subset of pl markers(fractionation markers), with one or more pl markers serving as both separation markers and fractionation markers.

[1103] The method 200, at 272 includes separating the plurality of pl markers and at least a subset of the plurality of analytes. For example, each pl marker and each analyte can be focused according to their isoelectric points, by applying a voltage across the capillary. In some embodiments, the method can include probing pl markers and / or analytes using a first modality during and / or after separation. Because separation markers are configured to fluoresce (e.g., natively and / or non-natively) in the predetermined range of wavelengths designated for analyte identification, separation markers can be probed, detected, and / or visualized at the same time and / or using the modalit(ies) (e.g.. UV absorbance or native fluorescence) as analytes.

[1104] At 273, the method includes eluting a portion of the sample that includes at least one of an analyte from the plurality of analytes or a pl marker from the subset of pl markers into a well.

[1105] At 274, the method includes probing the portion of the sample including the at least one of the analyte or the pl marker from the subset of pl markers with light within the known excitation range to cause the pl marker to emit the non-native fluorescence signal for example using the second modality. At 275, the method includes detecting the non-native fluorescence signal indicating the presence of the pl marker in the portion of the sample.

[1106] It should be understood that eluting at 273. the probing at 274. and detecting at 275 can performed in any order. For example, the probing the portion of the sample using the second modality can be performed at the distal end of the capillary (e.g., capillary 106 of system 100) prior to elution or at a sample well in a sample plate where a fraction of the portion of the sample has been eluted, as described herein.

[1107] As described herein, with reference to the system 100, in some implementations, the method 200 of elution of fractions can be fully or semi-automatic and the sample plate can include numerous well such that fractions can be serially eluted into subsequent wells of the sample plate. The portion eluted as described above at 273 can be a first portion, the pl marker can be a first pl marker (a first fractionation marker of having a first pl), the well can be a first well in a series of wells in a sample plate. The method can include eluting a second portion of the sample into a second well, eluting a third portion of the sample into a third well, and so on. The sample plate can be probed using the second modality as described herein.

[1108] The relative locations of fractionation markers can be used to determine or infer the location of sample fractions. As an example, if a first well contains a fractionation marker having a first known pl and a third well contains a fractionation marker having a third known pl, it can be determined or inferred that the second well contains analytes having a pl betw een the first pl and the third pl. Similarly, in a scenario where an analyte has been identified with a pl between the first and the third pl, the location of that analyte can be narrowed down to the first well, the second well, and the third well. This can reduce time and increase efficiency of monitoring elution of large number of fractions for further processing. In addition, probing the sample plate with the second modality results in detection of sample fractions being independent of the accuracy of the first modality and / or predictive techniques used to ascertain the likely elution time of sample fragments detected during a separation phase.

[1109] FIG. 3 illustrates an exemplary' method 300 of monitoring separation and fractionation of analytes in a sample and determining the location of analytes in the eluted fractions in a series of wells, according to an embodiment. The method 300 can be performed by any fractionation system described herein, for example the system 100 described with reference to FIG. 1.

[1110] The method 300 at 371 includes introducing a sample containing a plurality of analytes and a plurality' of pl markers in a conductive medium into a capillary, each pl marker from the plurality' of pl markers having a known isoelectric point from a plurality of isoelectric points and at least a subset of pl markers from the plurality' of pl markers having negligible native fluorescence in a predetermined range of wavelengths designated for analyte identification, each pl marker from the subset of pl markers being configured to be excited by light of a wavelength within a known excitation range of wavelengths to emit a non-native fluorescence signal at a wavelength within a range of emission wavelengths. The pl markers can include separation markers and / or fractionation markers as described herein.[HU] The method at 372 includes separating the plurality of pl markers and at least a subset of the plurality analytes, each pl marker and each analyte being focused according to their isoelectric points. This separation can be performed using any suitable techniques such as isoelectric focusing. In some embodiments, the method can include probing pl markers and / or analytes using a first modality during and / or after separation. Because separation markers are configured to fluoresce (e g., natively and / or non-natively) in the predetermined range of wavelengths designated for analyte identification, separation markers can be probed, detected,and / or visualized at the same time and / or using the modalit(ies) (e.g., UV absorbance or native fluorescence) as analytes.[1H2] The method includes eluting a first portion of the sample into a first well at 373, eluting a second portion of the sample into a second well at 374, and eluting a third portion of the sample into a third well at 375. The process of elution can proceed serially to any suitable number of wells as desired.[1H3] The method at 376 includes probing the first well, the second well, and the third well with light within the known excitation range to cause the pl markers from the subset of pl markers to emit the non-native fluorescence signal. The probing can be under the second modality as described herein, for example using a probe system 102 of the system 100 described above. Probing the wells at 376 results in detection of non-native fluorescence signal(s) indicating the presence of a first pl marker from the subset of pl markers in the first portion of the sample eluted into the first well and detecting a non-native fluorescence signal indicating the presence of a second pl marker from the subset of pl markers in the third portion of the sample eluted into the third well.[1H4] The first pl marker and the second pl marker can each have any suitable pl value. In some examples, a protein sample can be widely bracketed with a high and low pl markers such that there is a wide separation between the pl value associated with the first pl marker and the pl value associated with the second pl marker. In some examples, the first and second pl marker can be used to indicate a start and end of fractions with analytes of interest. In some implementations, the sample can be more tightly bracketed using pl markers more closely spaced to allow for higher resolution pl bracketing / identification.[1H5] In some implementations, the process of probing a sequence of wells in a sample plate also referred to as “read from top’?can be performed using a fluorescence plate reader, where the measured fluorescence can be used to identify wells that may contain specific charge variants. The fluorescence plate reader can typically be configured to only detect charge variant fractions of high concentrations, especially the main peak in a fraction consisting of several peaks associated with several focused analytes and / or pl markers. For example, the fluorescence plate reader may not have enough sensitivity' to locate low abundance acidic and basic charge variant fractions collected in a well. In such conditions, additional fractions from both acidic and basic sides may be needed for verification to cover a complete distribution ofcharge variants. The use of fractionation markers, as described herein, provides an alternative approach that uses a fluorescence plate reader under a second modality for locating the range of fraction wells of specific protein charge variant fractions.

[1116] Under this approach, in some implementations, the pl markers (fractionation markers) can be spiked or mixed into the sample loaded onto the fractionation cartridge. As the fractionation pl markers do not emit native fluorescence, they appear "transparent ’ and thus will not interfere with the native fluorescence detection under the first modality during the fractionation. However, when the sample plate is subjected to probing under the second modality using excitation on the plate reader (e g., excitation / emission: 550 / 580 nm), the fraction wells containing the eluted fractionation markers will emit strong fluorescence, providing references for easy identification of any protein charge variant fractions with pl values between these pl markers. In some embodiments, the fluorescence measurement could be carried out in a plate reader integrated into the instrument configured for separation and elution. This approach provides the advantage of allowing samples of interest to be directly analyzed on a Mass Spectrography instrument without further fraction checking with high confidence. In some embodiments, the fluorescence measurement could be carried out at a distal end of the capillary as fractions are eluted from the tip of the capillary. Real-time information (while fractionation is ongoing) regarding into which wells the pl markers were eluted can be used to improve peak tracking.[1H7] FIGS. 4A is an example plot showing results from separation by isoelectric focusing of a sample. The plot in FIG. 4A shows an icIEF electrogram of a sample including a specific analyte, a monoclonal antibody USP mAb2, that was processed by a fractionation system as described herein. The plot illustrates the use of separation markers which are pl markers with known pl that have individualized peaks that can be seen at the respective known pls on the electrogram. For example, the plot in FIG. 4A shows peaks corresponding to native fluorescence signals obtained from separation markers having a pl 6.14 and 10.17. The two separation markers have been used to bracket the analytes having a pl that is of intermediate value between the two separation markers (the main analyte USP mAb2, acidic, and basic charge variants are indicated).

[1118] FIG. 4B representations an elution of a series of fraction of the sample of FIG. 4A into a series of w ells in a sample plate. FIG. 4B shows a 3D plot of florescence intensities of 36 fractions obtained by probing fractions collected in a sample plate using a plate reader, asdescribed herein. The probing can be using a second modality to detect fractionation markers configured to be excited by light of a wavelength within a known excitation range of wavelengths (e.g., excitation at 550 nm) to emit a non-native fluorescence signal at a wavelength within a range of emission wavelengths (e.g., emission at 580 nm). Here the fractions were collected from Bl to B12 then C12 to Cl and then from DI to DI 2, in that sequential order. The significantly taller bars corresponding to wells Cl and C12 indicate nonnative florescence signals obtained from fractionation markers with pl values 6.0 and 8.4 as shown in FIG. 4B. The signals from the fractionation markers indicated the location of the charge variant or analyte of interest and highlighted the wells that may need further processing and / or verification. The fractions collected from C9 to C6 were identified to be requiring further analysis and verification.[U19] FIG. 4C illustrates experimental results from analysis of contents in a subset of wells C9 to C6 shown in FIG. 4B. Thus, based on the bracketing provided by the fractionation markers, a relatively small number of wells were analyzed to detect the full range of the basic, main, and acidic charge variants of the analyte. As illustrated by the low fluorescence of fraction C6, little to no analyte appears in wells eluted prior to well C6.

[1120] Embodiments disclosed herein present methods and systems to separate and collect charge variants from a sample using an icIEF process. The concentration of a collected fraction can be adjusted by adjusting a starting concentration of a sample, the inner diameter(s) of the capillary used, and / or the volume of the chemical mobilizer in the fraction collection wells. Chemicals with different negative ions, for example, acetate and phosphate, can be used as chemical mobilizers. The speed of mobilization can be adjusted by the concentrations of the mobilizer, the applied voltage, the composition of the separation buffer (mixture of sample, ampholyte, and additives), and the ID of the capillary tube. The collection time for a well can be adjusted along with the speed of mobilization to allow a single charge variant or multiple charge variants to be collected into a single well.[U21] Formulations of the assay buffers (e.g., “master mix”) (that may be used with other standard separation systems) can be used with the cartridges disclosed herein to be used for fractionation. Utilizing similar master mixes can minimize assay development time needed to transfer an existing assay from the standard icIEF Maurice™ cartridge to the sample fractionation cartridges and methods described herein (or vice versa).

[1122] The methods, apparatus, and systems disclosed herein enable high-performance sample fractionation in the easy-to-use Maurice™ icIEF cartridge format. In contrast to hyphenated methods such as cIEF-MS, a variety of downstream analyses can be performed on the fractions obtained from the fractionation methods disclosed here. For example, the collected fraction can be used directly without further sample preparation; or the collected fraction can be concentrated, diluted, or buffer exchanged before the downstream analysis; or the same fraction from multiple fractionation runs can be pooled together if a larger quantity is needed for certain downstream analysis; or the collected fraction can be cleaned-up to remove any components incompatible with certain downstream analysis.[H23] Some embodiments described herein relate to a computer storage product with a non-transitory computer-readable medium (also can be referred to as a non-transitory processor-readable medium) having instructions or computer code thereon for performing various computer-implemented operations. The computer-readable medium (or processor- readable medium) is non-transitory in the sense that it does not include transitory’ propagating signals per se (e.g., a propagating electromagnetic wave carrying information on atransmission medium such as space or a cable). The media and computer code (also can be referred to as code) may be those designed and constructed for the specific purpose or purposes. Examples of non-transitory computer-readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape; optical storage media such as Compact Disc / Digital Video Discs (CD / DVDs), Compact Disc-Read Only Memories (CD- ROMs), and holographic devices; magneto-optical storage media such as optical disks; carrier wave signal processing modules; and hardw are devices that are specially configured to store and execute program code, such as Application-Specific Integrated Circuits (ASICs), Programmable Logic Devices (PLDs), Read-Only Memory (ROM) and Random-Access Memory (RAM) devices. Other embodiments described herein relate to a computer program product, which can include, for example, the instructions and / or computer code discussed herein.[H24] Some embodiments and / or methods described herein can be performed by’ software (executed on hardw are), hardw are, or a combination thereof. Hardw are modules may include, for example, a general-purpose processor, a field programmable gate array (FPGA), and / or an application specific integrated circuit (ASIC). Software modules (executed on hardware) can be expressed in a variety of software languages (e.g., computer code), including C, C++,Java™, Ruby, Visual Basic™, and / or other object-oriented, procedural, or other programming language and development tools. Examples of computer code include, but are not limited to, micro-code or micro-instructions, machine instructions, such as produced by a compiler, code used to produce a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. For example, embodiments may be implemented using imperative programming languages (e.g., C, FORTRAN, etc ), functional programming languages (Haskell, Erlang, etc.), logical programming languages (e.g., Prolog), object- oriented programming languages (e.g., Java, C++, etc.) or other suitable programming languages and / or development tools. Additional examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

[1125] While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Where schematics and / or embodiments described above indicate certain components arranged in certain orientations or positions, the arrangement of components may be modified. While the embodiments have been particularly shown and described, it will be understood that various changes in form and details may be made. Although various embodiments have been described as having particular features and / or combinations of components, other embodiments are possible having a combination of any features and / or components from any of embodiments as discussed above.

[1126] Where methods and / or events described above indicate certain events and / or procedures occurring in certain order, the ordering of certain events and / or procedures may be modified. Additionally, certain events and / or procedures may be performed concurrently in a parallel process when possible, as well as performed sequentially as described above.

Claims

CLAIMSWhat is Claimed:

1. A method comprising: introducing, at a first time, a sample containing a plurality of analytes and a plurality of isoelectric point (pl) markers in a conductive medium into a capillary, each pl marker from the plurality of pl markers having a known isoelectric point from a plurality of isoelectric points, the plurality of pl markers including a subset of pl markers, each pl marker from the subset of pl markers being configured to be excited by light of a wavelength within a known excitation range of wavelengths to emit a non-native fluorescence signal at a wavelength within a range of emission wavelengths; separating, at a second time after the first time, the plurality of pl markers and at least a subset of the plurality of analytes, each pl marker and each analyte being focused according to their isoelectric points, by applying a voltage across the capillary: eluting a portion of the sample that includes at least one of an analyte from the plurality of analytes or a pl marker from the subset of pl markers into a well at a third time after the second time: probing the portion of the sample including the at least one of the analyte or the pl marker from the subset of pl markers with light within the known excitation range of wavelengths to cause the pl marker to emit the non-native fluorescence signal; and detecting the non-native fluorescence signal indicating a presence of the pl marker in the portion of the sample.2 . The method of claim 1, wherein each pl marker from the subset of pl markers is tagged with a fluorescent label the fluorescent label configured to be excited by light of the wavelength within the known excitation range of wavelengths to emit the non-native fluorescence signal at the wavelength within a range of emission wavelengths.

3. The method of claim 1. wherein: the subset of pl markers is a first subset of pl markers; each pl marker from the first subset of pl markers has negligible native fluorescence in a predetermined range of wavelength designated for analyte identification; the portion of the sample includes at least one analyte and a pl marker from the first subset of pl markers;the plurality of pl markers includes a second subset of pl markers; each pl marker from the second subset of pl markers has at least one of a native or a non-native fluorescence in the predetermined range of wavelengths designated for analyte identification.

4. The method of claim 1. wherein: the subset of pl markers is a first subset of pl markers; each pl marker from the first subset of pl markers has negligible native fluorescence in a predetermined range of wavelength designated for analyte identification; the plurality of pl markers includes a second subset of pl markers; each pl marker from the second subset of pl markers has an isoelectric point different than the isoelectric point associated with each analyte from the plurality of analytes; and each pl marker from the second subset of pl markers has at least one of a native or a non-native fluorescence in the predetermined range of wavelengths designated for analyte identification.

5. The method of claims 4, wherein the second subset of pl markers is different than the first subset of pl markers.

6. The method of claim 1, wherein the subset of pl markers is a first subset of pl markers and the plurality of pl markers includes a second subset of pl markers, each pl marker from the second subset of pl markers having an isoelectric point different than the isoelectric point associated with each analyte from the plurality7of analytes, and having at least one of a native or a non-native fluorescence in a predetermined range of wavelengths designated for analyte identification.

7. The method of claim 1, wherein the probing of the portion of the sample is performed after the portion of the sample is eluted.

8. The method of claim 1, wherein the probing of the portion of the sample is performed prior to the portion of the sample being eluted.

9. The method of claim 1. wherein:the portion is a first portion, the pl marker is a first pl marker, the well is a first well, and the presence of the first pl marker is detected in the first well by probing the portion of the sample in the first well, the method further comprising: eluting a second portion of the sample into a second well; eluting a third portion of the sample into a third well; and probing the second well and the third well; detecting a non-native fluorescence signal indicating a presence of a second pl marker from the plurality of pl markers in the third well.

10. The method of claim 1, wherein: the portion is a first portion, the pl marker is a first pl marker, the well is a first well, and the presence of the first pl marker is detected in the first well by probing the portion of the sample in the first well, the method further comprising: determining that the analyte is focused at a location between the first pl marker and the second pl marker by the separating; eluting a second portion of the sample into a third well; probing the third well; detecting a non-native fluorescence signal indicating a presence of a second pl marker from the plurality' of pl markers in the third well indicating that the analyte is disposed within at least one of the first well, the third well, or a second well disposed between the first well and the third well.

11. The method of claim 1, wherein the portion is a first portion and the analyte is a first analyte, the pl marker is a first pl marker, the well is a first well, and the first portion of the sample is probed in the first well, the method further comprising: eluting a second portion of the sample that includes a second analyte from the plurality of analytes and a second pl marker from the subset of pl markers into a second well at a fourth time after the third time; and probing the second portion of the sample in the second well with light of the wavelength within the known excitation range of wavelengths to cause the second pl marker to emit the non-native fluorescence at the wavelength within the range of emission wavelengths.

12. The method claim 11, wherein the first pl marker is configured to emit at a wavelength that is same as the wavelength of emission of the second pl marker, the detectingthe non-native florescence includes detecting the non-native fluorescence signal at the first well and at the second well, the detecting indicating the presence of the first analyte and the second analyte in at least one of the first well, the second well, or a well from the plurality of wells disposed between the first well and the second well.

13. The method of claim 1, wherein the portion is a first portion, and the analyte is a first analyte, and the well is a first well, the method further comprising: eluting each portion from a plurality of portions of the sample into each well from a plurality7of wells in a sample plate defining the plurality of wells; the probing the first portion of the sample is at the first well and includes probing the plurality of portions of the sample at the plurality of wells defined in the sample plate, each well containing a portion from the plurality of portions of the sample, the light of the wavelength within the known excitation range of wavelengths causing the subset of pl markers to emit the non-native fluorescence at the wavelength within the range of emission wavelengths; and the detecting the non-native fluorescence at the wavelength includes detecting the non-native fluorescence from a subset of wells from the plurality of wells, the subset of wells including the first well, the detecting the non-native fluorescence indicating a presence of a pl marker from the subset of pl markers in each well from the subset of wells; and determining, based on the wells from which the non-native fluorescence signal was detected and a relative location of each well from the subset of wells, a presence or absence of an analyte from the plurality of analytes in the portion of the sample contained in each well from the plurality of wells.

14. The method of claim 13, wherein the determining the presence or absence of the analyte from the plurality of analytes in the portion of the sample contained in each well from the plurality of wells includes determining a well containing an identified portion of the sample including a fraction with an isoelectric point between isoelectric points of two pl markers from the plurality of markers.

15. The method of claim 1, wherein the portion is a first portion, and the analyte is a first analyte, and the well is a first well, the method further comprising:probing the sample at a fourth time between the second and the third time with light in a predetermined range of wavelengths designated for analyte identification that is different from the known excitation range; determining, at the fourth time and based on at least one of native fluorescence or absorbance associated with the plurality7of analytes, a location in the capillary7of each analyte from the plurality of analytes that corresponds to an isoelectric point of that analyte; the eluting including eluting each portion from a plurality of portions of the sample into each well from a plurality of wells in a sample plate defining the plurality of wells; the probing the first portion of the sample is at the first well and includes probing the plurality7of portions of the sample at the plurality7of wells defined in the sample plate, each well containing a portion from the plurality of portions of the sample, the light of the wavelength within the known excitation range of wavelengths causing the subset of pl markers to emit the non-native fluorescence at the wavelength within the range of emission wavelengths; and the detecting the non-native fluorescence at the wavelength includes detecting the non-native fluorescence from a subset of wells from the plurality of wells, the subset of wells including the first well, the detecting the non-native fluorescence indicating the presence of the pl marker from the subset of pl markers in each well from the subset of wells; and determining, based on the wells from which the non-native fluorescence signal was detected, the isoelectric point of each analyte from the plurality of analytes, and a relative location of each well from the subset of wells, a presence or absence of an analyte from the plurality of analytes in the portion of the sample contained in each well from the plurality7of wells.

16. The method of claim 15, wherein the subset of pl markers is a first subset and the plurality of pl markers includes a second subset of pl markers different than the first subset, each pl marker from the second subset of pl markers having a native fluorescence in the predetermined range of wavelengths designated for analyte identification and an isoelectric point that is different than an isoelectric point associated with an analyte from the plurality7of analytes.

17. The method of claim 15, wherein the determining, at the fourth time the location in the capillary of each analyte from the plurality of analytes that corresponds to an isoelectricpoint of that analyte is further based on a native fluorescence associated with at least one pl marker from the second subset of pl markers.

18. The method of claim 15, wherein: the subset of pl markers is a first subset of pl markers; the plurality of pl markers includes a second subset of pl markers; each pl marker from the second subset of pl markers has at least one of a native or a non-native fluorescence in the predetermined range of wavelengths designated for analyte identification; and determining the location in the capillary of each analyte from the plurality of analytes at the fourth time includes determining a location of each pl marker from the second subset of pl markers.

19. The method of claim 15, wherein: the subset of pl markers is a first subset of pl markers; each pl marker from the first subset of pl markers has negligible native fluorescence in the predetermined range of wavelength designated for analyte identification such that probing the sample at the fourth time does not detect pl markers from the first subset of pl markers; the plurality of pl markers includes a second subset of pl markers; and each pl marker from the second subset of pl markers has at least one of a native or a non-native fluorescence in the predetermined range of wavelengths designated for analyte identification; and determining the location in the capillary of each analyte from the plurality of analytes at the fourth time includes determining a location of each pl marker from the second subset of pl markers.

20. The method of claim 15, wherein: the subset of pl markers is a first subset of pl markers; each pl marker from the first subset of pl markers has negligible native fluorescence in the predetermined range of wavelength designated for analyte identification such that probing the sample at the fourth time does not detect pl markers from the first subset of pl markers;the portion of the sample includes at least one analyte and a pl marker from the first subset of pl markers; the plurality of pl markers includes a second subset of pl markers; each pl marker from the second subset of pl markers has at least one of a native or a non-native fluorescence in the predetermined range of wavelengths designated for analyte identification; and determining the location in the capillary of each analyte from the plurality of analytes at the fourth time includes determining a location of each pl marker from the second subset of pl markers but does not include detecting the pl marker included in the potion of the sample.

21. The method of claim 1, wherein the probing with the light of the wavelength within the known excitation range of wavelengths is performed at the wavelength of 550 nm.

22. The method of claim 1, wherein each pl marker from the subset of pl markers is configured to emit the non-native florescence at the wavelength of 580 nm.

23. The method of claim 1, wherein each pl marker from the subset of pl markers is configured to emit induced florescence at a unique wavelength within the range of emission wavelengths.

24. The method of claim 1, further comprising: ceasing the eluting of the portion of the sample based on the detecting the non- native fluorescence at the wavelength indicating the presence of the pl marker and the presence of the analyte in the portion of the sample.

25. The method of claim 1, w herein the probing the portion of the sample is performed at the w ell and after the eluting at the third time.

26. The method of claim 1 , wherein the probing the portion of the sample is performed at the w ell during the eluting at the third time.

27. The method of claim 1. wherein the probing the portion of the sample is performed prior to the eluting at the third time when the portion of the sample is contained in the capillary.

28. The method of claim 1. further comprising: imaging at least a portion of the capillary for at least one of native fluorescence emission within a predetermined range of wavelengths designated for analyte identification or for ultraviolet absorption; and determining a location of focusing of each analyte from the plurality of analytes based on the imaging.

29. The method of claim 28, wherein each pl marker from the subset of pl markers has negligible native fluorescence in the predetermined range of wavelengths designated for analyte identification or for ultraviolet absorption.

30. The method of claim 28, wherein: the subset of pl markers is a first subset of pl markers having negligible native fluorescence in the predetermined range of wavelengths designated for analyte identification or for ultraviolet absorption; the plurality of pl markers includes a second subset of pl markers; and determining a location of focusing of each pl marker from the second subset of pl markers from the plurality of analytes based on the imaging.

31. The method of claim 1, further comprising: mixing each pl marker from the plurality of pl markers with an initial sample containing a plurality of analy tes to form the sample containing the plurality of analytes and the plurality of (pl) markers, each pl marker from the plurality of pl markers being a lyophilized protein form prior to the mixing.

32. The method of claim 1, wherein: the pl marker has a first isoelectric point, the analyte has a second isoelectric point, and the first isoelectric point is same as the second isoelectric point; and the portion of the sample that is eluted includes the analyte and the pl marker.

33. The method of claim 1, wherein the pl marker has a first isoelectric point and the analyte has a second isoelectric point different from the first isoelectric point.

34. An apparatus, comprising: a capillary configured to contain an electrically conductive sample including an analyte having a first isoelectric point and an isoelectric point (pl) marker associated with a second isoelectric point, the analyte associated with a first isoelectric point, the pl marker configured to emit an non-native fluorescence signal in response to receiving an excitation signal within an excitation wavelength range, the pl marker not exhibiting native fluorescence within a predetermined range of wavelengths designated for analyte identification; an electrical power source configured to apply a voltage across the capillary and between a first running buffer in ionic communication with a first end of the capillary and a second running buffer in ionic communication with a second end of the capillary, the voltage configured to effect separation and focusing of the pl marker and the analyte according to their respective isoelectric points; a sample plate defining a plurality of wells, the sample plate and the capillary collectively configured such that the second end of the capillary' can move between each well from the plurality’ of wells; and a first well from the plurality of wells configured to collect at least one of the analyte as it migrates from a first portion of the capillary or the pl marker as it migrates from the second portion of the capillary to be eluted into the first well.

35. The apparatus of claim 34, wherein the second isoelectric point is same as the first isoelectric point such that the first portion of the capillary is the same as the second portion of the capillary and the first yvell is configured to collect the analyte and the pl marker as they are eluted from the first portion of the capillary.

36. The apparatus of claim 34, wherein: the voltage is a first voltage and the first well is configured to contain a chemical mobilizer, and the electrical power source is configured to apply a second voltage, such that when the second end of the capillary is disposed in the first yvell and the second voltage is applied: the analyte migrates into the first yvell from the portion of the capillary associated with the first isoelectric point during the separation and focusing of the pl marker and the analyte according to their respective isoelectric points; andthe pl marker migrates into the first well from the portion of the capillary associated with the second isoelectric point during the separation and focusing of the pl marker and the analyte according to their respective isoelectric points.

37. The apparatus of claim 34, wherein the sample includes a plurality of analytes, the analyte being a first analyte from the plurality of analytes, each analyte from the plurality of analytes being associated with an isoelectric point that is different than the isoelectric point associated with the remaining analytes from the plurality of analytes, the sample further containing a plurality of pl markers, each pl marker from the plurality of pl markers being associated with an isoelectric point that is different than the isoelectric point associated with the remaining pl markers, the isoelectric point associated with at least two pl marker from the plurality of pl markers having isoelectric points that bracket at least one analyte from the plurality of analytes.

38. The apparatus of claim 37, wherein the isoelectric point associated with each pl marker from the plurality of pl markers is configured to match the isoelectric point associated with at least one analyte from the plurality7of analytes.

39. The apparatus of claim 34, wherein the pl marker is a lyophilized protein prior to being mixed with the sample.

40. The apparatus of claim 34, wherein the pl marker is configured to be excited with light of wavelength 550 nm.

41. The apparatus of claim 34, wherein the pl marker is configured to, upon being excited, emit the non-native fluorescence at a wavelength of 580 nm.

42. The apparatus of claim 34, wherein: the voltage is a first voltage and the analyte is a first analyte, and the pl marker is a first pl marker, the sample further includes a second analyte associated with a third isoelectric point and a second pl marker associated with a fourth isoelectric point the apparatus further comprising: a second well from the plurality of wells configured such that when the second end of the capillary is disposed in the second well, the second well is configured to collect the secondanalyte as it migrates into the second well from the portion of the capillary associated with the third isoelectric point, and the second well is further configured to collect the second pl marker that migrates into the second well from the portion of the capillary associated with the fourth isoelectric point.

43. The apparatus of claim 42, wherein the sample plate is configured to be probed by the light of the wavelength within the excitation wavelength range to cause excitation of the first pl marker in the first well and the second pl marker in the second well.

44. A non-transitory processor-readable medium storing code representing instructions to be executed by a processor, the instructions comprising code to cause the processor to: receive a first information associated with an induced emission of fluorescence signal in response to receiving an excitation signal within an excitation wavelength range by a set of isoelectric point (pl) markers included in a sample; receive a second information associated with at least one of a native emission of fluorescence signal by a set of analytes or a ultraviolet light absorbance associated with the set of analytes included in the sample; infer based on the first information and the second information, a relative location of a fraction eluted from the sample among a plurality of wells defined in a sample plate, each well from the plurality of wells containing an eluted fraction from the sample after separating the set of pl markers and the set of analytes, each pl marker and each analyte being focused according to their respective isoelectric points.

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