Device and method for sample characterization - Patent Application 20070122997

Abstract

To provide devices and methods for characterizing analyte mixtures.SOLUTION: Some methods described herein comprise performing enrichment steps on a device before expelling enriched analyte fractions from the device for subsequent analysis. Also included are devices for performing these enrichment steps.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a nonprovisional application of and claims the benefit of U.S. Provisional Patent Application No. 62 / 260,944, filed November 30, 2015, and U.S. Provisional Patent Application No. 62 / 338,074, filed May 18, 2016, each entitled "Devices, Methods, and Kits for Sample Characterization," the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Some embodiments described herein relate to devices and methods for the characterization of samples and various uses thereof. [Background technology]

[0003] Separating analyte components from a more complex analyte mixture based on their inherent properties and providing a set of fractions enriched in that property state is an important part of analytical chemistry. Simplifying the complex mixture in this way reduces the complexity of downstream analysis. It can be advantageous to perform two or more enrichment steps that are orthogonal (e.g., based on different and / or unrelated properties). However, in many cases, the process of performing orthogonal enrichment steps using known methods and / or devices is cumbersome and may dilute the analyte beyond the sensitivity of downstream analytical equipment. In addition, complications can arise when attempting to interface known enrichment methods and / or devices with analytical equipment and / or techniques.

[0004] Methods have been used to interface protein sample preparation techniques with downstream detection systems, such as mass spectrometers. A common method is to prepare samples using liquid chromatography and collect fractions for mass spectrometry (LC-MS). This has the disadvantage of requiring the protein sample to be digested into peptide fragments, leading to a large number of sample fractions to be analyzed and complex post-processing data reconstruction. While certain forms of liquid chromatography can be coupled to mass spectrometers, such as peptide map reversed-phase chromatography, these known techniques are limited to the use of peptide fragments rather than intact proteins, thereby limiting their usefulness.

[0005] Another method for introducing samples into a mass spectrometer is electrospray ionization (ESI). In ESI, small droplets of sample and solution at the distal end of a capillary or microfluidic device are ionized, inducing attraction to the charged plates of the mass spectrometer. The droplets then elongate into a conical shape (a "Taylor cone") within this induced electric field, and the droplets are then ejected and enter the mass spectrometer for analysis. This is typically done in a capillary, which provides a convenient volume and size for ESI. However, capillaries provide a linear flow path that does not allow for multi-step processing.

[0006] Other research has been pursued using microfluidic devices. Microfluidic devices can be fabricated by a variety of known techniques to provide fluid channels of predetermined widths, allowing the construction of channel networks designed to perform various fluidic operations. These devices offer an additional level of control and complexity compared to capillaries. In the context of ESI, known devices include outwardly tapered tips and conductive edges to enhance ESI in these devices. However, the outward taper of known microfluidic devices used for ESI exposes the fragile Taylor cone structure to potential disturbances from air turbulence and results in a contact surface geometry that supports only a limited range of conical arcs, limiting control over the volume introduced into the mass spectrometer via ESI. Additionally, electrolysis of water at the conductive edges can lead to the formation of gas bubbles, which can disrupt the cone's growth.

[0007] One application of protein mass spectrometry is the characterization during development and manufacturing of biopharmaceuticals and biosimilars, a class of drugs that includes, for example, recombinant proteins, antibodies, live viral vaccines, human plasma-derived proteins, cell-based drugs, naturally occurring proteins, antibody-drug conjugates, protein-drug conjugates, and other protein drugs.

[0008] Regulatory compliance dictates that biopharmaceuticals require extensive testing during development and manufacturing that is not required for small molecule drugs. This is because biopharmaceutical manufacturing is more complex due, for example, to the use of biological materials to generate the biopharmaceutical, the greater complexity of the biomolecules, and the more complex manufacturing process. Properties that must be defined include, for example, charge, potency, hydrophobicity, mass, and glycosylation. Currently, these tests are performed independently of each other, resulting in a very time-consuming and expensive process for determining the properties of biopharmaceuticals. Summary of the Invention

[0009] Some embodiments described herein relate to devices and methods that allow for the analysis of analytes in an analyte mixture. For example, specific characterization of biological proteins is often required by regulatory agencies. The methods and devices described herein may be suitable for characterizing proteins and / or other analytes. In some embodiments, the methods and devices described herein may relate to the characterization of an analyte mixture that includes one or more enrichment steps performed to separate the analyte mixture into enriched analyte fractions.

[0010] In some cases, these analytes may be, for example, glycans, carbohydrates, DNA, RNA, intact proteins, digested proteins, antibody-drug conjugates, protein-drug conjugates, peptides, metabolites, or other biologically relevant molecules. In some instances, these analytes may be small molecule drugs. In some instances, these analytes may be protein molecules in protein mixtures, such as biological protein pharmaceuticals and / or lysates, recovered from cells isolated from culture or in vivo.

[0011] Some embodiments described herein can include a first enrichment step in which fractions containing a subset of analyte molecules from the original analyte mixture are eluted one fraction at a time, and these enriched analyte fractions are then subjected to another enrichment step in which the enriched analyte fractions are released for further analysis.

[0012] In some embodiments, one or more of the enrichment steps is a solid phase separation. In some embodiments, one or more of the enrichment steps is a solution phase separation.

[0013] In some embodiments, a final step concentrates the enriched analyte fraction before release.

[0014] In some embodiments, substantially all of the enriched analyte fraction from the final enrichment step is output in a continuous stream. In some embodiments, a portion of the analyte mixture (e.g., the fraction of interest) is output from the microfluidic device through an outlet configured to interface with an analytical instrument, such as a mass spectrometer or another device configured to fractionate and / or enrich at least a portion of the sample. Another portion of the analyte mixture (e.g., containing fractions other than the fraction of interest) can be output through a waste channel.

[0015] In some embodiments, ejection is performed using pressure, electrical forces, or ionization, or a combination thereof.

[0016] In some embodiments, ejection is performed using electrospray ionization (ESI), for example into a mass spectrometer. In some embodiments, a sheath liquid is used as an electrolyte for electrophoretic separation. In some embodiments, a nebulizing gas is provided to break the analyte fraction into a fine spray. In some embodiments, other ionization methods are used, such as inductively coupled laser ionization, fast atom bombardment, soft laser desorption, atmospheric pressure chemical ionization, secondary ion mass spectrometry, spark ionization, or thermal ionization.

[0017] In some embodiments, the enriched fraction is deposited on a surface and further analyzed by matrix-assisted laser desorption / ionization, surface-enhanced laser desorption / ionization, immunoblotting, or the like.

[0018] Some embodiments described herein relate to devices and methods for visualizing analytes in an electrophoretic separation before and during ejection of the enriched fraction.

[0019] Some embodiments described herein relate to devices and methods for visualizing analytes during the enrichment step.

[0020] Some embodiments described herein relate to devices and methods for visualizing analytes in channels between enrichment zones.

[0021] In some embodiments, visualization of the analyte can be achieved via optical detection, such as ultraviolet absorbance, visible light absorbance, fluorescence, Fourier transform infrared spectroscopy, Fourier transform near infrared spectroscopy, Raman spectroscopy, optical spectroscopy, and the like.

[0022] Some embodiments described herein relate to devices that allow for the analysis of analyte mixtures in that they contain one or more enrichment zones and orifices for the exit of enriched analyte fractions. In some embodiments, these devices include at least one layer that is opaque to light of a particular wavelength and at least one layer that is transparent to the particular wavelength. One or more portions of the opaque layer can define one or more enrichment zones, such that the enrichment zones function as optical slits.

[0023] In some embodiments, the analyte mixture can be loaded into the device through tubing or capillaries that connect the device to an autosampler, hi some embodiments, the analyte mixture can be loaded directly into a reservoir on the device.

[0024] In some embodiments, the orifice through which at least a portion of the sample can exit the device is countersunk and / or shielded from airflow. In some embodiments, the orifice is not electrically conductive. As used herein, a countersunk hole should be understood to mean that a portion of the substrate defines a recess containing the orifice, regardless of the shape of the recess's sides or chamfer. Similarly, a countersunk hole should be understood to include a counterbore, a conical and / or frustoconical countersunk hole, a hemispherical hole, etc.

[0025] Some embodiments described herein relate to apparatus, such as a microfluidic device, that includes a substrate made of an opaque material (e.g., soda-lime glass, which is opaque to ultraviolet light). The substrate can define a microfluidic separation channel. Similarly, the microfluidic separation channel can be etched into the substrate or otherwise formed within the substrate. The microfluidic separation channel can have a depth equal to the thickness of the substrate. Similarly, the entire depth of the substrate (e.g., the entire top to bottom) can be etched into the microfluidic separation channel. In this manner, the microfluidic separation channel can define an optical slit through the substrate. A transparent layer (e.g., a top layer) can be disposed on the top surface of the substrate, e.g., sealing the top surface of the substrate. A transparent layer (e.g., a bottom layer) can also be disposed on the bottom surface of the substrate, sealing both the top and bottom of the microfluidic separation channel. In some embodiments, only a portion of the top layer and / or bottom layer can be transparent. For example, the top and / or bottom layers can define transparent windows in an otherwise opaque material, which can provide optical access to, for example, microfluidic separation channels.

[0026] Some embodiments described herein relate to a device, such as a microfluidic device, that includes a substrate. The substrate can define one or more enrichment zones or channels. For example, the substrate can define a first enrichment zone containing a medium configured to bind analytes. Such a first enrichment zone can be suitable for chromatographically separating an analyte mixture. The device can further include two electrodes electrically coupled to opposite ends of a second enrichment zone. Such a second enrichment zone can be suitable for electrophoretically separating an analyte mixture. The second enrichment zone can intersect with the first enrichment zone, thereby allowing analytes to be further separated, concentrated, and / or enriched in the second enrichment zone after a fraction of the analytes has been separated, concentrated, and / or enriched in the first enrichment zone. The device can also include a concave orifice. The orifice can be an outlet for the second enrichment channel and can be located on a countersunk or otherwise concave surface of the substrate. The device can be configured to eject a portion of the analyte mixture from the orifice via electrospray ionization. The recess may provide a stable environment for the formation of a Taylor cone associated with ESI and / or may be configured to receive the inlet port of a mass spectrometer.

[0027] Some embodiments described herein relate to methods that include introducing an analyte mixture into a microfluidic device containing a separation channel. An electric field can be applied across the separation channel to effect separation of the analyte mixture. The analyte mixture can be imaged through a transparent portion of the microfluidic device during separation. Similarly, a window and / or an optical slit can provide optical access to the separation channel, allowing the entire separation channel or a portion thereof to be imaged while separation is occurring. Fractions of the analyte mixture can be ejected through an orifice in fluid communication with the separation channel. For example, the fractions can be ejected via electrospray ionization (ESI). In some embodiments, the orifice can be positioned on a countersink surface of the microfluidic device such that a Taylor cone is formed within a recess defined by the countersink surface.

[0028] Some embodiments described herein relate to a method including injecting analytes into a microfluidic device containing a first separation channel and a second separation channel. The first separation channel can contain a medium configured to bind analytes from an analyte mixture. Accordingly, when the analyte mixture is injected into the microfluidic device, at least a fraction of the analyte mixture can bind to a matrix and / or be prevented from flowing through the first separation channel. For example, chromatographic separation can be performed in the first separation channel by injecting the analytes into the microfluidic device. An eluent can be injected into the microfluidic device so that at least a fraction of the analytes migrates from the medium. The first separation channel can be imaged while the analytes migrate. Imaging the first separation can include imaging the entire column (e.g., the entire channel) and / or imaging a portion of the channel. When imaging detects that a fraction is located at the intersection of the first and second separation channels, an electric field can be applied to the second separation channel to cause the fraction to migrate into the second separation channel. For example, in some embodiments, the first separation channel can be orthogonal to the second separation channel. Similarly, the first separation channel and the second separation channel can form a T-junction. Imaging can detect when a portion of the fraction (e.g., a portion of interest) is located at the junction. By applying an electric field, a portion of the fraction (and optionally other portions of the fraction that are not located at the junction) can be moved into the second separation channel for a second separation step. At least a portion of the fraction can be ejected from the microfluidic device. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic diagram of a device for two-dimensional separation and ESI of automatically loaded samples, according to one embodiment.

[0030] [Figure 2]FIG. 2 is a schematic exploded view of a device having three layers, according to one embodiment.

[0031] [Figure 3] FIG. 3 is a schematic diagram of an optical path through a microfluidic device, according to one embodiment.

[0032] [Figure 4] FIG. 4 is a schematic diagram of a device for IEF and ESI of automatically loaded samples, according to one embodiment.

[0033] [Figure 5] FIG. 5 is a schematic diagram of a microfluidic device, according to one embodiment.

[0034] [Figure 6] FIG. 6 is a flow chart of an exemplary method for analyte characterization.

[0035] [Figure 7] FIG. 7 is a schematic diagram of a microfluidic device, according to one embodiment.

[0036] [Figure 8] FIG. 8 is a schematic diagram of a microfluidic device, according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0037] It should be understood that both the general description above and the description below are exemplary and illustrative only and are not intended to limit the methods and devices described herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "or" means "and / or" unless specifically stated otherwise. Similarly, the terms "comprise," "comprises," "comprising," "include," and "including" are not intended to be limiting. (device) 1 is a schematic diagram of a device for automatically loaded two-dimensional separation and ESI of samples, according to one embodiment. A microfluidic network 100 is defined by a substrate 102. The substrate is fabricated from a material that is compatible with the enrichment step to be performed. For example, considerations related to material selection include chemical compatibility, pH stability, temperature, transparency at various wavelengths of light, mechanical strength, etc.

[0038] The substrate 102 can be made from glass, quartz, fused silica, plastic, polycarbonate, PFTE, PDMS, silicon, polyfluorinated polyethylene, polymethacrylate, cyclic olefin copolymer, cyclic olefin polymer, polyetheretherketone, and / or any other suitable material. If different properties are desired in different layers of the planar substrate and / or any other suitable material, a mixture of materials can be utilized. If different properties are desired in different layers of the planar substrate, a mixture of materials can be utilized.

[0039] Channels 106, 110, 114, 116, 118, 124, 122, 126, 132, 136, and 140 form microfluidic network 100 and are fabricated in substrate 102. Similarly stated, substrate 102 defines channels 106, 110, 114, 116, 118, 124, 122, 126, 132, 136, and / or 140.

[0040] The channels can be fabricated in the substrate by any channel fabrication method, such as photolithographic etching, molding, machining, additive (3D) printing, and the like.

[0041] The sample mixture and external reagents can be loaded through tube / conduit 112 and excess reagents / waste can be removed through tube / conduit 130 .

[0042] Tubing 112 and 130 may be made from any material compatible with the assay being performed, including, for example, fused silica, fused silica capillary tubing, silicone tubing, and / or PTFE tubing.

[0043] Channels 116 and 124 can be used to separate and / or enrich analytes and / or portions (e.g., fractions) of analytes. Channels 116 and / or 124 can be used to perform chromatographic separations (e.g., reverse-phase, immunoprecipitation, ion exchange, size exclusion, ligand affinity, chromatographic, hydrophobic interaction chromatography, hydrophilic interaction chromatography, pH gradient ion exchange, affinity, capillary electrokinetic chromatography, micellar electrokinetic chromatography, high performance liquid chromatography (HPLC), amino acid analysis HPLC, ultra-high performance liquid chromatography, peptide mapping HPLC, field-flow fractionation-multiangle light scattering), or electrophoretic separations (e.g., isoelectric focusing, capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow counterbalanced capillary electrophoresis, field gradient focusing, dynamic field gradient focusing). For example, the channel 116 can be derivatized or filled with a material to perform a first enrichment step.

[0044] The materials disposed in channels 116 and / or 124 can be selected to collect analytes based on, for example, hydrophobicity (reverse phase), immunoaffinity (immunoprecipitation), affinity (availability), size (size exclusion chromatography), charge (ion exchange), or other forms of liquid chromatography.

[0045] Many different methods can be used to place enrichment materials within channels 116 and / or 124. The walls can be directly derivatized with, for example, covalently bound or adsorbed molecules, or beads, glass particles, sol-gels, etc. can be derivatized and loaded into these channels.

[0046] After the sample is loaded into channel 116 , a wash solution and then an elution reagent can be introduced through tube 112 and channel 114 .

[0047] The elution process depends on the enrichment method implemented in channel 116. A suitable eluent can be selected to elute the fraction of bound analytes. Some enrichment options may not require an elution step (e.g., size exclusion chromatography, electrophoretic separation, etc.).

[0048] The eluate or flow-through flows through channel 118 into channel 124. Channel 124 can be used to perform either a chromatographic enrichment step or an electrophoretic enrichment step.

[0049] Electrophoretic separation can be performed in channel 124 by applying an electric field between reservoir 108 and reservoir 120 using a power supply. Similarly, device 100 can include electrodes in electrical contact with reservoir 108 and / or reservoir 120. An electrical ground of the power supply can be connected to an electrical ground of the mass spectrometer to provide continuity of the electric field from channel 124 to the mass spectrometer.

[0050] Any CE electrophoretic method, such as IEF, ITP, CGE, CZE, etc., can be performed in channel 124. Alternatively, a non-electrophoretic enrichment method can be performed in channel 124.

[0051] In the case of IEF or ITP, the concentrated purified sample band would be moved, for example, by pressure or electrical means to junction 126. Sheath solutions from reservoirs 108 and 134 can function as sheath and catholyte solutions.

[0052] The sheath / catholyte can be any basic solution compatible with electrophoretic separation and mass spectrometry (e.g., MeOH / N4OH / H2O). The anolyte can be any acidic solution (e.g., 10 mM phosphoric acid).

[0053] Alternatively, the electric field can be reversed and catholyte (NaOH) can fill reservoir 120 , and anolyte can be used as the sheath solution in reservoirs 108 and 134 .

[0054] Junction 126 is where the enriched analyte fraction mixes with the sheath solution. As the analyte fraction moves through channel 124, the solution is forced through junction 126 and out orifice 128.

[0055] Orifice 128 can be disposed within a recess defined by surface 127 of substrate 102. For example, surface 127 can be the ESI surface of a countersunk hole. For example, as shown in FIG. 1 , an enriched analyte solution electrically grounded through well 108 can form a Taylor cone emanating from orifice 128, which is disposed entirely within the recess defined by surface 127. Orifice 128 and / or surface 127 can be directed toward the inlet of a mass spectrometer, which can have a potential difference relative to well 108. As the spray separates from the conical structure toward the mass spectrometer, it can be flanked by a spray gas supplied through channels 106 and 140 before exiting substrate 102. The spray gas can be any inert or non-reactive gas (e.g., argon, nitrogen, etc.).

[0056] Additionally, the use of sheath liquid and / or nebulizing gas can enable the use of an ion depletion step as a final "on-device" step: the sheath liquid allows for the ionic potential lost during the IEF charged assay concentration step prior to ESI, and the nebulization provides the sample in a fine mist for offline analysis.

[0057] By creating the Taylor cone on surface 127, the cone is formed in a stable pocket or recess and is protected from disturbing airflow. Additionally, the conical shape surrounding the countersink has a naturally expanding contact surface to accommodate a wide range of Taylor cone radial cross sections, allowing a wider range of flow rates into the mass spectrometer.

[0058] Orifice 128 can be positioned proximate to an inlet port of a mass spectrometer. In some cases, surface 127 can be configured such that an inlet port of a mass spectrometer can be positioned within a recess defined by surface 127.

[0059] Figure 2 is a schematic exploded view of a device 212 having three layers, according to one embodiment. Figure 2A shows the top layer 202 of device 212, according to one embodiment. Figure 2B shows the middle layer 206 of device 212, according to one embodiment. Figure 2C shows the bottom layer 210 of device 212, according to one embodiment. Figure 2D shows the assembled device 212, according to one embodiment. Each of the three layers 202, 206, 210 may be made of any material compatible with the assay that device 212 is intended to perform.

[0060] In some embodiments, layer 202 is made of a material that is transparent to a particular wavelength or range of wavelengths of light. As used herein, "transparent" should be understood to mean having sufficient transmittance so that the amount of light having a particular wavelength or range of wavelengths on one side of the material can be quantified by a detector on the other side. In some cases, a material with a transmittance of 30%, 50%, 80%, 95%, or 100% is transparent. In some embodiments, the wavelength range of interest includes the mid-ultraviolet range (e.g., 200 nm to 300 nm), and materials such as glass, quartz, fused silica, and polycarbonate, polyfluorinated polyethylene, polymethacrylate, cyclic olefin polymers, cyclic olefin copolymers, and other ultraviolet-transparent materials can be used as transparent materials. In some embodiments, the light spectrum of interest extends beyond the visible spectrum (e.g., 200 to 900 nm).

[0061] Through-holes 204 are fabricated in layer 202 to allow pressure and electrical interface from the exterior of the device to the channel network in the underlying layer (eg, layer 208).

[0062] 2B shows the interior middle layer 206 of device 212 containing a channel network 208. The channel network is designed to interface with through-holes fabricated in the top layer 202. The channel network 208 includes inlet and outlet tubes / conduits 209, orifices 205 for ejecting enriched analyte fractions, and a visible enrichment zone 207. The enrichment zone 207 is fabricated such that its depth is the entire thickness of the layer 206. In other embodiments, the zone 207 may be less than the entire thickness of the layer 206.

[0063] In some embodiments, layer 206 is made from a material that is opaque and / or not transparent to a particular wavelength or range of wavelengths of light. As used herein, "opaque" should be understood to mean a material that has insufficient transmittance to allow the amount of light on one side to be quantified by a detector on the other side, effectively blocking this light except in zones within the channel network as deep as the entire thickness of layer 206.

[0064] 2C shows the bottom layer 210 of device 212. Bottom layer 210 can be, for example, a solid substrate. In some embodiments, bottom layer 210 can be made of a material that has the same transmittance as layer 202.

[0065] 2D shows device 212 including assembled top layer 202, middle layer 206, and bottom layer 210, according to one embodiment. Inlet and outlet tubes 209, reservoir 204, and orifice 205 remain accessible after device 210 is assembled. In some embodiments, the entire top layer 202 and / or the entire bottom layer 210 can be transparent. In other embodiments, a portion of top layer 202 and / or a portion of bottom layer 210 can be opaque, while another portion of top layer 202 and / or bottom layer 210 can be transparent. For example, top layer 210 and / or bottom layer 210 can define an optical window that aligns with at least a portion of enrichment zone 207 when device 212 is assembled.

[0066] FIG. 3 is a schematic diagram of an optical path through a microfluidic device 302, according to one embodiment. FIG. 3A shows a top view of the microfluidic device 302. FIG. 3B shows the microfluidic device 302 positioned between a light source 306 and a detector 308. The detector 308 is positioned to measure light passing through the device 302. Although not shown in FIG. 3, the microfluidic device 302 can have a channel structure similar to that described in FIGS. 1 and 2, although the channel structure is not shown for ease of reference. In some embodiments, portions of the top surface of the microfluidic device 302 are opaque, completely or substantially obscuring light projected from the light source 306 and preventing it from reaching the detector 308. The opaque portions of the top surface substantially prevent transmission of light through the device in portions where detection of sample properties is not desired. In some embodiments, the microfluidic device 302 is not opaque (e.g., allows some light to pass) across one or more channel regions 304, for example, because the channels 304 traverse the entire thickness of a non-transparent layer.

[0067] In some embodiments, the transparent channel region(s) 304 may be an enrichment zone, where optical detection can be used to detect analytes, monitor the progress of enrichment, and / or monitor the enriched analyte fraction(s) exiting the device. In some embodiments, changes in the amount of light passing through the transparent channel 304 are used to measure absorbance while the analyte fraction(s) are in the channel. Thus, in some embodiments, the channel region(s) 304 define an optical slit, such that a light source 306 located on one side of the microfluidic device 302 effectively illuminates the detector 308 only through the transparent channel region(s) 304. In this manner, stray light (e.g., light that does not completely pass through the transparent channel region(s) and / or sample) can be effectively blocked from the detector 308, thereby reducing noise and improving the detector 308's ability to observe the sample within the transparent channel region(s) 304. In some embodiments, a transparent channel region(s) 304 is between two enrichment zones and can be used to detect analyte fractions as they elute from the upstream enrichment zone.

[0068] (method) FIG. 6 illustrates a method of analyte mixture enrichment, according to one embodiment. The method includes, at 20, loading and / or introducing an analyte mixture into a microfluidic device. The microfluidic device can be similar to the microfluidic devices described above with reference to FIGS. 1-3. In some embodiments, the analyte mixture can be, for example, glycans, carbohydrates, DNA, RNA, intact proteins, digested proteins, peptides, metabolites, vaccines, viruses, and small molecules. In some embodiments, the analyte mixture can be a mixture of proteins such as a lysate of cultured cells, a therapeutic agent derived from somatic cells, or cells derived from a tumor or other tissue, recombinant proteins including biopharmaceuticals, blood-derived cells, perfusion, or a protein mixture from any other source. The analyte mixture can be loaded directly into the device or can be loaded into an autosampler for sequential analysis of multiple mixtures.

[0069] The microfluidic device can include a first separation channel and / or enrichment zone. In some embodiments, the first separation channel and / or enrichment zone can be configured for chromatographic separation. For example, the first separation channel and / or enrichment zone can contain a medium configured to bind analytes from the analyte mixture and / or otherwise perform chromatographic separation. At 21, a first enrichment can be performed, e.g., a chromatographic separation can be performed in the first separation channel and / or enrichment zone. In some embodiments, such as embodiments in which the analyte mixture is a protein mixture, the first enrichment at 21 can simplify the protein mixture. The first enrichment at 21 can be based on any distinguishable property of the analytes.

[0070] The enriched analyte fraction is then eluted at 22. For example, an elution fluid can be injected into the microfluidic device to displace the enriched analyte fraction from a medium disposed within the first separation channel and / or enrichment zone. In some embodiments, the enrichment and / or mobilization of the enriched analyte fraction can be imaged. For example, as described above, the first separation channel and / or enrichment zone can define an optical slit. Light can be projected onto the microfluidic device, and a detector can detect light passing through the first separation channel and / or enrichment zone. The sample, or a portion thereof, can be detected via absorbance and / or fluorescence imaging techniques.

[0071] The microfluidic device can include a second separation channel and / or enrichment zone. In some embodiments, the second separation channel and / or enrichment zone can be configured for electrophoretic separation. At 23, for example, a second enrichment can be performed on the eluate. For example, an electric field and / or potential can be applied across the second separation channel and / or enrichment zone.

[0072] In some embodiments, the second enrichment can begin when a fraction of the analyte mixture is disposed at the intersection of the first separation channel and / or enrichment zone and the second separation channel and / or enrichment zone at 23. For example, the first separation channel and / or enrichment zone can be monitored (e.g., imaged), and a potential and / or field can be applied when the fraction of interest reaches the intersection.

[0073] In some embodiments, the second enrichment at 23 can provide fractions enriched based on charge characteristics (charge isoforms). Such enrichment can include, for example, gel isoelectric focusing, isoelectric focusing with mobilization, isoelectric focusing with whole column imaging, ion exchange chromatography, pH gradient exchange chromatography, isotachophoresis, capillary zone electrophoresis, capillary gel electrophoresis, or other, e.g., charge-based, enrichment techniques.

[0074] Although the first enrichment at 21 is described as a chromatographic enrichment and the second enrichment at 23 is described as electrophoretic, it should be understood that any suitable enrichment can be performed in any suitable order. For example, the first enrichment at 21 and the second enrichment at 23 can both be chromatographic or both electrophoretic. As another example, the first enrichment at 21 can be electrophoretic and the second enrichment at 23 can be chromatographic.

[0075] In some embodiments, one or more enrichments can provide enriched fractions based on hydrophobicity changes, such as oxidation. Such enrichments can include, for example, reverse phase chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or other enrichment techniques based, for example, on hydrophobicity.

[0076] In some embodiments, one or more enrichments can provide fractions enriched based on post-translational modifications, such as galactosylation, fucosylation, sialylation, mannose derivatives and other glycosylation, as well as glycoforms including glycation, oxidation, reduction, phosphorylation, sulfonation, disulfide bond formation, deamidation, acylation, pegylation, cleavage, antibody-drug conjugates (ADCs), protein-drug conjugates, C-terminal lysine processing, other post-translational modifications of natural and non-natural origin, and other chemical and structural modifications introduced after modification of the protein, etc. Such enrichments can include, for example, binding assays, etc.

[0077] In some embodiments, one or more enrichments can provide enriched fractions based on hydrophobicity changes, such as oxidation. Such enrichments can include, for example, reverse phase chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or other hydrophobicity-based enrichment techniques.

[0078] In some embodiments, one or more enrichments can provide fractions enriched based on primary amino acid sequence, such as caused by mutations, amino acid substitutions during production, etc. Such enrichments can include, for example, separation by charge isoforms, hydrophobicity changes, or other enrichment techniques capable of distinguishing differences in primary amino acid sequence.

[0079] In some embodiments, one or more enrichments can provide an enriched fraction based on potency, such as, for example, a bioassay, an enzyme inhibition assay, an enzyme activation assay, a competitive assay, a fluorescence polarization assay, a scintillation proximity assay, or other potency-based enrichment techniques.

[0080] In some embodiments, one or more enrichments can provide an affinity-based enriched fraction, such as, for example, solution-phase target binding, bead-based target binding, surface-bound target binding, immunoprecipitation, Protein A binding, Protein G binding, etc.

[0081] In some embodiments, one or more enrichments can provide fractions enriched based on mass or size, such as polyacrylamide gel electrophoresis, capillary gel electrophoresis, size exclusion chromatography, gel permeation chromatography, or other mass-based enrichment techniques.

[0082] In some embodiments, the analyte mixture undergoes three or more rounds of enrichment before being discharged from the device.

[0083] The enriched analyte fraction can be discharged from the device at 24. In some embodiments, the enriched analyte fraction can be discharged via IEF. Discharging the enriched analyte fraction at 24 can concentrate the analyte fraction before discharge.

[0084] In some embodiments, the analyte fraction is ejected at 24 using an ionization technique such as electrospray ionization, atmospheric pressure chemical ionization, or the like.

[0085] In some embodiments, the analyte fraction is expelled at 24 using electrokinetic or hydrodynamic forces.

[0086] In some embodiments, the enriched protein fraction is output from the device at 24 in a form coupled to a mass spectrometer.

[0087] The mass of an analyte (e.g., biological or biosimilar) ejected from a microfluidic device can be measured by, for example, time-of-flight mass spectrometry, quadrupole mass spectrometry, ion trap or orbitrap mass spectrometry, distance-of-flight mass spectrometry, Fourier transform ion cyclotron resonance, resonance mass measurement, nanomechanical mass spectrometry.

[0088] In some embodiments, pI markers are used to map pI ranges within the visualized IEF channels (e.g., the first separation channel and / or enrichment zone and / or the second separation channel and / or enrichment zone). In some embodiments, pI markers or ampholytes can be used to determine the pI of an analyte by the presence of the pI marker or ampholyte in downstream mass spectrometry data.

[0089] In some embodiments, IEF can be monitored during mobilization and ESI. In this way, mass spectrometry data can be correlated with IEF peaks, and peak resolution can be maintained and / or improved.

[0090] In some embodiments, the analyte mixture and / or portions thereof can be moved within the microfluidic device using a pressure source. In some embodiments, the mobilization is performed by hydrostatic pressure. In some embodiments, the mobilization is chemical immobilization. In some embodiments, the mobilization is electrokinetic mobilization.

[0091] 7 is a schematic diagram of a microfluidic device, according to one embodiment. A microfluidic network 800 is disposed within and / or defined by a substrate 802. The substrate is fabricated from a material that is compatible with the enrichment step to be performed. For example, with respect to material selection, chemical compatibility, pH stability, temperature, transparency at various wavelengths of light, mechanical strength, etc. may be important when selecting a material.

[0092] The substrate 802 can be made from glass, quartz, fused silica, plastic, polycarbonate, PFTE, PDMS, silicon, polyfluorinated polyethylene, polymethacrylate, cyclic olefin copolymer, cyclic olefin polymer, polyetheretherketone, and / or any other suitable material. A mixture of materials can be utilized when different properties are desired in different layers of the planar substrate.

[0093] Channels 806, 808, 810, 811, 817, 814, 812 form a channel network and are fabricated in (eg, defined by) substrate 802.

[0094] The channels can be fabricated in the substrate by any channel fabrication method, such as photolithographic etching, molding, machining, additive (3D) printing, etc.

[0095] The sample mixture and external reagents can be loaded through tube 804 and excess reagents / waste can be removed through tubes 810 and 818 .

[0096] Tubing 804, 810, and / or 818 may be made from any material compatible with the assay being performed, including fused silica, fused silica capillary tubing, silicone tubing, PTFE tubing, and the like.

[0097] Channels 806 and 814 can be designated as separation / enrichment zones. Either channel 806 and / or 814 can be used to perform chromatographic separations (reverse-phase, immunoprecipitation, ion exchange, size exclusion, ligand affinity, chromaticity, hydrophobic interaction, affinity, capillary electrokinetic chromatography, micellar electrokinetic chromatography, and / or the like) or electrophoretic separations (isoelectric focusing, capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow counterbalanced capillary electrophoresis, field gradient focusing, dynamic field gradient focusing). For example, channel 806 can be derivatized or filled with a material to perform a first enrichment step, represented by the dark circle in channel 806.

[0098] The material disposed within channel 806 can be selected to collect analytes based on hydrophobicity (reverse phase), affinity (availability), size (size exclusion chromatography), charge (ion exchange), immunoaffinity (immunoprecipitation), protein-protein interactions, DNA-protein interactions, aptamer-base capture, small molecule-base capture, or other forms of liquid chromatography, etc.

[0099] Many different methods can be used to place enrichment material within channels 806 and / or 814. The walls can be directly derivatized with covalently bound or adsorbed molecules, or beads, glass particles, sol-gels, etc. can be derivatized and packed into these channels, or the channels can be filled with a sieving material such as linear polyacrylamide (LPA), polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), linear polymer solutions such as dextran, cross-linked polymer solutions such as polyacrylamide, liquid chromatography matrices, or other materials.

[0100] Depending on the particular assay being performed, a chemically reactive solution can be added. In some cases, derivatization of the material can occur after the material is loaded into channel 806 (or channel 814) by adding molecules that will adsorb or covalently bond to the loaded material or that can chemically crosslink to the material. For example, a material coated with an antibody-binding molecule, such as Protein A, Protein G, or epoxy, can be placed into channel 806. Subsequent rinsing with an antibody solution leaves the antibody-coated material available to participate in immunoaffinity capture. In some cases, the antibody can be mixed with a target analyte or lysate, allowing the antibody to bind to its target in free solution before being coated onto the material.

[0101] After the enrichment material is loaded into the device, the sample is loaded into channel 806 via tubing 804. Washing solutions and elution reagents can then be introduced into channel 806 through tubing 804.

[0102] In some cases, a detection reagent is added to bind to the captured material. Detection moieties, such as fluorophores, chromophores, or other detection molecules, can be covalently attached to the target protein at the end of the polypeptide, and numerous labeling reagents are available via attachment to amino acid side chains, such as lysine, cysteine, and other amino acid moieties. Covalently attached detection moieties allow the protein to be detected through fluorescence excitation, chromophoric assays, or other indirect means. In some cases, the target protein is left unlabeled and can be detected through natural absorbance at 220 nm, 280 nm, or any other wavelength at which the protein absorbs light, or natural fluorescence. In some cases, proteins are detected using non-covalently attached fluorogenic, chromogenic, fluorescent, or chromophoric labels, such as SYPRO® ruby, Coomassie blue, etc.

[0103] In some cases, detection reagents are added directly to channel 814 to facilitate detection.

[0104] The elution process depends on the enrichment method performed in channel 806. The elution process is selected to elute at least a fraction of the bound analytes. In some cases, the elution process can be achieved by a combination of heat and sodium dodecyl sulfate (SDS), or other detergents, glycine, urea, or any other method that induces the release of captured analytes. Some enrichment options may not require a direct elution step (e.g., size exclusion chromatography). In some cases, elution is followed by denaturation.

[0105] The eluate then flows through channel 808 into the next separation / enrichment zone, channel 814. Channel 814 can be used to perform either a chromatographic or an electrophoretic enrichment step.

[0106] Electrophoretic separation can occur in channel 814 by applying an electric field between reservoir 812 and reservoir 816 using a power supply. As eluate from channel 806 passes through the intersection of channels 808 and 814, the electric field can be enabled, causing the analyte to load into channel 814. In some cases, the analyte is negatively charged, as in standard gel electrophoresis modes where the protein analyte is saturated with a negatively charged detergent such as SDS. However, the polarity of channel 814 can be easily reversed to accommodate systems where the protein analyte is saturated with a positively charged detergent such as cetyltrimethylammonium bromide (CTAB). In other cases, the protein analyte can be coated with a neutral detergent or be detergent-free, as in native gel electrophoresis. In this case, the polarity is selected based on the expected charge of the protein target in the selected buffer system, and the protein analyte migrates into channel 814.

[0107] Any CE electrophoretic method, such as IEF, ITP, CGE, CZE, etc., can be performed in channel 814. Alternatively, non-electrophoretic enrichment methods can be performed in the channel.

[0108] Analytes within channel 814 can be viewed by full-column imaging, partial-column imaging, and / or single-point detection.

[0109] In some cases, the enriched material in channels 806, 814, or both, can be removed and refilled with new material, allowing the device to be used for another sample to be analyzed.

[0110] In some cases, a channel design such as that of Figure 7 can be repeated multiple times on a device, allowing two or more analyte samples to be analyzed in parallel.

[0111] (example) Aspects of the embodiments can be further understood in light of the following examples, which should not be construed as limiting in any way.

[0112] Example 1 - Characterizing Protein Charge on a Chip Prior to Mass Spectrometry (MS) In this example, the channel network shown in FIG. 4 is fabricated from a plate of soda-lime glass using standard photolithographic etching techniques, which has very low transmittance for 280 nm light. The depth of the enrichment channels 418 is the same as the thickness of the glass layer 402; that is, the enrichment channels 418 run entirely through the glass plate 402 from top to bottom. The device 400 can be illuminated by a light source located on one side of the device 400 and imaged by a detector located on the opposite side of the device 400. Although the substrate 402 is opaque, the enrichment channels 418 define an optical slit, allowing the substrate 402 to block light that does not pass through the enrichment channels 418, blocking stray light and improving the resolution of the imaging process.

[0113] The glass layer 402 is sandwiched between two fused silica plates that are transparent (e.g., transparent) to 280 nm light. As in Figure 2, the top plate contains through-holes for instruments and users to interface with the channel network, while the bottom plate is solid. The three plates are bonded at 520°C for 30 minutes. Inlet and outlet tubes are fabricated from cut capillaries (100 μm ID, polymicro) and bonded to the channel network.

[0114] The device is mounted on an instrument containing a nitrogen gas supply, a heater, a positive pressure pump (e.g., Parker, T5-1IC-03-1EEP), an electrophoresis power supply (Gamm High Voltage, MC30) terminating in two platinum-iridium electrodes (e.g., Sigma-Aldrich, 357383), a UV light source (e.g., LED, qphotonics, UVTOP280), a CCD camera (e.g., ThorLabs, 340UV-GE), and an autosampler for loading samples into the device. The power supply shares a common ground with the mass spectrometer. The instrument is controlled via software (e.g., LabView).

[0115] Protein samples are placed in vials and premixed with an ampholyte pH gradient and pI marker before being loaded into the autosampler, from which they are loaded into the microfluidic device 400 via inlet 412, passed through enrichment channel 418, and exited the device through outlet 434 to waste 430.

[0116] Sheath / catholyte (50% MeOH, N4OH / H2O) is filled into the two catholyte wells 404, 436, anolyte (10 mM H3PO4) is filled into the anolyte well 426, and heated nitrogen gas sources are attached to the two gas wells 408, 440.

[0117] After all reagents have been loaded, an electric field of +600 V / cm is applied from the anolyte well 426 to the catholyte wells 404, 436 by connecting electrodes to the anolyte well 426 and the catholyte wells 404, 436, and isoelectric focusing begins. A UV light source is aligned below the enrichment channel 418, and a camera is positioned above the enrichment channel 418 to measure the light that passes through the enrichment channel 418, thereby detecting the focused proteins by absorbance. The glass plate 402 is made of soda-lime glass and acts to block any stray light from the camera, preventing light that does not pass through the enrichment channel 418 from reaching the camera, increasing the sensitivity of the measurement.

[0118] Images of the focused proteins can be captured continuously and / or periodically during IEF. Once focusing is complete, low pressure is applied through inlet 412, driving the pH gradient toward orifice 424. The electric field can be maintained at this point to maintain high-resolution IEF separation. Continuing to image the enrichment channel 418 during the ESI process can be leveraged to determine the pI of each protein as it is ejected from orifice 424.

[0119] As the enriched protein fraction moves from the enrichment channel 418 into the junction 420, it mixes with sheath fluid, which can flow from the catholyte wells 404, 436 through the sheath / catholyte channels 406, 438 to the junction 420. Combining the enriched protein fraction with sheath fluid allows the protein fraction to be placed in a solution compatible with mass spectrometry and can restore charge to the focused proteins (IEF renders proteins uncharged).

[0120] The enriched protein fraction then continues to an orifice 424, which may be defined by a countersunk surface 422 of the glass plate 402. Once trapped in the electric field between the sheath fluid well and the mass spectrometer cathode, the enriched protein fraction can generate a Taylor cone.

[0121] As the solution continues to push through the Taylor cone from the enrichment channel 418, small droplets of fluid are ejected from the Taylor cone and travel toward the mass spectrometer inlet. Nitrogen gas (e.g., at 150°C) can flow from gas wells 408, 440 through gas channels 410, 432 to form a nitrogen gas jet adjacent to the Taylor cone, which can convert droplets ejected from the Taylor cone into a fine mist before exiting the microfluidic device, thereby aiding in detection in the mass spectrometer. By adjusting the pressure from inlet 412, the size of the Taylor cone can be adapted as needed to improve detection in the mass spectrometer.

[0122] Example 2 - Reversed Phase → IEF → MS Example 2 may be similar to Example 1, but will be described with reference to FIG. 1. Channel 116 may be a first enrichment zone filled with a C18-derivatized sol-gel. After protein loading, a volume of elution solution (MeCN / HO with IEF ampholytes and standards) may be loaded into channel 116 to elute the least hydrophobic proteins captured in the sol-gel. The elution solution is directed to channel 124, which may be a second enrichment zone where IEF, UV absorbance monitoring, and finally ESI are performed, as described in Example 1. Once ESI of the first elution solution is complete, a volume of a higher concentration of MeCN is then used to elute the protein fraction with the second least hydrophobicity.

[0123] Example 3—Efficacy → IEF → MS Example 3 can be similar to Example 2, but biological drug target derivatized beads can be packed into channel 116 and used to capture proteins. The affinity of the reaction is characterized through elution with solution phase targets (competitive), salt, pH, etc.

[0124] Example 4 - Reversed Phase → Capillary Zone Electrophoresis → MS Example 4 may be similar to Example 2, but will be described with reference to Figure 5. A protein mixture may be loaded and transferred through inlet 521 to enrichment zone 510, which may contain beads derivatized with C18 for reverse-phase chromatography. During loading, fluid passes through zone 510, through viewing region 511, and out outlet 522 to waste. Viewing region 510 traverses an inner layer made of soda-lime glass, which is opaque to 280 nm UV light, with top and bottom layers made of fused silica, which is transparent to 280 nm light.

[0125] A 280 nm light source is placed below the viewing area 511 and a CCD detector is placed above the viewing area 511 .

[0126] A 20% MeCN / HO solution is loaded through inlet 521 and passes through enrichment zone 510. This solution elutes a fraction enriched for the least hydrophobic proteins in the mixture. As the enriched protein fraction moves from enrichment zone 510 to outlet 522, the absorbance of the enriched protein fraction at 280 nm is monitored in viewing area 511. When the fraction is located at the intersection of enrichment zone 510 and enrichment zone 515, the power supply is turned on, creating an electric field between the positive electrode of reservoir 514 and the ground of reservoir 504. This polarity can be easily reversed by switching the polarity of the power supply. Once the electric field is present, the enriched protein fraction moves through enrichment zone 515, where the proteins are separated by capillary zone electrophoresis. The separated proteins mix with the sheath and electrolyte solution at junction 516, forming a Taylor cone on surface 518. Atomizing nitrogen gas lines connect to the device at ports 508 and 528 and travel through channels 512 and 530 to flank the material from the electrospray as it exits the device through orifice 520.

[0127] Alternatively, hydrodynamic pressure can be used to load the enriched protein fraction into enrichment zone 515 .

[0128] Example 5—Immunoprecipitation → Capillary Gel Electrophoresis of Protein Lysates In this example, the microfluidic channel layer represented by the layout of Figure 7 is fabricated from a cyclic olefin copolymer. Similarly stated, the substrate 802 of the microfluidic device 800 defines the channel network. In many applications, for example, where fluorescence detection is used, the microfluidic device 800 can be fabricated using a single material, provided that this material is transparent to light in the wavelength range required to detect the analyte.

[0129] Protein A-coated beads are loaded into channel 806. These beads are rinsed with a solution of antibodies against the target of interest that bind to the Protein A beads. To reduce antibody shedding, which interferes with analyte detection, the antibodies are then covalently crosslinked to the beads using commercially available crosslinking reagents such as dimethylpimelimidate (DMP) or bis(sulfosuccinimidyl)suberate (BS3). After the immunoprecipitation beads are prepared and loaded into channel 806, a lysate analyte sample can be loaded through tube 804. After sufficient time for the analyte to be captured by the immobilized antibody, unbound proteins are washed and removed as waste via tube 822.

[0130] The proteins can then be eluted from the antibody beads and analyzed. Elution is achieved by loading a solution of sodium dodecyl sulfate (SDS) and heating to 50°C for 10 minutes. Once released, the eluted analyte flows through channel 808 toward the intersection of channels 808 and 814. When the analyte plug reaches the intersection of channels 808 and 814, an electric field is turned on between the negative pole of reservoir 812 and the positive pole of reservoir 816, causing the negatively charged protein to migrate through a linear dextran polymer solution in channel 814, which is filled with the fluorescent protein dye SYPRO® Ruby.

[0131] Fluorescently labeled target proteins can be visualized using whole-column imaging during CGE in channel 814. Similarly, the entire channel 814 can be imaged while the SYPRO® ruby ​​dye is excited with 280 nm light and the emitted light at 618 nm is measured by a detector.

[0132] Example 6 - Variation of the microfluidic design without a mass spectrometer interface In some cases, it is advantageous to have two designs of microfluidic layers that differ in that they have a mass spectrometer interface and one with a mass spectrometer interface. Once the analyte is characterized, confirmatory characterization can be performed without mass spectrometry data. By performing confirmatory characterization with a microfluidic design that is nearly identical, when an anomaly is recognized, it becomes simple to return the assay to the chip with the mass spectrometer interface for mass identification. This eliminates the work that would otherwise be required to demonstrate that anomalies in the confirmatory data are analyzed with mass spectrometry data.

[0133] As an example, Figure 8 shows a microfluidic design similar to microfluidic device 400 shown in Figure 4, but without orifice 424 and countersunk surface 422. Analytes are still introduced into the chip via inlet 904 and channel 906 into enrichment channel 908, but after analysis, rather than performing electrospray ionization at an orifice, they flow out through outlet channel 910. This design can be run for normal operation, and then, when mass identification is required, the same enrichment can be performed with microfluidic device 400 shown in Figure 4, ensuring identification of analyte variants seen with microfluidic device 900 of Figure 8.

[0134] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. While various embodiments have been described as having particular features and / or combinations of elements, other embodiments are possible that have any combination of features and / or elements from any of the embodiments, as appropriate. The embodiments were chosen and described to best explain the principles of the invention and its practical application, thereby enabling those skilled in the art to best utilize the invention and various embodiments, with various modifications suited to the particular uses contemplated. It is intended that the scope of the invention be defined by the appended claims and their equivalents.

[0135] While the methods and / or schematic diagrams described above show particular events and / or flow patterns occurring in a particular order, the order of the particular events and / or flow patterns can be changed. In addition, particular events can be performed simultaneously in parallel processes where possible, or can be performed sequentially. While embodiments have been shown and described in detail, it will be understood that various changes in form and detail can be made.

[0136] All patents, patent applications, publications, and references cited in this specification are expressly incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method, comprising: (a) detecting separated analyte peaks by separating a mixture of analytes in a first fluidic channel while imaging the first fluidic channel or a portion thereof in a microfluidic device; (b) moving the separated analytes in the first fluidic channel while imaging the first fluidic channel or a portion thereof; (c) ejecting the transferred analyte through an orifice in fluid communication with the first fluid channel into a mass spectrometer via electrospray ionization; (d) correlating mass spectrometer data for the separated analytes with the separated analyte peaks; A method comprising:

2. 10. The method of claim 1, wherein the imaging comprises absorbance imaging to detect and monitor the separated analyte peaks, and / or the imaging comprises fluorescence imaging to detect and monitor the separated analyte peaks.

3. 3. The method of claim 1 or claim 2, wherein the separating in (a) is carried out using isoelectric focusing or capillary electrophoresis or chromatography.

4. 4. The method of claim 3, wherein in (a) the method further comprises introducing an isoelectric point (pI) marker into the first fluid channel before performing the isoelectric focusing, and wherein the imaging further comprises detecting and monitoring the position of the pI marker.

5. The method described in claim 4, wherein the position of the pI marker is used to determine the isoelectric point (pI) for one or more separated samples.

6. 6. The method of claim 1, wherein moving the separated analytes in (b) comprises chemically moving, electrokinetically moving, and / or moving using pressure.

7. 7. The method of claim 1, wherein the mixture of analytes is separated by applying a first electric field across a first fluidic channel, and the separated analytes are moved by applying a second electric field across the first fluidic channel, and / or the separated analytes are moved using pressure.

8. The method of claim 7 , further comprising applying the first electric field to the first fluid channel using a power source.

9. The method of any one of claims 1 to 8, wherein the mixture of analytes comprises proteins.

10. The method of claim 9, wherein the mixture of samples comprises intact proteins, biopharmaceuticals, and / or ampholytes.

11. 11. The method of claim 9 or claim 10, wherein the separating in (a) comprises separating a mixture of charge variants of one or more biopharmaceuticals and ampholytes via isoelectric focusing while imaging the first fluidic channel or a portion thereof.

12. The microfluidic device comprises: at least one inlet configured to introduce a mixture of analytes onto the first fluid channel; a second fluid channel in fluid communication with the first fluid channel; at least one gas delivery channel; a transparent portion configured to allow imaging of the mixture of analytes; and The method of any one of claims 1 to 11, further comprising:

13. (i) the transparent portion is configured to allow imaging of the focused analyte mixture and / or movement of the analyte mixture; (ii) the second fluid channel is configured to direct electrolytes into the separated analyte mixture; and / or 13. The method of claim 12, wherein (iii) the at least one gas delivery channel is configured to facilitate the electrospray ionization.

14. 14. The method of claim 12 or 13, wherein the second fluid channel is in fluid communication with a junction region downstream of the first fluid channel, and / or the first fluid channel and the second fluid channel intersect at a junction region.

15. 15. The method of claim 14, wherein the junction region is within the electric field of the first fluid channel and / or the junction region is along the first fluid channel and the orifice.

Images