Software for a Microfluidic System Interfacing with Mass Spectrometry

Abstract

To provide software for microfluidic systems interfacing with mass spectrometry.SOLUTION: Methods, devices, systems, and software for improving the quality of electrospray ionization mass spectrometry (ESI-MS) data are described, as are methods, devices, systems, and software for achieving improved correlation between chemical separation data and mass spectrometry data. The disclosed methods, devices, systems, and software have potential application in a variety of fields including, but not limited to, proteomics research, drug discovery and development, and clinical diagnostics.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 62 / 678,265, filed May 31, 2018, and U.S. Provisional Application No. 62 / 684,090, filed Jun. 12, 2018, both of which are hereby incorporated by reference in their entirety.

Background Art

[0002] The present disclosure relates to the field of chemical analysis, and more particularly to the separation of analytes in a mixture and their subsequent analysis by mass spectrometry (MS). Separating analyte components from more complex analyte mixtures based on the unique qualities of the analytes and providing a set of fractions that are concentrated with respect to their quality state is an important part of analytical chemistry. Simplifying complex mixtures in this way reduces the complexity of downstream analysis. However, problems can arise when attempting to interface devices and / or techniques with known concentration methods and / or analytical instrumentation.

[0003] For example, various methods have been used to interface protein sample preparation techniques with downstream detection systems such as mass spectrometers. A common method is to prepare a sample using liquid chromatography and collect fractions for mass spectrometry (LC-MS). This has the drawback that the protein sample needs to be digested into peptide fragments, which results in a large number of sample fractions that need to be analyzed and complex data reconstruction after execution. Certain forms of liquid chromatography, such as peptide map reverse-phase chromatography, can be coupled to a mass spectrometer, but these known techniques are limited to using peptide fragments rather than intact proteins, and thus their usefulness is limited.

[0004] Another method of introducing a sample into a mass spectrometer is electrospray ionization (ESI). In ESI, small droplets of the sample and solution are ejected from the tip of a capillary or microfluidic device with an electrospray function, such as an emitter tip or orifice, by the application of an electric field between the capillary tip or emitter tip and the source plate of the mass spectrometer. The droplets elongate and expand within this induced electric field, evaporate to form a conical emission (i.e., a "Taylor cone") containing increasingly smaller droplets that generate gas-phase ions for further separation and detection and are introduced into the mass spectrometer. Generally, the emitter tip is formed from a capillary that provides a convenient droplet volume for ESI. However, the capillary is limited to a straight flow path that does not permit multi-step sample processing. Also, ESI depends on the voltage in the ESI tip remaining constant throughout the analysis, which can be a problem in many analyses because the internal fluid resistance can change over time, thereby changing the voltage drop across various parts of the electrical circuit and, as a result, the voltage of the ESI tip.

[0005] Other research has been conducted regarding microfluidic devices. Microfluidic devices can be fabricated by various known techniques and provide fluid channels of a given dimension that can form a channel network designed to perform different fluid operations. These devices offer an additional level of control and complexity over capillaries and are thus a better choice for sample preparation. However, like capillaries, these tools often limit the characterization of separated analyte fractions, if any, prior to introduction into the mass spectrometer. Also, systems involving capillary devices or microfluidic devices generally do not provide tools for calibrating the system to re-establish the Taylor cone during operation.

[0006] Methods, devices, systems, and software for improving the quality of electrospray ionization mass spectrometry (ESI-MS) data are described, as well as methods, devices, systems, and software for realizing a more quantitative characterization of the correlation between chemical separation data and mass spectrometry data and for improving said correlation.

Summary of the Invention

Means for Solving the Problems

[0007] A method for maintaining an electrospray ionization (ESI) chip at a constant voltage with respect to ground while performing a separation reaction, comprising: a) applying a first voltage to the proximal end of a separation channel, wherein the distal end of the separation channel is in fluid communication and electrical communication with the ESI chip; b) applying a second voltage to the proximal end of an auxiliary fluid channel, wherein the distal end of the auxiliary fluid channel is in fluid communication and electrical communication with the distal end of the separation channel; c) performing a separation reaction to separate a mixture of analytes, wherein the separation reaction occurs within the separation channel; d) monitoring, in a feedback loop, a change in the resistance of the separation channel or a change in the voltage at the ESI chip, wherein the feedback loop adjusts the first and second voltages to maintain a constant voltage drop across the entire separation channel and a constant voltage at the ESI chip. In some embodiments, the separation channel is the lumen of a capillary. In some embodiments, the capillary comprises a microvial spray tip. In some embodiments, the separation channel is a fluid channel within a microfluidic device. In some embodiments, the separation reaction comprises an isoelectric focusing reaction. In some embodiments, the separation reaction is an electrophoretic separation reaction. In some embodiments, the first voltage is applied to the cathode and the second voltage is applied to the anode. In some embodiments, the voltage at the ESI chip is held at ground. In some embodiments, the voltage at the ESI chip is held at the second voltage. In some embodiments, the step of adjusting the first and second voltages comprises subtracting a transient voltage change measured at the ESI chip from the first and second voltages. In some embodiments, the voltage at the ESI chip is measured using a power supply that provides the first or second voltage. In some embodiments, the feedback loop operates at a frequency of at least 0.1 Hz. In some embodiments, the feedback loop operates at a frequency of at least 10 Hz. In some embodiments, the feedback loop maintains the voltage at the ESI chip within ±10% of a preset value.In some embodiments, the feedback loop maintains the voltage in the ESI chip within ±1% of a preset value. In some embodiments, the feedback loop maintains the voltage drop across the entire separation channel within ±10% of a preset value. In some embodiments, the feedback loop maintains the voltage drop across the entire separation channel within ±1% of a preset value.

[0008] Disclosed herein is a method comprising: a) preparing a sample comprising a mixture of two or more analytes; b) performing separation within a fluid channel containing the sample to resolve individual analyte peaks from the mixture of two or more analytes; c) calculating the velocity of an analyte peak upon mobilization of the contents of the fluid channel towards the fluid channel outlet; and d) determining the time at which the analyte peak reaches the fluid channel outlet using the velocity of the analyte peak. In some embodiments, the fluid channel is the lumen of a capillary. In some embodiments, the fluid channel is part of a microfluidic device. In some embodiments, the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, the velocity of the analyte peak is calculated from the time interval required for the analyte peak to move from a first position to a second position. In some embodiments, the first position, the second position, and the time interval are determined from a series of two or more images of the fluid channel. In some embodiments, the series of two or more images includes ultraviolet absorption images, visible light absorption images, or fluorescence images. In some embodiments, the fluid channel outlet comprises an electrospray interface with a mass spectrometer. In some embodiments, the time at which the analyte peak reaches the fluid channel outlet is used to correlate mass spectrometer data with the analyte peak. In some embodiments, the mobilization of the contents of the fluid channel includes the use of electroosmotic flow mobilization techniques, chemical mobilization techniques, hydrodynamic mobilization techniques, or any combination thereof. In some embodiments, the two or more analytes include proteins, protein-drug complexes, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, small organic compounds, or any combination thereof. In some embodiments, comparison of mass spectrometer data collected for samples of a biological drug candidate and a reference drug is used to determine biological similarity. In some embodiments, the velocity of the analyte peak is used in a feedback loop to adjust control parameters for separation or mobilization of the analyte peak.In some embodiments, the control parameter is voltage. In some embodiments, the feedback loop operates at a frequency of at least 0.1 Hz.

[0009] Disclosed herein is a method comprising: a) preparing a sample comprising a mixture of two or more analytes; b) performing separation within a fluid channel containing the sample to resolve individual analyte peaks from the mixture of two or more analytes; and c) collecting mass spectrometer data regarding two or more individual analyte peaks exiting the fluid channel via an electrospray interface with a mass spectrometer, wherein the data collection mode for the mass spectrometer alternates between a high mass scan and a low mass scan. In some embodiments, the mass spectrometer is switched between a high mass scan data collection mode and a low mass scan data collection mode at a frequency of at least 0.5 Hz. In some embodiments, the fluid channel is the lumen of a capillary. In some embodiments, the fluid channel is part of a microfluidic device. In some embodiments, the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, the high mass scan captures mass spectral data regarding biopolymers. In some embodiments, the biopolymers include proteins, protein-drug complexes, nucleic acid molecules, reduced proteins, fusion proteins, protein complexes, or any combination thereof. In some embodiments, the m / z ratio for the high mass scan ranges from 1500 to 6000. In some embodiments, the low mass scan captures mass spectral data regarding the liquid phase ampholytes used when performing isoelectric focusing separation. In some embodiments, the m / z ratio for the low mass scan ranges from 150 to 1500. In some embodiments, the mass spectra of one or more liquid phase ampholytes are used to calibrate the isoelectric point (pI) regarding the biopolymers identified by the high mass scan.

[0010] The present specification discloses a method comprising: a) performing separation within a fluid channel containing a sample, wherein the sample comprises a mixture of two or more analytes and the separation resolves individual analyte peaks from the mixture of two or more analytes; b) mobilizing the contents of the fluid channel towards a fluid channel outlet, wherein the fluid channel outlet comprises an electrospray interface with a mass spectrometer; c) (i) imaging at least a portion of the fluid channel simultaneously or alternately to monitor the position of the analyte peaks during (a) and (b), and (ii) imaging a Taylor cone flowing out between the fluid channel outlet and the inlet to the mass spectrometer simultaneously or alternately to monitor electrospray performance. In some embodiments, the positions of the analyte peaks in two or more images of at least a portion of the fluid channel are used to calculate the velocity with respect to the analyte peaks. In some embodiments, the velocity of the analyte peaks is used to determine the time at which the analyte peaks reach the fluid channel outlet. In some embodiments, the time at which the analyte peaks reach the fluid channel outlet is used to correlate mass spectrometer data with the analyte peaks. In some embodiments, data obtained from imaging the Taylor cone is used in a feedback loop to adjust electrospray performance. In some embodiments, the feedback loop operates at a frequency of at least 0.1 Hz. In some embodiments, the fluid channel is a capillary lumen. In some embodiments, the fluid channel is part of a microfluidic device. In some embodiments, the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, the imaging comprises ultraviolet light absorption imaging, visible light absorption imaging, or fluorescence imaging. In some embodiments, the mobilization of the contents of the fluid channel comprises the use of electroosmotic flow mobilization techniques, chemical mobilization techniques, hydrodynamic mobilization techniques, or any combination thereof.In some embodiments, the two or more analytes include proteins, protein-drug complexes, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, small organic compounds, or any combination thereof.

[0011] Disclosed herein is a computer-implemented method for maintaining an electrospray ionization (ESI) chip at a constant voltage relative to ground while performing a separation reaction, the method comprising: a) using a processor to receive a first measurement of the voltage in the ESI chip, wherein the tip of the separation channel is in fluid communication and electrical communication with the ESI chip; b) using the processor to receive a second measurement of the voltage in the ESI chip; c) using the processor to compare the second measurement with the first measurement, and if the second measurement is different from the first measurement, adjusting, by the processor, the voltage at the base of the separation channel and the voltage at the base of an auxiliary fluid channel having a tip in fluid communication and electrical communication with the tip of the separation channel such that the voltage in the ESI chip is returned to the first measurement; and d) repeating steps (a)-(c) at a particular frequency. In some embodiments, the separation channel comprises a capillary lumen or a fluid channel within a microfluidic device. In some embodiments, the separation reaction includes an isoelectric focusing reaction. In some embodiments, the separation reaction is an electrophoretic separation reaction. In some embodiments, the voltage in the ESI chip is held at ground. In some embodiments, the particular frequency is at least 1 Hz. In some embodiments, the voltage in the ESI chip is maintained within ±5% of a particular value.

[0012] In this specification, there are steps of: a) receiving image data including two or more images obtained using a detector configured to image all or part of a separation channel within a capillary or within a microfluidic device using a processor; b) processing the image data using the same processor or a different processor to determine the positions of analyte peaks within the separation channel in two or more images; c) calculating the velocity of the analyte peaks based on the positions of the analyte peaks in two or more images and a known time interval between acquisitions of the two or more images using the same processor or a different processor; and d) determining the time at which the analyte peaks reach the separation channel outlet using the same processor or a different processor. In some embodiments, the separation reaction carried out within the separation channel includes isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, the two or more images include ultraviolet absorption images, visible light absorption images, or fluorescence images. In some embodiments, the separation channel outlet is in fluid communication with a mass spectrometer or comprises an electrospray interface with a mass spectrometer. In some embodiments, the time at which the analyte peaks reach the fluid channel outlet is used to correlate mass spectrometer data with the analyte peaks. In some embodiments, the analyte is separated from a mixture and includes proteins, protein-drug complexes, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, or small organic compounds. In some embodiments, comparison of mass spectrometer data collected for samples of biological drug candidates and reference drugs is used to determine biological similarity. In some embodiments, the velocity of the analyte peaks is used in a feedback loop to adjust control parameters for the separation reaction carried out within the separation channel. In some embodiments, the control parameter is voltage. In some embodiments, the feedback loop operates at a frequency of at least 0.1 Hz. spray interface with a mass spectrometer. In some embodiments, the time at which the analyte peaks reach the fluid channel outlet is used to correlate mass spectrometer data with the analyte peaks. In some embodiments, the analyte is separated from a mixture and includes proteins, protein-drug complexes, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, or small organic compounds. In some embodiments, comparison of mass spectrometer data collected for samples of biological drug candidates and reference drugs is used to determine biological similarity. In some embodiments, the velocity of the analyte peaks is used in a feedback loop to adjust control parameters for the separation reaction carried out within the separation channel. In some embodiments, the control parameter is voltage. In some embodiments, the feedback loop operates at a frequency of at least 0.1 Hz.

[0013] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In case of conflict between the terms in this specification and those in the incorporated reference documents, the terms in this specification shall govern. The present invention provides, for example, the following items. (Item 1) A method for maintaining an electrospray ionization (ESI) chip at a constant voltage with respect to ground while performing a separation reaction, a) applying a first voltage to the proximal end of a separation channel, wherein the distal end of the separation channel is in fluid communication and electrical communication with the ESI chip; b) applying a second voltage to the proximal end of an auxiliary fluid channel, wherein the distal end of the auxiliary fluid channel is in fluid communication and electrical communication with the distal end of the separation channel; c) performing the separation reaction to separate a mixture of samples, wherein the separation reaction is carried out in the separation channel; d) monitoring, in a feedback loop, a change in resistance of the separation channel or a change in voltage in the ESI chip, wherein the feedback loop adjusts the first and second voltages to maintain a constant voltage drop across the entire separation channel and a constant voltage in the ESI chip. A method comprising the above. (Item 2) The method according to item 1, wherein the separation channel is the lumen of a capillary. (Item 3) The method according to item 2, wherein the capillary comprises a microvial spray tip. (Item 4) The method according to item 1, wherein the separation channel is a fluid channel in a microfluidic device. (Item 5) The method according to any one of items 1 to 4, wherein the separation reaction includes an isoelectric focusing reaction. (Item 6) The method according to any one of items 1 to 4, wherein the separation reaction includes an electrophoresis separation reaction. (Item 7) The method according to any one of items 1 to 6, wherein the first voltage is applied to the cathode and the second voltage is applied to the anode. (Item 8) The method according to any one of items 1 to 7, wherein the voltage in the ESI chip is held at ground. (Item 9) The method according to any one of items 1 to 7, wherein the voltage in the ESI chip is held at the second voltage. (Item 10) The method according to any one of items 1 to 9, wherein the step of adjusting the first and second voltages includes subtracting a transient voltage change measured in the ESI chip from the first and second voltages. (Item 11) The method according to any one of items 1 to 10, wherein the voltage in the ESI chip is measured using a power supply that provides the first voltage or the second voltage. (Item 12) The method according to any one of items 1 to 11, wherein the feedback loop operates at a frequency of at least 0.1 Hz. (Item 13) The method according to any one of items 1 to 11, wherein the feedback loop operates at a frequency of at least 10 Hz. (Item 14) The method according to any one of items 1 to 13, wherein the feedback loop maintains the voltage in the ESI chip within ±10% of a preset value. (Item 15) The method according to any one of items 1 to 13, wherein the feedback loop maintains the voltage in the ESI chip within ±1% of a preset value. (Item 16) The method according to any one of items 1 to 15, wherein the feedback loop maintains the voltage drop across the entire separation channel within ±10% of a preset value. (Item 17) The method according to any one of items 1 to 15, wherein the feedback loop maintains the voltage drop across the entire separation channel within ±1% of a preset value. (Item 18) a) preparing a sample containing a mixture of two or more analytes; b) performing separation in a fluid channel containing the sample to resolve individual analyte peaks from the mixture of two or more analytes; c) calculating the velocity of the analyte peak during mobilization of the contents of the fluid channel towards the fluid channel outlet; d) determining the time at which the analyte peak reaches the fluid channel outlet using the velocity of the analyte peak; A method comprising the steps of. (Item 19) The method according to item 18, wherein the fluid channel is the lumen of a capillary. (Item 20) The method according to item 18, wherein the fluid channel is part of a microfluidic device. (Item 21) The separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). The method according to any one of items 18 to 20. (Item 22) The method according to any one of items 18 to 21, wherein the velocity of the sample peak is calculated from the time interval required for the sample peak to move from a first position to a second position. (Item 23) The method according to item 22, wherein the first position, the second position, and the time interval are determined from a series of two or more images of the fluid channel. (Item 24) The method according to item 23, wherein the series of two or more images includes an ultraviolet absorption image, a visible light absorption image, or a fluorescence image. (Item 25) The method according to any one of items 18 to 24, wherein the fluid channel outlet comprises an electrospray interface with a mass spectrometer. (Item 26) The method according to any one of items 18 to 25, wherein the time at which the sample peak reaches the fluid channel outlet is used to correlate mass spectrometer data with the sample peak. (Item 27) The method according to any one of items 18 to 26, wherein the mobilization of the contents of the fluid channel includes the use of electroosmotic flow mobilization techniques, chemical mobilization techniques, hydrodynamic mobilization techniques, or any combination thereof. (Item 28) The method according to any one of items 18 to 27, wherein the two or more samples include proteins, protein-drug complexes, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, small organic compounds, or any combination thereof. (Item 29) The method according to any one of items 26 to 28, wherein a comparison of mass spectrometer data collected for samples of a biological drug candidate and a reference drug is used to determine biological similarity. (Item 30) The method according to any one of items 18 to 29, wherein the velocity of the sample peak is used in a feedback loop to adjust control parameters for the separation or mobilization of the sample peak. (Item 31) The method according to item 30, wherein the control parameter is voltage. (Item 32) The method according to item 30 or item 31, wherein the feedback loop operates at a frequency of at least 0.1 Hz. (Item 33) a) preparing a sample comprising a mixture of two or more samples; b) performing separation in a fluid channel containing the sample to resolve individual sample peaks from the mixture of the two or more samples; c)Collecting mass spectrometer data regarding the two or more individual analyte peaks released from the fluid channel via an electrospray interface with a mass spectrometer, wherein the data collection mode for the mass spectrometer is alternately performed between a high mass scan and a low mass scan; A method comprising. (Item 34) The method according to item 33, wherein the mass spectrometer is switched between the high mass scan data collection mode and the low mass scan data collection mode at a frequency of at least 0.5 Hz. (Item 35) The method according to item 33 or 34, wherein the fluid channel is the lumen of a capillary. (Item 36) The method according to item 33 or 34, wherein the fluid channel is part of a microfluidic device. (Item 37) The method according to any one of items 33 to 36, wherein the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). (Item 38) The method according to any one of items 33 to 37, wherein the high mass scan captures mass spectral data regarding biopolymers. (Item 39) The method according to item 39, wherein the biopolymers include proteins, protein-drug complexes, nucleic acid molecules, reduced proteins, fusion proteins, protein complexes, or any combination thereof. (Item 40) The method according to any one of items 33 to 39, wherein the m / z ratio for the high mass scan is in the range of 1500 to 6000. (Item 41) The method according to any one of items 33 to 40, wherein the low mass scan captures mass spectral data regarding liquid phase ampholytes used when performing isoelectric focusing separation. (Item 42) The method according to any one of items 33 to 42, wherein the m / z ratio for the low mass scan is in the range of 150 to 1500. (Item 43) The method according to item 41 or 42, wherein the mass spectra of one or more liquid phase ampholytes are used to calibrate the isoelectric point (pI) regarding the biopolymers identified in the high mass scan. (Item 44) a)Performing a separation within a fluid channel containing a sample, wherein the sample contains a mixture of two or more analytes, and the separation resolves individual analyte peaks from the mixture of the two or more analytes; b) A step of mobilizing the contents of the fluid channel toward the fluid channel outlet, wherein the fluid channel outlet comprises an electrospray interface with a mass spectrometer; c) (i) Imaging at least a part of the fluid channel simultaneously or alternately to monitor the position of the analyte peak during (a) and (b), and (ii) Imaging the Taylor cone flowing out between the fluid channel outlet and the inlet to the mass spectrometer simultaneously or alternately to monitor electrospray performance; A method comprising the steps of. (Item 45) The method according to item 44, wherein the position of the analyte peak in two or more images of at least a part of the fluid channel is used to calculate the velocity of the analyte peak. (Item 46) The method according to item 45, wherein the velocity of the analyte peak is used to determine the time when the analyte peak reaches the fluid channel outlet. (Item 47) The method according to item 46, wherein the time when the analyte peak reaches the fluid channel outlet is used to correlate mass spectrometer data with the analyte peak. (Item 48) The method according to any one of items 44 to 47, wherein data obtained from imaging the Taylor cone is used in a feedback loop to adjust electrospray performance. (Item 49) The method according to item 48, wherein the feedback loop operates at a frequency of at least 0.1 Hz. (Item 50) The method according to any one of items 44 to 49, wherein the fluid channel is the lumen of a capillary. (Item 51) The method according to any one of items 44 to 49, wherein the fluid channel is part of a microfluidic device. (Item 52) The separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC), according to any one of items 44 to 51. The method described. (Item 53) The method according to any one of items 44 to 52, wherein the imaging includes ultraviolet light absorption imaging, visible light absorption imaging, or fluorescence imaging. (Item 54) The mobilization of the contents of the fluid channel includes the use of electroosmotic flow mobilization technology, chemical mobilization technology, hydrodynamic mobilization technology, or any combination thereof, according to any one of items 44 to 53. The method described. (Item 55) The method according to any one of Items 44 to 54, wherein the two or more specimens include a protein, a protein-drug complex, a peptide, a nucleic acid molecule, a carbohydrate molecule, a lipid molecule, a metabolite molecule, a small organic compound, or any combination thereof. (Item 56) A computer-implemented method for maintaining an electrospray ionization (ESI) chip at a constant voltage with respect to ground while performing a separation reaction, comprising: a) using a processor to receive a first measurement of the voltage in the ESI chip, wherein a tip of the separation channel is in fluid communication and electrical communication with the ESI chip; b) using the processor to receive a second measurement of the voltage in the ESI chip; c) using the processor to compare the second measurement with the first measurement, and if the second measurement is different from the first measurement, adjusting, by the processor, the voltage at the base end of the separation channel and the voltage at the base end of an auxiliary fluid channel having a tip in fluid communication and electrical communication with the tip of the separation channel such that the voltage in the ESI chip is returned to the first measurement; d) repeating steps (a) to (c) at a specific frequency. A computer-implemented method comprising the above steps. (Item 57) The computer-implemented method according to Item 56, wherein the separation channel comprises a capillary lumen or a fluid channel in a microfluidic device. (Item 58) The computer-implemented method according to Item 56 or 57, wherein the separation reaction includes an isoelectric focusing reaction. (Item 59) The computer-implemented method according to Item 56 or 57, wherein the separation reaction includes an electrophoretic separation reaction. (Item 60) The computer-implemented method according to any one of Items 56 to 59, wherein the voltage in the ESI chip is held at ground. (Item 61) The computer-implemented method according to any one of Items 56 to 60, wherein the specific frequency is at least 1 Hz. (Item 62) The computer-implemented method according to any one of Items 56 to 61, wherein the voltage in the ESI chip is maintained within ±5% of a specific value. (Item 63) a) Receiving image data including two or more images obtained using a detector configured to image all or part of a separation channel within a capillary or within a microfluidic device using a processor; b) Processing the image data using the same processor or a different processor to determine the positions of analyte peaks within the separation channel in the two or more images; c) Calculating the velocity of the analyte peaks based on the positions of the analyte peaks in the two or more images and a known time interval between acquisitions of the two or more images using the same processor or a different processor; d) Determining the time at which the analyte peaks reach a separation channel outlet using the same processor or a different processor; A computer-implemented method comprising the steps of. (Item 64) The computer-implemented method according to item 63, wherein the separation reaction carried out within the separation channel comprises isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). (Item 65) The computer-implemented method according to item 63 or 64, wherein the two or more images include ultraviolet absorption images, visible light absorption images, or fluorescence images. (Item 66) The computer-implemented method according to any one of items 63 to 65, wherein the separation channel outlet is in fluid communication with an electrospray interface with a mass spectrometer or comprises an electrospray interface with a mass spectrometer. (Item 67) The computer-implemented method according to any one of items 63 to 66, wherein the time at which the analyte peaks reach the fluid channel outlet is used to correlate mass spectrometer data with the analyte peaks. (Item 68) The computer-implemented method according to any one of items 63 to 67, wherein the analyte is separated from a mixture and includes proteins, protein-drug complexes, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, or small organic compounds. (Item 69) The computer-implemented method according to any one of items 63 to 68, wherein comparison of mass spectrometer data collected for samples of a biological drug candidate and a reference drug is used to determine biological similarity. (Item 70) The computer-implemented method according to any one of items 63 to 69, wherein the velocity of the sample peak is used in a feedback loop to adjust control parameters for a separation reaction performed within the separation channel. (Item 71) The computer-implemented method according to item 70, wherein the control parameter is a voltage. (Item 72) The computer-implemented method according to item 70 or item 71, wherein the feedback loop operates at a frequency of at least 0.1 Hz.

[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description which sets forth illustrative embodiments in which the principles of the invention are utilized, and from the appended drawings.

Brief Description of the Drawings

[0015]

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DETAILED DESCRIPTION OF THE INVENTION

[0016] Some embodiments described herein relate to innovative software and systems for analyzing data from a separation system based on capillary and microfluidics integrated with mass spectrometry detection to direct the operation of the separation system. In some embodiments, samples are imaged during separation in a capillary or microfluidic device, and the molecular weight or mass-to-charge ratio is measured with a mass spectrometer after separation. The disclosed methods, devices, systems, and software provide more accurate characterization of separated sample peaks and achieve improved correlation between chemical separation data and mass spectrometry (MS) data. Methods, devices, systems, and software for improving the quality of electrospray ionization mass spectrometry (ESI-MS) data are also disclosed. The disclosed methods, devices, systems, and software have potential applications in various fields including, but not limited to, proteomics research, drug discovery and development, and clinical diagnostics. For example, in some embodiments, the disclosed methods, devices, systems, and software may be utilized for the characterization of biological and biosimilar pharmaceuticals during development and / or manufacturing, as discussed in more detail below. Biologics and biosimilars are drugs including, for example, recombinant proteins, antibodies, live virus vaccines, human plasma-derived proteins, cell-based drugs, naturally-derived proteins, antibody-drug conjugates, protein-drug conjugates, and other protein drugs.

[0017] A microfluidic device is described that is designed to perform any of a variety of chemical separation techniques and also includes an electrospray ionization interface for performing downstream mass spectrometry-based analysis. In a preferred embodiment, the disclosed device is designed to perform isoelectric focusing of proteins or other biopolymers. In other preferred embodiments, the disclosed device is designed to be used with imaging techniques. Devices and methods for integrating imaged microfluidic separations with mass spectrometry are already described, for example, in International Publication No. WO 2017 / 095813, which is a published PCT patent application, and U.S. Patent Application Publication No. 2017 / 0176386, and these are hereby incorporated by reference in their entirety for all purposes. These applications describe, inter alia, systems for performing imaging separations in conjunction with MS analysis. Such microfluidic systems represent an important advance in biological characterization. However, for such systems to provide maximum benefit, it is beneficial to have innovative software and systems that assist in the operation of these systems and the downstream integration of imaging data with MS data, as disclosed herein.

[0018] Accordingly, in a preferred embodiment, for example, to accurately determine the isoelectric point (pI) for one or more analytes that have been isoelectrically focused from a mixture of analytes in a separation channel and form a series of concentrated fractions containing substantially pure individual analyte components (also referred to herein as "peaks" or "bands"), the disclosed microfluidic device may be used in combination with imaging techniques. By imaging all or a portion of the separation channel, the positions of two or more pI standards (or pI markers) that have been injected with the sample to be separated can be determined, and thus a more accurate pI can be calculated by extrapolation for each peak of the separated analytes to determine the local pH. In some embodiments, imaging of the analyte mixture in the separation channel is performed while the separation is being carried out, and determination of the isoelectric point for one or more of the separated analytes may be performed while the separation is being carried out and repeatedly updated. In some embodiments, the imaging-based determination of the isoelectric point for one or more analytes that have been isoelectrically focused is performed after the separation is complete. In some embodiments, the imaging-based determination of the isoelectric point for one or more analytes that have been isoelectrically focused is performed after the separation is complete and before the separated analyte mixture is mobilized towards an electrospray tip. In some embodiments, the imaging-based method disclosed herein may be used in combination with a capillary-based ESI-MS system rather than a microfluidic device-based ESI-MS system. In some embodiments, determination of the isoelectric point for one or more analyte peaks may be performed by a computer-implemented method.

[0019] In another preferred embodiment, after mobilization of the separated sample mixture, i.e., when the peaks are moving from the separation channel towards the electrospray tip, the disclosed microfluidic device may be used in combination with imaging techniques to image the separated sample peaks. In some embodiments, the imaging mobilization step is the same step as the imaging separation step, such as when performing a separation step that includes capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, or any other separation technique that separates components of the sample mixture by differential velocity. In some embodiments, the imaging mobilization step may be analyzed to correlate the concentrated fraction in the imaging separation with a mass spectrum. Imaging of the mobilized sample peaks may be utilized, for example, to determine the velocity at one or more sample peaks based on their positions within a series of mobilization images, which velocity may then be used to determine the time point at which the (one or more) sample peaks flow out of the separation channel or are released by the electrospray tip, and thus may be used to correlate mass spectrometer data with a particular sample peak. In some cases, the velocity of the (one or more) sample peaks is calculated from the time interval required for the sample peaks to move a specific displacement value (e.g., move from a first position to a second position). In some embodiments, imaging of the mobilized sample peaks allows for direct monitoring of the (one or more) peaks as they move through the fluid channel and are released by the electrospray tip, and thus may be used to directly correlate mass spectrometer data with a particular sample peak. In some embodiments, the imaging-based methods disclosed herein may be used in conjunction with a capillary-based ESI-MS system rather than a microfluidic device-based ESI-MS system. In some embodiments, determination of the velocity for one or more sample peaks, the actual or predicted separation channel outflow time of the sample peaks, and / or the electrospray release time of the sample peaks may be performed by a computer-implemented method.

[0020] In some embodiments, the mobilization of the separated analyte peaks may be initiated by a change in the electric field or flow parameters within the microfluidic device. In some embodiments, one or more electrodes connecting a power source to the microfluidic device may be connected or disconnected to initiate mobilization by a computer-implemented method. In some embodiments, the Taylor cone formed by the electrospray tip may be imaged during the mobilization step. In some embodiments, image analysis implemented by a computer may be used to identify stable electrospray operating conditions. In some embodiments, the image analysis may be performed by an operator. In some embodiments, the image analysis may be performed using automated image processing software. In some embodiments, one or more operating parameters known to affect electrospray performance are adjusted to restore a stable electrospray operating state. Examples of operating parameters that may be adjusted include, but are not limited to, electrophoresis voltage, flow rate, distance from the electrospray tip to the MS inlet, MS voltage, and the like. In some embodiments, a computer-implemented method may be used to adjust the electrospray parameters.

[0021] In some embodiments, multiple power supplies may be used to generate an electrophoretic electric field. In some embodiments, two power supplies having a positive polarity may be used. In some embodiments, one or more power supplies may have a negative polarity. In some embodiments, the voltage settings in the power supplies may be changed simultaneously to maintain the same voltage gradient within the separation channel for electrophoretic separation. In some embodiments, the voltage settings in the power supplies may be changed to maintain a constant voltage in the electrospray chip. In some embodiments, the multiple power supplies may be different channels within a single multi-channel power supply. In some embodiments, isoelectric focusing may be performed within the separation channel, and the resistance within the channel may increase over time. In some embodiments, chemical mobilization may be performed within the separation channel, and the resistance within the channel may decrease over time. In some embodiments, pressure-driven mobilization may be performed, and the resistance within the channel may change over time as new reagents are pushed into the channel. In some embodiments, the electrospray chip may be held at ground. In some embodiments, the electrospray chip may be maintained at a specific voltage relative to a mass spectrometer. In some embodiments, the electrospray chip may be maintained at a specific voltage relative to ground. In some embodiments, a computer-implemented method may adjust the voltage to maintain a constant electric field strength (or a constant voltage drop between the anode and the cathode) within the separation channel and a constant voltage of the electrospray chip. In some embodiments, the voltage of the chip may be measured using a voltmeter. In some embodiments, the voltage of the chip may be measured using an electrode positioned on or inside the chip. In some embodiments, an additional power supply may be set to 0 μA using current control and may also be used as a voltmeter for reading the chip voltage. In some embodiments, a computer-implemented method reads the value of the voltage of the chip and adjusts the voltage to maintain a constant electric field strength (or a constant voltage drop between the anode and the cathode) within the separation channel and a constant voltage at the chip.In some embodiments, the computer-implemented method calculates the voltage of the ESI chip based on the flow rate of current through the separation electric field circuit. In some embodiments, the voltage drop across the entire separation channel is adjusted such that a constant power or maximum power is applied to the separation channel, in which case the power applied to the separation channel is calculated as follows. [Equation 1] Power = Voltage across the entire separation channel × Current in the separation channel

[0022] Here, the current can be measured continuously or periodically during separation, and the current measurement can be used to adjust the voltage across the entire separation channel. This method of controlling the power in the separation channel can help manage the temperature effects within the separation channel.

[0023] In some embodiments, the separation path is a predetermined length of a straight coated or uncoated capillary, tube, or line with an inlet inserted into a vial containing acidic anolyte and a positive electrode or basic catholyte and a negative electrode. In some embodiments, the outlet of the separation path is inserted into a junction sprayer. In some embodiments, the junction sprayer includes a tee for a secondary tube, line, or capillary that can introduce another conductive replenishing solution to the capillary outlet that brings the liquid to the liquid electrical contact, and accommodates both a liquid flow for supporting electrospray and transporting the analyte emerging from the separation channel to the chip for introduction into a mass spectrometer by electrospray ionization. In some embodiments, the system may be configured with anolyte and a positive electrode at the separation path inlet, and the catholyte may be filled at the junction or tip of the separation path immediately before focusing. After focusing is complete, the mobilizing substance with competing anions may be introduced into the junction by either hydrodynamic or electroosmotic forces. In some embodiments, the separation path inlet may be immersed in a vial with catholyte and a negative electrode, and the anolyte may be filled at the junction or tip of the capillary immediately before focusing. After focusing is complete, the mobilizing substance with competing cations may be introduced into the junction by either hydrodynamic or electroosmotic forces. In some embodiments, the separation channel is a predetermined length of a straight capillary with one end inserted into an anolyte reservoir connecting the capillary to a positive electrode and the other end inserted into a catholyte reservoir connecting the capillary to a negative electrode for isoelectric focusing. In some embodiments, after focusing, the catholyte end of the capillary is removed from the catholyte and inserted into a junction sprayer (e.g., a microvial sprayer) near the mass spectrometer as shown in FIG. 14A. In some embodiments, the junction sprayer may include a predetermined amount of mobilizing agent for charging the analyte in the ESI and mobilizing the focused analyte. In some embodiments, the junction sprayer may provide an electrical connection to the complete mobilization circuit.In some embodiments, the voltages of the anolyte and the junction sprayer are adjusted such that the change (ΔV) in the voltage or electric field between the anolyte and the junction sprayer remains constant and the voltage of the ESI chip remains constant.

[0024] In some embodiments, the separation channel (e.g., capillary) comprises a microvial that may facilitate the transfer of the mobilized effluent to the ESI. The microvial may be part of the capillary or may be added to and / or fused with the separation channel. The microvial may be part of the ESI chip. In some cases, the microvial may comprise a junction sprayer or may be part of the junction sprayer. The microvial may comprise a fluid flow path (e.g., for sheath fluid) in part of the channel or in the ESI chip.

[0025] In some embodiments, one power source may be connected to a resistor to form a current sink. In some embodiments, the resistor may sink current by connecting the electrophoresis circuit to ground. In some embodiments, the resistor is a field effect transistor (FET) adjustable resistor. In some embodiments, the resistor may be a precision variable resistor, a relay resistor network, a resistor ladder, or any other resistive element that can provide a path for sinking current. In some embodiments, the current sink may be an FET, in which case the FET is controlled to provide a constant current flowing through the FET, or can be controlled to function as an open circuit or a short circuit as needed. In some embodiments, a bipolar junction transistor (BJT) can be used for the current sink function. In some embodiments, the resistor may sink current by connecting the electrophoresis circuit to a current sink power source. In some embodiments, the voltage setting of the current sink power source is adjusted as the resistance of the separation channel changes over time. In some embodiments, the voltage of the current sink power source is adjusted to maintain a constant current across the resistor. In some embodiments, a resistor or set of resistors, a resistive circuit, etc. may be used as the current sink.

[0026] In some embodiments, the mass-to-charge (m / z) ratio range to be scanned may be changed during the mobilization / ESI step. In some embodiments, a computer-implemented method may be used to switch between a high m / z range and a low m / z range. In some embodiments, the mass spectrum of a 1 m / z range may be used as an internal standard for the separation of analytes in different mass ranges. This spectrum may include data regarding free solution isoelectric focusing ampholytes that may be used as a standard for the isoelectric point (pI), or alternatively, this spectrum may include data regarding electrophoretic mobility standards that may be used as a standard in electrophoresis, such as capillary zone electrophoresis. In some cases, this spectrum may include data regarding any molecule that can be resolved by, for example, pI, charge-to-mass ratio, gel-based assessment, electrophoretic mobility, etc. in a separation step and that is in a different mass range than the analyte of interest.

[0027] The system of the present disclosure includes (i) a capillary or microfluidic device designed to perform separation based on isoelectric focusing electrophoresis that results in, for example, an electrospray interface with a mass spectrometer, (ii) a mass spectrometer, (iii) an imaging device or system, (iv) a processor or computer, (v) software for coordinating the operation of sample separation based on the capillary or microfluidic device with image acquisition, (vi) software for processing images to determine the (one or more) positions of one or more pI standards or sample peaks in the separation channel during separation, after separation is complete, or after mobilization of the pI standards and sample peaks towards the electrospray chip, (vii) software for processing images and determining velocity, elution time, and / or electrospray emission time for one or more pI standards or sample peaks, (viii) software for simultaneously or alternately acquiring images of the separation channel to monitor the positions of the Taylor cone and sample peaks present between the electrospray chip and the inlet to the mass spectrometer and to monitor electrospray performance, (ix) software for processing images of the Taylor cone and adjusting one or more of the position of the electrospray chip relative to the inlet of the mass spectrometer, the flow rate of fluid through the electrospray chip, the voltage between the electrospray chip and the mass spectrometer, or any combination thereof to affect changes in the quality of the mass spectrometry data, (x) software for controlling the collection of mass spectrometry data for individual sample peaks emitted from the electrospray interface, wherein the data collection mode in the mass spectrometer alternates between high mass scans and low mass scans, (xi) software for reading the voltage of the electrospray chip and adjusting the separation channel voltage while keeping the voltage of the chip constant to maintain a constant electric field strength in the channel (or a constant voltage drop between the anode and cathode), or one or more of any combination thereof. In some embodiments, the system may comprise an integrated system in which the selection of these functional components is packaged in a predetermined form.In some embodiments, the system may comprise a modular system in which the selection of functional components may be changed to reconfigure the system for new uses. In some embodiments, some of these functional system components, such as capillary or microfluidic devices, are replaceable or disposable components.

[0028] It should be understood that both the foregoing general summary and the following description are exemplary and explanatory only and are not restrictive of the methods and devices described herein.

[0029] Definition: Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0030] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. References to "or" in this specification are intended to encompass "and / or" unless stated otherwise. Similarly, the terms "comprise", "comprises", "comprising", "include", "includes", and "including" are not intended to be limiting.

[0031] As used herein, a number accompanied by the term "about" refers to a number that is plus or minus 10% of that number. When the term "about" is used in connection with a range, it refers to that range - 10% of the minimum value of the range and + 10% of the maximum value of the range.

[0032] Samples: As described above, the disclosed methods, devices, systems, and software enable more accurate characterization of isolated sample peaks and enable improved correlation between chemical separation data and mass spectrometry data. In some cases, these samples may be, for example, released glycans, carbohydrates, lipids or their derivatives (e.g., extracellular vesicles, liposomes, etc.), DNA, RNA, intact proteins, digested proteins, protein complexes, antibody-drug conjugates, protein-drug conjugates, peptides, metabolites, organic compounds, or other biologically relevant molecules, or any combination thereof. In some cases, these samples may be small molecule drugs. In some cases, these samples may be protein molecules in a protein mixture, e.g., lysates collected from biological protein pharmaceuticals and / or cells isolated from cultures or in vivo.

[0033] Samples: The disclosed methods, devices, systems, and software may be used for the separation and characterization of samples obtained from any of a variety of biological or non-biological samples. Examples include, but are not limited to, tissue samples, cell culture samples, whole blood samples (e.g., venous blood, arterial blood, capillary blood samples), plasma, serum, saliva, interstitial fluid, urine, sweat, tears, protein samples obtained from industrial enzymes or biological drug manufacturing processes, environmental samples (e.g., air samples, water samples, soil samples, surface swipe samples). In some embodiments, the sample may be processed using any of a variety of techniques known to those of skill in the art prior to analysis using the disclosed methods and devices for integrated chemical separation and mass spectrometry characterization. For example, in some embodiments, the sample may be processed to extract proteins or nucleic acids. The sample may be collected from any of a variety of sources or subjects, such as bacteria, viruses, plants, animals, or humans.

[0034] Sample volume: In some embodiments of the disclosed methods and devices, the miniaturization achievable through the use of microfabrication techniques enables the processing of very small sample volumes. In some embodiments, the sample volume used for analysis may range from about 0.1 μl to about 1 ml. In some embodiments, the sample volume used for analysis may be at least 0.1 μl, at least 1 μl, at least 2.5 μl, at least 5 μl, at least 7.5 μl, at least 10 μl, at least 25 μl, at least 50 μl, at least 75 μl, at least 100 μl, at least 250 μl, at least 500 μl, at least 750 μl, or at least 1 ml. In some embodiments, the sample volume used for analysis may be at most 1 ml, at most 750 μl, at most 500 μl, at most 250 μl, at most 100 μl, at most 75 μl, at most 50 μl, at most 25 μl, at most 10 μl, at most 7.5 μl, at most 5 μl, at most 2.5 μl, at most 1 μl, or at most 0.1 μl. Any combination of the lower and upper limits described in this paragraph may form a range included within the present disclosure. For example, in some embodiments, the sample volume used for analysis may range from about 5 μl to about 500 μl. As would be appreciated by one of ordinary skill in the art, the sample volume used for analysis may have any value within this range, for example, about 10 μl.

[0035] Separation techniques: The disclosed methods, devices, systems, and software may utilize any of a variety of analyte separation techniques known to those of skill in the art. For example, in some embodiments, imaging separation may be electrophoretic separation such as isoelectric focusing electrophoresis, capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, field gradient focusing, dynamic field gradient focusing, etc., that produce one or more separated analyte fractions from an analyte mixture.

[0036] Capillary isoelectric focusing (CIEF): In some embodiments, the separation technique may include isoelectric focusing (IEF), for example, capillary isoelectric focusing (CIEF). Isoelectric focusing (or "focusing electrophoresis") is a technique for separating molecules based on the differences in their isoelectric points (pI), that is, the differences in the pH at which the molecules have a net zero charge. CIEF involves adding an ampholyte (amphoteric electrolyte) solution to the sample channel between reagent reservoirs containing an anode or a cathode, and generating a pH gradient within a separation channel (i.e., a fluid channel connecting wells containing electrodes) across which a separation voltage is applied. The ampholyte may be in the liquid phase or may be immobilized on the surface of the channel wall. Negatively charged molecules move towards the positive electrode across the pH gradient in the medium, while positively charged molecules move towards the negative electrode. A protein (or other molecule) in a pH region below its isoelectric point (pI) is positively charged and thus moves towards the cathode (i.e., the negatively charged electrode). The overall net charge of the protein decreases as it moves across the increasing pH gradient until it reaches the pH region corresponding to its pI (e.g., due to the protonation of carboxyl groups or other negatively charged functional groups), and at the point when it reaches the pH region corresponding to its pI, it has no net charge and thus stops moving. As a result, a mixture of proteins separates based on the relative content of acidic and basic residues, and each protein becomes focused into a sharp stationary band positioned at the point of the pH gradient corresponding to its pI. This technique provides very high resolution using different proteins as a single charge is fractionated into separate bands. In some embodiments, isoelectric focusing may be performed in a separation channel that is permanently or dynamically coated, for example, with a neutral and hydrophilic polymer coating, to eliminate electroosmotic flow (EOF).Examples of suitable coatings include amino modifiers, hydroxypropyl cellulose (HPC) and polyvinyl alcohol (PVA), Guarant (registered trademark) (Alcor Bioseparations), linear polyacrylamide, polyacrylamide, dimethylacrylamide, polyvinylpyrrolidine (PVP), methylcellulose, hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), triethylamine, proylamine, morpholine, diethanolamine, triethanolamine, diaminopropane, ethylenediamine, chitosan, polyethyleneimine, cadaverine, putrescine, spermidine, diethylenetriamine, tetraethylenepentamine, cellulose, dextran, polyethylene oxide (PEO), cellulose acetate, amylopectin, ethylpyrrolidine methacrylate, dimethyl methacrylate, didodecyldimethylammonium bromide, Brij 35, sulfobetaine, 1,2-dilauroyl-sn-phosphatidylcholine, 1,4-didecyl-1,4-diazoniabicyclo[2,2,2]octane dibromide, agarose, poly(N-hydroxyethylacrylamide), pore-323, hyperbranched polyaminoester, pullulan, glycerol, adsorption coatings, covalent coatings, dynamic coatings, etc., but are not limited thereto. In some embodiments, isoelectric focusing is performed in a separation medium using additives such as methylcellulose, glycerol, urea, formamide, surfactants (e.g., Triton-X 100, CHAPS, digitonin) (e.g., within an uncoated separation channel) to significantly reduce electroosmotic flow, enable better protein solubilization, and limit diffusion within the capillary of the fluid channel by increasing the viscosity of the electrolyte.

[0037] As described above, the pH gradient used for capillary isoelectric focusing technology is generated by the use of ampholytes, i.e., amphoteric molecules that contain both acidic and basic groups and exist mainly as zwitterions within a specific range of pH. The portion of the electrolyte solution on the anode side of the separation channel is known as the "anolyte". That portion of the electrolyte solution on the cathode side of the separation channel is known as the "catholyte". In the disclosed methods and apparatuses, various electrolytes may be used, including but not limited to phosphoric acid, sodium hydroxide, ammonium hydroxide, glutamic acid, lysine, formic acid, dimethylamine, triethylamine, acetic acid, piperidine, diethylamine, and / or any combination thereof. The electrolyte may be used at any suitable concentration, such as 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. The concentration of the electrolyte may be at least 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. The concentration of the electrolyte may be at most 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%. A concentration of the electrolyte within a predetermined range, for example, 0.1% - 2% may be used. The ampholyte may be selected from any commercially available or non-commercially available carrier ampholyte mixture (e.g., Servalyt pH 4 - 9 (Serva, Heidelberg, Germany), Beckman pH 3 - 10 (Beckman Instruments, Fullerton, California, USA), Ampholine 3.5 - 9.5 and Pharmalyte 3 - 10 (both provided by General Electric Healthcare, Orsay, France), AESlytes (AES), FLUKA ampholytes (Thomas Scientific, Swedesboro, New Jersey), Biolyte (Bio-Rad, Hercules, California), etc.).The carrier ampholyte mixture may comprise a mixture of small molecules (about 300-1,000 Da) containing a plurality of aliphatic amino groups and carboxylic acid groups having closely spaced pI values and good buffering capacity. In the presence of an applied electric field, the carrier ampholytes are separated into a smooth linear or non-linear pH gradient that gradually increases from the anode to the cathode.

[0038] For the disclosed methods and devices for calculating the isoelectric point for separated analyte peaks, any of a variety of pI standards may be used. For example, pI markers commonly used in CIEF applications, such as protein pI markers and synthetic small molecule pI markers, may be used. In some cases, the protein pI marker may be a specific protein having a generally accepted pI value. In some cases, the pI marker may be detectable, for example, by imaging. A variety of commercially available protein pI markers or synthetic small molecule pI markers or combinations thereof, such as small molecule pI markers available from Advanced Electrophoresis Solutions, Ltd (Cambridge, Ontario, Canada), ProteinSimple, peptide libraries designed by Shimura, and Slais dyes (Alcor Biosepartions) may be used.

[0039] Mobilization techniques: In some embodiments, for example, when isoelectric focusing is used, the separated analyte bands may be mobilized towards the end of the separation channel that interfaces with a downstream analysis device, such as an electrospray ionization interface with a mass spectrometer. In some embodiments, for example, when capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, or any other separation technique that separates components of an analyte mixture by differential velocity is used, the separation step may be considered a mobilization step.

[0040] In some embodiments, mobilization of the analyte band may be performed by applying a hydrodynamic pressure to one end of the separation channel. In some embodiments, mobilization of the analyte band may be performed by orienting the separation channel in a vertical position such that gravity can be used. In some embodiments, mobilization of the analyte band may be performed using EOF-assisted mobilization. In some embodiments, mobilization of the analyte band may be performed using chemical mobilization. In some embodiments, any combination of these mobilization techniques may be used.

[0041] In one embodiment, the mobilization step for the isoelectrically focused analyte band includes chemical mobilization. Compared to pressure-based mobilization, chemical mobilization has the advantage of exhibiting minimal band broadening by overcoming the hydrodynamic parabolic flow profile caused by the use of pressure. Chemical mobilization may be performed by introducing either the inlet or the outlet of the separation path, which includes a pH gradient that is fully or partially focused, into a conductive solution with ions that compete with either hydronium or hydroxyl with respect to electrophoresis within the separation path. This results in a stepwise electrokinetic displacement of the pH gradient component by disrupting the near-zero net charge state. In the case of cathodic chemical mobilization, a predetermined amount of hydroxyl cathode solution may be replaced with a mobilization solution containing competing anions. The competing anions may cause a decrease in the pH of the separation path that exhibits a positive charge with the pH gradient component, thereby allowing the pH gradient component to move towards the cathode. Correspondingly, in anodic mobilization, a predetermined amount of hydronium anode solution is replaced with a mobilization solution containing competing cations that increase the pH in the separation where the pH gradient component exhibits a negative charge, thereby allowing the pH gradient component to move towards the anode. In some embodiments, cathodic mobilization may be initiated using an acidic electrolyte such as formic acid, acetic acid, carbonic acid, phosphoric acid, etc. at any suitable concentration. In some embodiments, anodic mobilization may be initiated using a basic electrolyte such as ammonium hydroxide, dimethylamine, diethylamine, piperidine, sodium hydroxide, etc. In some embodiments, chemical mobilization may be initiated by adding a salt such as sodium chloride or any other salt to the anode solution or cathode solution.

[0042] In a preferred embodiment, the chemical mobilization step may be initiated within a microfluidic device designed to integrate CIEF with ESI-MS by changing the electric field within the device to electrophoretically mobilize the mobilization electrolyte into the separation channel. In some embodiments, the change in the electric field may be effected by connecting or disconnecting one or more electrodes attached to one or more power supplies, where the one or more electrodes are disposed within reagent wells on the device or integrated with the fluid channels of the device. In some embodiments, the connecting or disconnecting of the one or more electrodes may be controlled using a computer-implemented method and a programmable switch, whereby the timing and duration of the mobilization step can be coordinated with the separation step, the electrospray ionization step, and / or the mass spectrometry data collection. In some embodiments, the disconnecting of one or more electrodes from the separation circuit may be effected by setting the current to 0 μA using current control.

[0043] Capillary zone electrophoresis (CZE): In some embodiments, the separation technique may include capillary zone electrophoresis, a method for separating charged analytes in solution within an applied electric field. The net velocity of the charged analyte molecules is affected by both the electroosmotic flow (EOF) mobility μ EOF shown by the separation system, and the electrophoretic mobility μ EP (which depends on the size, shape, and charge of the molecule), whereby analyte molecules exhibiting different sizes, shapes, or charges exhibit different differential migration velocities and are separated into bands.

[0044] Capillary Gel Electrophoresis (CGE): In some embodiments, the separation technique may include capillary gel electrophoresis, a method for separating and analyzing macromolecules (e.g., DNA, RNA, and proteins) and their fragments based on the size and charge of the macromolecules. This method includes the use of a separation channel filled with a gel, where the gel acts as a convection prevention medium and / or a sieving medium during the electrophoretic migration of charged analyte molecules within an applied electric field. The gel functions to suppress thermal convection caused by the application of the electric field and also acts as a sieving medium that retards the passage of molecules, thereby resulting in differential migration rates for molecules of different sizes or charges.

[0045] Capillary Isotachophoresis (CITP): In some embodiments, the separation technique may include capillary isotachophoresis, a method for the separation of charged analytes using a discontinuous system of two electrolytes (known as the leading electrolyte and the terminating electrolyte) within a capillary or fluid channel of suitable dimensions. The leading electrolyte may contain ions with the highest electrophoretic mobility, while the terminating electrolyte may contain ions with the lowest electrophoretic mobility. The analyte mixture to be separated (i.e., the sample) can be sandwiched between these two electrolytes, and upon application of an electric field, the charged analyte molecules within the capillary or fluid channel are separated into closely adjacent zones in order of decreasing electrophoretic mobility. The zones move at a constant speed within the applied electric field such that the passage of the zones along the separation channel can be recorded using a detector, e.g., a conductivity detector, an optical detector, or an imaging device. Unlike capillary zone electrophoresis, the simultaneous determination or detection of anionic and cationic analytes cannot be achieved in a single analysis using capillary isotachophoresis.

[0046] Capillary electrochromatography (CEC): In some embodiments, the separation technique may include a method for separating a sample mixture based on capillary electrochromatography, a combination of liquid chromatography and electrokinetic separation methods. CEC provides both the efficiency of capillary electrophoresis (CE) and the selectivity and sample capacity of packed capillary high performance liquid chromatography (HPLC). Since the capillary used in CEC is filled with HPLC packing material, the various sample selectivities available in HPLC are also available in CEC. Due to the high surface area of these packing materials, CEC capillaries can accept relatively large amounts of sample, such that the detection of the subsequently eluted samples is a somewhat simpler operation than the detection operations in capillary zone electrophoresis (CZE).

[0047] Micellar electrokinetic chromatography (MEKC): In some embodiments, the separation technique may include a method for separating a sample mixture based on micellar electrokinetic chromatography, differential partitioning between surfactant micelles (pseudo-stationary phase) and the surrounding aqueous buffer (mobile phase). In MEKC, the buffer may contain surfactant at a concentration above the critical micelle concentration (CMC) such that the surfactant monomers are in equilibrium with the micelles. MEKC may be performed in an open capillary or fluid channel using alkaline conditions to generate a strong electroosmotic flow. Various surfactants such as sodium dodecyl sulfate (SDS) may be used in MEKC applications. For example, due to the anionic sulfate group of SDS, the surfactant and micelles have an electrophoretic mobility opposite to the direction of the strong electroosmotic flow. As a result, the surfactant monomers and micelles move slowly, but their net movement is still in the direction of the electroosmotic flow, i.e., towards the cathode. During MEKC separation, the sample may distribute between the hydrophobic interior of the micelle and the hydrophilic buffer. Hydrophilic samples that are insoluble inside the micelle move at the electroosmotic flow rate u o and are detected at the retention time t M of the buffer. Hydrophobic samples that are completely solubilized inside the micelle move at the micelle velocity u c and elute at the final elution time t c .

[0048] Flow-balanced capillary electrophoresis (FCCE): In some embodiments, the separation technique may include methods for actively retarding, stopping, or reversing the electrokinetic movement of a sample through a capillary using flow-balanced capillary electrophoresis, pressure-induced countercurrent to enhance the efficiency and resolution of capillary electrophoresis. By delaying, stopping, or moving the sample back and forth across the detection window, the target sample can be effectively confined in the separation channel for a much longer time than under normal separation conditions, thereby improving both the efficiency and resolution of the separation.

[0049] Separation time and separation resolution: Generally, the separation time required to achieve complete separation varies depending on the specific separation technique and the operating parameters utilized (separation channel length, microfluidic device design, buffer composition, applied voltage, etc.). In some embodiments, software determines when separation is complete based on an analysis based on imaging of the sample peak, as described in co-pending U.S. Patent Application No. 16 / 261,382. In some embodiments, the separation time may range from about 0.1 minute to about 30 minutes. In some embodiments, the separation time may be at least 0.1 minute, at least 0.5 minute, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, or at least 30 minutes. In some embodiments, the separation time may be at most 30 minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, at most 10 minutes, at most 5 minutes, at most 1 minute, at most 0.5 minute, or at most 0.1 minute. Any combination of the lower and upper limits described in this paragraph may form a range included within the present disclosure. For example, in some embodiments, the separation time may range from about 1 minute to about 20 minutes. The separation time may have any value within this range, for example, about 7 minutes.

[0050] Similarly, the separation efficiency and resolution achieved using the disclosed methods and devices can vary depending on the particular separation technique and the operating parameters utilized (e.g., separation channel length, microfluidic device design, buffer composition, applied voltage, etc.). In some embodiments, the separation efficiency achieved (e.g., number of theoretical plates) may range from about 1,000 to 1,000,000. In some cases, the separation efficiency may be at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 600,000, at least 700,000, at least 800,000, at least 900,000, or at least 1,000,000. The separation resolution of the efficiency can vary depending on one or more characteristics of the analytes in the mixture (e.g., molecular weight, diffusivity, electrophoresis or isoelectric mobility, etc.).

[0051] Design and manufacture of microfluidic devices: In some embodiments of the disclosed methods, devices, and systems, separation steps (e.g., steps as outlined above) involving one or more sample preparation steps (e.g., filtration steps, preconcentration steps, or extraction steps, etc.) and / or electrospray ionization steps are designed to integrate, and separation of analytes from a mixture may be performed using a microfluidic device, and subsequent analysis of the analytes using ESI-MS may be carried out.

[0052] In some embodiments, the disclosed microfluidic device may comprise one or more sample or reagent ports (also referred to as inlet ports, sample wells, or reagent wells), one or more waste ports (also referred to as outlet ports), one or more fluid channels connecting the inlet ports and outlet ports to each other or to an intermediate fluid channel (e.g., a separation channel), or any combination thereof. In some embodiments, the disclosed microfluidic device may further comprise one or more reaction chambers or mixing chambers, one or more microfabricated valves, one or more microfabricated pumps, one or more venting structures, one or more membranes (e.g., filtration membranes), one or more microcolumn structures (e.g., fluid channels or modified fluid channels filled with a chromatographic separation medium), or any combination thereof.

[0053] In a preferred embodiment, the disclosed microfluidic device incorporates an electrospray orifice or electrospray chip to provide an electrospray ionization interface with a mass spectrometer. One non-limiting example of such an interface is described in co-pending U.S. Patent Application Publications Nos. 2017 / 0176386 and 2018 / 0003674. FIG. 1 shows one non-limiting example of a microfluidic device designed to perform isoelectric focusing electrophoresis followed by ESI-MS characterization. The fluid channel network shown in FIG. 1 is fabricated from a soda lime glass plate having a very low transmittance of 280 nm light using standard photolithography etching techniques. The depth of the separation (or concentration) channel 418 is the same as the thickness of the glass layer 402, i.e., the concentration channel 418 extends all the way from the top to the bottom of the glass plate 402. The device 400 can be illuminated by a light source disposed on one side of the device 400 and imaged by a detector disposed on the opposite side of the device 400. Although the substrate 402 is opaque, since the concentration channel 418 defines an optical slit, the substrate 402 can block light that does not pass through the concentration channel 418, thereby blocking stray light and improving the resolution of the imaging process. The glass layer 402 is sandwiched between two fused silica glass plates that transmit 280 nm light (e.g., are transparent). The upper plate includes through holes for the instrument and the user to interface with the channel network, while the lower plate is solid. The three plates are bonded to each other at 520° C. for 30 minutes. The inlet and outlet tubes are fabricated from split capillaries (100 μm ID, Polymicro) that are coupled to the channel network. The operation of this device in performing protein isoelectric focusing electrophoresis and subsequent mass spectrometry characterization is described in Example 1 below.

[0054] The flow rate of the fluid of the sample and reagent through the device may be controlled using any of various fluid actuation mechanisms known to those skilled in the art. Examples of fluid actuation mechanisms suitable for use in the disclosed methods, devices, and systems include, but are not limited to, the application of positive or negative pressure to one or more inlet ports or outlet ports, gravity or centrifugal force, electrokinetic force, electro-wetting force, or any combination thereof. In some embodiments, the positive or negative pressure may be applied directly, for example, by using a mechanical actuator or piston coupled to the inlet port and / or outlet port to cause the flow of the sample or reagent through the fluid channel. In some embodiments, the mechanical actuator or piston may exert a force on a flexible membrane or partition used to seal the inlet port and / or outlet port. In some embodiments, the positive or negative pressure may be applied indirectly, for example, by using a pressurized gas line or vacuum line connected to one or more inlet ports and / or outlet ports. In some embodiments, a pump connected to one or more inlet ports and / or outlet ports, such as a programmable syringe pump, HPLC pump, or peristaltic pump, may be used to effect the flow of the fluid. In some embodiments, the electrokinetic force and / or electro-wetting force may be applied by using an electric field and controlling the surface properties within the device. The electric field may be applied by electrodes inserted into one or more inlet ports and / or outlet ports, or by electrodes incorporated into one or more fluid channels within the device. The electrodes may be connected to one or more DC or AC power sources to control the voltage and / or current within the device.

[0055] Generally, the inlet ports, outlet ports, fluid channels, or other components of the disclosed microfluidic devices, including the device body, may be manufactured using any of a variety of materials including, but not limited to, glass, fused silica, silicon, polycarbonate, polymethyl methacrylate, cyclic olefin copolymer (COC) or cyclic olefin polymer (COP), polydimethylsiloxane (PDMS), or other elastomeric materials. Suitable manufacturing techniques generally depend on the material selection, and vice versa. Examples include, but are not limited to, CNC machining, photolithography and chemical etching, laser ablation, injection molding, hot embossing, die cutting, 3D printing, etc. In some embodiments, the microfluidic device may comprise a layered structure in which a fluidics layer comprising fluid channels is sandwiched between an upper layer and / or a lower layer to seal the channels. The upper layer and / or the lower layer may comprise openings that are aligned with the fluid channels of the fluidics layer to form inlet ports and / or outlet ports, etc. Two or more device layers may be clamped together or permanently bonded to form a device that may be disassembled. Suitable bonding techniques generally depend on the material selection used to manufacture the layers. Examples include, but are not limited to, anodic bonding, thermal bonding, laser welding, or the use of curable adhesives (e.g., thermosetting or photocurable adhesives).

[0056] In some embodiments, all or part of the inlet ports, outlet ports, or fluid channels within the microfluidic device may comprise surface coatings (e.g., HPC or PVA coatings) used to alter electroosmotic flow properties and / or surface coatings (e.g., polyethylene glycol (PEG) coatings) used to alter the hydrophobicity / hydrophilicity properties of the inlet ports, outlet ports, or fluid channel walls.

[0057] The inlet port and / or outlet port of the disclosed device can be manufactured in various shapes and sizes. Suitable geometric forms of the inlet port and / or outlet port include, but are not limited to, spherical, cylindrical, elliptical, cubic, conical, hemispherical, rectangular, or polyhedral (a three-dimensional geometric form composed of several planes, such as a cuboid, hexagonal prism, octagonal prism, inverted triangular pyramid, inverted square pyramid, inverted pentagonal pyramid, inverted hexagonal pyramid, or inverted frustum of a pyramid), or any combination thereof.

[0058] The dimensions of the inlet port and / or outlet port may be characterized in terms of average diameter and depth. As used herein, the average diameter of the inlet port or outlet port refers to the largest circle that can be inscribed within the planar cross-section of the geometric form of the inlet port and / or outlet port. In some embodiments of the present disclosure, the average diameter of the inlet port and / or outlet port may range from about 0.1 mm to about 10 mm. In some embodiments, the average diameter of the inlet port and / or outlet port may be at least 0.5 mm, at least 1 mm, at least 2 mm, at least 4 mm, at least 8 mm, or at least 10 mm. In some embodiments, the average diameter may be at most 10 mm, at most 8 mm, at most 6 mm, at most 4 mm, at most 2 mm, at most 1 mm, or at most 0.5 mm. Any combination of the lower and upper limits described in this paragraph may form a range included in the present disclosure. For example, in some embodiments, the average diameter may range from about 2 mm to about 8 mm. As would be recognized by one of ordinary skill in the art, the inlet port and / or outlet port may have any value within this range, such as about 5.5 mm.

[0059] In some embodiments, the depth of the inlet port and / or the outlet port (e.g., sample or reagent well) may range from about 5 μm to about 500 μm. In some embodiments, the depth may be at least 5 μm, at least 10 μm, at least 25 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, or at least 500 μm. In some embodiments, the depth may be at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, at most 25 μm, at most 10 μm, or at most 5 μm. Any combination of the lower and upper limits described in this paragraph may form a range included in the present disclosure. For example, in some embodiments, the depth of the inlet and / or outlet port may range from about 50 μm to about 200 μm. As would be appreciated by one of ordinary skill in the art, the depth may have any value within this range, e.g., about 130 μm. In some embodiments, the depth of the inlet port and / or the outlet port (e.g., sample or reagent well) may range from about 500 μm to about 50 mm. In some embodiments, the depth may be at least 1 mm, at least 5 mm, at least 10 mm, at least 15 mm, at least 20 mm, or at least 50 mm. In some embodiments, the depth may be at most 50 mm, at most 20 mm, at most 15 mm, at most 10 mm, at most 5 mm, or at most 1 mm. Any combination of the lower and upper limits described in this paragraph may form a range included in the present disclosure. For example, in some embodiments, the depth of the inlet port and / or the outlet port may range from about 50 μm to about 5 mm.

[0060] In some embodiments, the fluid channels of the disclosed devices may have any of a variety of cross-sectional shapes, such as square, rectangular, circular, etc. Generally, the cross-sectional shape of the fluid channels depends on the manufacturing techniques used to form them, and vice versa. In some embodiments, the cross-sectional dimensions of the fluid channels (e.g., height, width, or average diameter for a fluid channel with a non-rectangular cross-section, where the average diameter is defined as the diameter of the largest circle that can be inscribed within the cross-sectional shape of the fluid channel) may range from about 5 μm to about 500 μm. In some embodiments, the dimensions of the fluid channels may be at least 5 μm, at least 10 μm, at least 25 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, or at least 1000 μm. In some embodiments, the dimensions of the fluid channels may be at most 1000 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, at most 25 μm, at most 10 μm, or at most 5 μm. Any combination of the lower and upper limits described in this paragraph may form a range included in the present disclosure. For example, in some embodiments, the dimensions of the fluid channels may range from about 75 μm to about 300 μm. As would be recognized by one of ordinary skill in the art, the dimensions may have any value within this range, e.g., about 95 μm. In some embodiments, the depth of the fluid channel may be equal to the depth of the inlet port and / or outlet port of the device.

[0061] Imaging technology: In some embodiments of the disclosed methods and devices, imaging of the analyte separation step and / or mobilization step may be performed using optical detection techniques such as ultraviolet (UV) light absorption, visible light absorption, fluorescence, Fourier transform infrared spectroscopy, Fourier transform near-infrared spectroscopy, Raman spectroscopy, and optical spectroscopy. In some embodiments, all or part of the separation (or concentration) channel, the junction or connection channel connecting the end of the separation channel to the downstream analytical instrument or electrospray orifice or chip, the electrospray orifice or chip itself, or any combination thereof may be imaged. In some embodiments, the separation (or concentration) channel may be the lumen of a capillary. In some embodiments, the separation (or concentration) channel may be a fluid channel within a microfluidic device.

[0062] The (one or more) wavelength ranges used for the detection of separated analyte bands generally depend on the choice of imaging technique and the materials used to manufacture the device or a part thereof. For example, if UV light absorbance is used to image all or part of the separation channel or other parts of the microfluidic device, detection at about 220 nm (due to the native absorbance of peptide bonds) and / or about 280 nm (due to the native absorbance of aromatic amino acid residues) can visualize protein bands during and / or mobilization if at least a part of the device, such as the separation channel, transmits light at these wavelengths. In some embodiments, the analyte to be separated and characterized via ESI-MS may be labeled prior to separation, for example, with a fluorescent probe, a chemiluminescent tag, or other suitable label, whereby the analyte can be imaged using fluorescence imaging or other suitable imaging techniques. In some embodiments, for example, in embodiments where the analyte comprises a protein produced by a commercial manufacturing process, the protein may be genetically engineered to incorporate a green fluorescent protein (GFP) domain or a variant thereof, whereby the protein can be imaged using fluorescence. In some embodiments, the protein may be tagged or labeled. The labeled protein may be configured such that the label does not interfere with or confound the analyte properties on which the selected separation technique is based.

[0063] For the purpose of implementing the disclosed methods, devices, and systems, any of a variety of imaging system components may be utilized. Examples include, but are not limited to, one or more light sources (e.g., light emitting diodes (LEDs), diode lasers, fiber lasers, gas lasers, halogen lamps, arc lamps, etc.), condenser lenses, objective lenses, mirrors, filters, beam splitters, prisms, image sensors (e.g., CCD image sensors or cameras, CMOS image sensors or cameras, diode arrays, thermal image sensors, FTIR, etc.), or any combination thereof. Depending on the imaging mode utilized, the light source and the image sensor may be positioned on opposite sides of the microfluidic device, for example, to enable acquisition of an image based on absorbance. In some embodiments, the light source and the image sensor may be positioned on the same side of the microfluidic device, for example, to enable acquisition of an epi-fluorescence image.

[0064] Images may be acquired continuously during separation, mobilization, and / or during the electrospray step, or randomly or at specific time intervals. In some embodiments, a series of one or more images are acquired continuously, at random time intervals, or at specific time intervals. In some embodiments, a series of one or more images may include video images.

[0065] Imaging of pI markers for determining the isoelectric point of proteins before electrospray: In some embodiments, as described above, the positions of two or more pI markers in an image of a separation channel containing a separated analyte mixture that has been subjected to CIEF may be used to determine the isoelectric point of one or more individual analyte peaks (such as protein analyte peaks). In some embodiments, the isoelectric point for one or more analyte peaks is calculated from the positions of two or more pI markers based on an assumed linear relationship between the local pH and the position along the separation channel. In some embodiments, the isoelectric point for one or more analyte peaks is calculated from the positions of three or more pI markers based on a non-linear fitting function (e.g., a non-linear polynomial) that represents the relationship between the local pH and the position along the separation channel. In some embodiments, the isoelectric point for one or more analytes is calculated based on the positions of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more pI standards determined from an image of the separation channel.

[0066] In some embodiments, the image used to determine the positions of two or more pI markers is acquired while the analyte mixture is being separated, and the calculation of the pI of each analyte band is repeatedly updated as the separation continues. In some embodiments, the image used to determine the positions of two or more pI markers is acquired after the separation is complete and before the start of the mobilization step. In some embodiments, the image used to determine the positions of two or more pI markers is acquired as the separated mixture is being mobilized and discharged through an electrospray tip or orifice. In some embodiments, the image used to determine the positions of two or more pI markers is acquired as the separated mixture is being mobilized and discharged through a fluid channel that connects the separation channel to a downstream analytical instrument.

[0067] In some embodiments, the images used to determine the positions of two or more pI markers and the position(s) of the analyte band(s) in the separated mixture are obtained using a computer-implemented method (e.g., a software package). In some embodiments, the positions of two or more pI markers and the position(s) of the analyte band(s) are determined using a computer-implemented method that includes automated image processing. In some embodiments, the computer-implemented method further includes performing a calculation of the isoelectric point of one or more analyte bands based on position data obtained from automated image processing.

[0068] FIG. 2 provides an example of a process flowchart for a computer-implemented method of obtaining (one or more) images of a separation channel (or other portion of a microfluidic device), determining the positions of pI markers and analyte bands in the (one or more) images (i.e., when the separation step includes CIEF), and calculating the pI for one or more analyte bands in the separated mixture of analytes. In some embodiments, the computer-implemented method may include controlling the acquisition of a series of one or more images, which are then processed to identify the positions of the pI markers and the separated analyte bands. Examples of suitable automated image processing algorithms are described in more detail below. In some embodiments, predetermined knowledge regarding the predicted position of the pI marker, e.g., the position of the pI marker determined from an image of a “control” sample containing only the pI marker, may be used to distinguish between the band corresponding to the pI marker and the bands corresponding to the separated analytes. In some embodiments, the image of the pI marker may be acquired at a different wavelength or using a different imaging mode than that used to acquire the image of the separated analyte bands. As shown in FIG. 2, if the image processing step cannot determine the positions regarding a known number of pI markers and / or the separated analyte bands, the system may be instructed to acquire (one or more) new images so that the image processing step can be repeated. When the positions of the pI markers and the separated analyte bands have been determined, the data regarding the positions of the pI markers are fitted to a model (e.g., a linear or non-linear model) selected by the user with respect to the pH gradient, and then the isoelectric points for one or more analyte bands are calculated using the resulting fit relationship between the local pH and the position along the separation channel.

[0069] In some embodiments, the computer-implemented method may be an iterative process in which the steps of detecting the positions of the pI markers and the analyte bands, fitting the position data to a pH gradient model, and calculating the isoelectric points for one or more analyte bands are repeated such that the isoelectric points are continuously updated and refined (e.g., by averaging several determinations). In some embodiments, a cycle including the steps of acquiring and processing an image, detecting the positions of the pI markers and the analyte bands, fitting the pI marker position data to a pH gradient model, and calculating the isoelectric points for one or more analyte bands may be completed in a sufficiently short time that the calculation of the isoelectric points is updated and refined at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz, or 1,000 Hz or any other relevant rate, e.g., at a rate of at least the Nyquist rate.

[0070] Imaging of the analyte band to determine velocity: In some embodiments, as described above, the position of one or more analyte bands can be determined from a series of two or more images of the separation channel (or other part of the microfluidic device) such that the velocity for one or more analyte bands can be calculated from the difference in the relative positions of the analyte bands in two or more images and the known time interval between the acquisition times for the two or more images. In some embodiments, two or more images of at least a portion of the separation channel may be acquired while the separation step is being performed. In some embodiments, two or more images may be acquired during the mobilization step. In some embodiments, the two or more images may be acquired while the sample separated through the fluid channel connecting the end of the separation channel to the downstream analytical instrument is being discharged. In some embodiments, the two or more images may be acquired while the sample separated to form a Taylor cone is being discharged through an electrospray tip or orifice. In some embodiments, the velocity determined for one or more analyte bands may be used to calculate the time at which a given analyte band flows out of the separation channel. In some embodiments, for example, when there are one or more interconnecting fluid junctions or fluid channels connecting the end of the separation channel to an outlet port, such as an electrospray orifice or tip, the velocity determined for one or more analyte bands may be used to calculate the time at which a given analyte band reaches the outlet port and flows out of the device. In some embodiments, the velocity determined for one or more analyte bands may be used to calculate the time at which a given analyte band flows out of an electrospray tip or electrospray orifice and enters the Taylor cone formed between the electrospray tip or orifice and the inlet of a mass spectrometer.

[0071] In some embodiments, the sequence of images used to determine the velocity for one or more analyte bands may be acquired using a computer-implemented method (e.g., a software package). In some embodiments, the velocity of one or more analyte bands is determined using a computer-implemented method that includes automated image processing. In some embodiments, the computer-implemented method further includes performing a calculation of the time at which a predetermined analyte band exits the separation channel. In some embodiments, the computer-implemented method further includes performing a calculation of the time at which a predetermined analyte band reaches the outlet port and exits the device. In some embodiments, the computer-implemented method further includes performing a calculation of the time at which a predetermined analyte band exits an electrospray tip or orifice and enters a Taylor cone formed between the electrospray tip or orifice and the inlet of a mass spectrometer. In some embodiments, the (one or more) outflow times determined for one or more analyte bands are used to correlate a particular analyte band with mass spectrometry data or data collected using other analytical instruments.

[0072] FIG. 3 provides another example of a process flow chart for a computer-implemented method for obtaining an image of a separation channel (or other portion of a microfluidic device), determining the velocity of one or more analyte bands, and calculating the time it takes for a given analyte band to reach a particular point within the device, such as the end of a separation channel, the junction between the separation channel and a secondary fluid channel, the outlet port of the device, or the outlet of an electrospray tip or orifice. In some embodiments, the computer-implemented method may include controlling the acquisition of a series of one or more images, which are then processed to identify the positions of the separated analyte bands. Examples of suitable automated image processing algorithms are described in more detail below. As shown in FIG. 3, if the image processing step is unable to determine the position of the separated analyte band, the system may be instructed to acquire one or more new images so that the image processing step can be repeated. When the position of the separated analyte band has been determined for a series of two or more images, the velocity is calculated for one or more of the analyte peaks based on the relative position of the analyte band in the two or more images and the known time interval(s) between the acquisition times of the two or more images. In some embodiments, tracking of one or more analyte bands from one image to the next in a series of images may be used to distinguish between some of the separated analyte bands and refine the velocity calculation (e.g., by averaging velocity values calculated from several pairs of images in the series of images). In some embodiments, a pI marker or other internal standard that can be detected using the selected imaging mode may be used as a "velocity standard". Using the analyte band velocity thus determined, the time it takes for a given band to reach a user-specified point within the device, such as the outlet end of a separation channel, a particular fluid junction within the device, the outlet port of the device, an electrospray ionization tip or orifice where the analyte enters a Taylor cone, etc., may be calculated.

[0073] In some embodiments, the computer-implemented method may be an iterative process in which the steps of detecting the analyte band position, determining the analyte band velocity, and calculating the elution time are repeated so that the elution time prediction is continuously updated and the correlation between the chemical separation data and the mass spectrometry data (or other types of downstream analysis data) is further improved. In some embodiments, the cycle including the image acquisition and processing step, the velocity calculation step, and the (one or more) elution time prediction steps is completed in a sufficiently short time such that the (one or more) elution time predictions are updated at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz, or 1,000 Hz, or at any other relevant rate, e.g., at least the Nyquist rate.

[0074] Correlation between separation data and mass spectrometry data: In some embodiments, the computer-implemented method described above for performing a determination based on imaging of the exact isoelectric point for an analyte band undergoing isoelectric focusing electrophoresis can correlate the isoelectric point data with specific m / z peaks in the mass spectrometry data (or other analysis data), thereby improving the information content of the data set (even in the case of a single run of the experiment) and enabling a more quantitative characterization of the analyte sample.

[0075] In some embodiments, the above-described computer-implemented method (using any of a variety of different separation techniques) for calculating a velocity with respect to a separated analyte band using data obtained from an image to predict an elution time can improve the temporal correlation between chemical separation data (e.g., retention time, electrophoretic mobility, isoelectric point, etc.) and a specific m / z peak in mass spectrometry data (or other analytical data), thereby improving the information content of the data set (even in the case of a single execution of an experiment) and correcting for run-to-run or instrument-to-instrument variations in separation time, enabling a more quantitative comparison between different sample runs, data collected on different devices for different samples, and data collected on different instruments. Thus, the disclosed methods, devices, and systems can be particularly advantageous for a variety of metabolomics, proteomics, and drug development or manufacturing applications.

[0076] In some embodiments (e.g., embodiments including a CIEF step), the computer-implemented method of the present disclosure may perform both a determination based on accurate isoelectric point imaging and a determination based on imaging of the velocity of the separated analyte band.

[0077] Mass Spectrometry and Electrospray Ionization: In some embodiments, the methods, devices, and systems of the present disclosure may be configured to perform electrospray ionization of a separated analyte mixture and injection thereof into a mass spectrometer. Mass spectrometry (MS) is an analytical technique that measures the "mass" of analyte molecules by ionizing the analyte molecules in a sample and sorting the resulting ions based on their mass-to-charge (m / z) ratio. In combination with a prior liquid or gas phase sample separation system, mass spectrometry provides, as is common, for example in biological samples, one of the most effective means available for analyzing a complex sample containing multiple small amounts of analytes.

[0078] All mass spectrometers share the requirement that ions be in the gas phase before introduction into the mass analyzer. A variety of sample ionization modes have been developed, including but not limited to matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). In MALDI technology, a sample (e.g., a biological sample containing a mixture of proteins) is mixed with an energy-absorbing matrix (EAM) such as sinapinic acid or α-cyano-4-hydroxycinnamic acid and crystallized on a metal plate. Surface-enhanced laser desorption ionization (SELDI) is a common variation of the technique that incorporates additional interfacial chemistry on the metal plate to facilitate specific binding of certain classes of proteins. The plate is inserted into a vacuum chamber, and the matrix crystals are irradiated with light pulses from a nitrogen laser. The energy absorbed by the matrix molecules is transferred to the proteins, causing the proteins to desorb, ionize, and generate a plume of ions in the gas phase. These ions are accelerated in the presence of an electric field and drawn into a flight tube, where they drift until they collide with a detector that records the time of flight. Subsequently, the time of flight can be used to calculate the m / z ratio of the ionized species. In some embodiments of the disclosed device, the outlet port of the device may be provided with a capillary or other functional component used to deposit a separated analyte band (or a portion thereof) on the MALDI plate for mass spectrometry, e.g., to correlate the isoelectric point for a particular analyte band with the MALDI mass spectrometer data.

[0079] Electrospray ionization (ESI; also simply referred to herein as "electrospray") is another technique widely used due to its inherent compatibility for interfacing liquid chromatography or electrokinetic chromatography separation techniques with a mass spectrometer. As described above, in electrospray ionization, small droplets of the sample and solution are ejected from the tip of a capillary or microfluidic device equipped with an electrospray function (e.g., an emitter tip or orifice) by the application of an electric field between the tip or orifice and the source plate of the mass spectrometer. The droplets then elongate and expand within this induced electric field, evaporate, and form a conical emission (i.e., a "Taylor cone") containing increasingly smaller droplets that generate gas-phase ions for further separation and detection and are introduced into the mass spectrometer. An emitter tip may be formed from a capillary or corner or ESI tip incorporated into a microfluidic chip form that provides a convenient droplet volume for ESI. The emitter tip may be sharpened using a lapping wheel, file, machining tools, CNC machining tools, waterjet cutting, or other tools or processes for forming the ESI tip to provide a small surface area, etc., such that the emitter tip provides a small surface area and droplet volume. In some embodiments, the tip may be drawn out by heating and elongating the tip portion of the tip. In some embodiments, the tip may then be cut to a desired length or diameter. In some embodiments, the electrospray tip may be coated with a hydrophobic coating that can minimize the size of the droplets formed on the tip. In some embodiments, the system may electrospray a mobilizing agent, catholyte, or any other liquid during the separation step when the analyte has not been eluted from the device.

[0080] In some embodiments of the disclosed methods, devices, and systems, other ionization methods such as inductively coupled laser ionization, fast atom bombardment, soft laser desorption, atmospheric pressure chemical ionization, secondary ion mass spectrometry, spark ionization, thermal ionization, and the like are used.

[0081] Regarding electrospray ionization, in some embodiments, the disclosed microfluidic device is adapted to facilitate a convenient interface with efficient electrospray ionization and downstream mass spectrometry, as shown in FIG. 1. The mass-to-charge ratio (or “mass”) of the analyte discharged from the microfluidic device and introduced into a mass spectrometer (e.g., biological or biosimilar) can be measured using any of a variety of different mass spectrometer configurations. Examples include, but are not limited to, time-of-flight mass spectrometry, quadrupole mass spectrometry, ion trap or orbitrap mass spectrometry, traveling distance mass spectrometry, Fourier transform ion cyclotron resonance, resonant mass measurement, and nanomechanical mass spectrometry.

[0082] In some embodiments, the electrospray function portion of the microfluidic device may be aligned with the separation channel. In some embodiments, the electrospray function portion of the microfluidic device may be oriented at a right angle or an intermediate angle to the separation channel. In some embodiments of the disclosed method, substantially all of the separated and / or concentrated analyte fraction from the final separation or concentration step performed in a capillary or microfluidic device is discharged from the electrospray tip or electrospray function portion in a continuous flow. In some embodiments, a portion of the analyte mixture (e.g., the fraction of interest) may be discharged from the microfluidic device through an outlet configured to interface with an analytical instrument, such as another device configured to fractionate and / or concentrate at least a portion of the mass spectrometer or sample. Other portions of the analyte mixture (e.g., including fractions other than the fraction of interest) can be discharged through a waste channel.

[0083] In some embodiments, ejection from a capillary or microfluidic device is performed using pressure, electrical force, ionization, or any combination thereof. In some embodiments, ejection coincides with the mobilization step as described above. In some embodiments, the sheath fluid used for electrospray ionization is used as an electrolyte for electrophoresis separation. In some embodiments, the spray gas is supplied to reduce the sample fraction to a fine spray.

[0084] Feedback Based on Imaging of Electrospray Ionization Performance: Conventional ESI-MS systems that use capillary or microfluidic devices generally do not have tools for calibrating the system to re-establish a Taylor cone during operation. Maintaining a stable Taylor cone can be complicated by the electrophoretic electric field applied across the separation channel within the microfluidic device or capillary. Changes in the conductivity of the reagent during or between runs can change the potential at the interface with the mass spectrometer. A change in potential at the interface can affect the Taylor cone and result in a loss of electrospray ionization efficiency. Disclosed herein are methods and systems for improving electrospray ionization performance and thus the quality of mass spectrometry data collected with respect to capillary-based or microfluidic device-based ESI-MS systems. In some embodiments, for example, imaging of the Taylor cone in an electrospray ionization setup may be used in a computer-implemented method to perform feedback control of one or more operating parameters so that the shape, density, or other characteristics of the Taylor cone are maintained within a particular range. In some embodiments, operating parameters that may be controlled by such a feedback process include alignment of the electrospray tip or orifice with the inlet of the mass spectrometer, the distance between the electrospray tip and the inlet of the mass spectrometer (e.g., by mounting a capillary tip or microfluidic device with an integrated electrospray function on a programmable precision X-Y-Z translation stage), the flow rate of the analyte sample through the electrospray tip (e.g., by adjusting the pressure, electric field strength, or a combination thereof used to effect discharge of the analyte sample), the voltage applied, for example, at the proximal end of the channel, for example, between the electrospray tip or orifice and the inlet of the mass spectrometer, the volumetric flow rate of the sheath liquid or sheath gas surrounding the discharged analyte sample, or any combination thereof, but are not limited thereto.

[0085] FIG. 4 provides an example of a process flowchart for a computer-implemented method used to (i) acquire an image of a Taylor cone (using any of a variety of image sensors, e.g., either a CCD image sensor or a CMOS image sensor), (ii) process the image to determine the shape, density, or other characteristics of the Taylor cone, (iii) compare the shape, density, or other characteristics of the Taylor cone to a set of specified or target values, and (iv) based on said comparison, determine an adjustment suitable for one or more operating parameters using a mathematical algorithm that associates the shape, density, or other characteristics of the Taylor cone with the one or more operating parameters, and restore the Taylor cone to the specified or target values. In some embodiments, in addition to data obtained from an image of the Taylor cone, data acquired from a mass spectrometer (e.g., total ion current data) may be used to monitor system performance and make adjustments to one or more operating parameters.

[0086] In some embodiments, the periodic process shown in FIG. 4, which includes the steps of acquiring and processing an image, identifying Taylor cone characteristics, comparing said Taylor cone characteristics to a set of target values, and calculating the adjustments necessary to one or more operating parameters of an ESI-MS system, may be completed in a time short enough to update the one or more operating parameters at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz, or 1,000 Hz, or at any other relevant rate, e.g., at least the Nyquist rate.

[0087] High quality / low quality scanning for interaction: In some embodiments of the disclosed methods, devices, and systems, the mass spectrometer may be set to alternate between a high mass scanning range (e.g., an m / z range of about 1500 - 6000) or "high mass scan" and a low mass scanning range (e.g., an m / z range of about 150 - 1500) or "low mass scan", whereby a low mass scan can be used to identify low mass markers, e.g., free solution ampholytes when an isoelectric focusing separation step has been performed. The low mass markers can be identified in the mass spectrometry data and can be used to calibrate it with respect to the characteristics indicated by the low mass markers (e.g., a specific range of isoelectric points when free solution ampholytes are detected, peptides, small molecule markers). The switching between the high mass scan and the low mass scan and the scan rate must be fast compared to the outflow of the analyte sample from the electrospray interface. In some cases, the switching rate between the high mass scan and the low mass scan may be in the range of about 0.5 Hz to about 50 Hz. In some cases, the switching rate may be at least 0.5 Hz, at least 1 Hz, at least 5 Hz, at least 10 Hz, at least 20 Hz, at least 30 Hz, at least 40 Hz, or at least 50 Hz.

[0088] Changing high separation / mobilization electrokinetic voltage and low separation / mobilization electrokinetic voltage to keep the ESI chip voltage constant: In some embodiments, the ESI ion source of the mass spectrometer has an adjustable power supply that can set a negative voltage with respect to the mass spectrometer. In some embodiments, the ESI ion source of the mass spectrometer has an adjustable power supply that can set a positive voltage with respect to the mass spectrometer. In some embodiments, the ESI ion source of the mass spectrometer is held at ground. In some embodiments, the ESI chip in the capillary or microfluidic device is held at ground or near ground to generate an electric field between the ESI chip and the charged ESI ion source of the mass spectrometer. In some embodiments, the ESI chip in the capillary or microfluidic device is held at a positive or negative voltage to generate an electric field between the ESI chip and the grounded ESI ion source of the mass spectrometer.

[0089] Figure 15 provides a typical flowchart of a computer control feedback loop for maintaining a constant voltage drop of 3000 V between the anode and the cathode while maintaining the ESI chip voltage at 0 V during mobilization. In some embodiments, this feedback loop may be implemented when the ESI ion source of the mass spectrometer is set to a positive voltage or a negative voltage (e.g., -3500 V) with respect to ground. In this example, in Figure 7A, by first setting the anode liquid port 110 to +3000 V and the mobilizing agent port 104 to 0 V, the ΔV between the anode liquid port 110 and the mobilizing agent port 104 is maintained at 3000 V. In some embodiments, a different ΔV may be set by setting the anode liquid port 110 to a different value. In some embodiments, anode mobilization may be used, and the port 110 may be a cathode liquid port set, for example, to -3000 V. In the example outlined in Figure 15, during mobilization, the resistance in the separation channel 112 decreases due to the analyte and ampholytes in the separation that recovers charge. As a result, the voltage drop across the entire channel 112 decreases, and as a result, an increase in the voltage of the ESI chip 116 is brought about according to Equation 1.

[0090] V 116 =(ΔV 110-104 )*(R 105 ) / (R 109 +R 112 +R 105 ) However, by measuring or calculating the ESI chip voltage 116, the voltage settings of the anode liquid port 110 and the mobilizing agent port 104 can be adjusted. By subtracting the ESI chip voltage 116 from both settings of the anode liquid port 110 and the mobilizing agent port 104, the ΔV 110-104 remains at 3000 V, thereby not affecting mobilization, but the voltage of the ESI chip 116 is set to 0 according to Equation 2.

[0091] V 116 =(ΔV 110-104 )*(R 105 ) / (R 109 +R112 +R 105 )+V 104 This feedback loop continues to operate until the mobilization is complete, whereby the voltage of the ESI chip 116 is adjusted to zero at a normal frequency, for example, the Nyquist rate or about 0.2 Hz. In some cases, the voltage of the ESI chip 116 may be adjusted to zero at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz, or 1,000 Hz. Maintaining a constant and stable voltage in the ESI chip 116 can be important for maintaining a stable electrospray during the mobilization process.

[0092] In some cases, the feedback loop operates to maintain the voltage of the ESI chip within a certain percentage of a preset value. For example, in some cases, the feedback loop operates to maintain the voltage of the ESI chip within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of a preset value. In some cases, the feedback loop operates to maintain the ESI chip voltage within a range of preset values of 1000 V, 500 V, 100 V, 75 V, 50 V, 25 V, 10 V, 5 V, or 1 V.

[0093] In some embodiments, the ESI ion source of the mass spectrometer is held at ground, and the ESI chip 116 needs to be maintained at a constant positive or negative voltage to generate an electric field between the ESI chip 116 and the mass spectrometer. In some embodiments, the ESI chip voltage (e.g., a preset value) may be about +5000V, about +4000V, about +3500V, about +3000V, about +2500V, about +2000V, about +1500V, about +1000V, about +500V, or about -5000V, about -4000V, about -3500V, about -3000V, about -2500V, about -2000V, about -1500V, about -1000V, or about -500V. FIG. 12 gives an example of a flowchart of a computer-controlled feedback loop for maintaining a constant voltage drop of 3000V between the anode and the cathode while maintaining the ESI chip voltage at 3000V during mobilization. The operation of the computer-controlled feedback loop is the same as that of FIG. 15, except that the voltages of the anode liquid port 110 and the mobilizing agent port 104 are offset by +3000V, thereby offsetting the voltage of the ESI chip 116 to +3000V and still conforming to Equation 2. In some embodiments, control of the electric field strength can be achieved using an analog circuit. In some embodiments, control of the voltage at one or more electrodes in contact with a capillary-based or microfluidic device-based separation system may be performed by using one, two, three, or four or more independent high-voltage power supplies. In some cases, control of the voltage at one or more electrodes in contact with a capillary-based or microfluidic device-based separation system may be performed, for example, by using a single multiplexed high-voltage power supply.

[0094] In some cases, the feedback loop operates to maintain the electric field strength in the separation channel or the voltage drop between the anode and the cathode within a specific percentage of a preset value. For example, in some cases, the feedback loop operates to maintain the electric field strength in the separation channel or the voltage drop between the anode and the cathode within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01% of a preset value. In some cases, the feedback loop operates to maintain the electric field strength in the separation channel or the voltage drop between the anode and the cathode within a range of preset values of 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V.

[0095] System Hardware: FIG. 5 provides a schematic diagram of a system hardware block diagram in one embodiment of the disclosed method, device, and system. As shown, the system of the present disclosure may comprise one or more of the following hardware components, namely, (i) a chemical separation system (e.g., a capillary or microfluidic device designed to perform separation of a sample, e.g., separation based on isoelectric focusing electrophoresis, and one or more high voltage power supplies), (ii) an electrospray interface for a mass spectrometer which may optionally (as shown by the dashed line) be directly integrated with the separation system, (iii) a mass spectrometer, (iv) an imaging device or system, (v) a processor or computer, and (vi) a computer memory device, or any combination thereof. In some embodiments, the system may further comprise one or more capillary or microfluidic device flow controllers (e.g., programmable syringe pumps, peristaltic pumps, HPLC pumps, etc.), a temperature controller configured to maintain a specific temperature for all or part of the capillary or microfluidic device, additional photo sensors or image sensors (e.g., photodiodes, avalanche photodiodes, CMOS image sensors and cameras, CCD image sensors and cameras, etc.), a light source (e.g., light emitting diodes (LEDs), diode lasers, fiber lasers, gas lasers, halogen lamps, arc lamps, etc.), other types of sensors (e.g., temperature sensors, flow sensors, pH sensors, conductivity sensors, etc.), a computer memory device, a computer display device (e.g., having a graphical user interface), a digital communication device (e.g., an intranet, the Internet, WiFi, Bluetooth®, or other wired or wireless communication hardware), etc.

[0096] In some embodiments, the system may comprise an integrated system in which the selection of functional hardware components is packaged in a predetermined form. In some embodiments, the system may comprise a modular system in which the selection of functional hardware components may be changed to reconfigure the system for new uses. In some embodiments, some of these functional system components, such as capillary or microfluidic devices, are replaceable or disposable components.

[0097] As described above, any of a variety of different mass spectrometers may be utilized in different embodiments of the disclosed system, including, but not limited to, time-of-flight mass spectrometers, quadrupole mass spectrometers, ion traps or orbitrap mass spectrometers, flight distance spectrometers, Fourier transform ion cyclotron resonance spectrometers, resonance mass spectrometry spectrometers, and nanomechanical mass spectrometers.

[0098] System and application software: As shown in FIG. 6, the system of the present disclosure may comprise a plurality of software modules. For example, the system may comprise a system control software module, a data acquisition software module, a data processing software module, or any combination thereof. Generally, these software modules are configured to operate within or in the context of an operating system hosted by a computer processor and may communicate with each other and / or with the operating system to share data.

[0099] In some embodiments, the system control software module may include software for (i) coordinating the operation of a sample separation system based on a capillary or microfluidic device with image acquisition by an imaging system, (ii) coordinating the operation of a sample separation system based on a capillary or microfluidic device with data acquisition by a mass spectrometry system, (iii) coordinating image acquisition by an imaging system with the operation of a sample separation system based on a capillary or microfluidic device and / or a mass spectrometry system, (iv) performing feedback control of one or more operating parameters of an electrospray ionization setup and / or a mass spectrometer based on data obtained from imaging of a separation channel and / or a Taylor cone, (v) controlling data acquisition by a mass spectrometer while switching between a high-mass scan range and a low-mass scan range in an alternating manner, (vi) monitoring the voltage of an ESI chip and adjusting the voltage of a separation circuit to maintain a constant separation electric field strength (or voltage drop between an anode and a cathode) and a constant voltage at the ESI chip, or any combination thereof.

[0100] In some embodiments, the data acquisition module may include software for (i) controlling image acquisition by one or more image sensors or an imaging system, storing the image data, and providing a software interface to a system control and / or data processing software module, (ii) controlling data acquisition by one or more mass spectrometry systems, storing the mass spectrometry data (or other downstream analytical instruments), and providing a software interface to system control and / or data processing software, or any combination thereof.

[0101] In some embodiments, the data processing module processes an image to determine the (one or more) positions of one or more pI standards or analyte peaks in the separation channel while separation is being performed, after separation is complete, or after mobilization of the pI standards and analyte peaks towards the separation channel outlet or the electrospray tip, (ii) processes an image to determine velocity, elution time, and / or electrospray ejection time for one or more pI standards or analyte peaks, (iii) processes an image of the separation channel to monitor the position of the analyte peak and the image of the Taylor cone and monitor electrospray performance, where the images of the separation channel and the Taylor cone are acquired simultaneously or alternately, (iv) processes an image of the Taylor cone to determine the shape, density, or other characteristics of the Taylor cone and calculate adjustments to be made to one or more operating parameters including, but not limited to, the position of the electrospray tip or orifice relative to the inlet of the mass spectrometer (i.e., alignment and / or separation distance), the flow rate of fluid through the electrospray tip or orifice, the voltage between the electrospray tip or orifice and the mass spectrometer, etc., or any combination thereof, that affect changes in the quality of the mass spectrometry data, or may comprise software for any combination thereof.

[0102] The disclosed systems and application software may be implemented using any of a variety of programming languages and environments known to those skilled in the art. Examples include, but are not limited to, C, C++, C#, PL / I, PL / S, PL / 8, PL-6, SYMPL, Python, Java, LabView, Visual Basic,.NET, etc.

[0103] Image processing software: In some embodiments, as described above, the data processing module may include image processing software for determining the position of pI markers or separated analyte bands in order to characterize shapes, densities, or other visual metrics such as the Taylor cone function. Any of a variety of image processing algorithms known to those of skill in the art for image preprocessing or image processing may be utilized in implementing the disclosed methods and systems. By way of example, the Canny edge detection method, the Canny-Deriche edge detection method, the first-order gradient edge detection method (e.g., Sobel operator, etc.), the second-order differential edge detection method, the phase coherence edge detection method, other image segmentation algorithms (e.g., intensity thresholding, intensity clustering method, intensity histogram-based method, etc.), feature and pattern recognition algorithms (e.g., generalized Hough transform for detecting any shape, circular Hough transform, etc.), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation, Savitzky-Golay smoothing, Eigen analysis, etc.), or any combination thereof, may be used, but are not limited thereto.

[0104] Processors and Computer Systems: One or more processors or computers may be used to implement the methods disclosed herein. The one or more processors may comprise a hardware processor such as a central processing unit (CPU), a graphics processing unit (GPU), a general-purpose processing unit, or a computing platform. The one or more processors may be composed of any of a variety of suitable integrated circuits (application-specific integrated circuits (ASICs) specially designed to implement deep learning network architectures, or field-programmable gate arrays (FPGAs) for accelerating computing time and / or facilitating deployment), microprocessors, emerging next-generation microprocessor forms (e.g., memory transistor-based processors), logic devices, etc. Although the present disclosure has been described in relation to processors, other types of integrated circuits and logic devices may also be applicable. The processor can have any suitable data manipulation capabilities. For example, the processor can execute 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data manipulations. The one or more processors may be a single-core or multi-core processor, or a plurality of processors configured for parallel processing.

[0105] One or more processors or computers used to implement the disclosed method may be part of a larger computer system and / or may be operatively coupled to a computer network (“network”) using a communication interface to facilitate data transmission and sharing of data. The network may be a local area network, an intranet and / or an extranet, an intranet and / or an extranet communicating with the Internet, or the Internet. In some cases, the network is a telecommunications and / or data network. The network may optionally include one or more computer servers that enable distributed computing such as cloud computing. The network may optionally implement a peer-to-peer network using a computer system, whereby devices coupled to the computer system may act as clients or servers.

[0106] Also, the computer system may include a memory or storage location (e.g., random access memory, read only memory, flash memory, Intel® Optane™ technology), an electronic storage unit (e.g., a hard disk), a communication interface (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices such as a cache, other memory, data storage, and / or an electronic display adapter. The memory, storage unit, interface, and peripheral devices may communicate with one or more processors, e.g., a CPU, via a communication bus, as may be seen, for example, on a motherboard. The (one or more) storage units may be (one or more) data storage units (or data repositories) for storing data.

[0107] One or more processors, such as a CPU, execute a series of machine-readable instructions embodied in a program (or software). The instructions are stored in a storage location. The instructions are directed to the CPU, and then the CPU programs or otherwise configures the CPU to implement the methods of the present disclosure. Examples of operations performed by the CPU include fetch, decode, execute, and write-back. The CPU may be part of a circuit such as an integrated circuit. One or more other components of the system may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0108] The storage unit stores files such as drivers, libraries, and saved programs. The storage unit stores user data, such as user-specified preferences and user-specified programs. The computer system may include one or more additional data storage units external to the computer system, such as being located on a remote server that communicates with the computer system via an intranet or the Internet in some cases.

[0109] Some aspects of the methods and systems provided herein are implemented by machine (e.g., processor) executable code stored in an electronic storage location of a computer system, such as in memory or an electronic storage unit. The machine executable code or machine readable code is provided in the form of software. In use, the code is executed by one or more processors. In some cases, the code is retrieved from the storage unit and stored in memory for access by one or more processors at any time. In some cases, the electronic storage unit is excluded and the machine executable instructions are stored in memory. The code may be pre-compiled and configured for use with a machine having one or more processors that are adapted to execute the code, or may be compiled at runtime. The code may be provided in a programming language selected to enable the code to be executed in a manner in which it is pre-compiled or compiled.

[0110] Various aspects of the disclosed methods and devices may be considered a "product" or "manufactured article", e.g., a "computer program or software product" in the form of machine (or processor) executable code and / or associated data generally stored on some kind of machine-readable medium, in which case the executable code includes a plurality of instructions for controlling a computer or computer system when executing one or more of the methods disclosed herein. The machine executable code may be stored in an optical storage unit including an optically readable medium such as an optical disk, CD-ROM, DVD, or Blu-ray disk. The machine executable code may also be stored in an electronic storage unit such as a memory (e.g., read-only memory, random access memory, flash memory) or on a hard disk. A "storage" type medium includes any or all of the tangible memories of a computer, processor, etc., or their associated modules, e.g., various semiconductor memory chips, optical drives, tape drives, disk drives, etc., which can perform temporary storage at any time for software encoding the methods and algorithms disclosed herein.

[0111] All or part of the software code may be communicated via the Internet or various other telecommunications networks. Such communication enables, for example, the loading of software from one computer or processor to another, such as from an administrative server or host computer to an application server's computer platform. Thus, other types of media used to convey software-encoded instructions include those used across the physical interface between local devices, via wired and optical fixed telephone networks, and across various atmospheric links, such as optical, electrical, and electromagnetic waves. Physical elements that convey such waves, such as wired or wireless links, optical links, etc., are also considered media for conveying software-encoded instructions for carrying out the methods disclosed herein. The terms such as "computer or machine readable medium" used herein refer to any medium involved in providing instructions to a processor for execution, if not limited to persistent tangible "memory" media.

[0112] A computer system may generally include or communicate with an electronic display, for example, to provide an image captured by a machine vision system. The display can generally also provide a user interface (UI). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs), web-based user interfaces, etc.

[0113] Use: The disclosed methods, devices, systems, and software have potential uses in various fields including, but not limited to, proteomics research, drug discovery and development, and clinical diagnostics. For example, the improved information content and data quality achievable for ESI-MS analysis based on the separation of specimen samples using the disclosed methods can be highly beneficial for the characterization of biological and biosimilar pharmaceuticals during development and / or manufacturing. Other uses include, but are not limited to, the analysis of environmental contaminants, pesticides, small molecules, metabolites, peptides, post-translational modifications, glycoforms, antibody-drug conjugates, fusion proteins, viruses, allergens, single-celled organisms, and other applications.

[0114] Biological products and biosimilars are drugs that include, for example, recombinant proteins, antibodies, live virus vaccines, human plasma-derived proteins, cell-based drugs, naturally-derived proteins, antibody-drug conjugates, protein-drug conjugates, and other protein drugs. The FDA and other regulatory authorities require the use of a stepwise approach to demonstrate biological similarity that may include a comparison of the proposed product to the reference product with respect to structure, function, animal toxicity, human pharmacokinetics (PK), and pharmacodynamics (PD), clinical immunogenicity, and clinical safety and efficacy (see "Scientific Considerations in Demonstrating Biosimilarity to a Reference Product: Industry Guidance," U.S. Food and Drug Administration, April 2015). Examples of structural property data that may be required for protein products include primary structure (i.e., amino acid sequence), secondary structure (i.e., degree of folding to form an alpha helix or beta sheet structure), tertiary structure (i.e., three-dimensional shape of the protein generated by folding of the polypeptide backbone and secondary structure domains), and quaternary structure (e.g., number of subunits required to form an active protein complex or the aggregation state of the protein). Often, this information may not be available without using expensive techniques that are cumbersome and time-consuming, such as X-ray crystallography. Thus, there is a need for experimental techniques that enable convenient, real-time, relatively high-throughput characterization of protein structures for the purpose of establishing biological similarity between a candidate biological drug and a reference drug.

[0115] In some embodiments, the disclosed methods, devices, and systems may be used to provide structural comparison data regarding a biological drug candidate (e.g., a monoclonal antibody (mAb)) and a reference biological drug for the purpose of establishing biological similarity. For example, in some instances, isoelectric point data and / or mass spectrometry data regarding the drug candidate and the reference drug may provide important evidence to support the demonstration of biological similarity. In some embodiments, isoelectric point data and / or mass spectrometry data regarding a drug candidate and a reference drug, both processed with a site-specific protease under the same reaction conditions, may provide important evidence to support the demonstration of biological similarity. In some embodiments, the disclosed methods, devices, and systems are used to monitor a biological drug manufacturing process and ensure product quality and consistency by analyzing samples taken at different points in the manufacturing process or samples taken from different manufacturing runs. In some embodiments, the disclosed methods, devices, and systems are used to evaluate the stability of a formulation buffer. In some embodiments, the disclosed methods, devices, and systems may be used to evaluate a cloned cell line with respect to the production and quality of a biological drug candidate.

[0116] [Examples] These examples are provided for illustrative purposes only and do not limit the claims set forth herein.

[0117] Example 1 - Characterization of Protein Charge on Chip before Performing Mass Spectrometry The manufacture of the microfluidic device shown in FIG. 1 has been described above. To operate, the device is mounted on equipment including a nitrogen gas source, a heater, a positive pressure pump (e.g., Parker, T5-1IC-03-1EEP), an electrophoresis power supply (Gamm High Voltage, MC30) terminated with 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 earth ground with the mass spectrometer. The equipment is controlled by software (e.g., Lab View).

[0118] The protein sample is premixed with an ampholyte pH gradient and a pI marker before being placed in a vial and loaded into the autosampler. The protein sample is continuously loaded from the autosampler through the inlet 412 into the microfluidic device 400 through the concentration channel 418 and sent from the device to the waste 430 through the outlet 434.

[0119] The sheath / catholyte (50% MeOH, N40H / H20) is loaded into the two catholyte wells 404, 436, the anolyte (10 mM H3P04) is loaded into the anolyte well 426, and a source of heated nitrogen gas is attached to the two gas wells 408, 440.

[0120] After all the reagents have been incorporated, an electric field of +600 V / cm is applied from the anolyte well 426 to the catholyte wells 404, 436 by connecting the electrodes to the anolyte well 426 and the catholyte wells 404, 436, and isoelectric focusing electrophoresis is initiated. The UV light source is aligned under the concentration channel 418, and a camera is arranged above the concentration channel 418 to measure the light passing through the concentration channel 418, thereby detecting the focused proteins by their absorbance. The glass plate 402 composed of soda-lime glass acts to block any stray light from the camera, so that the light not passing through the concentration channel 418 is prevented from reaching the camera, thereby increasing the sensitivity of the measurement.

[0121] Images of the focused proteins can be captured continuously and / or periodically during IEF. When focusing is complete, a low voltage is applied from the inlet 412, thereby mobilizing the pH gradient towards the orifice 424. At this point, the electric field can be maintained to maintain high-resolution IEF separation. By continuing to image the concentration channel 418 during the ESI process, the pI of each protein can be determined when the protein is discharged from the orifice 424.

[0122] When the concentrated protein fraction moves from the concentration channel 418 to the junction 420, the concentrated protein fraction is mixed with the sheath fluid, and the sheath fluid can flow from the catholyte wells 404, 436 to the junction 420 through the sheath / catholyte channels 406, 438. By mixing the concentrated protein fraction with the sheath fluid, the protein fraction can be placed in a mass spectrometry-compatible solution and the charge to the focused proteins can be restored (IEF brings the proteins to an uncharged state), thereby improving ionization.

[0123] Thereafter, the concentrated protein fraction continues to proceed to the orifice 424, and the orifice 424 can be defined by the dish surface 422 of the glass plate 402. The concentrated protein fraction can form a Taylor cone when it is trapped within the electric field between the sheath fluid well ground and the negative electrode of the mass spectrometer.

[0124] As the solution continues to push the Taylor cone out of the concentration channel 418, small droplets of the fluid are ejected from the Taylor cone and fly towards the inlet of the mass spectrometer. Nitrogen gas (e.g., 150 °C) can flow down the gas channels 410, 432 from the gas wells 408, 440 and form nitrogen gas jets located on both sides of the Taylor cone that can convert the droplets emerging from the Taylor cone into a fine mist before leaving the microfluidic device, which can be useful for detection in the mass spectrometer. By adjusting the pressure from the inlet 412, the size of the Taylor cone can be adjusted as needed, improving detection in the mass spectrometer.

[0125] Example 2 - Tracking the velocity when the analyte peak exits the microfluidic chip and enters the mass spectrometer In this example, the microfluidic channel network 100 of FIG. 7A is formed in a 250 micron thick layer of an opaque cyclic olefin polymer. The depth of channel 112 is 250 microns, and thus the channel completely penetrates the 250 micron layer. All other channels are 50 microns deep. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer as in the case of FIG. 7B to produce a planar microfluidic device. Ports 102, 104, 106, 108, 110 provide access to the channel network for reagent introduction from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, whereby channel 103 can act as a waste channel, such that priming of other reagents through the channel network can be "wasted". Acid (1% formic acid) is primed into channels 109, 112, 114, 103 via port 108 and discharged to port 102. Samples (4% Pharmalyte 3-10, 12.5 mM pI standard 3.38 (purified peptide, sequence: Trp-Asp-Asp-Asp), 12.5 mM pI standard 10.17 (purified peptide, sequence: Trp-Tyr-Lys-Arg), NIST monoclonal antibody standard (part number 8671, NIST)) are primed into channels 107, 112, 114, 103 via port 106 and discharged to port 102. This leaves channel 112 containing the sample analyte. Base (1% dimethylamine) is primed into channels 105, 114, 103 via port 104 and discharged to port 102. Mobilizing agent (1% formic acid, 49% methanol) is primed into channels 111, 114, 103 via port 110 and discharged from channel 103 to port 102.

[0126] Electrophoresis of the sample in channel 112 is performed by applying 4000 V to port 108 and connecting port 110 to ground. Ampholytes in the sample establish a pH gradient extending across channel 112. Absorbance imaging of the separation is performed by using a 280 nm light source aligned with channel 112 and measuring the transmittance of 280 nm light passing through channel 112 with a CCD camera. Software calculates the absorbance by comparing the light transmittance during separation or mobilization with a “blank” reference measurement performed when there is no focused sample prior to the sample being run, and then displays the absorbance pixel by pixel across the length of channel 112. As shown in FIGS. 9A - 9F, locations where standards or samples are focused are displayed as peaks.

[0127] When the sample has completed focusing, a final focused absorbance image is captured. Software identifies the spatial positions of the pI markers, interpolates between the markers, and calculates the pI of the focused sample fraction peaks. At this point, the control software triggers a relay, which disconnects ground at port 110, connects port 104 to ground, and sets the pressure acting on the mobilization reservoir connected to port 104 to establish a flow of 100 nL / min of mobilization reagent solution entering channels 105 and 114 through port 104 and exiting the chip at orifice 116. Orifice 116 is positioned 2 mm away from the ESI inlet of a mass spectrometer where the inlet voltage is between - 3500 V and - 4500 V.

[0128] While the pressure - driven flow directs the mobilization reagent from port 104 to orifice 116, a portion of the formic acid in the mobilization reagent electrophoreses in the form of formate from channel 105 through channel 112 to the anode at port 108. As the formate moves through channel 112, it disturbs the isoelectric pH gradient, whereby the ampholytes, standards, and sample increase in charge and electrophoretically move from channel 112 to channel 114, where the pressure - driven flow from port 110 carries them from orifice 116 to the ESI spray.

[0129] While mobilization is occurring, the software continues to capture absorbance images and identify peaks, thereby tracking the movement of the peaks from imaging channel 112 to channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and the flow rate within channel 114, the software calculates the time at which the peak is introduced into the mass spectrometer via orifice 116 across channel 114, thereby enabling a direct correlation between the initial focused peak and the resulting mass spectrum.

[0130] Figures 9A - 9F give examples of a series of absorbance traces acquired at 1 - minute intervals and show the mobilization of isoelectric point (pI) standards determined from images of the separation channel. Figure 9A shows a plot of absorbance 910 versus channel distance 905 after isoelectric focusing of five pI standards (peaks 915, 920, 925, 930, 935) before mobilization. As shown in Figure 9B, after 1 minute of mobilization, peak 915 corresponding to the pI = 9.99 standard is at the edge of the field of view of the imaging system. As shown in Figure 9C, after 2 minutes of mobilization, peak 915 (pI = 9.99 standard) has exited the portion of the channel being imaged. As shown in Figure 9D, after 3 minutes of mobilization, peak 920 (pI = 8.40 standard) has exited the portion of the channel being imaged. As shown in Figure 9E, after 4 minutes of mobilization, peak 925 (pI = 7.00 standard) has exited the portion of the channel being imaged. As shown in Figure 9F, after 5 minutes of mobilization, peak 930 (pI = 4.05 standard) has left the portion of the channel being imaged.

[0131] Example 3 - Adjusting MS Parameters and ESI Parameters Using Feedback In Example 3, the chip, device, and software all perform the same procedures as in Example 2. Further, as shown in Figure 8, a second CCD camera is used to image the Taylor cone during ESI. These images are used to evaluate the quality and consistency of the Taylor cone. By evaluating the images and / or total counts with a mass spectrometer, it becomes possible to identify defects and diagnose the causes of ESI Taylor cone problems.

[0132] The formation of a Taylor cone in ESI depends on maintaining an input flow into the cone that matches the velocity of the fluid being lost to evaporation and ESI. The size of the Taylor cone depends on the flow rate, the voltage gradient between the microfluidic device and the MS, the distance between the microfluidic device and the MS, and subtle changes in the ESI tip of the microfluidic device and the local environment.

[0133] Imaging of the Taylor cone can diagnose the causes of ESI malfunctions. For example, loss of the Taylor cone indicates insufficient flow, and software can increase the flow of mobilizing agent to the microfluidic device. Similarly, corona discharge indicates that the voltage is too high, and software can lower the voltage. Expansion of the ESI cloud indicates that the voltage is too high, while forming droplets instead of a Taylor cone indicates that the voltage is too low. These differences and other visual differences can be identified in the image, and software can automatically compensate to re-establish the Taylor cone.

[0134] Example 4 - Low mass scanning as a marker for separation In Example 4, the chip, equipment, and software all perform the same procedures as in Example 2. Further, when mobilization occurs and the analyte peak begins to move to the MS, the MS is set to alternate between m / z ranges of 1500 - 6000 and 150 - 1500. The range of 1500 - 6000 is used to identify the NIST antibody analyte fraction peaks when they are introduced into the MS. The 150 - 1500 m / z range scan is used to identify the ampholytes of the free solution (Pharmalytes) when they are introduced into the MS. Since the presence of specific ampholytes defines part of the isoelectric point pH gradient being analyzed at any given time by the MS, the ampholytes can be identified by mass scanning and used to calibrate the total ion chromatogram from the MS.

[0135] Example 5 - Change the high and low voltages to maintain the electric field strength and a constant voltage in the chip.

[0136] In the case of this example, the microfluidic channel network 100 of FIG. 7A is formed in a 250 - micron - thick layer of an opaque cyclic olefin polymer. The depth of channel 112 is 250 microns, so the channel completely penetrates the 250 - micron layer. All other channels are 50 microns deep. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer as in the case of FIG. 7B to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, 110 provide access to the channel network for reagent introduction from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, whereby channel 103 can act as a waste channel, so that the priming of other reagents through the channel network can be made into "waste". Acid (1% formic acid) is primed into channels 109, 112, 114, 103 through port 108 and discharged to port 102. Samples (4% Pharmalyte 3 - 10, 12.5 mM pI standard 3.38 (purified peptide, sequence: Trp - Asp - Asp - Asp), 12.5 mM pI standard 10.17 (purified peptide, sequence: Trp - Tyr - Lys - Arg), NIST monoclonal antibody standard (part number 8671, NIST)) are primed into channels 107, 112, 114 through port 106 and discharged to port 102. Thereby, channel 112 containing the sample analyte remains. Base (1% dimethylamine) is primed into channels 105, 114, 103 through port 104 and discharged to port 102. Mobilizer (1% formic acid, 49% methanol) is primed into channels 110, 114, 103 through port 110 and discharged from port 102 to port 102. Pressure is applied to the base reservoir to generate a flow of 100 nL / min entering channels 105, 114 through port 104 and exiting through orifice 116.

[0137] Isoelectric focusing of the sample sample in channel 112 is initiated by applying 2000 V to port 108 using power supply 1005 and connecting port 110 to high voltage power supply 1010 to apply -2000 V. This establishes the circuit shown in Figure 10A that includes high voltage power supply 1005 and high voltage power supply 1010 (in some cases, power supply 1005 and power supply 1010 may comprise two channels of a single multiplexed high voltage power supply) to create a 4000 V voltage drop between the anode and the cathode. The electrical resistance of the channel depends on the dimensions of the channel and the conductivity of the reagent. In this example, the electrical resistance R109 of the acid channel corresponding to channel 109 (see Figure 7A) is 10 MΩ, the electrical resistance R112 of the sample channel corresponding to channel 112 (see Figure 7A) starts at 40 MΩ, and the base resistance R111 in the channel corresponding to channel 111 (see Figure 7A) is 50 MΩ. The resistance R113 of the electrospray ionization (ESI) interface between orifice 116 (see Figure 7A) and mass spectrometer 1015 is 2 GΩ. The total voltage drop between channels 109, 112, 111 (see Figure 7A) is 4000 V, and since these channels correspond to three resistors in series, the voltage of the chip (V 116 ) is calculated according to Equation 1.

[0138] V 116 =ΔV 108-110 *(R 111 ) / (R 109 +R 112 +R 111 )+(high voltage - power supply 1010 voltage setting).

[0139] At the start of isoelectric focusing, V 116 = 0 volts. Orifice 116 (see Figure 7A) is positioned 2 mm away from the ESI inlet of the mass spectrometer, and in this case, the inlet voltage is -3500 V to -4500 V to form a Taylor cone. Figure 10B shows another embodiment of the circuit shown in Figure 10A that includes the resistance R105 of channel 105.

[0140] The ampholytes in the sample establish a pH gradient reaching channel 112. The absorbance imaging of the separation is performed by using a 280 nm light source aligned with channel 112 and measuring the transmittance of the 280 nm light passing through channel 112 by a CCD camera. Software calculates the absorbance by comparing the light transmittance during separation or mobilization with a "blank" reference measurement performed when there is no focused sample before the sample is run, and then displays the absorbance for each pixel along the length of channel 112. As shown in FIGS. 9A-9F, the locations where the standard or sample is focused are displayed as peaks.

[0141] As the sample focuses, the ampholytes, antibody isoforms, and standards reach their isoelectric points and lose their charges, increasing the resistance of sample channel 112, while the resistances in channels 109, 111, and the ESI interface remain constant. The computer-implemented method can monitor the current at power supply 1005 and calculate the resistance at any point within channel 112. The computer-implemented method uses this information to adjust power supplies 1005, 1010. For example, if the resistance of channel 112 rises to 140 megaohms, if the power supplies are not adjusted, the voltage at orifice 116 will reach -1000 V and the Taylor cone will be disrupted. However, by adjusting power supply 1005 to +3000 V and power supply 1010 to -1000 V, the chip remains at 0 V and the total voltage drop across channels 109, 112, 111 remains at 4000 V. These adjustments are made on the fly as the resistance of channel 112 changes.

[0142] When the sample has completed focusing, a final focused absorbance image is captured. The software identifies the spatial positions of the pI markers, interpolates between the markers, and calculates the pI of the focused sample fraction peaks. At this point, the control software triggers a relay, which disconnects power 1010 at port 110, connects port 104 to power 1010, and sets the pressure acting on the mobilization reagent reservoir connected to port 104 to establish a flow of 100 nL / min of mobilization reagent solution entering channels 105, 114 through port 104 and exiting the chip at orifice 116 (see the chip schematic of FIG. 7A and the electrical circuit shown in FIG. 10B). Orifice 116 is positioned 2 mm away from the ESI inlet of a mass spectrometer where the inlet voltage is between -3500 V and -4500 V.

[0143] While the pressure-driven flow directs the mobilization reagent from port 104 towards orifice 116, a portion of the formic acid in the mobilization reagent electrophoreses in the form of formate to the anode at port 108 through channel 105 and channel 112. As the formate moves through channel 112, the formate disrupts the isoelectric pH gradient, whereby the ampholytes, standards, and sample samples increase in charge and electrophoretically move from channel 112 to channel 114, where the pressure-driven flow from port 110 carries them from orifice 116 to the ESI spray.

[0144] While mobilization is occurring, the resistance of channel 112 decreases. FIGS. 11A - 11B show examples of voltage and current data for channel 112 that may be used to derive the resistance of the channel. FIG. 11A shows a plot of voltage as a function of time. FIG. 11B shows a plot of current as a function of time. The software monitors the change in current, adjusts the power supply, maintains the voltage drop between the anode and cathode at 3000 V, and maintains 0 V at chip 116, as shown in FIG. 15. The voltage change may be transient or stable.

[0145] While mobilization is occurring, the software continues to capture absorbance images and identify peaks, thereby tracking the movement of peaks from imaging channel 112 to channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and the flow rate within channel 114, the software calculates the time at which the peak is introduced into the mass spectrometer through orifice 116 across channel 114, thereby enabling a direct correlation between the initial focused peak and the resulting mass spectrum.

[0146] Figure 13A gives a representative circuit diagram in the microfluidic device shown in FIG. 7A during chemical mobilization where an additional resistor R120 is used to sink current to ground and the ESI chip is held at a positive voltage. The circuit may comprise a high voltage power supply 1305 that may be substantially similar to 1005 and a high voltage power supply 1310 that may be substantially similar to 1010 to produce a specific voltage drop (e.g., 4000V) between the anode and the cathode. The circuit may further comprise a third high voltage power supply 1307. The electrical resistance of the channels depends on the dimensions of the channels and the conductivity of the reagent. The circuit incorporates the electrical resistance R109 of the acid channel corresponding to channel 109 (see FIG. 7A), the electrical resistance R112 of the sample channel corresponding to channel 112 (see FIG. 7A), and the base resistance R111 in the channel corresponding to channel 111 (see FIG. 7A), as well as the resistance R113 of the electrospray ionization (ESI) interface between orifice 116 (see FIG. 7A) and the voltage source of the mass spectrometer 1315, which may be substantially similar to 1015. Also, the circuit may include the electrical resistance R105 of channel 105. Power supply 1307 is connected to channel 111 (see FIG. 7A) and can use current control set to 0 μA during mobilization. This power supply may be used to read the voltage of the chip and implement a computer-controlled feedback loop to maintain the voltage of the chip constant.

[0147] FIG. 13B shows a representative circuit diagram in the microfluidic device shown in FIG. 7A during chemical mobilization where the ESI chip is held at a positive voltage using resistor R120 to sink current to power supply 1320. FIG. 13C shows a representative circuit diagram in the microfluidic device shown in FIG. 7A during chemical mobilization where the ESI chip is held at a positive voltage using field effect transistor (FET) 1325 to sink current. The electrical circuit may further comprise amplifier 1330, voltage reference 1335, and further resistor R200. FIG. 13D shows a representative circuit diagram in the microfluidic device shown in FIG. 7A during chemical mobilization where the ESI chip is held at a positive voltage using bipolar junction transistor (BJT) 1340 to sink current. Power supply 1307 is connected to channel 111 (see FIG. 7A) and can use current control set to 0 μA. This power supply may be used to read the voltage of the chip and implement a computer-controlled feedback loop to maintain the voltage of the chip constant. FIG. 13E gives a representative circuit diagram in the microfluidic device shown in FIG. 7A during chemical mobilization of a separated analyte mixture where the ESI chip is held at or near ground. Power supply 1307 is connected to channel 111 (see FIG. 7A) and can use current control set to 0 μA. This power supply may be used to read the voltage of the chip and implement a computer-controlled feedback loop to maintain the voltage of the chip constant.

[0148] Example 6 - Changing high and low voltages to maintain a constant voltage and electric field strength in the chip based on measurement of the chip voltage In this example, the microfluidic channel network 100 of FIG. 7A is formed in a 250 micron thick layer of an opaque cyclic olefin polymer. The depth of channel 112 is 250 microns, and thus the channel completely penetrates the 250 micron layer. All other channels are 50 microns in depth. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer as in the case of FIG. 7B to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, 110 provide access to the channel network for reagent introduction from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, whereby channel 103 can act as a waste channel, such that priming of other reagents through the channel network can be "wasted". Acid (1% formic acid) is primed into channels 109, 112, 114, 103 through port 108 and discharged to port 102. Samples (4% Pharmalyte 3-10, 12.5 mM pI standard 3.38 (purified peptide, sequence: Trp-Asp-Asp-Asp), 12.5 mM pI standard 10.17 (purified peptide, sequence: Trp-Tyr-Lys-Arg), NIST monoclonal antibody standard (part number 8671, NIST)) are primed into channels 107, 112, 114 through port 106 and discharged to port 102. Thereby, channel 112 containing the sample analyte remains. Base (1% dimethylamine) is primed into channels 105, 114, 103 through port 104 and discharged to port 102. Mobilizer (1% formic acid, 49% methanol) is primed into channels 110, 114, 103 through port 110 and discharged from port 102 to port 102 (see the chip schematic of FIG. 7A and the electrical circuit shown in FIG. 13E).

[0149] Electrophoresis of the sample analyte in channel 112 is initiated by applying 1500 V to port 108 using power supply 1305 and connecting to power supply 1307 set to 0 V at port 110. After 5 minutes, power supply 1305 is increased to 3000 V over 3 minutes to complete focusing.

[0150] The ampholytes in the sample establish a pH gradient extending to channel 112. Absorbance imaging for separation is performed by using a 280 nm light source aligned with channel 112 and measuring the transmittance of 280 nm light passing through channel 112 with a CCD camera. Software calculates the absorbance by comparing the light transmittance during separation or mobilization with a "blank" reference measurement performed when there is no focused sample prior to the sample being run, and then displays the absorbance for each pixel over the length of channel 112. As shown in FIGS. 9A - 9F, the locations where the standard or sample is focused are displayed as peaks.

[0151] When the sample has completed focusing, a final focused absorbance image is captured. Software identifies the spatial positions of the pI markers, interpolates between the markers, and calculates the pI of the focused sample fraction peaks. At this point, the control software triggers a relay, and the relay connects port 104 to power supply 1310 and sets the pressure acting on the mobilization material reservoir connected to port 104 to establish a flow of 100 nL / min of mobilization material solution entering channels 105 and 114 through port 104 and exiting the chip at orifice 116. Orifice 116 is positioned 2 mm away from the ESI inlet 1315 of a mass spectrometer where the inlet voltage is -3500 V to -4500 V. Power supply 1307 is set to 0 μA using current control, power supply 1305 is set to 3000 V, power supply 1310 is set to 0 V, and the MS ESI ion source is set to -3500 V to -4500 V.

[0152] While the pressure-driven flow directs the mobilizing material from port 104 towards orifice 116, a portion of the formic acid in the mobilizing material reagent electrophoreses in the form of formate to the anode of port 108 through channel 105 and channel 112. As the formate moves through channel 112, the formate disturbs the isoelectric pH gradient, whereby the ampholytes, standards, and sample specimens increase their charge and electrophoretically move from channel 112 to channel 114, where the pressure-driven flow from port 110 transports them from orifice 116 to the ESI spray.

[0153] While mobilization is occurring, the resistance of channel 112 decreases. Since the voltage drop across channel 111 is zero at this point (ΔV = IR = 0 * R111 = 0V), the power supply 1307 set to 0 μA is equal to the voltage of V116 in FIG. 13E. As shown in the data of FIGS. 11A - 11B, 8 minutes (480 seconds) after focusing is completed, the software monitors the change in current and adjusts the power supply to maintain a constant voltage drop of 3000V between the anode and the cathode and to maintain 0 volts at the chip 116, as described in FIG. 15. The voltage of the chip (V116) is represented by Equation 2.

[0154] V 116 =ΔV 108-110 *(R 111 ) / (R 109 +R 112 +R 105 )+(power supply 1310 voltage setting).

[0155] While mobilization is occurring, the software continues to capture absorbance images to identify peaks, thereby tracking the movement of the peaks from imaging channel 112 to channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and the flow rate within channel 114, the software calculates the time the peak is introduced into the mass spectrometer through orifice 116 across channel 114, thereby enabling a direct correlation between the initial focused peaks and the resulting mass spectra.

[0156] Example 7 - Measuring chip voltage and resistors to change high and low voltages to maintain electric field strength and a constant voltage in the chip In this example, the microfluidic channel network 100 of FIG. 7A is formed in a 250 - micron - thick layer of an opaque cyclic olefin polymer. The depth of channel 112 is 250 microns, so the channel completely penetrates the 250 - micron layer. All other channels are 50 microns deep. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer as in the case of FIG. 7B to manufacture a planar microfluidic device. Ports 102, 104, 106, 108, 110 provide access to the channel network for reagent introduction from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, whereby channel 103 can act as a waste channel, and as a result, the priming of other reagents through the channel network can be made into "waste". Acid (1% formic acid) is primed into channels 109, 112, 114, 103 through port 108 and discharged to port 102. Samples (4% Pharmalyte 3 - 10, 12.5 mM pI standard 5.52 (purified peptide, sequence: Trp - Glu - His), 12.5 mM pI standard 8.4 (purified peptide, sequence: Trp - Tyr - Lys), infliximab biosimilar monoclonal antibody standard (part number MCA6090, Bio - rad)) are primed into channels 107, 112, 114 through port 106 and output to port 102. Thereby, channel 112 containing the sample specimen remains. Base (1% dimethylamine) is primed into channels 105, 114, 103 through port 104 and discharged to port 102. The mobilizing agent (1% formic acid, 49% methanol) is primed into channels 110, 114, 103 through port 110 and discharged from port 102 to port 102 (see the chip schematic of FIG. 7A and the electrical circuit shown in FIG. 13B).

[0157] Electrophoresis of the sample in channel 112 is initiated by applying 1500 V to port 108 using power supply 1305 and connecting it to power supply 1307 with port 110 set to 0 V. After 5 minutes, power supply 1305 is increased to 3000 V.

[0158] The ampholytes in the sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed by using a 280 nm light source aligned with channel 112 and measuring the transmittance of the 280 nm light passing through channel 112 with a CCD camera. Software calculates the absorbance by comparing the light transmittance during separation or mobilization with a "blank" reference measurement performed when there is no focused sample prior to the sample being run, and then displays the absorbance pixel by pixel across the length of channel 112. As shown in FIGS. 9A - 9F, the locations where the standard or sample is focused are displayed as peaks.

[0159] When the sample has completed focusing, the charge variants of infliximab are separated as shown in Figure 16A, and the final focused absorbance image is captured. The software identifies the spatial positions of the pI markers, interpolates between the markers, and calculates the pI of the focused sample fraction peaks. At this point, the control software triggers a relay, which connects port 104 to power supply 1310 and sets the pressure acting on the mobilization material reservoir connected to port 104 to establish a flow of 100 nL / min of mobilization material solution entering channels 105 and 114 through port 104 and exiting the chip at orifice 116. Orifice 116 is positioned 2 mm away from the ESI inlet 1315 of the mass spectrometer. Power supply 1307 is set to 0 μA using current control, power supply 1305 is set to 7000 V, power supply 1310 is set to 4000 V, and the MS ESI ion source 1315 is held at ground. As shown in Figure 13B, an additional resistor R120 is connected to the system between power supply 1310 and channel 105 (R current sink), and the opposite side of resistor R120 is connected to power supply 1320. Power supply 1320 is set at least 4000 V lower than power supply 1310 to act as a current sink. Resistor R120 can alternatively connect the electrical circuit to ground as in the case of Figure 13A, can become a field effect transistor (FET) as shown in Figure 13C, can become a bipolar junction transistor (BJT) as shown in Figure 13D, or can be any other resistive element capable of sinking current from power supply 1310 to form a functioning electrophoresis circuit.

[0160] While the pressure-driven flow directs the mobilization material from port 104 towards orifice 116, a portion of the formic acid in the mobilization material reagent electrophoreses in the form of formate from channel 105 through channel 112 to the anode at port 108. As the formate moves through channel 112, the formate disrupts the isoelectric pH gradient, whereby the ampholytes, standards, and sample samples increase their charge and electrophoretically move from channel 112 to channel 114, where the pressure-driven flow from port 110 transports them from orifice 116 to the ESI spray.

[0161] While mobilization occurs, the resistance of channel 112 decreases. Since the voltage drop across channel 111 is zero at this point (ΔV = IR = 0 * R111 = 0V), the power supply 1307 set to 0 μA is equal to the voltage of V116. As shown in the data of FIGS. 11A - 11B, the software monitors the change in current as described in FIG. 12, adjusts the power supply, and maintains a constant voltage drop of 3000V between the anode and the cathode and 3000 volts at chip 116. The voltage of the chip (V116) is represented by Equation 2.

[0162] V 116 = ΔV 108-110 * (R 111 ) / (R 109 + R 112 + R 105 ) + (power supply 1310 voltage setting).

[0163] While mobilization occurs, the software continues to capture the absorbance image to identify the peak, thereby tracking the movement of the peak from imaging channel 112 to channel 114. By tracking the time each peak leaves imaging channel 112, its speed, and the flow rate within channel 114, the software can calculate the time the peak is introduced into the mass spectrometer through orifice 116 across channel 114, thereby enabling a direct correlation between the initial focused peak and the resulting mass spectrum. For example, FIG. 16B shows the mass of the glycoform electrosprayed into the mass spectrometer included in the acidic peak of the electropherogram shown in FIG. 16A. FIG. 16C shows the mass of the glycoform at the major infliximab peak from FIG. 16A. FIGS. 16D and 16E show the mass of the base peak from the electropherogram shown in FIG. 16A.

[0164] Example 8 - 2 Changing High and Low Voltages to Maintain Electric Field Strength and Constant Voltage in Step - Capillary IEF In Example 8, as outlined in FIG. 14A, two-step IEF (isoelectric focusing followed by mobilization) is performed in a 60 cm capillary and mobilized to ESI-MS via a junction sprayer. The separation capillary 1808 is immersed in the anolyte vial 1806. The high voltage power supply 1802 is connected to the anolyte vial 1806 via the electrode 1804. The other end of the capillary 1808 is connected to the junction sprayer 1814 via the tee union 1812. The capillary 1808 is inserted into the junction sprayer 1814 such that the capillary outlet is in proximity to the ESI tip 1824. The third arm of the tee union 1812 is connected to the mobilization capillary 1816 that is immersed in the pressurized mobilization vial 1818. Also, the pressurized mobilization vial 1818 is grounded via the electrode 1817 such that it acts as a current sink. Further, the junction sprayer 1814 is connected to the power supply 1810 via the wiring 1820 that connects outside the sprayer 1814. In this example, the ion source of the mass spectrometer is held at ground.

[0165] The reagents are prepared as follows. The anolyte vial 1806 is filled with 1% formic acid in water, the separation capillary 1808 is filled with an aqueous sample (250 μg / mL NIST mAb, 1.5% Pharmalyte 5-8 ampholyte, 1.5% Pharmalyte 8-10.5, 5 mg / mL pI standard 7.00 and 10.17), the junction sprayer chamber 1826 and the mobilization capillary 1816 are filled with 1% diethylamine in water, and the pressurized mobilization vial 1818 is filled with 1% formic acid, 50% acetonitrile, and 49% water.

[0166] In this example, the ion source of the mass spectrometer is held at ground. To initiate focusing, the power supply 1802 is set to +30 kV and the power supply 1810 is set to 4 kV. Then, the pressure-driven flow from the mobilization vial 1818 is initiated at 100 nL / min. In this way, ESI is initiated at the junction sprayer cavity 1826 using diethylamine, which also acts as the catholyte for the isoelectric focusing step.

[0167] As focusing progresses in capillary 1808, the sample loses its charge - carrying ability and the resistance in capillary 1808 increases. When the ESI chip is electrically positioned between capillary 1808 and the diethylamine in chamber 1826 (see Figure 14B), the ESI chip voltage (V 1824 ) decreases according to Equation 3.

[0168] V 1824 =ΔV 1806-1814 *R 1826 / (R 1808 +R 1826 )+V 1814 Furthermore, as the resistance of capillary 1808 increases, the current passing through the capillary that can be measured at power supply 1802 decreases. The increased current is directly related to the change in the resistance of capillary 1808 by Equation 4.

[0169] I 1806 =ΔV 1806-1814 / (R 1808 +R 1826 ) Using a computer - controlled feedback loop as described in Figure 12, the system can calculate the change in the resistance of capillary 1808 (and thus the change in the voltage drop across capillary 1808 that defines the voltage at ESI chip 1824), and the system can adjust power supplies 1802, 1810 to maintain a ΔV of 26 kV and an ESI chip voltage of 4000 kV.

[0170] After the bundling is completed (about 30 minutes), the mobilizing agent solution in the pressurized mobilizing agent vial 1818 has replaced the diethylamine in the joining nebulizer chamber 1826, whereby the mobilization of the NIST mAb protein isoform in the capillary 1808 is initiated. In a similar manner but opposite to isoelectric focusing electrophoresis, as the mobilization proceeds, the resistance of the capillary 1808 decreases, thereby affecting the voltage of the ESI chip 1824. Also in this case, the computer-controlled feedback loop uses Equations 3 and 4 to calculate the necessary changes to the power supplies 1802, 1810 to maintain an electric field of 26 kV while maintaining the voltage of the ESI chip 1824 at 4 kV.

[0171] Preferred embodiments of the invention have been illustrated and described herein, but as will be apparent to those skilled in the art, such embodiments are provided by way of example only. Without departing from the invention, numerous variations, modifications, and substitutions may occur to those skilled in the art. It should be understood that various alternatives to the embodiments of the invention described herein may be used arbitrarily in combination when practicing the invention. The following claims define the scope of the invention, and it is intended thereby to cover the methods and structures within these claims and their equivalents.

Claims

1. A method comprising: a) providing a sample comprising a mixture of two or more analytes; b) separating individual analyte peaks from the mixture of two or more analytes by performing separation within a fluid channel containing the sample; c) calculating the velocity of an analyte peak during transfer of the contents of the fluid channel towards a fluid channel outlet; d) determining the time taken for the analyte peak to reach the fluid channel outlet using the velocity of the analyte peak. A method as described above.

2. The method according to claim 1, wherein the fluid channel is the lumen of a capillary.

3. The method according to claim 1, wherein the fluid channel is part of a microfluidic device.

4. The method according to any one of claims 1 to 3, wherein the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC).

5. The method according to any one of claims 1 to 4, wherein the velocity of the analyte peak is calculated from the time interval required for the analyte peak to move from a first position to a second position.

6. The method according to claim 5, wherein the first position, the second position, and the time interval are determined from a series of two or more images of the fluid channel.

7. The method according to claim 6, wherein the series of two or more images includes ultraviolet absorption images, visible light absorption images, or fluorescence images.

8. The method according to any one of claims 1 to 7, wherein the fluid channel outlet comprises an electrospray interface with a mass spectrometer.

9. The method according to claim 8, wherein the time taken for the analyte peak to reach the fluid channel outlet is configured to be used when correlating mass spectrometer data with the analyte peak.

10. The method according to any one of claims 1 to 9, wherein the transfer of the contents of the fluid channel includes the use of electroosmotic flow techniques, chemical mobilization techniques, hydrodynamic mobilization techniques, or any combination thereof.

11. The method according to any one of claims 1 to 10, wherein the two or more samples include proteins, protein-drug complexes, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, small organic compounds, or any combination thereof.

12. The method according to any one of claims 1 to 10, further comprising adjusting control parameters for a separation reaction performed in the fluid channel using a feedback loop that uses the velocity of the sample peak.

13. The method according to claim 12, wherein the control parameter is voltage.

14. The method according to claim 12 or claim 13, wherein the feedback loop operates at a frequency of at least 0.1 Hz.

15. A computer-implemented method comprising: a) receiving, using a processor, image data including two or more images acquired using a detector, the detector being configured to image all or a portion of a separation channel within a capillary or a microfluidic device; b) determining, using the same or a different processor, the positions of sample peaks within the separation channel in the two or more images by processing the image data; c) calculating, using the same or a different processor, the velocity of the sample peaks based on the positions of the sample peaks in the two or more images and a known time interval between acquisitions of the two or more images; d) determining, using the same or a different processor, the time taken for the sample peaks to reach the separation channel outlet A computer-implemented method comprising.

16. The computer-implemented method according to claim 15, wherein the separation reaction performed in the separation channel includes isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC).

17. The computer-implemented method according to claim 15 or claim 16, wherein the two or more images include ultraviolet absorption images, visible light absorption images, or fluorescence images.

18. The computer-implemented method according to any one of claims 15 to 17, wherein the separation channel outlet is in fluid communication with a mass spectrometer or comprises an electrospray interface with the mass spectrometer.

19. The time taken for the analyte peak to reach the separation channel outlet is configured to be used when correlating mass spectrometer data with the analyte peak. The computer-implemented method according to claim 18.

20. The analyte peak is separated from a mixture, and the analyte peak comprises a protein, a protein-drug complex, a peptide, a nucleic acid molecule, a carbohydrate molecule, a lipid molecule, a metabolite molecule, or a small organic compound.

21. The computer-implemented method according to any one of claims 15 to 20, further comprising adjusting control parameters for a separation reaction performed in the separation channel using a feedback loop that uses the velocity of the analyte peak.

22. The control parameter is a voltage.

23. The feedback loop operates at a frequency of at least 0.1 Hz.

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