Software for microfluidic systems that interface with mass spectrometry

Abstract

To provide software for microfluidic systems interfacing with mass spectrometry.SOLUTION: Methods, devices, and systems for improving the quality of electrospray ionization mass spectrometer (ESI-MS) data are described, as are methods, devices, and systems for achieving improved correlation between chemical separation data and mass spectrometry data. Some embodiments described herein relate to innovative software and systems for analyzing data from and directing the operation of capillary- and microfluidic-based separation systems integrated with mass spectrometric detection. In some embodiments, analytes are imaged during separation in capillaries or on microfluidic devices, and the molecular weight or mass / charge ratio is measured in a mass spectrometer after the separation.SELECTED DRAWING: Figure 18

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 078,856, filed on September 15, 2020; U.S. Provisional Patent Application No. 63 / 021,020, filed on May 6, 2020; U.S. Provisional Patent Application No. 62 / 966,372, filed on January 27, 2020; and U.S. Provisional Patent Application No. 62 / 940,071, filed on November 25, 2019, each of which is incorporated herein by reference in its entirety for all purposes.

Background Art

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

[0003] Various methods have been used, for example, 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 requires that the protein sample be broken down into peptide fragments and has the disadvantage of leading to a large number of sample fractions that must be analyzed and complex post - run data reconstruction. 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, which limits their usefulness.

[0004] Another method for introducing a sample into a mass spectrometer is electrospray ionization (ESI). In ESI, small droplets of the sample and solution are emitted from the distal end of a capillary or microfluidic device with electrospray features, such as an emitter tip or orifice, by applying an electric field between the capillary tip or emitter tip and the mass spectrometer source plate. The droplets stretch and expand within this induced electric field, forming a conical emission (i.e., a "Taylor cone"), which contains increasingly smaller droplets that evaporate and generate gaseous ions introduced into the mass spectrometer for further separation and detection. Typically, the emitter tip is formed from a capillary, which provides a convenient droplet volume for ESI. However, the capillary is limited to a linear channel, which does not allow for multi-stage sample processing. ESI is also dependent on the voltage at the ESI tip, as it remains constant throughout the analysis, which can be a challenge in many assays, as the internal fluid resistance changes over time, altering the voltage drop in different parts of the electrical circuit and thereby changing the voltage at the ESI tip.

[0005] Other research has been pursued using microfluidic devices. Microfluidic devices can provide fluid channels of defined dimensions that can be produced by various known techniques and constitute a network of channels designed to perform different fluid operations. These devices offer an additional level of control and complexity than capillaries, making them a better choice for sample preparation. However, like capillaries, these tools often provide, if limited, characterization of separated analyte fractions prior to introduction into a mass spectrometer. Furthermore, systems involving capillaries or microfluidic devices generally do not provide any 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 are methods, devices, systems, and software for achieving more quantitative characterization of chemical separation data and mass spectrometry data, as well as improved correlation between them. [Overview of the Initiative] [Means for solving the problem]

[0007] In one aspect, what is disclosed herein is a computer implementation method comprising: (a) using a processor to receive a time-series imaging dataset comprising a plurality of images of isoelectric focusing separation performed in a separation channel, wherein each of the plurality of images corresponds to a different time point; (b) using a processor to convert each of the plurality of images into intensity or absorbance measurements as a function of position along the length of the separation channel with respect to the corresponding time point; and (c) using a processor to generate a heatmap or three-dimensional plot of the intensity or absorbance measurements as a function of position along the length of the separation channel and as a function of time.

[0008] In some embodiments, the time-series imaging dataset further comprises multiple images of the mobilization of separated analyte peaks in the separation channel. In some embodiments, the time-series imaging dataset comprises multiple ultraviolet (UV) absorbance images. In some embodiments, the time-series imaging dataset comprises multiple fluorescence images. In some embodiments, the fluorescence images comprise images of innate fluorescence. In some embodiments, the time-series imaging dataset comprises multiple images acquired at a frame rate of at least one image per minute. In some embodiments, the time-series imaging dataset comprises multiple images acquired at a frame rate of at least one image per 30 seconds. In some embodiments, the time-series imaging dataset comprises multiple images acquired at a frame rate of at least one image per 10 seconds. In some embodiments, the method further comprises the step of imaging the separation channel while isoelectric focusing separation is performed, and steps (a)-(c) are performed iteratively as images are acquired. In some embodiments, a heatmap or three-dimensional plot is used to perform one or more tasks selected from the group consisting of steps of comparing isoelectric focusing separation with additional isoelectric focusing separation, comparing a recruitment reaction with isoelectric focusing separation, comparing a recruitment reaction with an additional recruitment reaction, determining the completion of isoelectric focusing separation, monitoring the progress of the recruitment reaction, determining the presence of electroosmotic flow, and determining separation performance parameters. In some embodiments, the separation performance parameter is the separation resolution.

[0009] In another aspect, provided herein is a computer implementation method comprising the steps of (a) using a processor to receive a first dataset comprising (i) a plurality of intensity or absorbance measurements as a function of length along the separation channel from isoelectric focusing electrophoresis separation performed in the separation channel, and (ii) a second dataset comprising a plurality of mass spectrometer total ion measurements as a function of time, (b) using a processor to convert the second dataset into a third dataset comprising ion count measurements as a function of mass, and (c) using a processor to overlay plots of the first dataset and the third dataset.

[0010] In some embodiments, (c) further includes the step of using a processor to overlay a plot of the second dataset with plots of the first and third datasets. In some embodiments, (c) includes the step of undoing the convolution of the second dataset to generate the third dataset. In some embodiments, in (c), a first peak in intensity or absorbance of the first dataset is mapped to a set of peaks in the third dataset. In some embodiments, the second dataset is used to map the first dataset to the set of peaks in the third dataset. In some embodiments, the computer implementation method further includes the step of using a processor to correlate the first peak to a set of peaks and determine the mass distribution and isoelectric point of at least one analyte of the first peak. In some embodiments, the first peak corresponds to an analyte peak and provides information about the isoelectric point of one or more analytes in the analyte peak. In some embodiments, the set of peaks corresponds to the mass distribution of one or more analytes in the analyte peak. In some embodiments, the computer implementation method further includes the step of using a processor to determine the identification of one or more analytes in the analyte peak with respect to a given isoelectric point. In some embodiments, one or more analytes include different protein isoforms. In some embodiments, protein isoforms include different post-translational modifications of proteins. In some embodiments, the overlay plot shows a time series of (i) multiple intensity or absorbance measurements as a function of length along the separation channel and (ii) ion count measurements as a function of mass. In some embodiments, the computer implementation method further includes the step of performing isoelectric focusing separation, recruitment, and electrospray ionization using a single integrated microfluidic device coupled to a mass spectrometer to obtain a first and second dataset. In some embodiments, (b) and (c) are performed within one minute of or in parallel with ESI-MS.In some embodiments, (b) or (c) is performed automatically as part of a software package for obtaining or processing electrospray ionization mass spectrometry (ESI-MS) data.

[0011] In another aspect, what is provided herein is a method comprising the step of assigning post-translational modifications to one or more analytes using (i) mass spectrometry data for one or more analytes and (ii) isoelectric focusing electrophoresis data for one or more analytes.

[0012] In some embodiments, post-translational modifications are selected from the group consisting of hydroxylation, methylation, lipidization, acetylation, disulfide bonding, smoylation, ubiquitination, glycosylation, glycation, amino acid addition or removal, amidation, deamidation, isomerization, oxidation, fucosylation, sialylation, and phosphorylation. In some embodiments, the method further includes the steps of performing isoelectric focusing separation on a mixture of analytes containing one or more analytes to generate isoelectric focusing data, recruiting one or more analytes, and performing electrospray ionization mass spectrometry (ESI-MS) to generate mass spectrometry data. In some embodiments, isoelectric focusing separation and recruitment are performed using a single microfluidic device comprising a separation channel and an integrated electrospray tip. In some embodiments, isoelectric focusing electrophoresis data comprises one or more intensity or absorbance measurements as a function of distance along the separation channel, and the peaks in the intensity or absorbance measurements correspond to analyte peaks comprising one or more analytes having the same given isoelectric point. In some embodiments, the method further includes the step of using mass spectrometry data with respect to a given isoelectric point to distinguish at least one post-translational modification of one or more analytes. In some embodiments, the isoelectric focusing electrophoresis data comprises information on the isoelectric point of one or more analytes, and the mass spectrometry data comprises information on the mass of one or more analytes. In some embodiments, the method further includes the step of using known values ​​of isoelectric point shifts and mass shifts of a plurality of post-translational modifications to assign the post-translational modifications to at least one of one or more analytes. In some embodiments, the assignment of post-translational modifications is performed within one minute of obtaining the ESI-MS data.In some embodiments, isoelectric focusing electrophoresis data comprises information on the isoelectric point of one or more analytes, mass spectrometry data comprises information on the mass of one or more analytes, and post-translational modifications are assigned by matching one or more isoelectric points and one or more masses to a reference comprising multiple known isoelectric point and mass values ​​for multiple post-translational modifications. In some embodiments, the reference comprises publicly available data.

[0013] In another aspect, provided herein is a method for maintaining a constant voltage difference between an electrospray ionization (ESI) tip and a mass spectrometer inlet, the method comprising: (a) applying a first voltage to the proximal end of a separation channel, the distal end of the separation channel being in fluid and electrical communication with the ESI tip; (b) applying a second voltage to the proximal end of an auxiliary fluid channel, the distal end of the auxiliary fluid channel being in fluid and electrical communication with the distal end of the separation channel; (c) carrying out a separation reaction to separate a mixture of analytes, the separation reaction taking place within the separation channel; and (d) maintaining a constant voltage difference between the ESI tip and the mass spectrometer inlet by monitoring changes in the resistance of the separation channel or changes in the voltage at the ESI tip in a feedback loop that modulates a third voltage applied to the mass spectrometer inlet. In some embodiments, the separation channel is the lumen of a capillary tube. In some embodiments, the capillary tube 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 electrophoresis reaction. In some embodiments, the separation reaction comprises an electrophoretic separation reaction. In some embodiments, a first voltage is applied at the cathode coupled to the separation channel, and a second voltage is applied at the anode coupled to the separation channel. In some embodiments, the voltage at the ESI tip or mass spectrometer inlet is held at ground. In some embodiments, the voltage at the mass spectrometer inlet is held at a third voltage. In some embodiments, the third voltage is adjusted by adding a transient voltage change measured at the ESI tip to the third voltage. In some embodiments, the voltage at the ESI tip is measured using a power source. In some embodiments, the power source is coupled to the separation channel. In some embodiments, the power source is coupled to another channel coupled to the separation channel. In some embodiments, the power source is set to 0 microamperes.In some embodiments, the voltage at the ESI tip is measured using an electrode placed at the ESI tip, which is configured to output a current of 0 microamperes. 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 tip within ±10% of a preset value. In some embodiments, the feedback loop maintains the voltage at the ESI tip within ±1% of a preset value. In some embodiments, the feedback loop maintains a constant voltage difference between the ESI tip and the mass spectrometer inlet within ±10% of a preset value. In some embodiments, the feedback loop maintains a constant voltage difference between the ESI tip and the mass spectrometer inlet within ±1% of a preset value.

[0014] In another aspect, what is disclosed herein is a constant voltage difference (ΔV) between the electrospray ionization (ESI) tip and the mass spectrometer inlet. TIP-MS This method is for maintaining (a)ΔV TIP-MS Target value (ΔV) TARGET (b) setting a first voltage at the ESI tip, wherein the ESI tip is in fluid and electrical communication with the isolation channel, and (c) ΔV TIP-MS (d) Using a feedback loop, calculate the instantaneous value related to ΔV TIP-MS =ΔV TARGETincluding periodically or continuously adjusting a second voltage at the mass spectrometer inlet so as to achieve. 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 carried out in the separation channel comprises an isoelectric focusing reaction. In some embodiments, the separation reaction is carried out in the separation channel, and the separation reaction comprises an electrophoresis separation reaction. In some embodiments, the first voltage at the ESI tip or the second voltage at the mass spectrometer inlet is held at ground. In some embodiments, the first voltage at the ESI tip is monitored using an electrode disposed at the ESI tip. In some embodiments, the electrode disposed at the ESI tip is configured to output a current of 0 microamperes. In some embodiments, the first voltage at the ESI tip is monitored using a power supply that is in electrical communication with a fluid channel that intersects the separation channel at a location near the ESI tip and is configured to output a current of 0 microamperes. 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 operates at a frequency of at least 100 Hz. In some embodiments, the feedback loop maintains ΔV TIP-MS within ±10% of ΔV TARGET . In some embodiments, the feedback loop maintains ΔV TIP-MS within ±1% of ΔV TARGET .

[0015] In another aspect, disclosed herein is a computer implementation method for maintaining a constant voltage difference between an electrospray ionization (ESI) tip and the inlet of a mass spectrometer, the method comprising: (a) using a processor to receive a measurement of a first voltage at the ESI tip, the ESI tip being in fluid and electrical communication with a separation channel; (b) using a processor to receive a measurement of a second voltage at the inlet of the mass spectrometer; and (c) using a processor to compare the first voltage with the second voltage, the processor adjusting the voltage at the inlet of the mass spectrometer or the ESI tip if the second voltage is different from the first voltage, such that the difference between the voltage at the ESI tip and the voltage at the inlet of the mass spectrometer remains constant. In some embodiments, the method further comprises (d) using a feedback loop to repeat steps (a)–(c) at a specified frequency. In some embodiments, the separation channel comprises (i) a capillary lumen or (ii) a fluid channel in a microfluidic device. In some embodiments, the capillary tube is equipped with a microvial spray tip. In some embodiments, the separation reaction is carried out in a separation channel, and the separation reaction comprises an isoelectric focusing electrophoresis reaction. In some embodiments, the voltage at the ESI tip or the voltage at the inlet of the mass spectrometer is held at ground. In some embodiments, the voltage at the ESI tip is measured using an electrode placed at the ESI tip. In some embodiments, the voltage at the ESI tip is monitored using a power source that is electrically connected to a fluid channel intersecting the separation channel near the ESI tip and configured to output a current of 0 microamperes. 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.

[0016] Also disclosed herein is a method for maintaining an electrospray ionization (ESI) tip at a constant voltage relative to ground while carrying out a separation reaction, the method comprising: a) applying a first voltage to the proximal end of a separation channel, the distal end of the separation channel being in fluid and electrical communication with the ESI tip; b) applying a second voltage to the proximal end of an auxiliary fluid channel, the distal end of the auxiliary fluid channel being in fluid and electrical communication with the distal end of the separation channel; c) carrying out a separation reaction to separate a mixture of analytes, the separation reaction taking place within the separation channel; and d) monitoring changes in the resistance of the separation channel or changes in the voltage at the ESI tip in a feedback loop that modulates the first and second voltages, and maintaining a constant voltage drop across the separation channel and a constant voltage at the ESI tip. 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 in a microfluidic device. In some embodiments, the separation reaction comprises an isoelectric focusing reaction. In some embodiments, the separation reaction comprises an electrophoretic separation reaction. In some embodiments, a first voltage is applied at the cathode and a second voltage is applied at the anode. In some embodiments, the voltage at the ESI tip is held at ground. In some embodiments, the voltage at the ESI tip is held at a second voltage. In some embodiments, the adjustment of the first and second voltages includes the step of subtracting the transient voltage change measured at the ESI tip from the first and second voltages. In some embodiments, the voltage at the ESI tip is measured using a power source 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 tip within ±10% of a preset value.In some embodiments, the feedback loop maintains the voltage at the ESI tip within ±1% of a preset value. In some embodiments, the feedback loop maintains the voltage drop across the isolation channel within ±10% of a preset value. In some embodiments, the feedback loop maintains the voltage drop across the isolation channel within ±1% of a preset value.

[0017] Also disclosed herein is a method comprising the steps of: a) providing a sample comprising a mixture of two or more analytes; b) performing separation in a fluid channel containing the sample to decompose individual analyte peaks from the mixture of two or more analytes; c) calculating the velocity of the analyte peaks in accordance with the recruitment of the contents of the fluid channel toward the fluid channel exit; and d) using the velocity of the analyte peaks to determine the time it takes for the analyte peaks to reach the fluid channel exit. In some embodiments, the fluid channel is the lumen of a capillary tube. 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 isokinetic electrophoresis (CITP), or micellar electrodynamic chromatography (MEKC). In some embodiments, the velocity of the analyte peaks is calculated from the time interval required for the analyte peaks to move from a first position to a second position. In some embodiments, the first position, second position, and 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 comprises ultraviolet absorbance images, visible light absorbance images, or fluorescence images. In some embodiments, the fluid channel exit comprises an electrospray interface with a mass spectrometer. In some embodiments, the time it takes for the analyte peak to reach the fluid channel exit is used to correlate the mass spectrometer data with the analyte peak. In some embodiments, the recruitment of the contents of the fluid channel comprises the use of electroosmotic recruitment techniques, chemical recruitment techniques, hydrodynamic recruitment 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 with respect to 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 a control parameter related to the separation or recruitment 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.

[0018] Disclosed herein is a method comprising the steps of: a) providing a sample comprising 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; and c) collecting mass spectrometer data relating to two or more individual analyte peaks emitted from the fluid channel via an electrospray interface to a mass spectrometer, wherein the data acquisition mode relating to the mass spectrometer is alternated between high-mass scanning and low-mass scanning. In some embodiments, the mass spectrometer is switched between high-mass scanning and low-mass scanning data acquisition modes at a frequency of at least 0.5 Hz. In some embodiments, the fluid channel is the lumen of a capillary tube. 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 isokinetic electrophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, high-mass scanning captures mass spectral data relating to biomacromolecules. In some embodiments, biomacromolecules 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 high-mass scanning ranges from 1,500 to 6,000. In some embodiments, low-mass scanning captures mass spectral data relating to solution-phase amphoteric electrolytes used when performing isoelectric focusing separation. In some embodiments, the m / z ratio for low-mass scanning ranges from 150 to 1,500. In some embodiments, the mass spectra of one or more solution-phase amphoteric electrolytes are used to calibrate the isoelectric point (pI) relating to the biomacromolecules identified in high-mass scanning.

[0019] Disclosed herein is a method comprising: a) performing separation in a fluid channel containing a sample, wherein the sample comprises a mixture of two or more analytes, and the separation decomposes individual analyte peaks from the mixture of two or more analytes; b) mobilizing the contents of a fluid channel toward a fluid channel exit, wherein the fluid channel exit comprises an electrospray interface to a mass spectrometer; and c) simultaneously or alternately imaging at least a portion of the fluid channel for monitoring the position of the analyte peak between (i)(a) and (b), and (ii) imaging a Taylor cone located between the fluid channel exit and the inlet to the mass spectrometer for monitoring electrospray performance. In some embodiments, the position of the analyte peak in two or more images of at least a portion of the fluid channel is used to calculate the velocity of the analyte peak. In some embodiments, the velocity of the analyte peak is used to determine the time it will take for the analyte peak to reach the fluid channel exit. In some embodiments, the time it takes for the analyte peak to reach the fluid channel exit is used to correlate the mass spectrometer data with the analyte peak. In some embodiments, data derived from imaging of the Taylor cone is used in a feedback loop to adjust the 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 the lumen of a capillary tube. In some embodiments, the fluid channel is part of a microfluidic device. In some embodiments, separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isokinetic electrophoresis (CITP), or micellar electrodynamic chromatography (MEKC). In some embodiments, imaging comprises ultraviolet absorbance imaging, visible light absorbance imaging, or fluorescence imaging. In some embodiments, recruitment of the contents of the fluid channel comprises the use of electroosmotic recruitment techniques, chemical recruitment techniques, hydrodynamic recruitment techniques, or any combination thereof.In some embodiments, 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.

[0020] Disclosed herein is a computer-implemented method for maintaining an electrospray ionization (ESI) tip at a constant voltage relative to ground while a separation reaction is being carried out, the method comprising: a) using a processor to receive a first measurement of voltage at the ESI tip, wherein the distal end of a separation channel is in fluid and electrical communication with the ESI tip; b) using a processor to receive a second measurement of voltage at the ESI tip; c) using a processor to compare the second measurement with the first measurement, wherein if the second measurement differs from the first measurement, the processor adjusts the voltage at the proximal end of the separation channel and the voltage at the proximal end of an auxiliary fluid channel having a distal end in fluid and electrical communication with the distal end of the separation channel, such that the voltage at the ESI tip returns to the first measurement; and d) repeating steps (a)–(b) at a specified frequency. In some embodiments, the separation channel comprises a fluid channel in the lumen of a capillary or a microfluidic device. In some embodiments, the separation reaction comprises an isoelectric focusing reaction. In some embodiments, the separation reaction comprises an electrophoretic separation reaction. In some embodiments, the voltage at the ESI tip is maintained at ground. In some embodiments, the specified frequency is at least 1 Hz. In some embodiments, the voltage at the ESI tip is maintained within ±5% of the specified value.

[0021] Also disclosed herein is a computer-implemented method comprising: a) using a processor to receive image data comprising two or more images obtained using a detector configured to image all or part of a separation channel in a capillary or microfluidic device; b) using the same or different processor to process the image data and determine the position of the analyte peak in the separation channel in two or more images; c) using the same or different processor to calculate the velocity of the analyte peak based on the position of the analyte peak in two or more images and a known time interval between the acquisition of the two or more images; and d) using the same or different processor to determine the time at which the analyte peak will reach the separation channel exit. In some embodiments, the separation reaction carried out in the separation channel comprises isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isokinetic electrophoresis (CITP), or micellar electrodynamic chromatography (MEKC). In some embodiments, two or more images comprise ultraviolet absorbance images, visible light absorbance images, or fluorescence images. In some embodiments, the separation channel outlet is in fluid communication with, or comprises, an electrospray interface to, a mass spectrometer. In some embodiments, the time it takes for the analyte peak to reach the separation channel outlet is used to correlate the mass spectrometer data with the analyte peak. 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 with respect to samples of a biological drug candidate and a reference drug is used to determine biosimilarity. In some embodiments, the rate of the analyte peak is used in a feedback loop to adjust a control parameter relating to the separation reaction carried out in 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. The present invention provides, for example, the following: (Item 1) A computer implementation method, (a) Using a processor, receive a time-series imaging dataset comprising multiple images of isoelectric focusing separation performed in a separation channel, wherein each of the multiple images corresponds to a different time point in time. (b) Using the processor, convert each of the plurality of images into an intensity or absorbance measurement as a function of position along the length of the separation channel with respect to the corresponding time point, (c) Using the processor to generate a heatmap or three-dimensional plot of the intensity or absorbance measurements as a function of position along the length of the isolation channel and as a function of time. Computer implementation methods, including those mentioned above. (Item 2) The computer implementation method according to item 1, further comprising the time-series imaging dataset, a plurality of images of the mobilization of separated analyte peaks in the separation channel. (Item 3) The computer implementation method according to item 1 or item 2, wherein the time-series imaging dataset comprises multiple ultraviolet (UV) absorbance images. (Item 4) The aforementioned time-series imaging dataset comprises multiple fluorescence images, as described in any one of items 1-3, and is a computer implementation method according to item 1-3. (Item 5) The aforementioned fluorescence image comprises a natural fluorescence image, as described in item 4, in the computer implementation method. (Item 6) The computer implementation method according to any one of items 1-5, wherein the time-series imaging dataset comprises multiple images obtained at a frame rate of at least one image per minute. (Item 7) The computer implementation method according to item 6, wherein the time-series imaging dataset comprises multiple images obtained at a frame rate of at least one image per 30 seconds. (Item 8) The computer implementation method according to item 7, wherein the time-series imaging dataset comprises multiple images obtained at a frame rate of at least one image per 10 seconds. (Item 9) A computer implementation method according to any one of items 1-8, further comprising imaging the separation channel while the isoelectric focusing separation is being performed, wherein (a)-(c) are performed iteratively when the images are obtained. (Item 10) A computer implementation method according to any one of items 1-9, wherein the heatmap or the three-dimensional plot is used to perform one or more tasks selected from the group consisting of comparing the isoelectric focusing separation with additional isoelectric focusing separation, comparing the recruitment reaction with the isoelectric focusing separation, comparing the recruitment reaction with an additional recruitment reaction, determining the completion of the isoelectric focusing separation, monitoring the progress of the recruitment reaction, determining the presence of electroosmotic flow, and determining separation performance parameters. (Item 11) The separation performance parameter is the separation resolution, as described in item 10, for the computer implementation method. (Item 12) A computer implementation method, (a) Using a processor, (i) A first dataset comprising a plurality of intensity or absorbance measurements as a function of length along the separation channel from isoelectric focusing electrophoresis separation performed in the separation channel, (ii) A second dataset comprising multiple mass spectrometer total ion measurements as a function of time and Receiving and (b) Using the processor, convert the second dataset into a third dataset comprising ion count measurements as a function of mass, (c) Using the processor, overlay plots of the first dataset and the third dataset. Computer implementation methods, including those mentioned above. (Item 13) (c) The computer implementation method according to item 12, further comprising using the processor to overlay a plot of the second dataset with plots of the first dataset and the third dataset. (Item 14) (c) a computer implementation method according to item 12 or 13, comprising unfolding the convolution of the second dataset to generate the third dataset. (Item 15) (c) The computer implementation method according to any one of items 12-14, wherein a first peak in intensity or absorbance of the first dataset is mapped to a set of peaks of the third dataset. (Item 16) The computer implementation method described in item 15, wherein the second dataset is used to map the first dataset to a set of peaks in the third dataset. (Item 17) The computer implementation method according to item 15, further comprising using the processor to correlate the first peak with the set of peaks and to determine the mass distribution and isoelectric point of at least one analyte of the first peak. (Item 18) The computer implementation method according to item 15, wherein the first peak corresponds to the analyte peak and provides information regarding the isoelectric point of one or more analytes in the analyte peak. (Item 19) The computer implementation method according to item 18, wherein the set of peaks corresponds to the mass distribution of one or more analytes in the analyte peaks. (Item 20) The computer implementation method according to item 19, further comprising using the processor to determine the identification of one or more analytes in the analyte peak with respect to a given isoelectric point. (Item 21) The computer implementation method described in item 20, wherein one or more of the aforementioned analytes include different protein isoforms. (Item 22) The computer implementation method according to item 21, wherein the protein isoform includes different post-translational modifications of the protein. (Item 23) The computer implementation method according to any one of items 12-22, wherein the overlay plot shows a time series of (i) intensity or absorbance measurements among the plurality of intensity or absorbance measurements as a function of length along the separation channel and (ii) ion count measurements as a function of mass. (Item 24) A computer implementation method according to any one of items 12-23, further comprising using a single integrated microfluidic device coupled to a mass spectrometer to perform the isoelectric focusing separation, recruitment, and electrospray ionization to obtain the first and second datasets. (Item 25) (b) and (c) are the computer implementation methods described in item 24, performed within one minute of or concurrently with the ESI-MS. (Item 26) (b) or (c) is a computer implementation method described in any one of items 12–25, which is performed automatically as part of a software package for obtaining or processing electrospray ionization mass spectrometry (ESI-MS) data. (Item 27) (b) and (c) are computer implementations described in any one of items 12-26, which are performed automatically as part of a software package for obtaining or processing electrospray ionization mass spectrometry (ESI-MS) data. (Item 28) It is a method, (i) assigning post-translational modifications to one or more analytes using mass spectrometry data for one or more analytes and (ii) isoelectric focusing electrophoresis data for the one or more analytes. Methods that include... (Item 29) The method according to item 28, wherein the post-translational modification is selected from the group consisting of hydroxylation, methylation, lipidization, acetylation, disulfide bond formation, smoylation, ubiquitination, glycosylation, glycation, amino acid addition or removal, amidation, deamidation, isomerization, oxidation, fucosylation, sialylation, and phosphorylation. (Item 30) The method according to item 28 or 29, further comprising: performing isoelectric focusing separation on a mixture of analytes containing one or more of the aforementioned analytes to generate isoelectric focusing data; recruiting the one or more of the aforementioned analytes; and performing electrospray ionization mass spectrometry (ESI-MS) to generate mass spectrometry data. (Item 31) The method according to item 30, wherein the isoelectric focusing electrophoresis separation and recruitment are carried out using a single microfluidic device comprising a separation channel and an integrated electrospray tip. (Item 32) The method according to any one of items 28-31, wherein the isoelectric focusing electrophoresis data comprises one or more intensity or absorbance measurements as a function of distance along the separation channel, and the peaks in the intensity or absorbance measurements correspond to analyte peaks comprising one or more analytes having the same given isoelectric point. (Item 33) The method according to item 32, further comprising using the mass spectrometry data with respect to a given isoelectric point to distinguish at least one post-translational modification of the one or more analytes. (Item 34) The method according to any one of items 28-33, wherein the isoelectric focusing electrophoresis data comprises information relating to the isoelectric focus of one or more analytes, and the mass spectrometry data comprises information relating to the mass of one or more analytes. (Item 35) The method according to any one of items 28-34, further comprising using known values ​​of isoelectric point shifts and mass shifts of multiple post-translational modifications and assigning the post-translational modifications to at least one of the one or more analytes. (Item 36) The assignment of the post-translational modifications is performed within one minute of obtaining the ESI-MS data, as described in any one of items 28-35. (Item 37) The method according to any one of items 28-36, wherein the isoelectric focusing electrophoresis data comprises information relating to the isoelectric point of one or more of the one or more analytes, the mass spectrometry data comprises information relating to the mass of one or more of the one or more analytes, and the post-translational modification is assigned by matching the isoelectric point and mass of one or more of the one or more to a reference comprising multiple known isoelectric point and mass values ​​of multiple post-translational modifications. (Item 38) The above reference is the method described in item 37, which includes publicly available data. (Item 39) A method for maintaining a constant voltage difference between an electrospray ionization (ESI) tip and a mass spectrometer inlet, (a) Applying a first voltage to the proximal end of the isolation channel, wherein the distal end of the isolation channel is in fluid and electrical communication with the ESI tip, (b) Applying a second voltage to the proximal end of the auxiliary fluid channel, wherein the distal end of the auxiliary fluid channel is in fluid and electrical communication with the distal end of the separation channel, (c) Carrying out a separation reaction to separate the mixture of analytes, wherein the separation reaction is carried out within the separation channel, (d) In a feedback loop that adjusts the third voltage applied to the mass spectrometer inlet, the change in the resistance of the isolation channel or the change in the voltage at the ESI tip is monitored, and the constant voltage difference between the ESI tip and the mass spectrometer inlet is maintained. Methods that include... (Item 40) The separation channel is the lumen of a capillary, as described in item 39. (Item 41) The method according to item 40, wherein the capillary tube is equipped with a microvial spray tip. (Item 42) The method according to item 39, wherein the separation channel is a fluid channel in a microfluidic device. (Item 43) The separation reaction is the method according to any one of items 39-42, comprising an isoelectric focusing electrophoresis reaction. (Item 44) The separation reaction is the method according to any one of items 39-42, comprising an electrophoretic separation reaction. (Item 45) The method according to any one of items 39-44, wherein the first voltage is applied at the cathode coupled to the isolation channel, and the second voltage is applied at the anode coupled to the isolation channel. (Item 46) The method according to any one of items 39-45, wherein the voltage at the ESI tip or the mass spectrometer inlet is held in ground. (Item 47) The method according to any one of items 39-46, wherein the voltage at the mass spectrometer inlet is maintained at the third voltage. (Item 48) The method according to any one of items 39-47, wherein the third voltage is adjusted by adding a transient voltage change measured at the ESI tip to the third voltage. (Item 49) The voltage at the ESI tip is measured using a power source, as described in any one of items 39-48. (Item 50) The power supply is coupled to the isolated channel, as described in item 49. (Item 51) The power supply is coupled to another channel which is coupled to the isolated channel, according to item 50. (Item 52) The power supply is set to 0 microamperes, as described in item 11. (Item 53) The method according to any one of items 39-48, wherein the voltage at the ESI tip is measured using an electrode positioned at the ESI tip, the electrode configured to output a current of 0 microamperes. (Item 54) The feedback loop operates at a frequency of at least 0.1 Hz, as described in any one of items 39-53. (Item 55) The feedback loop operates at a frequency of at least 10 Hz, as described in any one of items 39-53. (Item 56) The feedback loop maintains the voltage at the ESI tip within ±10% of a preset value, as described in any one of items 39-55. (Item 57) The feedback loop maintains the voltage at the ESI tip within ±1% of a preset value, as described in any one of items 39-55. (Item 58) The feedback loop maintains the constant voltage difference between the ESI tip and the mass spectrometer inlet within ±10% of a preset value, as described in any one of items 39-55. (Item 59) The method according to any one of items 39-55, wherein the feedback loop maintains the constant voltage difference between the ESI tip and the mass spectrometer inlet within ±1% of a preset value. (Item 60) A constant voltage difference (ΔV) between the electrospray ionization (ESI) tip and the mass spectrometer inlet. TIP-MS ) A method for maintaining, the method is (a)ΔV TIP-MS Target value (ΔV) TARGET ) setting, (b) Periodically or continuously monitoring a first voltage at the ESI tip, wherein the ESI tip is in fluid and electrical communication with the isolation channel, (c)ΔV TIP-MS Calculating instantaneous values ​​related to, (d) Using a feedback loop, ΔV TIP-MS =ΔV TARGET The second voltage at the mass spectrometer inlet is adjusted periodically or continuously to achieve the following: Methods that include... (Item 61) The separation channel is the lumen of a capillary, according to the method of item 60. (Item 62) The method according to item 61, wherein the capillary tube is equipped with a microvial spray tip. (Item 63) The method according to item 60, wherein the separation channel is a fluid channel in a microfluidic device. (Item 64) The separation reaction carried out in the separation channel is the method according to any one of items 60-63, comprising an isoelectric focusing electrophoresis reaction. (Item 65) The separation reaction carried out in the separation channel is the method according to any one of items 60-63, comprising an electrophoretic separation reaction. (Item 66) The method according to any one of items 60-65, wherein the first voltage at the ESI tip or the second voltage at the mass spectrometer inlet is held in ground. (Item 67) The method according to any one of items 60-66, wherein the first voltage at the ESI tip is monitored using an electrode positioned at the ESI tip. (Item 68) The method according to item 67, wherein the electrode positioned at the tip of the ESI is configured to output a current of 0 microamperes. (Item 69) The method according to any one of items 60-30, wherein the first voltage at the ESI tip is monitored using a power source configured to electrically communicate with a fluid channel intersecting the isolation channel at a location near the ESI tip and to output a current of 0 microamperes. (Item 70) The feedback loop operates at a frequency of at least 0.1 Hz, as described in any one of items 60-69. (Item 71) The feedback loop operates at a frequency of at least 10 Hz, as described in any one of items 60-70. (Item 72) The feedback loop operates at a frequency of at least 100 Hz, as described in any one of items 60-71. (Item 73) The aforementioned feedback loop is ΔV TIP-MS ΔV TARGET Maintain within ±10% of the limit, as described in any one of items 60-72. (Item 74) The aforementioned feedback loop is ΔV TIP-MS Maintain within ±1% of 61, as described in any one of items 60-73. (Item 75) A computer implementation method for maintaining a constant voltage difference between an electrospray ionization (ESI) tip and the inlet of a mass spectrometer, wherein the method is: (a) Using a processor to receive a measurement of a first voltage at the ESI tip, wherein the ESI tip is in fluid and electrical communication with the isolation channel, (b) Using the processor, receive a measurement of the second voltage at the input of the mass spectrometer, (c) Using the processor to compare the first voltage with the second voltage, and if the second voltage is different from the first voltage, the processor adjusts the voltage at the input of the mass spectrometer or the ESI tip such that the difference between the voltage at the ESI tip and the voltage at the input of the mass spectrometer remains constant. Methods that include... (Item 76) (d) A computer implementation method as described in item 75, further comprising repeating steps (a)-(c) at a specified frequency using a feedback loop. (Item 77) The computer implementation method according to item 75 or 76, wherein the separation channel comprises (i) the lumen of a capillary or (ii) a fluid channel in a microfluidic device. (Item 78) The computer implementation method according to item 77, wherein the capillary tube is equipped with a microvial spray tip. (Item 79) A computer implementation method according to any one of items 75-78, wherein the separation reaction is carried out in the separation channel, and the separation reaction comprises an isoelectric focusing electrophoresis reaction. (Item 80) The computer implementation method according to any one of items 75-79, wherein the voltage at the ESI tip or the voltage at the input of the mass spectrometer is held in ground. (Item 81) The computer implementation method according to any one of items 75-80, wherein the voltage at the ESI tip is measured using an electrode installed at the ESI tip. (Item 82) The computer implementation method according to any one of items 75-80, wherein the voltage at the ESI tip is monitored using a power source configured to electrically communicate with a fluid channel intersecting the isolation channel near the ESI tip and to output a current of 0 microamperes. (Item 83) The computer implementation method according to item 76, wherein the feedback loop operates at a frequency of at least 0.1 Hz. (Item 84) The computer implementation method according to item 76 or item 83, wherein the feedback loop operates at a frequency of at least 10 Hz. (Integrated by reference)

[0022] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein by reference as a whole. In the event of any conflict between the terminology herein and the terminology in any incorporated reference, the terminology herein shall prevail. [Brief explanation of the drawing]

[0023] Novel features of the present invention are described in detail in the appended claims. A deeper understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description, which describes illustrative embodiments in which the principles of the present invention are utilized, and the accompanying drawings.

[0024] [Figure 1] Figures 1A-B provide schematic diagrams of a device for isoelectric focusing (IEF) and electrospray ionization (ESI) of an automatically loaded sample according to one embodiment of the present disclosure. Figure 1A shows a schematic diagram of the device. Figure 1B shows another schematic diagram of the device.

[0025] [Figure 2] Figure 2 provides an illustrative flowchart of a computer implementation method for calculating the isoelectric point of a separated analyte band.

[0026] [Figure 3]Figure 3 provides another illustrative flowchart of a computer implementation method for determining the velocity for one or more separated analyte zones and calculating the exit time.

[0027] [Figure 4] Figure 4 provides another illustrative flowchart of a computer implementation method for implementing imaging-based feedback and control of one or more operating parameters for an ESI-MS analysis system.

[0028] [Figure 5] Figure 5 provides a schematic block diagram of hardware components relating to one embodiment of the disclosed system.

[0029] [Figure 6] Figure 6 provides a schematic block diagram of software components relating to one embodiment of the disclosed system.

[0030] [Figure 7] Figures 7A-B illustrate microfluidic devices for use in several embodiments of the present invention. Figure 7A provides a schematic diagram of the fluid channel network of an exemplary microfluidic device. Figure 7B provides a computer-aided design (CAD) drawing of an assembled microfluidic device. The fluid channel layer shown in Figure 7A is sandwiched between two transparent layers to seal the fluid channels.

[0031] [Figure 8] Figure 8 provides images of the Taylor cone and electrospray ionization (ESI) plume during the recruitment of separated samples.

[0032] [Figure 9-1]Figures 9A–F provide non-limiting examples of data regarding sample recruitment following the separation of analytes in a mixture of analytes using isoelectric focusing. Figure 9A shows the absorbance trace at t=0 min (completion of isoelectric focusing). Figure 9B shows the absorbance trace at t=1 min. Figure 9C shows the absorbance trace at t=2 min. Figure 9D shows the absorbance trace at t=3 min. Figure 9E shows the absorbance trace at t=4 min. Figure 9F shows the absorbance trace at t=5 min. [Figure 9-2] Figures 9A–F provide non-limiting examples of data regarding sample recruitment following the separation of analytes in a mixture of analytes using isoelectric focusing. Figure 9A shows the absorbance trace at t=0 min (completion of isoelectric focusing). Figure 9B shows the absorbance trace at t=1 min. Figure 9C shows the absorbance trace at t=2 min. Figure 9D shows the absorbance trace at t=3 min. Figure 9E shows the absorbance trace at t=4 min. Figure 9F shows the absorbance trace at t=5 min.

[0033] [Figure 10] Figures 10A-B provide representative schematics for a microfluidic device designed to perform isoelectric focusing electrophoresis for separating analytes and subsequent mobilization of the separated analyte mixture. Figure 10A provides a representative schematic for the microfluidic device shown in Figure 7A during isoelectric focusing electrophoresis, where the ESI tip will be held at or near ground. Figure 10B shows a representative schematic for the microfluidic device shown in Figure 7A during chemical mobilization of the separated analyte mixture. The resistance of channel 114 (shown in Figure 7A) is assumed to be negligible in this embodiment.

[0034] [Figure 11] Figures 11A-B provide representative data on mobilization while the ESI tip is kept at 0V. Figure 11A shows a plot of voltage as a function of time. Figure 11B shows a plot of current as a function of time.

[0035] [Figure 12] Figure 12 provides an illustrative flowchart of a voltage feedback loop in which the ESI tip is held at +3,000V.

[0036] [Figure 13A] Figures 13A–E provide examples of typical circuit diagrams for the microfluidic devices of this disclosure. Figure 13A provides a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to ground, and the ESI tip will be held at a positive voltage. Figure 13B shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to a third power source, and the ESI tip will be held at a positive voltage. Figure 13C shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a field-effect transistor (FET) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13D shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a bipolar junction transistor (BJT) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13E provides a representative schematic of the microfluidic device shown in Figure 7A during the chemical mobilization of the separated analyte mixture, in which the ESI tip will be held at or near ground. [Figure 13B]Figures 13A–E provide examples of typical circuit diagrams for the microfluidic devices of this disclosure. Figure 13A provides a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to ground, and the ESI tip will be held at a positive voltage. Figure 13B shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to a third power source, and the ESI tip will be held at a positive voltage. Figure 13C shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a field-effect transistor (FET) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13D shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a bipolar junction transistor (BJT) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13E provides a representative schematic of the microfluidic device shown in Figure 7A during the chemical mobilization of the separated analyte mixture, in which the ESI tip will be held at or near ground. [Figure 13C]Figures 13A–E provide examples of typical circuit diagrams for the microfluidic devices of this disclosure. Figure 13A provides a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to ground, and the ESI tip will be held at a positive voltage. Figure 13B shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to a third power source, and the ESI tip will be held at a positive voltage. Figure 13C shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a field-effect transistor (FET) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13D shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a bipolar junction transistor (BJT) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13E provides a representative schematic of the microfluidic device shown in Figure 7A during the chemical mobilization of the separated analyte mixture, in which the ESI tip will be held at or near ground. [Figure 13D]Figures 13A–E provide examples of typical circuit diagrams for the microfluidic devices of this disclosure. Figure 13A provides a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to ground, and the ESI tip will be held at a positive voltage. Figure 13B shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to a third power source, and the ESI tip will be held at a positive voltage. Figure 13C shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a field-effect transistor (FET) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13D shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a bipolar junction transistor (BJT) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13E provides a representative schematic of the microfluidic device shown in Figure 7A during the chemical mobilization of the separated analyte mixture, in which the ESI tip will be held at or near ground. [Figure 13E]Figures 13A–E provide examples of typical circuit diagrams for the microfluidic devices of this disclosure. Figure 13A provides a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to ground, and the ESI tip will be held at a positive voltage. Figure 13B shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which an additional resistor is used to sink the current to a third power source, and the ESI tip will be held at a positive voltage. Figure 13C shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a field-effect transistor (FET) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13D shows a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, in which a bipolar junction transistor (BJT) is used to sink the current, and the ESI tip will be held at a positive voltage. Figure 13E provides a representative schematic of the microfluidic device shown in Figure 7A during the chemical mobilization of the separated analyte mixture, in which the ESI tip will be held at or near ground.

[0037] [Figure 14A] Figure 14-B provides a schematic diagram of a capillary joint sprayer. Figure 14A provides a typical schematic diagram of a capillary joint sprayer. Figure 14B shows a typical resistor circuit diagram related to the capillary joint sprayer diagram in Figure 14A. [Figure 14B] Figure 14-B provides a schematic diagram of a capillary joint sprayer. Figure 14A provides a typical schematic diagram of a capillary joint sprayer. Figure 14B shows a typical resistor circuit diagram related to the capillary joint sprayer diagram in Figure 14A.

[0038] [Figure 15] Figure 15 provides an illustrative flowchart of a computer-controlled voltage feedback loop in which the ESI tip is held at 0V.

[0039] [Figure 16]Panels A and E of Figure 16 provide examples of analyte separation data and corresponding mass spectrometry data for the separated analyte species. Panel A of Figure 16 shows the electrophoresis of the separated analyte mixture. Panel B of Figure 16 shows the mass spectrum of the acidic peak of the separated species. Panel C of Figure 16 shows the mass spectrum of the major peak present in the electrophoresis of Panel A of Figure 16. Panels D and E of Figure 16 show the mass spectra of the two basic peaks from the electrophoresis shown in Panel A of Figure 16.

[0040] [Figure 17] Figures 17A and 17B provide examples of separation data. Figure 17A shows a typical example of the electrophoretic map of the captured isoelectric focusing. Figure 17B provides a typical example of the dynamic heatmap display of separation and recruitment within the separation channel.

[0041] [Figure 18] Figure 18 provides a typical example of a multi-axis plot displaying the electrophoresis diagram of combined isoelectric focusing, the total ion chromatogram of the mass spectrometer, and the individual mass spectra.

[0042] [Figure 19] Figure 19 provides a typical example of a dynamic heatmap display of isolated and recruited data, shown as a three-axis graph with the x-axis plotting distance, the y-axis plotting time, and the z-axis plotting absorbance (arbitrary units).

[0043] [Figure 20] Panels A and B of Figure 20 provide representative examples of monoclonal antibody data in isoelectric focusing and mass spectrometry. Panel A of Figure 20 provides isoelectric focusing data and mass spectral chromatograms of charge deformation. Panel B of Figure 20 shows an example of unconvoluted mass data.

[0044] [Figure 21]Panels A and B of Figure 21 provide tables illustrating exemplary post-translational modifications and the expected changes in protein mass and charge. Panel A of Figure 21 provides a representative table listing post-translational modifications and the expected changes in protein mass and charge resulting from the modifications. Panel B of Figure 21 provides representative examples of modifications that result in the same mass change but may have different effects on protein charge.

[0045] [Figure 22] Panels A and B of Figure 22 provide examples of mass spectra. Panel A of Figure 22 provides a representative example of a deconvolved mass spectrum obtained from the analysis of charge deformations separated by isoelectric focusing. Panel B of Figure 22 provides another representative example of a deconvolved mass spectrum obtained from the analysis of charge deformations separated by isoelectric focusing.

[0046] [Figure 23] Panels A and B of Figure 23 provide representative examples of comparing the unfolded masses of major protein charge deformation versus acidic and basic deformation. Panel A of Figure 23 provides examples of the unfolded masses of major protein charge deformation and acidic deformation. Panel B of Figure 23 provides examples of the unfolded masses of major protein charge deformation and basic deformation. [Modes for carrying out the invention]

[0047] Detailed explanation Several embodiments described herein relate to innovative software and systems for analyzing data from capillary and microfluidic-based separation systems integrated with mass spectrometry detection and directing their operation. In some embodiments, the analyte is imaged during separation in a capillary or on a microfluidic device, and its molecular weight or mass / charge ratio is measured in a mass spectrometer after separation. The disclosed methods, devices, systems, and software provide more accurate characterization of separated analyte peaks and achieve improved correlation between chemical separation data and mass spectrometry (MS) data. Also disclosed are methods, devices, systems, and software for improving the quality of electrospray ionization mass spectrometry (ESI-MS) data. The disclosed methods, devices, systems, and software have potential applications in a variety of fields, including, but are 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 used for characterizing biologics and biosimilars during development and / or manufacturing, as will be discussed in more detail below. Biologics and biosimilars are a class of drugs that include, for example, recombinant proteins, antibodies, live viral vaccines, human plasma-derived proteins, cell-based drugs, naturally occurring proteins, antibody-drug conjugates, protein-drug conjugates, and other protein-based drugs.

[0048] Microfluidic devices are described that are designed to perform any of a variety of chemical separation techniques and also have 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 biomolecules. In another preferred embodiment, the disclosed device is designed to be used in conjunction with imaging techniques. Devices and methods for integrating imaged microfluidic separation with mass spectrometry have been previously described, for example, in the published PCT Patent Application Publication WO 2017 / 095813 and U.S. Patent Application Publication US 2017 / 0176386 (incorporated herein by reference for all purposes). These applications, among other things, describe systems for performing separation imaged in conjunction with MS analysis. Such microfluidic systems represent a significant advance in biopharmaceutical characterization. However, in order for such systems to provide the greatest benefit, it would be beneficial to have innovative software and systems, as disclosed herein, to assist in the operation of these systems and the downstream integration of imaged and MS data.

[0049] Therefore, in preferred embodiments, the disclosed microfluidic device may be used in combination with imaging techniques to accurately determine, for example, the isoelectric point (pI) of one or more analytes isoelectrically separated from the analyte mixture in the separation channel, forming a series of concentrated fractions containing substantially pure individual analyte components (also referred to herein as “peaks” or “bands”). Imaging all or part of the separation channel allows for the determination of the locations of two or more pI standards (or pI markers) injected with the sample to be separated, thus enabling the calculation of a more accurate pI for each separated analyte peak by extrapolation and the determination of 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 optionally, the determination of the isoelectric point of one or more of the analytes being separated is performed and iteratively updated while the separation is being carried out. In some embodiments, the imaging-based determination of the isoelectric point of one or more isoelectrically focused analytes is performed after the separation is complete. In some embodiments, the imaging-based determination of the isoelectric point for one or more isoelectrically focused analytes is performed after separation is complete and before the separated analyte mixture is recruited toward the electrospray tip. 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, the determination of the isoelectric point for one or more analyte peaks may be performed by a computer-implemented method.

[0050] In another preferred embodiment, the disclosed microfluidic device may be used in combination with an imaging technique to image the separated analyte peaks after the recruitment of the separated analyte mixture, i.e., as the peaks move out of the separation channel toward the electrospray tip. In some embodiments, the imaged recruitment step is the same step as the imaged separation step, such as when implementing a separation step comprising capillary gel electrophoresis, capillary zone electrophoresis, isokinetic electrophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow-balanced capillary electrophoresis, or any other separation technique that separates the components of the analyte mixture by velocity difference. In some embodiments, the imaged recruitment step would be analyzed to correlate the enriched fraction in the imaged separation with a mass spectrum. Imagery of recruited analyte peaks may be used, for example, to determine the velocity of one or more analyte peaks based on their positions in a series of recruited images, which may then be used to determine the time at which the analyte peaks will exit the separation channel or be emitted by the electrospray tip, and thus may be used to correlate mass spectrometer data with specific analyte peaks. In some cases, the velocity of an analyte peak is calculated from the time interval required for the analyte peak to move a certain displacement value (e.g., from a first position to a second position). In some embodiments, imagery of recruited analyte peaks may allow direct monitoring of the peaks as they progress through the fluid channel and are emitted by the electrospray tip, and thus may be used to directly correlate mass spectrometer data with specific analyte peaks. In some embodiments, the image-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, the determination of the rates for one or more analyte peaks, their actual or predicted separation channel exit times, and / or their electrospray release times may be performed by a computer implementation method.

[0051] In some embodiments, the recruitment of separated analyte peaks may be initiated by a change in the electric field or flow parameters in the microfluidic device. In some embodiments, one or more electrodes connecting a power source to the microfluidic device will be connected or disconnected through a computer-implemented method to initiate recruitment. In some embodiments, the Taylor cone formed at the electrospray tip may be imaged during the recruitment step. In some embodiments, computer-implemented image analysis 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 of the operating parameters known to affect electrospray performance will be adjusted to restore stable electrospray operating conditions. Examples of operating parameters that can be adjusted include, but are not limited to, electrophoretic voltage, flow rate, distance from the electrospray tip to the MS inlet, MS voltage, and equivalents. In some embodiments, a computer-implemented method may be used to adjust the electrospray parameters.

[0052] In some embodiments, more than one power source may be used to generate the electrophoretic electric field. In some embodiments, two power sources having positive polarity may be used. In some embodiments, one or more power sources may have negative polarity. In some embodiments, the voltage settings for the power sources may be changed simultaneously to maintain the same voltage gradient in the separation channel for electrophoretic separation. In some embodiments, the voltage settings for the power sources may be changed to maintain a constant voltage at the electrospray tip. In some embodiments, the multiple power sources may be different channels in a single multi-channel power source. In some embodiments, isoelectric focusing may be performed in the separation channel, and the resistance in the channel may increase over time. In some embodiments, chemical recruitment may be performed in the separation channel, and the resistance in the channel may decrease over time. In some embodiments, pressure-driven recruitment may be performed, and the resistance in the channel may change over time as new reagent is pushed into the channel. In some embodiments, the electrospray tip may be kept at ground. In some embodiments, the electrospray tip may be kept at a specific voltage relative to the mass spectrometer. In some embodiments, the electrospray tip may be maintained at a specific voltage relative to ground. In some embodiments, a computer implementation method may regulate the voltage to maintain a constant electric field strength in the isolation channel (or a constant voltage drop between the anode and cathode) and a constant voltage at the electrospray tip. In some embodiments, the voltage at the tip may be measured using a voltmeter. In some embodiments, the voltage at the tip may be measured using electrodes located at or inside the tip. In some embodiments, an additional power source may be set to 0 μA using current control and used as a voltmeter for reading the tip voltage.In some embodiments, a computer implementation method would read the voltage value at the tip and adjust the voltage to maintain a constant electric field strength (or a constant voltage drop between the anode and cathode) in the isolation channel and to maintain a constant voltage at the tip. In some embodiments, a computer implementation method would calculate the voltage at the ESI tip based on the current flow through the isolation electric field circuit. In some embodiments, the voltage drop across the isolation channel is adjusted so that a constant or maximum power is applied in the isolation channel, and the power applied in the isolation channel is calculated as follows: Power = Voltage across the isolated channel × Current in the isolated channel The current can be measured continuously or periodically during isolation, and the current measurement can be used to regulate the voltage across the isolation channel. This method of controlling power in the isolation channel may be useful for managing temperature effects in the isolation channel.

[0053] In some embodiments, the separation path is a linear coated or uncoated capillary, tube, or line of a certain length, and the inlet will be inserted into a vial containing an acidic anode and positive electrode or a basic cathode and negative electrode. In some embodiments, the outlet of the separation path will be inserted into a junction sprayer. In some embodiments, the junction sprayer houses both T-tubes for a secondary tube, line, or capillary that can introduce another conductive supplement fluid to the capillary outlet, providing liquid-liquid electrical contact and liquid flow to assist electrospray and transport the analyte emerging from the separation channel to the tip for introduction into a mass spectrometer by electrospray ionization. In some embodiments, the system may be configured with an anode and positive electrode at the separation path inlet, and the junction or distal portion of the separation path may be loaded with cathode fluid immediately before focusing. After focusing is complete, a recruiter with competing anions may be introduced into the junction by either hydrodynamic force or electroosmotic force. In some embodiments, the separation channel inlet may be immersed in a vial containing the cathode solution and negative electrode, and the junction or distal portion of the capillary may be loaded with the anode solution immediately before focusing. After focusing is complete, a recruiter with competing cations may be introduced into the junction by either hydrodynamic force or electroosmotic force. In some embodiments, the separation channel will be a linear capillary of a certain length, with one end inserted into an anode solution reservoir connecting the capillary to the positive electrode and the other end inserted into a cathode solution reservoir connecting the capillary to the negative electrode for isoelectric focusing. In some embodiments, after focusing, the cathode solution end of the capillary will be removed from the cathode solution and inserted into a junction sprayer (e.g., a microvial sprayer) adjacent to the mass spectrometer, as shown in Figure 14A. In some embodiments, the junction sprayer may provide a certain volume of recruiter to charge the analyte in ESI and to mobilize the focused analyte. In some embodiments, the joining sprayer may provide electrical connections to complete the mobilization circuit.In some embodiments, the voltages in the anodelime and junction sprayer will be adjusted so that the change in voltage (ΔV) or electric field between the anodelime and junction sprayer remains constant, and the voltage at the ESI tip remains constant. In some embodiments, the ΔV or electric field between the anodelime and junction sprayer may increase or decrease over time, and the voltage at the ESI tip may fluctuate. In such cases, the voltage or potential applied to the mass spectrometer inlet will be the voltage difference (ΔV) between the ESI tip and the mass spectrometer inlet. TIP-MS ) may be adjusted so that it remains constant.

[0054] In some embodiments, the separation channel (e.g., a capillary) comprises a microvial that can facilitate the transfer of mobilized effluent to the ESI. The microvial may be part of the capillary or may be added to and / or fused to the separation channel. The microvial may be part of the ESI tip. In some cases, the microvial may comprise or be part of a joining sprayer. The microvial may provide a fluid channel (e.g., for sheath fluid) in part of the channel or at the ESI tip.

[0055] In some embodiments, one power source may be connected to a resistor to create a current sink. In some embodiments, the resistor may sink the current by connecting the electrophoretic 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 capable of providing a path for sinking current. In some embodiments, the current sink may be an FET, which may be controlled to function as an open circuit or short circuit when it is required or necessary to provide a constant current flow through the FET. In some embodiments, a bipolar junction transistor (BJT) may be used for the current sink function. In some embodiments, the resistor may sink the current by connecting the electrophoretic circuit to a current sink power source. In some embodiments, the voltage setting of the current sink power source will be adjusted as the resistance in the isolation channel changes over time. In some embodiments, the voltage to the current sink power source will be adjusted to maintain a constant current across the resistor. In some embodiments, a resistor, a set of resistors, a resistor circuit, or an equivalent may be used as a current sink.

[0056] In some embodiments, the scanned mass / charge (m / z) range may be changed during the recruitment / ESI step. In some embodiments, a computer implementation method may be used to switch between high and low m / z ranges. In some embodiments, a mass spectrum within one m / z range may be used as an internal standard for separating analytes in different mass ranges. This spectrum may contain data on amphoteric electrolytes with free solution isoelectric gradients, which can be used as a standard for isoelectric point (pI), or it may contain data on electrophoretic mobility standards, which can be used as a standard in electrophoresis, e.g., capillary zone electrophoresis. In some cases, this spectrum may contain data on any molecules that can be degraded in the separation step, e.g., by pI, charge-to-mass ratio, evaluation through gel, electrophoretic mobility, etc., which are in a different mass range from the analyte of interest.

[0057] In some embodiments, correlation between charge deformation peaks and mass spectral data may allow for the identification of post-translational modifications or other protein or peptide modifications. For example, a mass difference between two molecules may be detected during mass spectrometer analysis. In some cases, multiple modifications may exist that could result in the detected mass difference. In some cases, it may be known that certain modifications cause a specific charge shift (or change in isoelectric point) related to the molecule. In some cases, grasping the charge shift associated with the mass difference may allow for the exclusion of a particular modification or set of modifications. In some embodiments, grasping the charge shift and detecting identical or similar masses (within the mass accuracy limits of the mass spectrometer) may allow for the exclusion of a particular modification or set of modifications. In some cases, grasping the charge shift and detecting identical or similar masses (within the mass accuracy limits of the mass spectrometer) may allow for the assignment of a particular modification or set of modifications to a molecule. In some cases, grasping the charge shift associated with the mass difference may allow for the assignment of a particular modification or set of modifications to a molecule.

[0058] The system of this disclosure includes: (i) a capillary or microfluidic device designed to perform analyte separation, e.g., isoelectric focusing-based separation, providing 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 capillary or microfluidic device-based analyte separation with image acquisition; (vi) software for processing images and determining the position 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 pI standards and analyte peaks toward the electrospray tip; (vii) software for processing images and determining the rate, exit time, and / or electrospray release time for one or more pI standards or analyte peaks; and (viii) images of the separation channel for monitoring the position of analyte peaks and a Taylor present between the electrospray tip and the inlet to the mass spectrometer for monitoring electrospray performance. (ix) Software for simultaneously or alternately capturing images of the cone, software for processing images of the Taylor cone and adjusting one or more of the position of the electrospray tip relative to the mass spectrometer inlet, the fluid flow through the electrospray tip, the voltage between the electrospray tip and the mass spectrometer, or any combination thereof, to affect changes in the quality of the mass spectrometer data, (x) Software for controlling the collection of mass spectrometer data relating to individual analyte peaks emitted from the electrospray interface, wherein the data collection mode relating to the mass spectrometer is alternated between high-mass scanning and low-mass scanning, software, (xi) Software for reading the voltage at the electrospray tip and / or the mass spectrometer inlet, (a) adjusting the isolation channel voltage to maintain a constant electric field strength in the channel (or a constant voltage drop between the anode and cathode) while maintaining a constant voltage at the tip, and / or (b) adjusting the voltage applied to the mass spectrometer inlet to maintain a constant voltage between the tip and the mass spectrometer inlet,Alternatively, the system may include one or more software for adjusting any combination thereof. In some embodiments, the system may include an integrated system in which the selection of these functional components is packaged in a fixed configuration. In some embodiments, the system may include a modular system in which the selection of functional components can be changed to reconfigure the system for new applications. In some embodiments, some of these functional system components, such as capillaries or microfluidic devices, are replaceable or disposable components.

[0059] Please understand that both the general overview above and the following description are illustrative and explanatory only, and do not limit the methods and devices described herein.

[0060] Definitions: Unless otherwise defined, all technical terms used herein have the same meaning as those commonly understood by those skilled in the art within the field to which this disclosure belongs.

[0061] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context otherwise clearly determines. Any reference to “or” herein is intended to include “and / or” unless otherwise stated. Similarly, the phrases “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are not intended to be restrictive.

[0062] As used herein, the term “about” refers to that number ± 10% of that number. When used in the context of a range, the term “about” refers to that range - 10% of its lowest value + 10% of its highest value.

[0063] Analytes: As described above, the disclosed methods, devices, systems, and software enable more accurate characterization of separated analyte peaks and improved correlation between chemical separation data and mass spectrometry data. In some cases, these analytes 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 complexes, antibodies, antibody fragments, protein-drug complexes, peptides, metabolites, organic compounds, or other biologically relevant molecules, or any combination thereof. In some cases, these analytes may be small molecule drugs. In some cases, these analytes may be protein molecules in protein mixtures such as biological protein preparations and / or lysates collected from cells cultured or isolated in vivo.

[0064] Samples: The disclosed methods, devices, systems, and software may be used for the separation and characterization of analytes 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, or capillary blood samples), plasma, serum, saliva, interstitial fluid, urine, sweat, tears, protein samples derived from industrial enzyme or biological drug manufacturing processes, environmental samples (e.g., air samples, water samples, soil samples, surface swipe samples), and equivalents. In some embodiments, the sample may be treated using any of a variety of techniques known to those skilled 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 treated 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.

[0065] Sample Volume: In some embodiments of the disclosed methods and devices, miniaturization, which can be achieved 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 up to 1 ml, up to 750 μl, up to 500 μl, up to 250 μl, up to 100 μl, up to 75 μl, up to 50 μl, up to 25 μl, up to 10 μl, up to 7.5 μl, up to 5 μl, up to 2.5 μl, up to 1 μl, or up to 0.1 μl. Any of the lower and upper limits described in this paragraph may be combined to form a range included in this disclosure, for example, in some embodiments, the sample volume used for analysis may range from about 5 μl to about 500 μl. Those skilled in the art will recognize that the sample volume used for analysis may have any value within this range, for example, about 10 μl.

[0066] Separation Techniques: The disclosed methods, devices, systems, and software may utilize any of the various analyte separation techniques known to those skilled in the art. For example, in some embodiments, the separation being imaged may be electrophoretic separation such as isoelectric focusing, capillary gel electrophoresis, capillary zone electrophoresis, isokinetic electrophoresis, capillary electrodynamic chromatography, micellar electrodynamic chromatography, flow-balanced capillary electrophoresis, electric field gradient focusing, dynamic field gradient focusing, and equivalents, which produce one or more separated analyte fractions from the analyte mixture.

[0067] Capillary Isoelectric Focusing (CIEF): In some embodiments, the separation technique may include isoelectric focusing (IEF), such as capillary isoelectric focusing (CIEF). Isoelectric focusing (or "focused electrophoresis") is a technique for separating molecules by their isoelectric points (pI), i.e., the pH difference at which they have a net zero charge. CIEF involves adding an amphoteric electrolyte (amphoteric electrolyte) solution to a sample channel between reagent reservoirs containing an anode or cathode, and generating a pH gradient within the separation channel (i.e., a fluid channel connecting electrode-containing wells) across which a separation voltage is applied. The amphoteric electrolyte can be in solution phase or immobilized on the surface of the channel wall. Negatively charged molecules migrate through the pH gradient in the medium toward the positive electrode, while positively charged molecules migrate toward the negative electrode. Proteins (or other molecules) within a pH range below their isoelectric point (pI) will be positively charged and therefore will migrate toward the cathode (i.e., the negatively charged electrode). The overall net charge of a protein will decrease as it migrates through an increasing pH gradient (e.g., due to protonation of carboxyl groups or other negatively charged functional groups) until it reaches the pH range corresponding to its pI, at which point it has no net charge and therefore migration ceases. As a result, the mixture of proteins separates based on their relative content of acidic and basic residues, converging into a steep steady-state band where each protein is positioned at a point in the pH gradient corresponding to its pI. This technique allows for extremely high resolution, where proteins are fractionated into distinct bands based on a single charge. In some embodiments, isoelectric focusing may be performed in separation channels permanently or dynamically coated, for example, with neutral and hydrophilic polymer coatings, to eliminate electroosmotic flow (EOF).Examples of suitable coatings include, but are not limited to, amino-modifying factors, hydroxypropyl cellulose (HPC) and polyvinyl alcohol (PVA), Guarant® (Alcor Bioseparations), linear polyacrylamide, polyacrylamide, dimethylacrylamide, polyvinylpyrrolidine (PVP), methylcellulose, hydroxyethylcellulose (HEC), hydroxypropyl methylcellulose (HPMC), triethylamine, propylamine, morpholine, diethanolamine, triethanolamine, diaminopropane, ethylenediamine, chitosan, polyethyleneimine, cadaverine, putrescine, spermidine, diethylenetriamine, tetraethylenepentamine, cellulose, dextran, This includes polyethylene oxide (PEO), cellulose acetate, amylopectin, ethylpyrrolidine methacrylate, dimethyl methacrylate, didodecyldimethylammonium bromide, Brij35, sulfobetaine, 1,2-dilauroyl sn-phosphatidylcholine, 1,4-didecyl-1,4-diazoniabicyclo[2,2,2]octanedibromide, agarose, poly(N-hydroxyethylacrylamide), POL-323, hyperbranched polyaminoesters, pullulan, glycerol, adsorption coating, covalent coating, dynamic coating, etc. In some embodiments, isoelectric focusing may be performed using additives in the separation medium such as methylcellulose, glycerol, urea, formamide, and surfactants (e.g., Triton®-X100, CHAPS, digitonin) to significantly reduce electroosmotic flow, enable better protein solubilization, and limit diffusion inside the capillaries of the fluid channels by increasing the viscosity of the electrolyte (e.g., in uncoated separation channels).

[0068] As described above, the pH gradient used for capillary isoelectric focusing techniques is generated through the use of amphoteric electrolytes, i.e., amphoteric molecules that contain both acidic and basic groups and exist mostly as zwitterions within a certain pH range. The portion of the electrolyte solution on the anode side of the separation channel is known as the "anodic solution." The portion of the electrolyte solution on the cathode side of the separation channel is known as the "cathode solution." A variety of electrolytes, 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, may be used in the disclosed methods and devices. The electrolytes may be used in any preferred concentration such as 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. The electrolyte concentration may be at least 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The electrolyte concentration may be up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, 0.01%, 0.001%, or 0.0001%. A range of electrolyte concentrations, for example, 0.1% to 2%, may be used. The amphoteric electrolyte is any commercially or non-commercial carrier amphoteric electrolyte mixture (e.g., Servalyt pH4-9 (Serva, Heildelberg, Germany), Beckman pH3-10 (Beckman Instruments, Fullerton, CA, USA), Ampholine 3.5-9.5 and Pharmalyte 3-10 (both from General Electric Healthcare, Orsay, France), AESlytes (AES), FLUKA amphoteric electrolyte (Thomas Scientific, Swedesboro, NJ), Biolyte (Bio-Rad, Hercules, CA)), and equivalents can be selected. The carrier amphoteric electrolyte mixture may also contain a mixture of small molecules (about 300-1,000 Da) containing multiple aliphatic amino and carboxylate groups, having closely spaced pI values ​​and good buffer capacity. In the presence of an applied electric field, the carrier amphoteric electrolyte is divided into a smooth linear or nonlinear pH gradient that gradually increases from anode to cathode.

[0069] Any of the various pI standards may be used in the disclosed methods and devices to calculate the isoelectric point for the separated analyte peak. 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 with a generally accepted pI value. In some cases, the pI marker may be detectable, for example, by imaging. Various 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, a peptide library designed by Shimura, and Slais dyes (Alcor Biosepartions), may be used.

[0070] Mobilization Technique: In some embodiments, for example, isoelectric focusing electrophoresis is employed, in those cases the separated analyte band may be mobilized toward the end of the separation channel that interfaces with an electrospray ionization interface to a downstream analytical device, such as a mass spectrometer. In some embodiments, for example, capillary gel electrophoresis, capillary zone electrophoresis, isokinetic electrophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow-balanced capillary electrophoresis, or any other separation technique that separates components of an analyte mixture by velocity difference is employed, in those cases the separation step may be considered a mobilization step.

[0071] In some embodiments, analyte beam mobilization may be implemented by applying hydrodynamic pressure to one end of the separation channel. In some embodiments, analyte beam mobilization may be implemented by orienting the separation channel to a vertical position so that gravity can be employed. In some embodiments, analyte beam mobilization may be implemented using EOF-assisted mobilization. In some embodiments, analyte beam mobilization may be implemented using chemical mobilization. In some embodiments, any combination of these mobilization techniques may be employed.

[0072] In one embodiment, the recruitment step relating to an isoelectrically focused analyte band includes chemical recruitment. Compared to pressure-based recruitment, chemical recruitment has the advantage of exhibiting minimal band spreading by overcoming the hydrodynamic parabolic flow profile induced by the use of pressure. Chemical recruitment may be implemented by introducing either the inlet or outlet of a separation pathway containing a fully or partially focused pH gradient for electrophoresis into the separation pathway into a conductive solution containing ions that compete with either hydronium or hydroxyl. This results in stepwise electrokinetic displacement of the pH gradient components by disturbing a near-zero net charge state. In the case of cathode chemical recruitment, the supply of hydroxyl, which is the cathode solution, may be replaced with a recruitment solution containing the competing anions. The competing anions can cause a decrease in pH in the separation pathway that develops a positive charge relative to the pH gradient components, allowing them to migrate toward the cathode. Correspondingly, in anode mobilization, the supply of hydronium, which is the anode solution, is replaced with a mobilization solution containing competing cations, which increases the pH of the separation, developing the negative charge of the pH gradient components and allowing them to migrate toward the anode. In some embodiments, cathode mobilization may be initiated using an acidic electrolyte such as formic acid, acetic acid, carbonic acid, phosphoric acid, and equivalents at any preferred concentration. In some embodiments, anode mobilization may be initiated using a basic electrolyte such as ammonium hydroxide, dimethylamine, diethylamine, piperidine, sodium hydroxide, and equivalents. 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.

[0073] In a preferred embodiment, the chemical recruitment step may be initiated within a microfluidic device designed to integrate CIEF with ESI-MS by altering the electric field within the device to electrophores the recruited electrolyte into a separation channel. In some embodiments, the change in the electric field may be implemented by connecting or disconnecting one or more electrodes attached to one or more power sources, the one or more electrodes being located in a reagent well on the device or integrated with the fluid channel of the device. In some embodiments, the connecting or disconnecting of one or more electrodes may be controlled using a computer implementation method and programmable switches so that the timing and duration of the recruitment step can be coordinated with the separation step, the electrospray ionization step, and / or mass spectrometry data acquisition. In some embodiments, the disconnection of one or more electrodes from the separation circuit may be implemented using current control, by setting the current to 0 μA.

[0074] Capillary zone electrophoresis (CZE): In some embodiments, the separation technique may include capillary zone electrophoresis, a method for separating charged analytes in a solution within an applied electric field. The net velocity of charged analyte molecules is the electroosmotic flow (EOF) mobility exhibited by the separation system for each analyte (depending on the size, shape, and charge of the molecule), i.e., μ, such that analyte molecules exhibiting different sizes, shapes, or charges exhibit differences in migration velocity and separate into bands. EOF , and electrophoretic mobility, i.e., μ EP It is affected by both.

[0075] Capillary gel electrophoresis (CGE): In some embodiments, the separation technique may include capillary gel electrophoresis, a method for the separation and analysis of macromolecules (e.g., DNA, RNA, and proteins) and their fragments based on their size and charge. The method involves the use of a gel-packed separation channel, in which the gel acts as a countercurrent and / or sieve medium during the electrophoretic migration of charged analyte molecules in an applied electric field. The gel functions to suppress thermal convection caused by the application of the electric field and also acts as a sieve medium that slows the passage of molecules, thereby resulting in differences in migration velocities for molecules of different sizes or charges.

[0076] Capillary isophatic electrophoresis (CITP): In some embodiments, the separation technique may include capillary isophatic electrophoresis, a method for separating charged analytes that uses a discontinuous system of two electrolytes (known as a leading electrolyte and a terminal electrolyte) in a capillary or fluid channel of suitable dimensions. The leading electrolyte may contain ions with the highest electrophoretic mobility, while the terminal 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 the application of an electric field results in the separation of charged analyte molecules in the capillary or fluid channel into closely adjacent zones in order of decreasing electrophoretic mobility. The zones move at a constant speed within the applied electric field so that a detector, e.g., a conductivity detector, a photodetector, or an imaging device, can be used to record their passage along the separation channel. Unlike capillary isoelectric electrophoresis, simultaneous determination or detection of anionic and cationic analytes is not possible in a single analysis performed using capillary isoelectric electrophoresis.

[0077] Capillary electrokinetic chromatography (CEC): In some embodiments, the separation technique may include capillary electrokinetic chromatography, a method for separating analyte mixtures based on a combination of liquid chromatography and electrophoretic separation methods. CEC offers both the efficiency of capillary electrophoresis (CE) and the selectivity and sample volume of packed capillary high-performance liquid chromatography (HPLC). Because the capillaries used in CEC are packed with HPLC packing material, the wide variety of analyte selectivity available in HPLC is also available in CEC. The large surface area of ​​these packing materials allows CEC capillaries to accommodate relatively large sample volumes, making the subsequent detection of eluted analytes a somewhat simpler task than in capillary zone electrophoresis (CZE).

[0078] Micelle electrokinetic chromatography (MEKC): In some embodiments, the separation technique may include micelle electrokinetic chromatography, a method for separating analyte mixtures based on differential separation between surfactant micelles (pseudosteady phase) and a surrounding aqueous buffer solution (mobile phase). In MEKC, the buffer solution may contain the surfactant at a concentration above the critical micelle concentration (CMC) so that the surfactant monomer is in equilibrium with the micelles. MEKC may be carried out in an open capillary tube 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, the anionic sulfate group of SDS gives the surfactant and micelles electrophoretic mobility opposite to the direction of the strong electroosmotic flow. As a result, the surfactant monomer and micelles migrate slowly, but their net migration is still in the direction of the electroosmotic flow, i.e., toward the cathode. During MEKC separation, the analyte may be distributed between the hydrophobic interior of the micelle and the hydrophilic buffer solution. The hydrophilic analyte, which is insoluble inside the micelle, is then subjected to electroosmotic flow rate u o It migrates in the buffer solution and remains there for a certain amount of time. M It will be detected in the micelle. Hydrophobic analytes that are completely solubilized within the micelle will be detected at the micelle velocity uc It migrates in the area, and the final elution time t c It elutes in that state.

[0079] Flow-balanced capillary electrophoresis (FCCE): In some embodiments, the separation technique may include flow-balanced capillary electrophoresis, a method for increasing the efficiency and resolution of capillary electrophoresis by utilizing pressure-induced backflow to actively slow, stop, or reverse the electrokinetic migration of the analyte through the capillary. By slowing, stopping, or moving the analyte back and forth across the detection window, the analyte of interest can be effectively confined in the separation channel for a much longer time period than under normal separation conditions, thereby increasing both the efficiency and resolution of the separation.

[0080] Separation Time and Resolution: Generally, the separation time required to achieve complete separation will vary depending on the specific separation technique used and operating parameters (e.g., separation channel length, microfluidic device design, buffer composition, applied voltage, etc.). In some embodiments, the software will determine when separation is complete based on an imaging-based analysis of the analyte peak, as described in concurrently pending U.S. Patent Application No. 16 / 261,382. In some embodiments, the separation time may range from about 0.1 minutes to about 30 minutes. In some embodiments, the separation time may be at least 0.1 minutes, at least 0.5 minutes, 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 up to 30 minutes, up to 25 minutes, up to 20 minutes, up to 15 minutes, up to 10 minutes, up to 5 minutes, up to 1 minute, up to 0.5 minutes, or up to 0.1 minutes. Any of the lower and upper limits described in this paragraph may be combined to form a range included in this disclosure, for example, in some embodiments the isolation time may range from about 1 minute to about 20 minutes. The isolation time may have any value within this range, for example, about 7 minutes.

[0081] Similarly, the separation efficiency and resolution achieved using the disclosed methods and devices may vary depending on the specific separation technique and operating parameters used (e.g., separation channel length, microfluidic device design, buffer composition, applied voltage, etc.). In some embodiments, the achieved separation efficiency (e.g., number of theoretical stages) may range from approximately 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 may vary depending on the properties of one or more of the analytes in the mixture (e.g., molecular mass, diffusivity, electrophoresis, or isoelectric mobility).

[0082] Microfluidic Device Design and Fabrication: In some embodiments of the methods, devices, and systems disclosed, the separation of analytes from a mixture, and optionally their subsequent analysis using ESI-MS, may be carried out using a microfluidic device designed to integrate one or more sample preparation steps (e.g., filtration, pre-concentration, or extraction steps, and equivalents) and / or separation steps (e.g., as outlined above) with an electrospray ionization step.

[0083] 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 and outlet ports to each other or to intermediate fluid channels (e.g., separation channels), or any combination thereof. In some embodiments, the disclosed microfluidic device may further comprise one or more reagent chambers or mixing chambers, one or more microfabricated valves, one or more microfabricated pumps, one or more vent structures, one or more membranes (e.g., filtration membranes), one or more microcolumn structures (e.g., fluid channels or modified fluid channels filled with chromatographic separation medium), or any combination thereof.

[0084] In a preferred embodiment, the disclosed microfluidic device incorporates an electrospray orifice or electrospray tip to provide an electrospray ionization interface with a mass spectrometer. One non-limiting embodiment of such an interface is described in concurrently pending U.S. Patent Applications Publications US 2017 / 0176386 A1 and US 2018 / 0003674 A1. Figures 1A and 1B illustrate one non-limiting embodiment of a microfluidic device designed to perform isoelectric focusing, followed by ESI-MS characterization. The fluid channel network shown in Figures 1A and 1B is fabricated from a soda-lime glass plate having very low transmittance of 280 nm light using a standard photolithography etching technique. The device comprises a sample inlet channel 414 connected to an inlet 412, a concentration channel 418, and a recruitment channel 438. An anode 416 is set in electrical contact with an anodic acid well 426. The depth of the separation (or concentration) channel 418 is the same as the thickness of the glass layer 402; that is, the concentration channel 418 extends from the top to the bottom of the glass plate 402. The device 400 can be illuminated by a light source positioned on one side of the device 400 and imaged by a detector positioned on the opposite side of the device 400. The substrate 402 is opaque, but because the concentration channel 418 defines an optical slit, the substrate 402 can block light that does not pass through the concentration channel 418, block stray light, and improve the resolution of the imaging process. The glass layer 402 is sandwiched between two fused silica plates that are transparent (e.g., clear) to 280 nm light. The top plate contains through holes for the instrument and user to interface with the channel network, while the bottom plate is solid. The three plates are bonded together at 520°C for 30 minutes. The inlet and outlet tubes are fabricated from cleavable capillary tubes (100 μm inner diameter, Polymicro) that are joined to the channel network. The operation of this device during isoelectric focusing and subsequent mass spectrometry characterization of proteins will be described in Example 1 below.

[0085] Any of the various fluid actuation mechanisms known to those skilled in the art may be used to control the fluid flow of samples and reagents through the device. Suitable fluid actuation mechanisms 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 or outlet ports, gravity or centrifugal force, kinetic power, electrowetting force, or any combination thereof. In some embodiments, positive or negative pressure may be applied directly, for example, through the use of mechanical actuators or pistons coupled to the inlet and / or outlet ports to actuate the flow of samples or reagents through the fluid channel. In some embodiments, the mechanical actuators or pistons may apply force to flexible membranes or septa used to seal the inlet and / or outlet ports. In some embodiments, positive or negative pressure may be applied indirectly, for example, through the use of pressurized gas lines or vacuum lines connected to one or more inlet and / or outlet ports. In some embodiments, a pump, such as a programmable syringe pump, HPLC pump, or peristaltic pump, connected to one or more inlet and / or outlet ports, may be used to drive fluid flow. In some embodiments, dynamic power and / or electrowetting force may be applied through the use of an electric field and control of surface properties within the device. The electric field may be applied using electrodes inserted into one or more inlet and / or outlet ports, or using electrodes integrated into one or more fluid channels within the device. The electrodes may be connected to one or more DC or AC power sources for controlling voltage and / or current within the device.

[0086] In general, the inlet ports, outlet ports, fluid channels, or other components of the disclosed microfluidic device, including the main body of the device, may be fabricated 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 elastomer materials. The preferred fabrication technique will generally depend on the selection of the material, and vice versa. Examples include, but not limited to, CNC machining, photolithography and chemical etching, laser photoablation, injection molding, hot embossing, die cutting, 3D printing, and equivalents. In some embodiments, the microfluidic device may have a layered structure in which, for example, a fluid layer having 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 have openings that align with the fluid channels in the fluid layer to create inlet and / or outlet ports, etc. Two or more device layers may be sandwiched together or permanently joined to form a device that can be disassembled. The preferred joining technique will generally depend on the selection of materials used to process the layers. Examples include, but are not limited to, anodic bonding, thermal bonding, laser welding, or the use of curable adhesives (e.g., thermal or photocurable adhesives).

[0087] In some embodiments, all or part of the inlet ports, outlet ports, or fluid channels within a microfluidic device may be provided with a surface coating used to modify the electroosmotic properties (e.g., HPC or PVA coating) and / or hydrophobic / hydrophilic properties (e.g., polyethylene glycol (PEG) coating) of the inlet port, outlet port, or fluid channel walls.

[0088] The inlet and / or outlet ports of the disclosed device can be fabricated in a variety of shapes and sizes. Suitable inlet and / or outlet port geometric shapes include, but are not limited to, spheres, cylinders, ellipses, cuboids, cones, hemispheres, rectangles, or polygons (e.g., three-dimensional geometric shapes consisting of several planes, e.g., rectangular cuboids, hexagonal prisms, octagonal prisms, inverted triangular pyramids, inverted square pyramids, inverted pentagonal pyramids, inverted hexagonal pyramids, or inverted truncated pyramids), or any combination thereof.

[0089] Inlet and / or outlet port dimensions may be characterized in terms of average diameter and depth. As used herein, the average diameter of an inlet or outlet port refers to the largest circle that can be inscribed in the planar cross-section of the inlet and / or outlet port geometry. In some embodiments of this disclosure, the average diameter of the inlet and / or outlet port may range from about 0.1 mm to about 10 mm. In some embodiments, the average diameter of the inlet 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 up to 10 mm, up to 8 mm, up to 6 mm, up to 4 mm, up to 2 mm, up to 1 mm, or up to 0.5 mm. Any of the lower and upper limits described in this paragraph may be combined to form a range included in this disclosure, for example, in some embodiments, the average diameter may range from about 2 mm to about 8 mm. Those skilled in the art will recognize that the average diameter of the inlet and / or outlet ports may be any value within this range, for example, about 5.5 mm.

[0090] In some embodiments, the depth of the inlet and / or outlet ports (e.g., sample or reagent wells) 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 up to 500 μm, up to 400 μm, up to 300 μm, up to 200 μm, up to 100 μm, up to 50 μm, up to 25 μm, up to 10 μm, or up to 5 μm. Any of the lower and upper limits described in this paragraph may be combined to form a range included in this disclosure, for example, in some embodiments, the depth of the inlet and / or outlet ports may range from about 50 μm to about 200 μm. Those skilled in the art will recognize that the depth may have any value within this range, for example, about 130 μm. In some embodiments, the depth of the inlet and / or outlet ports (e.g., sample or reagent wells) 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 up to 50 mm, up to 20 mm, up to 15 mm, up to 10 mm, up to 5 mm, or up to 1 mm. Any of the lower and upper limits described in this paragraph may be combined to form a range included in this disclosure, for example, in some embodiments, the depth of the inlet and / or outlet ports may range from about 50 μm to about 5 mm.

[0091] In some embodiments, the fluid channels of the disclosed devices may have any of a variety of cross-sectional geometric shapes, such as square, rectangular, circular, and equivalent. Generally, the cross-sectional geometric shape of the fluid channels will depend on the fabrication technique used to create them, and vice versa. In some embodiments, the segment dimensions of the fluid channels (e.g., height, width, or average diameter for fluid channels 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 geometric 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 1,000 μm. In some embodiments, the dimensions of the fluid channel may be up to 1,000 μm, up to 500 μm, up to 400 μm, up to 300 μm, up to 200 μm, up to 100 μm, up to 50 μm, up to 25 μm, up to 10 μm, or up to 5 μm. Any of the lower and upper limits described in this paragraph may be combined to form a range included in the disclosure, for example, in some embodiments, the dimensions of the fluid channel may range from about 75 μm to about 300 μm. Those skilled in the art will recognize that the dimensions may have any value within this range, for example, about 95 μm. In some embodiments, the depth of the fluid channel may be equal to that with respect to the inlet and / or outlet ports of the device.

[0092] Imaging Techniques: In some embodiments of the disclosed methods and devices, imaging of the analyte separation step and / or recruitment step may be performed using optical detection techniques such as ultraviolet (UV) light absorbance, visible light absorbance, fluorescence, Fourier transform infrared spectroscopy, Fourier transform near-infrared spectroscopy, Raman spectroscopy, optical spectroscopy, and equivalents. In some embodiments, all or part of the separation (or concentration) channel, the end of the separation channel and the junction or connecting channel connecting the downstream analyzer or electrospray orifice or tip, the electrospray orifice or tip 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 in a microfluidic device.

[0093] The wavelength range used for detecting the separated analyte band will typically depend on the imaging technique and the selection of the material from which the device or a part thereof is fabricated. 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 approximately 220 nm (due to the intrinsic absorbance of peptide bonds) and / or approximately 280 nm (due to the intrinsic absorbance of aromatic amino acid residues) may allow visualization of the protein band during separation and / or recruitment, assuming that at least a part of the device, e.g., the separation channel, is transparent to light at these wavelengths. In some embodiments, the analytes to be separated and characterized via ESI-MS may be labeled prior to separation, for example, with fluorophores, chemiluminescent tags, or other suitable labels, so that they can be imaged using fluorescence imaging or other suitable imaging techniques. For example, in some embodiments, where the analytes include proteins produced by commercial manufacturing processes, the proteins may be genetically engineered to incorporate a green fluorescent protein (GFP) domain or a variation thereof, so that they can be imaged using fluorescence. In some embodiments, proteins may be tagged or labeled. Labeled proteins may be configured such that the labeling does not interfere with or disturb the analyte properties on which the selected separation technique is based. In some embodiments, no modification is required for imaging, and fluorescence imaging for UV imaging may be performed on specific proteins, peptides, or other analytes.

[0094] Any of the various imaging system components may be used for the purpose of implementing the disclosed methods, devices, and systems. Embodiments 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.), focusing 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 imaging sensors, FTIR, etc.), and equivalents, or any combination thereof. Depending on the imaging mode used, the light sources and image sensors may be positioned on opposite sides of the microfluidic device so, for example, absorbance-based images can be obtained. In some embodiments, the light sources and image sensors may be positioned on the same side of the microfluidic device so, for example, epifluorescence images can be obtained.

[0095] Images may be obtained sequentially during the separation, recruitment, and / or electrospray steps, or at random or defined time intervals. In some embodiments, one or more images in a series are obtained sequentially, at random or defined time intervals. In some embodiments, one or more images in a series may include video images.

[0096] Imaging of pI markers for determining protein isoelectric points prior to 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 undergone CIEF may be used to determine the isoelectric point of one or more individual analyte peaks (e.g., protein analyte peaks). In some embodiments, the isoelectric point of one or more analyte peaks is calculated from the positions of two or more pI markers based on an assumed linear relationship between local pH and position along the separation channel. In some embodiments, the isoelectric point of one or more analyte peaks is calculated from the positions of three or more pI markers based on a nonlinear fitting function (e.g., a nonlinear polynomial) that describes the relationship between local pH and position along the separation channel. In some embodiments, the isoelectric point of one or more analytes is calculated based on the positions of two, three, four, five, six, seven, eight, nine, or ten or more pI standards determined from the image of the separation channel.

[0097] In some embodiments, images used to determine the locations of two or more pI markers are obtained while the analyte mixture is being separated, and the pI calculation for each analyte strip is updated iteratively as the separation continues. In some embodiments, images used to determine the locations of two or more pI markers are obtained after the separation is complete and prior to the commencement of the recruitment step. In some embodiments, images used to determine the locations of two or more pI markers are obtained when the separated mixture is recruited and discharged through an electrospray tip or orifice. In some embodiments, images used to determine the locations of two or more pI markers are obtained when the separated mixture is recruited and discharged through a fluid channel connecting the separation channel to a downstream analytical instrument.

[0098] In some embodiments, images used to determine the positions of two or more pI markers and analyte bands in the separated mixture are obtained using a computer implementation method (e.g., a software package). In some embodiments, the positions of two or more pI markers and analyte bands are determined using a computer implementation method that includes automated image processing. In some embodiments, the computer implementation method further includes the step of performing isoelectric point calculations for one or more analyte bands based on position data derived from automated image processing.

[0099] Figure 2 provides an illustrative process flowchart of a computer implementation method for obtaining an image of a separation channel (or other part of a microfluidic device), determining the locations of pI markers and analyte bands in the image (i.e., if the separation step includes CIEF), and calculating the pI for one or more analyte bands in a mixture of separated analytes. In some embodiments, the computer implementation method may include a step of controlling the acquisition of a series of one or more images, which are then processed to identify the locations of pI markers and separated analyte bands. Examples of preferred automated image processing algorithms will be discussed in more detail below. In some embodiments, predetermined knowledge of the pI marker locations, such as those determined from an image of a “control group” sample containing only pI markers, may be used to distinguish between bands corresponding to pI markers and bands corresponding to separated analytes. In some embodiments, images of pI markers may be obtained at different wavelengths or using a different imaging mode than that used to obtain images of separated analyte bands. As illustrated in Figure 2, if the image processing step cannot determine the positions of a known number of pI markers and / or the separated analyte bands, the system may be instructed to obtain new images so that the image processing step can be repeated. Once the positions of the pI markers and the separated analyte bands are determined, the data on the positions of the pI markers are fitted to a user-selected model (e.g., a linear or nonlinear model) for the pH gradient, and the resulting fitted relationship between the local pH and the position along the separation channel is then used to calculate the isoelectric point for one or more analyte bands.

[0100] In some embodiments, the computer implementation method may be an iterative process in which the steps of detecting pI marker and analyte zone locations, fitting the location data to a pH gradient model, and calculating the isoelectric point for one or more analyte zones are repeated so that the latter is continuously updated and refined (e.g., through averaging several decisions). In some embodiments, a cycle including the steps of image acquisition and processing, detection of pI marker and analyte zone locations, fitting pI marker location data to a pH gradient model, and calculating the isoelectric point for one or more analyte zones may be completed in a sufficiently short time so that the isoelectric point calculation can be 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, for example, at least the Nyquist rate.

[0101] Image Separation and Mobilization: In some embodiments, the movement of peaks through the separation channel (e.g., during separation, during mobilization, etc.) can be monitored. Imaging may be UV imaging, fluorescence imaging, transmitted light imaging, or another imaging mode. In some embodiments, images of separation and mobilization may be recorded at a desired rate. For example, the imaging rate may be one image per minute, one image per 30 seconds, one image per 10 seconds, one image per 5 seconds, one image per second, one image per millisecond, etc. In some embodiments, the individual images may be combined as individual frames in a “movie” showing peak formation and mobilization. Figures 9A-F show a subset of images that can be combined to generate a movie. In some embodiments, this movie may be stored as GIF, AVI, MOV, MP4, or any other digital format capable of storing digital video data. In some embodiments, imaging may be performed in real time, for example, when separation is performed, when mobilization is performed, when electrospray is performed, etc.

[0102] In some embodiments, a dynamic heatmap, such as the one shown in Figure 17B, may be used to display a series of images (e.g., a time-series imaging dataset of separation and / or recruitment performed in a separation channel, where each image in the series corresponds to a different time point). For example, in Figure 17B, instead of displaying a graphical representation of the peaks together (e.g., overlaying multiple intensity or absorbance plots as a function of length (i.e., pixel position) along the separation channel on a single plot), the peaks are represented as an imaged analyte band (each containing intensity or absorbance measurements) along the length of the imaged channel and plotted as a function of time. Each row in the image (heatmap) of Figure 17B displays the position of the analyte band at a single time point between focusing and recruitment. For example, line 1704 shows an exemplary row of pixels in the dynamic heatmap, each row corresponding to an image of the separation channel and may be used (or generated from) an electrophoresis diagram (e.g., as shown in Figure 17A) for a given time point. The analyte peak 1702 in Figure 17A is represented by a bright pixel (also labeled as 1702) in Figure 17B. For each analyte peak in Figure 17A, Figure 17B displays the time course of the analyte peak migration. For example, during IEF separation, the analyte (or multiple analytes) may migrate from both ends of the channel to the isoelectric point of the analyte (or multiple analytes). At the isoelectric point, once focusing is complete (e.g., at the time corresponding to line 1704), the analyte may be concentrated, resulting in a bright peak (pixel 1702). Furthermore, at the beginning of recruitment (in this case, the portion above line 1704 in Figure 17B), accelerated migration of each peak toward the tip orifice and the mass spectrometer may occur. The time-series images are stacked vertically, so that the column axis (Y-axis) in Figure 17B represents time, while the X-axis represents the spatial resolution of the band at each time point (e.g., the position of the analyte as a function of position along the length of the separation channel). In some embodiments, the relative intensity of the bands may be represented by a grayscale or a color scale.In some embodiments, increasing or decreasing the color scale or grayscale may correlate with increasing the signal, intensity, or absorbance. In some embodiments, the speed of the bands may be determined by measuring the slope of the band progression over time across the modified heatmap. In some embodiments, the modified heatmap may be used to display imaging data from focusing, recruitment, or both. Such a display of separation data may be particularly useful in steps of comparing or characterizing inter-run variability of separation and / or recruitment; comparing separation and recruitment reactions (e.g., time scales relating to the completion of separation and recruitment reactions, separation resolution achieved during separation, separation resolution maintained during recruitment, etc.); determining when separation is complete; monitoring or detecting failures in the separation channel; monitoring the presence of electroosmotic flow; and / or determining separation performance characteristics (e.g., separation resolution, linearity of pH gradient, etc.).

[0103] In some embodiments, time-series imaging data may be plotted on a three-dimensional or three-axis graph, as shown in Figure 19. One axis of the graph may represent distance (e.g., physical distance along the length of the isolation channel or pixel position), and another axis may represent time. In some cases, a third axis may be used to represent signal intensity, intensity, or absorbance, which can be represented by a color scale or grayscale, either alternatively or in addition. In some embodiments, the x-axis may be used to represent distance, the y-axis to represent time, and the z-axis to represent signal or absorbance. It should be understood that the axes are used to represent any of the parameters (e.g., distance or position along the channel, pI, intensity or absorbance, time, etc.).

[0104] The imaging data and data plots (or other image processing) may be performed after the completion of separation and mass spectrometry, or in some cases, while separation, recruitment, and mass spectrometry are being performed. For example, a computer implementation or software may be configured to receive the imaging data as it is acquired, process the imaging data (e.g., to obtain intensity plots as a function of channel length), and plot the IEF data (e.g., iteratively or incrementally in a 3D plot or heatmap).

[0105] As described herein, computer implementations or software can be used to collect mass spectra at a specified scan rate. In some embodiments, computer implementations can be used to summarize mass spectrometry data in chromatogram form. For example, a plot may be generated in which the X-axis represents time and the Y-axis represents the sum of signals in the mass spectral data (e.g., total ion count), such as in line trace 1834 of Figure 18. The Y-axis can represent the sum of all signals in individual mass spectra (total ion chromatogram), the sum of signals for a specific reference peak or extracted ion (reference peak chromatogram, extracted ion chromatogram), or any other subset of the mass spectral data.

[0106] Imaging of analyte beams to determine velocity: In some embodiments, as described above, the position of one or more analyte beams may 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 with respect to one or more analyte beams can be calculated from the difference in a known time interval between its relative position in two or more images and the acquisition time in 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 recruitment step. In some embodiments, two or more images may be acquired while the separated sample is being discharged through a fluid channel connecting the end of the separation channel to a downstream analyzer. In some embodiments, two or more images may be acquired while the separated sample is being discharged through an electrospray tip or orifice to form a Taylor cone. In some embodiments, the velocity determined with respect to one or more analyte beams may be used to calculate the time it takes for a given analyte beam to exit the separation channel. In some embodiments, when there are one or more interconnected fluid joints or fluidized channels, for example, connecting the end of a separation channel to an outlet port, such as an electrospray orifice or tip, the velocity determined with respect to one or more analyte strips may be used to calculate the time it takes for a given analyte strip to reach the outlet port and exit the device. In some embodiments, the velocity determined with respect to one or more analyte strips may be used to calculate the time it takes for a given analyte strip to enter the electrospray tip or electrospray orifice and enter the Taylor cone formed between the electrospray tip or orifice and the inlet of the mass spectrometer.

[0107] In some embodiments, the sequence of images used to determine the velocity of one or more analyte beams may be obtained using a computer implementation method (e.g., a software package). In some embodiments, the velocity of one or more analyte beams is determined using a computer implementation method that includes automated image processing. In some embodiments, the computer implementation method further includes the step of performing a calculation of the time at which a given analyte beam will exit the separation channel. In some embodiments, the computer implementation method further includes the step of performing a calculation of the time at which a given analyte beam will reach the exit port and exit the device. In some embodiments, the computer implementation method further includes the step of performing a calculation of the time at which a given analyte beam will exit the electrospray tip or electrospray orifice and enter the Taylor cone formed between the electrospray tip or orifice and the inlet of the mass spectrometer. In some embodiments, the exit times determined for one or more analyte beams are used to correlate specific analyte beams with mass spectrometry data or data collected using other analytical instruments.

[0108] Figure 3 provides another exemplary process flowchart of a computer implementation method for acquiring images of a separation channel (or other part of a microfluidic device), determining the velocity of one or more analyte bands, and calculating the time it will take for a given analyte band to reach a defined point in the device, such as the end of the separation channel, the junction between the separation channel and the secondary fluid channel, the outlet port of the device, or the electrospray tip or orifice outlet. In some embodiments, the computer implementation method may include a step of controlling the acquisition of a series of one or more images, which are then processed to identify the location of the separated analyte band. Examples of preferred automated image processing algorithms will be discussed in more detail below. If the image processing step is unable to determine the location of the separated analyte band, as illustrated in Figure 3, the system may be instructed to acquire new images so that the image processing step can be repeated. Once the location of the separated analyte band is determined with respect to a series of two or more images, the velocity is calculated with respect to one or more of the analyte peaks, based on their relative positions in the two or more images and a known time interval between the acquisition times of the two or more images. In some embodiments, tracking one or more analyte bands from one image to the next in a series of images may be used to distinguish several isolated analyte bands and to refine velocity calculations (e.g., by averaging velocity values ​​calculated from several pairs of images in a series). In some embodiments, a pI marker or other internal standard that can be detected using a selected imaging mode may be used as a “velocity standard”. The analyte band velocity thus determined may be used to calculate the time it will take for a given band to reach a user-defined point in the device, e.g., the outlet end of a separation channel, a specific fluid junction in the device, an outlet port of the device, an electrospray ionization tip or orifice into which the analyte enters a Taylor cone, and equivalents.

[0109] In some embodiments, the computer implementation method may be an iterative process in which the steps of detecting the analyte zone position, determining the analyte zone velocity, and calculating the exit time are repeated so that the exit 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 steps of image acquisition and processing, velocity calculation, and exit time prediction may be completed in a sufficiently short time so that the exit time prediction can be 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, for example, at least the rate of the Nyquist rate.

[0110] In some embodiments (e.g., those including a CIEF step), the computer implementation method of the present disclosure may perform both the determination of the imaging base for a precise isoelectric point and the determination of the imaging base for the velocity of the separated analyte band.

[0111] Correlation between separation data and mass spectrometry data: In some embodiments, the computer implementation methods described above for performing accurate imaging-based determination of isoelectric points with respect to an isoelectrically focused analyte band allow for correlation of isoelectric point data with specific m / z peaks in mass spectrometry data (or other analytical data), thereby improving the informational content of the dataset (even with respect to a single run) and enabling a more quantitative characterization of the analyte sample. The computer implementation method may be configured to receive IEF data (e.g., multiple intensity or absorbance measurements as a function of length along the separation channel, or pl data for specific peaks) and MS data (e.g., a total ion chromatogram, multiple ion measurements as a function of mass, etc.) (e.g., using a processor).

[0112] In some embodiments, the imaged analyte peaks can be correlated with mass spectrometer data to provide information about the mass and charge (or isoelectric point) of one or more analytes in the analyte peaks. For example, during separation, images of one or more of the separation channels may be obtained at any useful imaging rate, thereby generating a time-series imaging dataset. The time-series imaging dataset may consist of multiple images of the separation channels, each image corresponding to a different point in time. In some cases, the IEF data (e.g., each image in the time series) may be plotted as an electrophoretic graph, which may show signal, intensity, or absorbance as a function of position along the length of the separation channel. In some cases, as described elsewhere in this specification, the IEF data may be used to generate a heatmap or three-dimensional plot (see, e.g., Figures 17A–B and 19).

[0113] IEF data may be plotted together with MS data (for example, using one or more processors). For example, line trace 1832 in Figure 18 represents an IEF electrophoresis map of NIST monoclonal antibody separation. The X-axis of line trace 1832 represents the spatial resolution or position (which may be expressed in units of distance, pixels, or isoelectric point) along the length of the separation channel, and the Y-axis of line trace 1832 represents the relative signal intensity (e.g., absorbance, intensity, etc.). As shown in Figure 18, the IEF electrophoresis map is plotted on a graph together with one or more chromatograms representing MS data. For example, one or more MS scans, averaged scans, processed data, or unconvolved data may be plotted together with the IEF electrophoresis map by aligning the time axis of the MS data (e.g., total ion chromatogram 1834) with the isoelectric focus peak in line trace 1832. In one such embodiment, line trace 1836 represents an embodiment of the unconvoluted mass spectrum collected by the mass spectrometer at equal time intervals corresponding to the time segment of the total ion chromatogram 1834 and the pI segment of the IEF electrophoresis diagram 1832. The unconvoluted mass spectrum can, in some cases, be unconvoluted from the total ion chromatogram 1834 or otherwise generated. Each line trace 1836 is aligned with the x-axis of the mass spectrometry total ion chromatogram 1834 and the IEF electrophoresis diagram 1832. The line trace 1836 shows the mass (represented by the vertical Y-axis) and the signal intensity (e.g., ion count) (along the X-axis) for each unconvoluted mass spectrum. Each mass spectrum line trace 1836 may also be shown on two reference scales, where a darker line is normalized to the highest signal across all mass spectra, and a lighter trace is normalized within each individual mass spectrum. Such a plot provides a display of signal intensity, but also allows low signals to be displayed in line trace 1836.

[0114] In some embodiments, correlating IEF data and MS data can be particularly useful in identifying or distinguishing one or more analytes that have similar properties (e.g., identical charge or isoelectric point, or identical mass) and / or different properties. For example, two molecules with different masses may be identified in mass spectrometer data. The two molecules may have different isoelectric points (pI) and be concentrated in different regions of the pH gradient in the IEF, or the two molecules may have the same isoelectric point (pI) and be concentrated in the same region of the pH gradient in the IEF. In cases where the two molecules have the same pI and different masses, the correlation of IEF and MS data may be used to distinguish the two molecules (e.g., to identify them as different species or isoforms).

[0115] As an example, two molecules having the same pI and different masses (or, alternatively, the same mass but different pIs) may be two protein isoforms, for example, isoforms where the difference in pI or mass is caused by post-translational modifications, translation errors (e.g., incorrect amino acid addition, folding, disulfide bond rearrangement, or other translational modifications), transcription, or encoding in the DNA sequence. In such examples, the correct post-translational modification may be identified by examining the difference (or similarity) of both mass and charge or pI. For example, an analyte peak in a separation channel (e.g., an IEF analyte peak) may contain one or more analytes having the same isoelectric point but different masses. The different masses may be determined by performing MS on the analyte peaks as described herein. By using information on both pI and mass, some isoforms that deviate from the known or expected pI and mass of a particular isoform can be excluded. Thus, one or more analytes may be identified using the difference in mass, even though they have the same isoelectric point. Alternatively, or in conjunction with other analytes, one or more analytes may be identified using differences in pI, even if they have the same mass.

[0116] Overlaying IEF data and MS data (e.g., total ion chromatogram and time-series ion measurements as a function of mass) on a single plot can be useful in identifying protein isoforms by mapping pI to the mass of one or more analytes. For example, referring to Figure 18, IEF data (e.g., IEF electrophoresis diagram 1832) may be mapped to a total ion chromatogram 1834. Each point (e.g., time interval) in the total ion chromatogram 1834 may be unconvoluted to generate a line trace 1836 showing the mass distribution and relative intensity. Thus, for each time interval in the total ion chromatogram 1834, the corresponding unconvoluted mass distribution data and isoelectric point may be determined. Similarly, for each isoelectric point, the corresponding mass distribution may be obtained.

[0117] For example, Figure 20 shows exemplary isoelectric focusing and mass spectrometry data from the analysis of NIST monoclonal antibodies. Panel A of Figure 20 shows isoelectric focusing data for charge deformations (labeled as Acidic 1, Acidic 2, Major, Basic 1, and Basic 2) in an inserted plot, while the larger graph shows the mass spectral reference peak chromatograms of the charge deformations introduced into the mass spectrometer, corresponding to the charge deformations in the inserted plot. Panel B of Figure 20 shows unconvoluted mass data displaying the mass assignments for the sample in the reference peak chromatograms for each time interval, each of which can be mapped back to a position along the length of the separation channel (e.g., from the acidic end to the basic end). The differences in peak profiles in Panel B of Figure 20 indicate differences in species mass and relative abundance in different charge deformation peaks separated in the isoelectric focusing process.

[0118] IEF and MS data may be used to assign post-translational modifications. Panel A of Figure 21 lists examples of post-translational modifications and the charge and mass changes expected by the modifications. These values ​​may be obtained from publicly available sources (e.g., published data, protein databases, etc.) or may be derived empirically. In the examples, in Panel A of Figure 21, both glycation and galactose (glycosylation) modifications result in the addition of 162 daltons to the molecular mass. However, glycation events can be distinguished from glycosylation events because glycation will make the molecule more acidic, shifting it to a lower isoelectric point in IEF or a different elution time in CZE. Many other combinations of modifications are possible in the protein analyte, although this and other examples are outlined in Panel B of Figure 21. For example, post-translational modifications may include hydroxylation, methylation, lipidation, acetylation, disulfide bonding, smoylation, ubiquitination, glycosylation, glycation, amino acid addition or removal, amidation, deamidation, isomerization, oxidation, fucosylation, sialylation, phosphorylation, or combinations thereof, or other post-translational modifications. Known post-translational modification properties (e.g., charge, mass, pI changes, etc.) may be obtained from publicly available sources (e.g., UniProt, BLAST, or other protein databases).

[0119] Panel A of Figure 22 shows the unconvoluted masses calculated from mass spectrometry of the Acid 2, Basic 1, and Major peaks (analyte peaks from IEF separation). The peaks in both Basic 1 and Major show a continuous increase of 162 daltons per molecule, indicating that the sequential glycosylation steps result in a mass difference but no charge (or pI) difference. As shown in Panel B of Figure 23, the relative abundance of molecules of different masses does not change between Basic 1 and Major, and only the 128 dalton offset shown in Panel A of Figure 22 exists, indicating additional lysine present on the molecule in the Basic 1 peak. Similarly, in Panel B of Figure 22, comparing the unconvoluted masses calculated from mass spectrometry of the Major, Acid 1, and Acid 2 peaks (analyte peaks from IEF separation), a consistent series of 162 dalton shifts in the molecule at each charge deformation peak can be observed. However, when the major acidic 1 and acidic 2 profiles are overlaid in panel A of Figure 23, a relative increase in greater mass is observed in the acidic charge deformation peak, which may indicate glycation in addition to the glycosylation series seen in basic 1 and the major peak.

[0120] Computer Implementation Methods: As described herein, one or more data presentation and analysis algorithms may be implemented using computer implementation methods. Computer algorithms may be employed to perform a variety of functions, including, but are not limited to, receiving IEF and MS data, transforming or processing the data, generating graphs or plots of the data, overlaying the IEF and MS data, and analyzing the IEF data, such as assessing charge and mass changes to correctly assign post-translational modifications. In some embodiments, neural networks or other artificial intelligence algorithms may be employed to assess charge and mass changes or similarities to correctly assign post-translational modifications. In some cases, the computer implementation method may be configured to store multiple reference values ​​(e.g., expected or known charge and / or mass changes for different post-translational modifications). These stored reference values ​​may then be used by one or more processors to match the IEF and MS data and determine or identify post-translational modifications in analyte peaks.

[0121] One or more computer implementations may be configured to perform one or more functions automatically (e.g., without human interaction). For example, one or more computer implementations may be part of a software package for acquiring data (e.g., performing imaging), receiving data (e.g., separation, mobilization, and / or MS data), processing data, displaying data, etc. Data processing or presentation may be performed substantially concurrently with imaging or after imaging is complete. Similarly, data processing or presentation may be performed during or immediately following ESI-MS. For example, post-translational modification determination or assignment may be performed within one second, one minute, ten minutes, one hour, etc., of acquiring ESI-MS data.

[0122] In some embodiments, the computer implementation methods described above for using image-derived data to calculate the velocity of separated analytes (using any of various different separation techniques) and predict exit times enable the improvement of the time correlation between chemical separation data (e.g., residence time, electrophoretic mobility, isoelectric point, etc.) and specific m / z peaks in mass spectrometry data (or other analytical data), thereby improving the informational content of both datasets (even with respect to a single run experiment) and enabling more quantitative comparisons of different sample runs, data collected with respect to different samples, or data collected on different instruments, due to the ability to compensate for inter-experimental or inter-instrumental variability in separation time.

[0123] The methods, devices, and systems disclosed may therefore be particularly advantageous for various metabolomics, proteomics, and drug development or manufacturing applications. While the above examples relate to isoelectric focusing coupled to mass spectrometry, it should be understood that the separation performed prior to introduction into the mass spectrometer may be electrophoresis, chromatography, or other separation, as described elsewhere in this specification.

[0124] 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 its injection into a mass spectrometer. Mass spectrometry (MS) is an analytical technique that measures the "mass" of analyte molecules in a sample by ionizing them and classifying the resulting ions based on their mass / charge (m / z) ratio. Combined with a preceding liquid-phase or gas-phase sample separation system, mass spectrometry provides one of the most effective means available for analyzing complex samples containing multiple low-abundance analytes, as is common in biological samples, for example.

[0125] All mass spectrometers share the requirement that ions are in the gas phase prior to their introduction into the mass spectrometer. Various sample ionization modes have been developed, but are not limited to matrix-assisted laser desorption and ionization (MALDI) and electrospray ionization (ESI). In the MALDI technique, a sample (e.g., a biological sample containing a mixture of proteins) is mixed with an energy-absorbing matrix (EAM) such as sinapic acid or α-cyano-4-hydroxycinnamic acid and crystallized on a metal plate. Surface-enhanced laser desorption and ionization (SELDI) is a common variation of the technique that incorporates additional surface chemistry onto a metal plate to facilitate the specific binding of a certain class of proteins. The plate is placed in a vacuum chamber, and the matrix crystal is impacted using light pulses from a nitrogen laser. The energy absorbed by the molecules is transferred to proteins, causing them to desorb, ionize, and generate plumes of ions in the gas phase, which are accelerated in the presence of radio waves and drawn into a flight tube, where they drift until they collide with a detector that records their time of flight. The time of flight may, in turn, be used to calculate the m / z ratio with respect to the ionized species. In some embodiments of the disclosed device, the exit port of the device may comprise a capillary tube or other feature used, for example, to deposit the separated analyte (or fraction thereof) onto a MALDI plate in preparation for mass spectrometry, in order to correlate the isoelectric point with MALDI mass spectrometer data with respect to a specific analyte.

[0126] Electrospray ionization (ESI, also simply referred to herein as “electrospray”) can also be used due to its intrinsic compatibility with liquid chromatography or electrodynamic chromatography separation techniques for interface with mass spectrometers. As described above, in electrospray ionization, small droplets of sample and solution are emitted from the distal end of a capillary or microfluidic device equipped with electrospray features (e.g., emitter tip or orifice) by applying an electric field between the tip or orifice and the mass spectrometer source plate. The droplets then stretch and expand within this induced electric field, forming a conical emission (i.e., a “Taylor cone”), which contains increasingly smaller droplets that evaporate and generate gaseous ions introduced into the mass spectrometer for further separation and detection. The emitter tip may be formed from a capillary or corner or ESI tip built into a microfluidic chip design, which provides a convenient droplet volume for ESI. The emitter tip may be sharpened to provide a small surface and droplet volume using a lapping wheel, file, machining tool, CNC machining tool, water jet cutting, or other tools or processes for shaping an ESI tip and providing a small surface volume, and equivalents. In some embodiments, the tip may be stretched by heating and stretching 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 droplets formed on the tip. In some embodiments, the system may electrospray a recruiter, cathode solution, or any other liquid during the separation step when no analyte has eluted from the device.

[0127] In some embodiments of the disclosed methods, devices, and systems, other ionization methods such as inductively coupled laser ionization, fast atomic bombardment, soft laser desorption, atmospheric pressure chemical ionization, secondary ion mass spectrometry, spark ionization, thermal ionization, and equivalents are also used.

[0128] With respect to electrospray ionization, in some embodiments, the disclosed microfluidic devices, as illustrated in Figure 1, feature characteristics designed to facilitate efficient electrospray ionization and a convenient interface with downstream mass spectrometry. The mass / charge ratio (or "mass") of the analyte discharged from the microfluidic device (e.g., a biopharmaceutical or biosimilar) and introduced into the mass spectrometer can be measured using one of a variety of different mass spectrometer designs. Examples include, but are not limited to, time-of-flight mass spectrometry, quadrupole mass spectrometry, ion trap or orbit trap mass spectrometry, distance-of-flight mass spectrometry, Fourier transform ion cyclotron resonance, resonance mass spectrometry, and nanomechanical mass spectrometry.

[0129] In some embodiments, the electrospray feature of the microfluidic device may be aligned with the separation channel. In some embodiments, the electrospray feature of the microfluidic device may be oriented perpendicular to the separation channel or at an intermediate angle. 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 the capillary or microfluidic device is discharged from the electrospray tip or feature 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 via an outlet configured to interface with an analytical instrument such as a mass spectrometer or another device configured to fractionate and / or concentrate at least a portion of the sample. Another portion of the analyte mixture (e.g., containing fractions other than the fraction of interest) may be discharged via a waste channel.

[0130] In some embodiments, discharge from the capillary or microfluidic device is carried out using pressure, electric force, ionization, or any combination thereof. In some embodiments, discharge coincides with the recruitment step as described above. In some embodiments, the sheath fluid used for electrospray ionization is used as the electrolyte for electrophoretic separation. In some embodiments, a nebulating gas is provided to reduce the analyte fraction to a fine spray.

[0131] Image-Based Feedback of Electrospray Ionization Performance: Conventional ESI-MS systems using capillary or microfluidic devices generally do not provide any tools for calibrating the system to re-establish the Taylor cone during operation. Maintaining a stable Taylor cone can be complicated by the electrophoretic electric field applied across the separation channel in the microfluidic device or capillary. Changes in the conductivity of reagents between or during runs can change the voltage potential at the interface with the mass spectrometer. Changes in potential at the interface can adversely affect the Taylor cone and lead to a loss of electrospray ionization efficiency. Disclosed herein are methods and systems for improving electrospray ionization performance and, therefore, the quality of mass spectrometry data collected for capillary-based or microfluidic device-based ESI-MS systems. In some embodiments, for example, imaging of the Taylor cone in the electrospray ionization setting may be used in a computer implementation method to provide feedback control of one or more operating parameters so that the shape, density, or other properties of the Taylor cone are maintained within a specified range. In some embodiments, operating parameters that can be controlled through such feedback processes include, but are not limited to, the alignment between the electrospray tip or orifice and the mass spectrometer inlet; the distance between the electrospray tip and the mass spectrometer inlet (e.g., by mounting a capillary tip or microfluidic device with integrated electrospray features on a programmable precision XYZ conversion stage); the flux of the analyte sample through the electrospray tip (e.g., by adjusting the pressure, electric field strength, or a combination thereof used to drive the discharge of the analyte sample); for example, the voltage applied between the electrospray tip or orifice and the mass spectrometer inlet at the proximal end of the channel; the volumetric flux of the sheath fluid or sheath gas surrounding the discharged analyte sample; or any combination thereof.

[0132] Figure 4 provides an illustrative process flowchart of a computer implementation method used to determine appropriate tuning of one or more operating parameters to restore the Taylor cone to a defined or target value, using a mathematical algorithm that associates the Taylor cone's shape, density, or other properties with one or more operating parameters based on the comparison. In some embodiments, data obtained from a mass spectrometer (e.g., total ion current data) may be used in addition to the data derived from the Taylor cone image to monitor system performance and tune one or more operating parameters.

[0133] In some embodiments, the periodic process illustrated in Figure 4, which includes the steps of image acquisition and processing, identification of Taylor cone characteristics, comparison of said Taylor cone characteristics with a set of target values, and calculation of adjustments required for one or more ESI-MS system operating parameters, may be completed in a sufficiently short time such that one or more operating parameters can be 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 any other relevant rate, for example, at least the rate of the Nyquist rate.

[0134] Alternating High-Mass / Low-Mass Scanning: In some embodiments of the disclosed methods, devices, and systems, the mass spectrometer may be configured to alternate between a high-mass scanning range (e.g., a m / z range of about 1,500 to 6,000) or "high-mass scanning" and a low-mass scanning range (e.g., a m / z range of about 150 to 1,500) or "low-mass scanning," so that the low-mass scanning can be used to calibrate the low-mass markers identified in the mass spectrometry data and indicated by the low-mass markers (e.g., specific ranges of isoelectric points, peptides, small molecule markers, in cases where amphoteric electrolytes in free solution are detected), for example, to identify amphoteric electrolytes in free solution in cases where an isoelectric focusing separation step is performed. The switching and scanning rate between high-mass scanning and low-mass scanning should be fast relative to the outflow of the analyte sample from the electrospray interface. In some cases, the switching rate between high-mass scanning and low-mass scanning may range from 0.5 Hz to about 50 Hz. In some cases, the switching rate may be at least 0.5Hz, at least 1Hz, at least 5Hz, at least 10Hz, at least 20Hz, at least 30Hz, at least 40Hz, or at least 50Hz.

[0135] Modified high and low separation / mobilization voltages to maintain a constant ESI tip voltage: In some embodiments, the ESI ion source of the mass spectrometer will have a controllable power source capable of setting a negative voltage for the mass spectrometer. In some embodiments, the ESI ion source of the mass spectrometer will have a controllable power source capable of setting a positive voltage for the mass spectrometer. In some embodiments, the ESI ion source of the mass spectrometer will be held at ground. In some embodiments, the ESI tip on a capillary or microfluidic device will be held at ground or close to ground to generate an electric field between the ESI tip and the charged ESI ion source of the mass spectrometer. In some embodiments, the ESI tip on a capillary or microfluidic device will be held at a positive or negative voltage to generate an electric field between the ESI tip and the grounded ESI ion source of the mass spectrometer.

[0136] Figure 15 provides an illustrative flowchart of a computer-controlled feedback loop for maintaining a constant voltage drop of 3,000V between the anode and cathode while keeping the ESI tip voltage at 0V during recruitment. In some embodiments, this feedback loop may be implemented when the mass spectrometer ESI ion source is set to a positive or negative voltage (e.g., -3,500V) relative to ground. In this embodiment, the ΔV between the anolyte port 108 and the recruiter port 104 is initially maintained at 3,000V by setting the anolyte port 108 to +3,000V and the recruiter port 104 to 0V in Figure 7A. In some embodiments, different ΔVs may be set by setting the anolyte port 108 to different values. In some embodiments, anode recruitment may be used, where port 108 is the cathode ray port, set to, for example, -3,000V. In the embodiment outlined in Figure 15, during recruitment, the resistance in the separation channel 112 decreases due to the analyte and amphoteric electrolyte regaining charge during separation. This causes a decrease in the voltage drop across channel 112, leading to an increase in voltage at the ESI tip 116 according to equation 1. V 116 =(ΔV 108-104 )×(R 105 ) / (R 109 +R 112 +R 105 However, by measuring or calculating the ESI tip voltage 116, the voltage settings at the anolyte port 108 and recruiter port 104 can be adjusted. By subtracting the ESI tip voltage 116 from both the anolyte port 108 and recruiter port 104 settings, ΔV 108-104 The voltage remains at 3,000V so that the mobilization is not affected, but the ESI tip 116 voltage is set to 0 according to equation 2. V 116 =(ΔV 108-104 )×(R 105 ) / (R 109 +R 112 +R 105 )+V104 This feedback loop continues to operate until mobilization is complete, adjusting the ESI tip 116 voltage to zero at regular frequencies, e.g., the Nyquist rate, or approximately 0.2 Hz. In some cases, the voltage at the ESI tip 116 may be adjusted to zero at rates 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 at the ESI tip 116 may be important for maintaining a stable electospray during the mobilization process.

[0137] In some cases, the feedback loop operates to maintain the voltage at the ESI tip within a specified percentage of a preset value. For example, in some cases, the feedback loop operates to maintain the voltage at the ESI tip 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 tip voltage within 1,000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V of a preset value.

[0138] In some embodiments, the mass spectrometer ESI ion source may be held in ground, and the ESI tip 116 may need to be held at a constant positive or negative voltage to create an electric field between the ESI tip 116 and the mass spectrometer. In some embodiments, the ESI tip voltage (e.g., a preset value) may be approximately +5,000V, approximately +4,000V, approximately +3,500V, approximately +3,000V, approximately +2,500V, approximately +2,000V, approximately +1,500V, approximately +1,000V, approximately +500V, or approximately -5,000V, approximately -4,000V, approximately -3,500V, approximately -3,000V, approximately -2,500V, approximately -2,000V, approximately -1,500V, approximately -1,000V, or approximately -500V. Figure 12 provides an illustrative flowchart of a computer-controlled feedback loop for maintaining a constant 3,000V voltage drop between the anode and cathode while keeping the ESI tip voltage at 3,000V during recruitment. The operation of the computer-controlled feedback loop is identical to that in Figure 15, except that the voltages at the anodic liquid port 108 and recruiter port 104 are offset by +3,000V, which still offsets the voltage at the ESI tip 116 to +3,000V according to equation 2. In some embodiments, control of the electric field strength can be performed using analog circuits. In some embodiments, control of the voltage at one or more electrodes in contact with a capillary-based or microfluidic device-based isolation system may be provided by using one, two, three, or four or more independent high-voltage power sources. In some cases, control of the voltage at one or more electrodes in contact with a capillary-based or microfluidic device-based isolation system may be provided, for example, by using a single multiplexed high-voltage power source.

[0139] In some cases, the feedback loop operates to maintain the electric field strength in the isolation channel or the voltage drop between the anode and cathode within a specified percentage of a preset value. For example, in some cases, the feedback loop operates to maintain the electric field strength in the isolation channel or the voltage drop between the anode and 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 isolation channel or the voltage drop between the anode and cathode within 1,000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V of a preset value.

[0140] The mass spectrometer inlet potential is modified to maintain a constant voltage difference between the ESI tip and the mass spectrometer inlet: In some embodiments, the mass spectrometer may have an adjustable power source coupled to it that can set a negative voltage (e.g., at the inlet) of the mass spectrometer. In some embodiments, the mass spectrometer may have an adjustable power source coupled to it that can set a positive voltage (e.g., at the inlet) of the mass spectrometer. In various embodiments, the mass spectrometer or the mass spectrometer inlet may be held at ground. In some cases, the ESI tip on a capillary tube or microfluidic device may be held at ground or close to it to generate an electric field between the ESI tip and the charged ESI ion source of the mass spectrometer. In some cases, the ESI tip on a capillary tube or microfluidic device may be held at a positive or negative voltage to generate an electric field between the ESI tip and the mass spectrometer (e.g., at the inlet). In various cases, the potential applied to the mass spectrometer is, for example, a constant voltage difference (ΔV) between the ESI tip and the mass spectrometer inlet. TIP-MS To maintain the voltage difference (ΔV), it may be adjusted in the feedback loop. In some cases, the voltage difference (ΔV) may be adjusted. TIP-MS ) is the target value (ΔV TARGET) or may be set to a range of target values. In some cases, both the potential of the ESI tip and the voltage or potential of the mass spectrometer (e.g., at the inlet) may be adjusted, for example, to keep the voltage drop between the ESI tip and the mass spectrometer constant or within a range of target values.

[0141] In various cases, a computer-controlled feedback loop can be used to maintain a constant voltage drop between the ESI tip and the mass spectrometer. In some embodiments, this feedback loop may be implemented when the mass spectrometer ESI ion source is set to a positive or negative voltage (e.g., -3,500V) relative to ground. In this embodiment, referring to Figure 7A, the ΔV between the anodelitic port 108 and the recruiter port 104 may be set to an initial voltage of 3,000V by setting the anodelitic port 108 to +3,000V and the recruiter port 104 to 0V. In some embodiments, different ΔVs may be set by setting the anodelitic port 108 to different values. In some embodiments, anode recruitment may be used, where port 108 is the cathodelitic port, set to, for example, -3,000V. In some cases, during recruitment, the resistance in the separation channel 112 may decrease due to the analyte and amphoteric electrolyte in the separation channel regaining charge. This can cause a voltage drop across channel 112, which can lead to an increase in voltage at the ESI tip 116 according to the following equation. V 116 =(ΔV 108-104 )×(R 105 ) / (R 109 +R 112 +R 105 The mass spectrometer voltage can be adjusted by measuring or calculating the voltage at the ESI tip 116. For example, an increase in voltage at the ESI tip 116 corresponds to the voltage difference between the mass spectrometer inlet and the ESI tip 116, i.e., ΔV TIP-MSThe voltage applied to the mass spectrometer can be adjusted in addition to the voltage of the ESI tip 116 so that it remains the same. In some cases, the voltages of both the ESI tip 116 and the mass spectrometer may be adjusted. The feedback loop continues to operate until recruitment is complete and can adjust the mass spectrometer voltage (e.g., at the inlet) to match a regular frequency, e.g., the Nyquist rate, or about 0.2 Hz, to match that of the ESI tip 116. In some cases, the voltage applied to the mass spectrometer may be adjusted to 0 at rates 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 difference between the ESI tip 116 and the mass spectrometer inlet can maintain a stable electrospray during the analyte recruitment process to the mass spectrometer.

[0142] In some cases, the feedback loop may operate to maintain the voltage at the mass spectrometer inlet, ESI tip, or both within a specified percentage of a preset value. For example, in some cases, the feedback loop controls the voltage at the mass spectrometer to a preset value (e.g., ΔV TARGET ) may operate to maintain within at least 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of ). In another embodiment, in some cases, the feedback loop may operate to maintain the voltage drop between the ESI tip and the mass spectrometer within a preset value (e.g., ΔV TARGET ) may operate to maintain the voltage within at least 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the value. In some cases, the feedback loop may operate to maintain the mass spectrometer voltage within a preset value (e.g., ΔV TARGET ) may operate to maintain the voltage within at least 1,000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V. In some cases, the feedback loop adjusts the voltage difference between the mass spectrometer and the ESI tip to a preset value (e.g., ΔV). TARGET) may operate to maintain a voltage within at least 1,000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V.

[0143] In some embodiments, the ESI tip 116 can be held in ground, and the mass spectrometer or mass spectrometer inlet can be held at a constant positive or negative voltage to create an electric field between the ESI tip 116 and the mass spectrometer inlet. In some embodiments, the mass spectrometer inlet voltage (e.g., a preset value) may be approximately 5,000V, approximately +4,000V, approximately +3,500V, approximately +3,000V, approximately +2,500V, approximately +2,000V, approximately +1,500V, approximately +1,000V, approximately +500V, or approximately -5,000V, approximately -4,000V, approximately -3,500V, approximately -3,000V, approximately -2,500V, approximately -2,000V, approximately -1,500V, approximately -1,000V, or approximately -500V. Figure 12 illustrates an exemplary flowchart of a computer-controlled feedback loop for maintaining a constant voltage drop of 3,000V between the anode and cathode while keeping the ESI tip voltage at 3,000V during mobilization. However, it should be understood that a similar computer-controlled feedback loop can be used to maintain a constant voltage drop, e.g., a voltage difference of 3,000V, between the ESI tip and the mass spectrometer inlet during mobilization. In such cases, the voltage at the ESI tip may be measured (e.g., by measuring resistance, current, or voltage on or at the ESI tip), and the voltage at the mass spectrometer inlet may be adjusted to match the change in voltage at the ESI tip. In some embodiments, the measurement of the potential at the tip can be used to predict the change in potential obtained and in subsequent runs. The mass spectrometer and / or tip potential can then be adjusted based on the predicted change to maintain a constant voltage between the ESI tip and the mass spectrometer. In some embodiments, voltage and current measurements in channels within the chip can be used to calculate electrical resistance, which can then be used in subsequent runs to calculate voltage at the tip or in specific channels.

[0144] The voltage at the ESI tip may be measured using various approaches or mechanisms, including the use of a power source or electrodes. For example, the microfluidic device may have additional channels that intersect with the isolation channel (e.g., near the ESI tip) or that can be fluidly or electrically connected. For example, additional channels may cross the separation channels at positions of approximately 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, and 10 mm from the ESI tip. An additional power source may be connected to this additional channel and set to a current of 0 microamperes. The additional power source may be used to measure the potential at the ESI tip without introducing additional electrical circuitry into the microfluidic device. Alternatively, or in addition, electrodes may be placed near the ESI tip. For example, electrodes may be placed at positions approximately 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, and 10 mm from the tip of the ESI. The electrodes also do not supply any power or current, thereby enabling measurements at the ESI tip without adding any additional electrical circuitry, and thus regulating the potential applied to the mass spectrometer inlet, the ESI tip (e.g., via the potential applied to the anode and cathode ports or the anode and mobilization ports), or both, and a constant ΔVTIP-MS It may be configured to allow maintenance.

[0145] In some embodiments, electric field strength control can be performed using analog circuits. In some embodiments, voltage control at one or more electrodes in contact with a capillary-based or microfluidic device-based isolation system may be provided by using one, two, three, four, or more independent high-voltage power sources. In some cases, voltage control at one or more electrodes in contact with a capillary-based or microfluidic device-based isolation system may be provided, for example, by using a single multiplexed high-voltage power source.

[0146] In some cases, the feedback loop may operate to maintain the electric field strength in the isolation channel, the voltage drop between the anode and cathode, or the voltage drop between the device (e.g., at the ESI tip) and the mass spectrometer inlet within a specified percentage of a preset value. For example, in some cases, the feedback loop may operate to maintain the electric field strength in the isolation channel, the voltage drop between the anode and cathode, or the voltage drop between the device (e.g., at the ESI tip) and the mass spectrometer inlet within at least 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 may operate to maintain the electric field strength in the isolation channel, the voltage drop between the anode and cathode, or the voltage drop between the device (e.g., at the ESI tip) and the mass spectrometer within a preset value of at least 1,000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V.

[0147] System Hardware: Figure 5 provides a schematic diagram of a system hardware block diagram relating to 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: (i) a chemical separation system (e.g., a capillary or microfluidic device designed to perform analyte separation, e.g., isoelectric focusing-based separation, and one or more high-voltage power sources); (ii) an electrospray interface for a mass spectrometer (as shown by dashed lines), which may be directly integrated with the separation system in some cases; (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 include one or more capillary or microfluidic device flow controllers (e.g., programmable syringe pumps, peristaltic pumps, HPLC pumps, etc.), temperature controllers configured to maintain a specified temperature for all or part of the capillary or microfluidic device, additional optical or image sensors (e.g., photodiodes, avalanche photodiodes, CMOS image sensors and cameras, CCD image sensors and cameras, etc.), light sources (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.), computer memory devices, computer display devices (e.g., with a graphical user interface), digital communication devices (e.g., intranets, the Internet, WiFi, (registered trademark), or other wired or wireless communication hardware), and equivalents.

[0148] In some embodiments, the system may comprise an integrated system in which a selection of functional hardware components is packaged in a fixed configuration. In some embodiments, the system may comprise a modular system in which a selection of functional hardware components can be changed to reconfigure the system for new applications. In some embodiments, some of these functional system components, such as capillaries or microfluidic devices, are replaceable or disposable components.

[0149] As described above, any of the various different mass spectrometers may be used in different embodiments of the disclosed system, including, but not limited to, time-of-flight mass spectrometers, quadrupole mass spectrometers, ion trap or orbit trap mass spectrometers, distance-of-flight mass spectrometers, Fourier transform ion cyclotron resonance spectrometers, resonance mass measurement spectrometers, and nanomechanical mass spectrometers.

[0150] System and Application Software: As illustrated in Figure 6, the system of this 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 an operating system or environment hosted by a computer processor and may communicate with and share data with each other and / or with the operating system.

[0151] In some embodiments, the system control software module (i) coordinates the operation of the capillary or microfluidic device-based analyte separation system with image acquisition by the imaging system; (ii) coordinates the operation of the capillary or microfluidic device-based analyte separation system with data acquisition by the mass spectrometer system; (iii) coordinates the image acquisition by the imaging system with the operation of the capillary or microfluidic device-based analyte separation system and / or the mass spectrometer system; and (iv) based on data derived from imaging of the separation channel and / or Taylor cone, the electrospray ionization settings and / or one or the mass spectrometers (v) providing feedback control of operating parameters that exceed this; (vi) controlling data acquisition by the mass spectrometer while switching between a high-mass scanning range and a low-mass scanning range in an alternating manner; (vi) monitoring the voltage at the ESI tip and adjusting the isolation circuit voltage to maintain a constant isolation electric field strength (or voltage drop between the anode and cathode) and a constant voltage at the ESI tip; (vii) monitoring the voltage at the ESI tip and adjusting the isolation circuit voltage and / or mass spectrometer circuit voltage to maintain a constant electric field strength (or voltage drop) between the ESI tip and the mass spectrometer (e.g., at the input); or software for any combination thereof.

[0152] In some embodiments, the data acquisition module may include software for (i) controlling image acquisition by one or more image sensors or imaging systems, storing the image data, and providing a software interface with a system control and / or data processing software module; and (ii) controlling data acquisition by one or more mass spectrometer systems, storing the mass spectrometer data (or other downstream analytical instruments), and providing a software interface with a system control and / or data processing software module, or any combination thereof.

[0153] In some embodiments, the data processing module (i) processes images while separation is being performed, after separation is complete, or after the recruitment of pI standards and analyte peaks toward the separation channel exit or electrospray tip to determine the position of one or more pI standards or analyte peaks in the separation channel; (ii) processes images to determine the velocity, exit time, and / or electrospray release time for one or more pI standards or analyte peaks; and (iii) images of the separation channel for monitoring the position of analyte peaks and images of the Taylor cone for monitoring electrospray performance, wherein the separation channel and the The software may include steps to process the images of the Iller cone, which are obtained either simultaneously or alternately; (iv) process the images of the Taylor cone to determine the shape, density, or other properties of the Taylor cone; and calculate adjustments to be made to one or more operating parameters, such as the position of the electrospray tip or orifice relative to the mass spectrometer inlet (i.e., matching and / or separation distance), the fluid flow rate through the electrospray tip or orifice, the voltage between the electrospray tip or orifice and the mass spectrometer, or any combination thereof, which will affect the quality of the mass spectrometer data; and software for any combination thereof.

[0154] The disclosed system and application software may be implemented using any of the various 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, and equivalents.

[0155] 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 the shape, density, or other visual indicators of a Taylor cone function. Any of the various image processing algorithms known to those skilled in the art may be used for image preprocessing or image processing when implementing the disclosed methods and systems. The examples include, but are not limited to, Canny edge detection methods, Canny-Delice edge detection methods, first-order gradient edge detection methods (e.g., Sobel operator), second-order difference edge detection methods, phase-congruent (phase-coincident) edge detection methods, other image segmentation algorithms (e.g., intensity thresholding, intensity clustering methods, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., generalized Hough transform, circular Hough transform for detecting arbitrary shapes, etc.), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation, Savitsky-Gorey smoothing, eigenvalue analysis, etc.), or any combination thereof.

[0156] Processor and Computer System: One or more processors or computers may be employed to implement the methods disclosed herein. One or more processors may comprise hardware processors such as a central processing unit (CPU), a graphics processing unit (GPU), a general-purpose processing unit, or a computing platform. One or more processors may comprise any of various suitable integrated circuits (e.g., application-specific integrated circuits (ASICs) specifically designed to implement deep learning network architectures or field-programmable gate arrays (FPGAs) for speeding up computation time and / or facilitating deployment), microprocessors, newly emerging next-generation microprocessor designs (e.g., memristor-based processors), logic devices, and equivalents. While this disclosure is described with reference to processors, other types of integrated circuits and logic devices may also be applicable. The processor may have any suitable data arithmetic capability. For example, the processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data arithmetic. One or more processors may be single-core or multi-core processors, or multiple processors configured for parallel processing.

[0157] One or more processors or computers used to implement the disclosed methods may be part of a larger computer system and / or operationally coupled to a computer network ("Network") via communication interfaces to facilitate the transmission and sharing of data. The Network may be a local area network, an intranet and / or extranet, an intranet and / or extranet communicating with the Internet, or the Internet. In some cases, the Network may be a telecommunications and / or data network. The Network may, in some cases, include one or more computer servers enabling distributed computing, such as cloud computing. The Network may, in some cases, implement a peer-to-peer network using a computer system, which may enable devices coupled to the computer system to behave as clients or servers.

[0158] A computer system may also include memory or memory locations (e.g., random access memory, read-only memory, flash memory, Intel® Optane® technology), electronic storage units (e.g., hard disks), communication interfaces for communicating with one or more other systems (e.g., network adapters), and peripheral devices such as caches, other memory, data storage devices, and / or electronic display adapters. Memory, storage units, interfaces, and peripheral devices may communicate with one or more processors, e.g., CPUs, through communication buses, such as those found on a motherboard. Storage units may be data storage units (or data repositories) for storing data.

[0159] One or more processors, for example, a CPU, execute a sequence of machine-readable instructions, which are embodied in a program (or software). The instructions are stored in memory locations. The instructions are directed to the CPU, which in turn programs or otherwise configures the CPU to implement the methods of this disclosure. Embodiments of the operations performed by the CPU include fetching, decoding, executing, and writing 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).

[0160] The storage unit stores files such as drivers, libraries, and saved programs. The storage unit also stores user data, such as user preferences and user-defined programs. In some cases, the computer system may include one or more additional data storage units located outside the computer system, such as those located on remote servers that communicate with the computer system via an intranet or the internet.

[0161] Some aspects of the methods and systems provided herein are implemented using machine-executable code (e.g., processor) stored in electronic storage locations of a computer system, such as memory or electronic storage units. The machine-executable or machine-readable code is provided in software form. During use, the code is executed by one or more processors. In some cases, the code is read from a storage unit and stored in memory for quick access by one or more processors. In some situations, electronic storage units are excluded, and 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 adapted to execute the code, or it may be compiled at runtime. The code may be supplied in a programming language selected to enable the code to be executed pre-compiled or as-compiled.

[0162] Various aspects of the disclosed methods and devices may typically be considered as “products” or “manufactured goods,” e.g., “computer programs or software products,” in the form of machine (or processor) executable code and / or associated data stored in a certain type of machine-readable medium, the executable code comprising a number of instructions for controlling a computer or computer system when implementing one or more of the methods disclosed herein. The machine-executable code may be stored in an optical storage unit comprising an optical-readable medium such as an optical disc, CD-ROM, DVD, or Blu-ray® disc. The machine-executable code may be stored in an electronic storage unit such as memory (e.g., read-only memory, random-access memory, flash memory) or on a hard disk. The “storage” type medium includes any or all of the following: tangible memory of a computer, processor, or equivalent, or various semiconductor memory chips, optical drives, tape drives, disk drives, and equivalents, which can provide non-transient storage at any time for software encoding the methods and algorithms disclosed herein.

[0163] All or part of the software code may be communicated from time to time via the Internet or various other telecommunications networks. Such communication enables, for example, the loading of software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media used to convey software encoded instructions include optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical fixed telephone networks, and over various atmospheric links. Physical elements that carry such waves, such as wired or wireless links, optical links, or equivalents, are also considered media for conveying software encoded instructions to implement the methods disclosed herein. Unless limited to non-transient tangible “storage” media as used herein, the terms computer or machine “readable media,” etc., refer to any medium involved in providing instructions to a processor for execution.

[0164] A computer system may typically include, for example, an electronic display for providing images captured by a machine vision system, or communicate with such a display. The display may also typically provide a user interface (UI). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs), web-based user interfaces, and equivalents.

[0165] Applications: As described above, the disclosed methods, devices, systems, and software have potential applications in a variety of fields, including, but are not limited to, proteomics research, drug discovery and development, and clinical diagnostics. For example, the improved information content and data quality that can be achieved using the disclosed methods for separation-based ESI-MS analysis of analyte samples may be highly beneficial for the characterization of biologics and biosimilars during development and / or manufacturing. Other applications may 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, multicellular organisms, and other applications.

[0166] Biologics and biosimilars are a class of drugs that include, for example, recombinant proteins, antibodies, live viral vaccines, human plasma-derived proteins, cell-based drugs, naturally occurring proteins, antibody-drug conjugates, protein-drug conjugates, and other protein drugs. The FDA and other regulatory agencies require the use of a stepwise approach to demonstrating biosimilarity, which may include comparisons of the proposed product and a reference product regarding structure, function, animal toxicity, human pharmacokinetics (PK) and pharmacodynamics (PD), clinical immunogenicity, and clinical safety and efficacy (see, for example, "Scientific Considerations in Demonstrating Biosimilarity to a Reference Product: Guidance for Industry," U.S. Department of Health and Human Services, Food and Drug Administration, April 2015). Examples of structural characterization 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., the three-dimensional shape of the protein produced by the folding of the polypeptide backbone and secondary structure domains), and quaternary structure (e.g., the number of subunits required to form an active protein complex or protein aggregate state). In many cases, this information may not be available without employing laborious, time-intensive, and expensive techniques such as X-ray crystallography. Therefore, there is a need for experimental techniques that enable convenient, real-time, and relatively high-rate characterization of protein structures for the purpose of establishing biosimilarity between candidate biopharmaceuticals and reference drugs.

[0167] In some embodiments, the disclosed methods, devices, and systems may be used to provide structural comparison data for a biopharmaceutical candidate (e.g., a monoclonal antibody (mAb)) and a reference biopharmaceutical for the purpose of establishing biosimilarity. For example, in some cases, isoelectric focusing data and / or mass spectrometry data for the drug candidate and the reference drug may provide important evidence supporting the demonstration of biosimilarity. In some embodiments, isoelectric focusing data and / or mass spectrometry data for the drug candidate and the reference drug, both treated with site-specific proteases under the same reaction conditions, may provide important evidence supporting the demonstration of biosimilarity. In some embodiments, the disclosed methods, devices, and systems may be used to monitor the biopharmaceutical manufacturing process and ensure product quality and consistency by analyzing samples taken at different points in the production process or from different production steps. In some embodiments, the disclosed methods, devices, and systems may be used to evaluate the stability of formulation buffers. In some embodiments, the disclosed methods, devices, and systems may be used to evaluate cloned cell lines with respect to the production and quality of biopharmaceutical candidates. [Examples]

[0168] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. [Examples]

[0169] Characterization of protein charge on a chip before performing mass spectrometry. The fabrication of the microfluidic device illustrated in Figure 1 has been described above. To operate the device, it is mounted on an instrumentation containing a nitrogen gas source, a heater, a positive pressure pump (e.g., Parker, T5-1IC-03-1EEP), an electrophoretic power source (Gamm High Voltage, MC30) terminated at 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 onto the device. The power source shares a common ground with the mass spectrometer. The instrumentation is controlled via software (e.g., LabView).

[0170] Protein samples are placed in vials and pre-mixed with amphoteric electrolyte pH gradients and pI markers before being loaded onto the autosampler. They are then continuously loaded from the autosampler via inlet 412 onto the microfluidic device 400, through concentration channel 418, and out of the device through outlet 434 to waste 430.

[0171] A sheath / cathodeliquid fluid (50% MeOH, N40H / H2O) is loaded into two cathodeliquid wells 404 and 436, an anolyte (10 mM H3P04) is loaded into an anolyte well 426, and a source of heated nitrogen gas is attached to two gas wells 408 and 440.

[0172] After all reagents are loaded, an electric field of +600 V / cm is applied from the anode well 426 to the cathode wells 404 and 436 by connecting electrodes to the anode well 426 and cathode wells 404 and 436 to initiate isoelectric focusing. A UV light source is aligned below the concentration channel 418, and a camera is positioned above the concentration channel 418 to measure the light passing through the concentration channel 418, thereby detecting the focused protein using its absorbance. A glass plate 402 constructed from soda-lime glass acts to block any stray light from the camera, and therefore, light that does not pass through the concentration channel 418 is prevented from reaching the camera, increasing the sensitivity of the measurement.

[0173] Images of the focusing proteins can be captured continuously and / or periodically during the IEF. Once focusing is complete, a low pressure will be applied from the inlet 412 to recruit a pH gradient toward the orifice 424. The electric field can be maintained at this point to maintain high-resolution IEF separation. Continuing to image the enrichment channel 418 during the ESI process can be used to determine the pI of each protein as it is effluxed from the orifice 424.

[0174] As the concentrated protein fraction moves from the concentration channel 418 into the confluence point 420, it is mixed with the sheath fluid, which can then flow from the cathode fluid wells 404 and 436 through the sheath / cathode fluid channels 406 and 438 to the confluence point 420. Mixing the concentrated protein fraction with the sheath fluid allows the protein fraction to be placed in a mass spectrometry-compatible solution, returning the charge to the focused protein (the IEF makes the protein uncharged), and improving ionization.

[0175] The concentrated protein fraction then proceeds to the orifice 424, which can be defined by the dish surface 422 of the glass plate 402. Once the concentrated protein fraction is drawn into the electric field between the sheath liquid well ground and the mass spectrometer negative electrode, it can form a Taylor cone.

[0176] As the solution continues to push the Taylor cone from the concentration channel 418, small droplets of fluid will be ejected from the Taylor cone and fly towards the mass spectrometer inlet. Nitrogen gas (e.g., at 150 °C) can flow from the gas wells 408, 440 through the gas channels 410, 432 to form a nitrogen gas injection located on the side of the Taylor cone, which can convert the droplets emitted from the Taylor cone into a fine mist before leaving the microfluidic device, which can assist detection in the mass spectrometer. Adjusting the pressure from the inlet 412 can adapt the Taylor cone size as needed to improve detection in the mass spectrometer.

Example

[0177] Tracking their velocities as analyte peaks leave the microfluidic chip and enter the mass spectrometer In this embodiment, the microfluidic channel network 100 in Figure 7A is fabricated in a 250-micron thick layer of opaque cyclic olefin polymer. Channel 112 is 250 microns deep and therefore traverses the entire 250-micron layer. All other channels are 50 microns deep. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer, as shown in Figure 7B, to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, and 110 provide access to the channel network for reagent introduction from an external reservoir and for electrical contact. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel and enabling priming of other reagents through the channel network to the "waste". An acid (1% formic acid) is primed through port 108 to channels 109, 112, 114, and 103, and up to port 102. The sample (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)) is primed through port 106 into channels 107, 112, 114, and 103, and up to port 102. This brings channel 112 into a state containing the sample analyte. The base (1% dimethylamine) is primed through port 104 into channels 105, 114, and 103, and up to port 102. The recruiter (1% formic acid, 49% methanol) is primed through port 110 into channels 111, 114, and 103, and out of channel 103, and up to port 102.

[0178] Electrophoresis of the analyte sample within channel 112 is performed by applying 4,000 V to port 108 and connecting port 110 to ground. The ampholytes within the analyte sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed using a 280 nm light source aligned with channel 112 and measuring the transmission of 280 light through channel 112 using a CCD camera. Software calculates the absorbance by comparing the light transmission during separation or mobilization to a “blank” reference measurement obtained in the absence of the focused analyte prior to the analyte being flowed, and then displays the absorbance per pixel across the length of channel 112. The location where the standard or analyte is focused is displayed as a peak, as shown in FIGS. 9A-9F.

[0179] Once the analyte has completed focusing, a final focused absorbance image is captured. The software will identify the spatial position of the pI markers, interpolate between the markers, and calculate the pI of the focused analyte fraction peak. At this point, the control software will disconnect the ground connection at port 110, trigger a relay connecting port 104 to ground, and set the pressure on the mobilizer reservoir connected to port 104 to establish a flow of mobilizer solution at 100 nL / min into channels 105 and 114 through port 104 and out of the chip at orifice 116. Orifice 116 is positioned 2 mm away from the mass spectrometer ESI inlet with an inlet voltage of -3,500 V to -4,500 V.

[0180] A pressure-driven flow directs the recruiter from port 104 to orifice 116, while a portion of the formic acid in the recruiter reagent will electrophoretically move from channel 105 through channel 112 to the anode at port 108 in the form of formate. As the formate moves through channel 112, this disturbs the isoelectric pH gradient, increasing the charge on the amphoteric electrolyte, standard, and analyte sample, causing them to electrophoretically migrate from channel 112 into channel 114, where the pressure-driven flow from port 104 will transport them into the ESI spray and out of orifice 116.

[0181] While mobilization occurs, the software continues to capture absorbance images, identify peaks, and track their migration from imaging channel 112 into channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and its flow rate within channel 114, the software can calculate the time it takes for the peaks to traverse channel 114 and be introduced into the mass spectrometer via the orifice 116, enabling a direct correlation between the original focused peaks and the resulting mass spectrum.

[0182] Figures 9A–F provide examples of a series of absorbance traces obtained at 1-minute intervals, illustrating the mobilization of isoelectric focusing (pI) standards as determined from the images of the separated channels. Figure 9A shows a plot of absorbance 910 as a function of channel distance 905 after isoelectric focusing electrophoresis of five pI standards (peaks 915, 920, 925, 930, 935) is completed prior to mobilization. As shown in Figure 9B, 1 minute after mobilization, peak 915, corresponding to pI=9.99 standard, is at the edge of the imaging system's field of view. As shown in Figure 9C, 2 minutes after mobilization, peak 915 (pI=9.99 standard) has moved out of the portion of the channel being imaged. As shown in Figure 9D, 3 minutes after mobilization, peak 920 (pI=8.40 standard) has moved out of the portion of the channel being imaged. As shown in Figure 9E, four minutes after mobilization, peak 925 (pI=7.00 standard) has moved out of the portion of the channel being imaged. As shown in Figure 9F, five minutes after mobilization, peak 930 (pI=4.05 standard) has moved away from the portion of the channel being imaged. [Examples]

[0183] Use feedback to adjust MS and ESI parameters. In Example 3, the tip, instrument, and software are all identical to those in Example 2. In addition, a second CCD camera is used to image the Taylor cone during ESI, as shown in Figure 8. These images are used to evaluate the quality and consistency of the Taylor cone. Evaluating the images and / or total ion counts from the mass spectrometer allows for the identification and diagnosis of failures in the ESI Taylor cone.

[0184] Taylor cone shape in ESI depends on maintaining input flow to the cone that matches the rate of fluid lost due 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 variations in the ESI tip and local environment of the microfluidic device.

[0185] Imaging the Taylor cone allows for the diagnosis of the cause of ESI failure. For example, loss of the Taylor cone indicates insufficient fluidity, and the software can increase the flow of recruiter into the microfluidic device. Similarly, corona discharge indicates that the voltage is too high, and the software can reduce the voltage. Expansion of the EIS cloud indicates that the voltage is too high, while the formation of droplets instead of Taylor cones indicates that the voltage is too low. These differences, and any other visual differences, can be identified in the image, and the software can automatically compensate by re-establishing the Taylor cone. [Examples]

[0186] Low-mass scanning as a marker for separation In Example 4, the tips, instruments, and software are all the same as in Example 2. In addition, once recruitment occurs and analyte peaks begin to migrate to the MS, the MS is set to alternate between the 1,500–6,000 and 150–1,500 m / z ranges. The 1,500–6,000 range is used to identify the NIST antibody analyte fraction peaks as they are introduced into the MS. The 150–1,500 m / z range scan is used to identify the free solution amphoteric electrolytes (pharlytes) as they are introduced into the MS. Since the presence of specific amphoteric electrolytes defines a portion of the isoelectric pH gradient being analyzed in the MS at any given time, amphoteric electrolytes can be identified in the mass scan and used to calibrate the total ion chromatogram from the MS. [Examples]

[0187] The high and low voltages are modified to maintain the electric field strength and a constant voltage at the tip. In this embodiment, the microfluidic channel network 100 in Figure 7A is fabricated in a 250-micron thick layer of opaque cyclic olefin polymer. Channel 112 is 250 microns deep and therefore traverses the entire 250-micron layer. All other channels are 50 microns deep. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer, as shown in Figure 7B, to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, and 110 provide access to the channel network for reagent introduction from an external reservoir and for electrical contact. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel and enabling priming of other reagents through the channel network to the "waste". An acid (1% formic acid) is primed through port 108 to channels 109, 112, 114, and 103, and up to port 102. The sample (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)) is primed through port 106 into channels 107, 112, and 114, and up to port 102. This brings channel 112 into a state containing the sample analyte. The base (1% dimethylamine) is primed through port 104 into channels 105, 114, and 103, and up to port 102. The recruiter (1% formic acid, 49% methanol) is primed through port 110 into channels 111, 114, and 103, and out of channel 102, and up to port 102. Pressure is applied to the base reservoir, generating a flow of 100 nL / min into channels 105 and 114 through port 104 and out through orifice 116.

[0188] Isoelectric focusing of the analyte sample in channel 112 is initiated by applying 2,000V to port 108 using power source 1005 and connecting port 110 to high-voltage power source 1010 and applying -2,000V. This establishes the circuit shown in Figure 10A, which includes high-voltage power sources 1005 and 1010 (in some cases, sources 1005 and 1010 may comprise two channels of a single multiplexed high-voltage power source) and generates a voltage drop of 4,000V between the anode and cathode. The electrical resistance of the channel depends on the channel dimensions and the conductivity of the reagent. In this embodiment, the electrical resistance R109 of the acidic channel corresponding to channel 109 (see Figure 7A) is 10 megaohms, the electrical resistance R112 of the sample channel corresponding to channel 112 (see Figure 7A) starts at 40 megaohms, and the resistance R111 of the base in the channel corresponding to channel 111 (see Figure 7A) is 50 megaohms. The resistance R113 of the electrospray ionization (ESI) interface between orifice 116 (see Figure 7A) and mass spectrometer 1015 is 2 gigaohms. The total voltage drop across channels 109, 112, and 111 (see Figure 7A) is 4,000V, and since these channels represent three resistors in series, the voltage at the tip (V) 116 ) is calculated according to the following equation 1. V 116 =ΔV 108-110 ×(R 111 ) / (R 109 +R 112 +R 111 ) + (High voltage power supply source 1010 voltage setting) At the start of isoelectric focusing, V 116 = 0 volts. Orifice 116 (see Figure 7A) is positioned 2 mm away from the ESI mass spectrometer inlet and forms a Taylor cone with an inlet voltage of -3,500 V to -4,500 V. Figure 10B shows another embodiment of the circuit shown in Figure 10A, with resistor R105 of channel 105.

[0189] The amphoteric electrolytes in the analyte sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed using a 280 nm light source matched to channel 112, and the transmission of 280 nm light through channel 112 is measured using a CCD camera. The software calculates absorbance by comparing the light transmission during separation or recruitment to a "blank" reference measurement obtained in the absence of the focused analyte before the analyte is flowed, and then displays the absorbance per pixel over the length of channel 112. The locations where the standard or analyte is focused are displayed as peaks, as illustrated in Figures 9A–9F.

[0190] As the sample is focused, the amphoteric electrolyte, antibody isoform, and standard reach their isoelectric points and lose their charge, so the resistance of sample channel 112 increases, while the resistances in channels 109 and 111, as well as in the ESI interface, remain unchanged. The computer implementation can monitor the current in power source 1005 and calculate the resistance in channel 112 at any given time. The computer implementation uses this information to adjust power sources 1005 and 1010. For example, when the resistance in channel 112 rises to 140 megaohms, if the power sources are not adjusted, the voltage at orifice 116 will be -1,000V, which will disturb the Taylor cone. However, by adjusting power source 1005 to +3,000V and power source 1010 to -1,000V, the tip will remain at 0V, and the total voltage drop across channels 109, 112, and 111 will remain at 4,000V. These adjustments are made on the fly as the resistance in channel 112 changes.

[0191] Once the analyte has completed focusing, the final focused absorbance image is captured. The software will identify the spatial positions of the pI markers, interpolate between the markers, and calculate the pI of the focused analyte fraction peak. At this point, the control software will deconnect the power source 1010 at port 110, trigger a relay to connect port 104 to the power source 1010, and set the pressure on the recruiter reservoir connected to port 104 to establish a flow of recruiter solution at 100 nL / min through port 104 into channels 105 and 114 and out of the tip at orifice 116 (see schematic diagram of the tip in Figure 7A and the electrical circuit shown in Figure 10B). Orifice 116 is positioned 2 mm away from the mass spectrometer ESI inlet with an inlet voltage of -3,500 V to -4,500 V.

[0192] A pressure-driven flow directs the recruiter from port 104 to orifice 116, while a portion of the formic acid in the recruiter reagent will electrophoretically move from channel 105 through channel 112 to the anode at port 108 in the form of formate. As the formate moves through channel 112, this disturbs the isoelectric pH gradient, increasing the charge on the amphoteric electrolyte, standard, and analyte sample, causing them to electrophoretically migrate from channel 112 into channel 114, where the pressure-driven flow from port 104 will transport them into the ESI spray and out of orifice 116.

[0193] During mobilization, the resistance of channel 112 will decrease. Figures 11A-B show examples of voltage and current data for channel 112 that can be used to derive the channel's resistance. Figure 11A shows a plot of voltage as a function of time. Figure 11B shows a plot of current as a function of time. The software monitors the current changes and adjusts the power source to maintain a voltage drop between the anode and cathode at tip 116 of 3,000V and 0V, as described in Figure 15. The voltage changes may be transient or stable.

[0194] While mobilization occurs, the software continues to capture absorbance images, identify peaks, and track their migration from imaging channel 112 into channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and its flow rate within channel 114, the software can calculate the time it takes for the peaks to traverse channel 114 and be introduced into the mass spectrometer via the orifice 116, enabling a direct correlation between the original focused peaks and the resulting mass spectrum.

[0195] Figure 13A provides a typical circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization, where the ESI tip will be held at a positive voltage using an additional resistor R120 to sink the current to ground. The circuit may also include a high-voltage power source 1305 which may be substantially similar to 1005 and a high-voltage power source 1310 which may be substantially similar to 1010, generating a defined voltage drop (e.g., 4,000V) between the anode and cathode. The circuit may also include a third high-voltage power source 1307. The electrical resistance of the channel depends on the dimensions of the channel and the conductivity of the reagent. Also integrated into the circuit are the electrical resistance R109 of the acidic channel corresponding to channel 109 (see Figure 7A), the electrical resistance R112 of the sample channel corresponding to channel 112 (see Figure 7A), and the resistance R111 of the base in the channel corresponding to channel 111 (see Figure 7A), as well as the electrospray ionization (ESI) resistance R113 between the orifice 116 (see Figure 7A) and the voltage source of the mass spectrometer 1315, which may be substantially similar to 1015. The circuit may also include the electrical resistance R105 of channel 105. The power source 1307 is connected to channel 111 (see Figure 7A) and can use current control set to 0 μA during mobilization. This power source may be used to read the voltage at the tip and implement a computer-controlled feedback loop to maintain a constant voltage at the tip.

[0196] Figure 13B shows a representative circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization. The ESI tip will be held at a positive voltage using resistor R120 to sink current to power supply 1320. Figure 13C shows a representative circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization. The ESI tip will be held at a positive voltage using field effect transistor (FET) 1325 to sink current. The electrical circuit may additionally comprise amplifier 1330, voltage reference 1335, and additional resistor R200. Figure 13D shows a representative circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization. The ESI tip will be held at a positive voltage using bipolar junction transistor (BJT) 1340 to sink current. Power supply 1307 is connected to channel 111 (see Figure 7A) and can use current control set to 0 μA. This power supply may be used to implement a computer-controlled feedback loop to read the voltage at the tip and maintain a constant voltage at the tip. Figure 13E provides a representative circuit diagram for the microfluidic device shown in Figure 7A during chemical mobilization of a separated analyte mixture. The ESI tip will be held at or near ground. Power supply 1307 is connected to channel 111 (see Figure 7A) and can use current control set to 0 μA. This power supply may be used to implement a computer-controlled feedback loop to read the voltage at the tip and maintain a constant voltage at the tip.

Example

[0197] Based on measuring the tip voltage, modify the high and low voltages to maintain the electric field strength and a constant voltage at the tip In this embodiment, the microfluidic channel network 100 in Figure 7A is fabricated in a 250-micron thick layer of opaque cyclic olefin polymer. Channel 112 is 250 microns deep and therefore traverses the entire 250-micron layer. All other channels are 50 microns deep. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer, as shown in Figure 7B, to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, and 110 provide access to the channel network for reagent introduction from an external reservoir and for electrical contact. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel and enabling priming of other reagents through the channel network to the "waste". An acid (1% formic acid) is primed through port 108 to channels 109, 112, 114, and 103, and up to port 102. The sample (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)) is primed through port 106 into channels 107, 112, and 114, and up to port 102. This brings channel 112 into a state containing the sample analyte. The base (1% dimethylamine) is primed through port 104 into channels 105, 114, and 103, and up to port 102. The mobilizer (1% formic acid, 49% methanol) is primed through port 110 into channels 110, 114, and 103, and exiting channel 102 back to port 102 (see the schematic chip diagram in Figure 7A and the electrical circuit diagram in Figure 13E).

[0198] Electrophoresis of the analyte sample in channel 112 is initiated by applying 1,500V to port 108 using power source 1305 and connecting port 110 to power source 1307, which is set to 0V. After 5 minutes, power source 1305 is increased to 3,000V over 3 minutes to complete focusing.

[0199] The amphoteric electrolytes in the analyte sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed using a 280 nm light source matched to channel 112, and the transmission of 280 nm light through channel 112 is measured using a CCD camera. The software calculates absorbance by comparing the light transmission during separation or recruitment to a "blank" reference measurement obtained in the absence of the focused analyte before the analyte is flowed, and then displays the absorbance per pixel over the length of channel 112. The locations where the standard or analyte is focused are displayed as peaks, as illustrated in Figures 9A–9F.

[0200] Once the analyte has completed focusing, the final focused absorbance image is captured. The software will identify the spatial positions of the pI markers, interpolate between the markers, and calculate the pI of the focused analyte fraction peak. At this point, the control software will trigger a relay connecting port 104 to the power source 1310, and set the pressure on the recruiter reservoir connected to port 104 to establish a flow of recruiter solution at 100 nL / min through port 104 into channels 105 and 114 and out of the tip at orifice 116. Orifice 116 is positioned 2 mm away from the mass spectrometer ESI inlet 1315 with an inlet voltage of -3,500 V to -4,500 V. Power source 1307 is set to 0 μA using current control, power source 1305 is set to 3,000 V, power source 1310 is set to 0 V, and the MS ESI ion source is set to -3,500 V to -4,500 V.

[0201] A pressure-driven flow directs the recruiter from port 104 to orifice 116, while a portion of the formic acid in the recruiter reagent will electrophoretically move from channel 105 through channel 112 to the anode at port 108 in the form of formate. As the formate moves through channel 112, this disturbs the isoelectric pH gradient, increasing the charge on the amphoteric electrolyte, standard, and analyte sample, causing them to electrophoretically migrate from channel 112 into channel 114, where the pressure-driven flow from port 104 will transport them into the ESI spray and out of orifice 116.

[0202] While mobilization occurs, the resistance of channel 112 will decrease. Since the voltage drop across channel 111 is 0 here (ΔV=IR=0×R111=0V), the power source 1307, set to 0μA, will be equal to the voltage at V116 in Figure 13E. As shown in the data in Figure 11A-B, at 8 minutes (480 seconds) after focusing is complete, the software monitors the current change and adjusts the power source to maintain a constant voltage drop between the anode and cathode at tip 116 of 3,000V and 0 volts, as described in Figure 15. The voltage at tip (V116) is described by equation 2 below. V 116 =ΔV 108-110 ×(R 111 ) / (R 109 +R 112 +R 105 ) + (Power supply source 1310 voltage setting)

[0203] While mobilization occurs, the software continues to capture absorbance images, identify peaks, and track their migration from imaging channel 112 into channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and its flow rate within channel 114, the software can calculate the time it takes for the peaks to traverse channel 114 and be introduced into the mass spectrometer via the orifice 116, enabling a direct correlation between the original focused peaks and the resulting mass spectrum. [Examples]

[0204] By measuring the tip voltage and using a resistor, the high and low voltages are modified to maintain the electric field strength and a constant voltage at the tip. In this embodiment, the microfluidic channel network 100 in Figure 7A is fabricated in a 250-micron thick layer of opaque cyclic olefin polymer. Channel 112 is 250 microns deep and therefore traverses the entire 250-micron layer. All other channels are 50 microns deep. The channel layer is sandwiched between two transparent layers of cyclic olefin polymer, as shown in Figure 7B, to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, and 110 provide access to the channel network for reagent introduction from an external reservoir and for electrical contact. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel and enabling priming of other reagents through the channel network to the "waste". An acid (1% formic acid) is primed through port 108 to channels 109, 112, 114, and 103, and up to port 102. The sample (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)) is primed through port 106 into channels 107, 112, and 114, and up to port 102. This brings channel 112 into a state containing the sample analyte. The base (1% dimethylamine) is primed through port 104 into channels 105, 114, and 103, and up to port 102. The mobilizer (1% formic acid, 49% methanol) is primed through port 110 into channels 110, 114, and 103, and exiting channel 102 back to port 102 (see the schematic chip diagram in Figure 7A and the electrical circuit diagram in Figure 13B).

[0205] Electrophoresis of the analyte sample in channel 112 is initiated by applying 1,500V to port 108 using power source 1305 and connecting port 110 to power source 1307, which is set to 0V. After 5 minutes, power source 1305 is increased to 3,000V.

[0206] The amphoteric electrolytes in the analyte sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed using a 280 nm light source matched to channel 112, and the transmission of 280 nm light through channel 112 is measured using a CCD camera. The software calculates absorbance by comparing the light transmission during separation or recruitment to a "blank" reference measurement obtained in the absence of the focused analyte before the analyte is flowed, and then displays the absorbance per pixel over the length of channel 112. The locations where the standard or analyte is focused are displayed as peaks, as illustrated in Figures 9A–9F.

[0207] Once the analyte has completed focusing, the charge deformation of infliximab is separated as shown in panel A of Figure 16, and the final focused absorbance image is captured. The software will identify the spatial positions of the pI markers, interpolate between the markers, and calculate the pI of the focused analyte fraction peak. At this point, the control software will trigger a relay connecting port 104 to the power source 1310, and set pressure on the recruiter reservoir connected to port 104 to establish a flow of recruiter solution at 100 nL / min through port 104 into channels 105 and 114 and out of the tip at orifice 116. Orifice 116 is positioned 2 mm away from the mass spectrometer ESI inlet 1315. Power source 1307 is set to 0 μA using current control, power source 1305 is set to 7,000 V, power source 1310 is set to 4,000 V, and MS ESI ion source 1315 is held to ground. An additional resistor R120 is connected to the system between power source 1310 and channel 105 (R current sink), and the other side of resistor R120 is connected to power source 1320 as shown in Figure 13B. Power source 1320 will be set to a minimum of 4,000 V, below power source 1310, in order to act as a current sink. The resistor R120 could instead be a field-effect transistor (FET) as shown in Figure 13C, which could connect the electrical circuit to ground as shown in Figure 13A, a bipolar junction transistor (BJT) as shown in Figure 13D, or any other resistive element that can sink current from the power source 1310 to create a working electrophoretic circuit.

[0208] A pressure-driven flow directs the recruiter from port 104 to orifice 116, while a portion of the formic acid in the recruiter reagent will electrophoretically move from channel 105 through channel 112 to the anode at port 108 in the form of formate. As the formate moves through channel 112, this disturbs the isoelectric pH gradient, increasing the charge on the amphoteric electrolyte, standard, and analyte sample, causing them to electrophoretically migrate from channel 112 into channel 114, where the pressure-driven flow from port 104 will transport them into the ESI spray and out of orifice 116.

[0209] While mobilization occurs, the resistance of channel 112 will decrease. Since the voltage drop across channel 111 is 0 here (ΔV=IR=0×R111=0V), the power source 1307, set to 0μA, will be equal to the voltage at V116. As shown in the data in Figures 11A and 11B, the software monitors the change in current and adjusts the power source to maintain a constant voltage drop of 3,000V and 3,000V between the anode and cathode at tip 116, as illustrated in Figure 12. The voltage at tip (V116) is described by equation 2 below. V 116 =ΔV 108-110 ×(R 111 ) / (R 109 +R 112 +R 105 ) + (Power supply source 1310 voltage setting)

[0210] While recruitment occurs, the software continues to capture absorbance images, identify peaks, and track their migration from imaging channel 112 into channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and its flow rate within channel 114, the software can calculate the time it takes for the peak to traverse channel 114 and be introduced into the mass spectrometer via the orifice 116, allowing for a direct correlation between the original focused peak and the resulting mass spectrum. For example, panel B of Figure 16 shows the mass of the glycoform electrosprayed into the mass spectrometer that was contained within the acidic peak in the electrophoresis diagram shown in panel A of Figure 16. Panel C of Figure 16 shows the mass of the glycoform in the primary infliximab peak from panel A of Figure 16. Panels D and E of Figure 16 show the masses of the basic peaks from the electrophoresis diagram shown in panel A of Figure 16. [Examples]

[0211] In a two-step capillary IEF, high and low voltages are modified to maintain electric field strength and a constant voltage. In Example 8, as outlined in Figure 14A, a two-step IEF (isoelectric focusing electrophoresis followed by recruitment) is performed in a 60 cm capillary tube and recruited into the ESI-MS through a joining sprayer. Separation capillary tube 1808 is immersed in an anodeliquid vial 1806. A high-voltage power source 1802 is connected to the anodeliquid vial 1806 through an electrode 1804. The other end of capillary tube 1808 is connected to a joining sprayer 1814 through a T-connector union 1812. Capillary tube 1808 is inserted into the joining sprayer 1814, so that the capillary outlet is close to the ESI tip 1824. A third arm of the T-connector union 1812 is connected to a recruiter capillary tube 1816, which is immersed in a pressurized recruiter vial 1818. The pressurized mobilizer vial 1818 is also grounded via electrode 1817, and therefore can act as a current sink. In addition, the junction sprayer 1814 is connected to the power supply 1810 via wire 1820 which connects to the outside of the sprayer 1814. In this embodiment, the mass spectrometer ion source is held in ground.

[0212] The reagents are prepared as follows: Anode liquid vial 1806 is filled with 1% formic acid in water; separation capillary tube 1808 is filled with aqueous sample (250 μg / mL NIST mAb, 1.5% Pharmalyte 5-8 amphoteric electrolyte, 1.5% Pharmalyte 8-10.5, 5 mg / mL pI standards 7.00 and 10.17); junction sprayer chamber 1826 and recruiter capillary tube 1816 are filled with 1% diethylamine in water; and pressurized recruiter vial 1818 is filled with 1% formic acid, 50% acetonitrile, and 49% water.

[0213] In this embodiment, the mass spectrometer ion source is held at ground. To initiate focusing, power source 1802 is set to +30kV and power source 1810 is set to 4kV. A pressure-driven flow from recruiter vial 1818 is also started at 100nL / min. Thus, ESI is initiated using diethylamine in bonding sprayer cavity 1826, and the diethylamine also acts as the cathode solution for the isoelectric focusing step.

[0214] As focusing progresses in capillary tube 1808, the sample loses its charge-carrying ability and its resistance increases in capillary tube 1808. When the ESI tip is electrically positioned between capillary tube 1808 and the diethylamine in chamber 1826 (see Figure 14B), the ESI tip voltage (V 1824 ) will descend according to the following equation 3. V 1824 =ΔV 1806-1814 ×R 1826 / (R 1808 +R 1826 )+V 1814

[0215] In addition, as the resistance in capillary tube 1808 increases, the current passing through the capillary tube decreases, which can be measured at the power source 1802. The increased current will be directly related to the change in resistance in capillary tube 1808 by the following equation 4. I 1806 =ΔV 1806-1814 / (R 1808 +R 1826 )

[0216] Using a computer-controlled feedback loop as illustrated in Figure 12, the system can calculate the change in resistance in capillary tube 1808 (and thus the change in voltage drop across capillary tube 1808, which defines the voltage at ESI tip 1824), and the system can adjust power sources 1802 and 1810 to reserve a ΔV of 26kV and maintain an ESI tip voltage of 4,000kV.

[0217] After focusing is complete (approximately 30 minutes), the recruiter solution in pressurized recruiter vial 1818 will replace the diethylamine in the bonding sprayer chamber 1826, initiating the recruitment of NIST mAb protein isoforms in capillary tube 1808. In a similar manner, but contrary to isoelectric focusing, as recruitment progresses, the resistance in capillary tube 1808 will decrease, affecting the voltage at ESI tip 1824. Again, the computer-controlled feedback loop will use equations 3 and 4 to calculate the changes in power sources 1802 and 1810 required to maintain a 26kV electric field while keeping the ESI tip 1824 voltage at 4kV.

[0218] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous modifications, alterations, and substitutions will be conjured upon those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in any combination when practicing the present invention. The following claims define the scope of the present invention, and methods and structures within the scope of these claims, as well as their equivalents, are intended to be covered thereby.

Claims

1. A computer implementation method, wherein the computer implementation method is (a) Using a processor, (i) A first dataset comprising a plurality of intensity or absorbance measurements as a function of length along the separation channel from isoelectric focusing electrophoresis separation performed in the separation channel, (ii) A second dataset comprising multiple mass spectrometer total ion measurements as a function of time and Receiving and (b) Using the processor, convert the second dataset into a third dataset comprising multiple ion count measurements as a function of mass, (c) Using the processor to overlay the plot of the first dataset and the plot of the third dataset Computer implementation methods, including those mentioned above.

2. (c) The computer implementation method according to claim 1, further comprising using the processor to overlay a plot of the second dataset with the plot of the first dataset and the plot of the third dataset.

3. (b) Converting the second dataset to the third dataset comprises undoing the convolution of the second dataset, the third dataset being the undoed convolution of the second dataset, according to claim 1 or 2.

4. (c) The computer implementation method according to any one of claims 1 to 3, wherein a first peak in intensity or absorbance of the first dataset is mapped to a set of peaks of the third dataset.

5. The computer implementation method according to claim 4, wherein the second dataset is used to map the first dataset to the set of peaks of the third dataset.

6. The computer implementation method according to claim 4, further comprising determining the mass distribution and isoelectric point of at least one analyte of the first peak by correlating the first peak with a set of peaks using the processor.

7. The computer implementation method according to claim 4, wherein the first peak corresponds to the analyte peak and provides information regarding the isoelectric point of one or more analytes in the analyte peak.

8. The computer implementation method according to claim 7, wherein the set of peaks corresponds to the mass distribution of one or more analytes in the analyte peaks.

9. The computer implementation method according to claim 8, further comprising using the processor to determine the identification of one or more analytes in the analyte peak with respect to a given isoelectric point.

10. The computer implementation method according to claim 9, wherein the one or more analytes include different protein isoforms.

11. The protein isoform comprises different post-translational modifications of the protein. The computer mounting method according to claim 10, wherein the modification is selected from the group consisting of hydroxylation, methylation, lipidization, acetylation, disulfide bond formation, smoylation, ubiquitination, glycosylation, glycation, amino acid addition or removal, amidation, deamidation, isomerization, oxidation, fucosylation, sialylation, and phosphorylation.

12. The computer implementation method according to any one of claims 1 to 11, wherein the overlay plot shows a time series of (i) intensity or absorbance measurements among the plurality of intensity or absorbance measurements as a function of the length along the separation channel, and (ii) a plurality of ion count measurements as a function of mass.

13. The computer implementation method according to any one of claims 1 to 12, further comprising obtaining the first dataset and the second dataset by performing isoelectric focusing separation and recruitment and electrospray ionization using a single integrated microfluidic device coupled to a mass spectrometer.

14. (b) and (c) are performed within one minute from the ESI-MS, or in parallel with the ESI-MS, the computer implementation method according to claim 13.

15. (b) and / or (c) are performed automatically as part of a software package for obtaining or processing electrospray ionization mass spectrometry (ESI-MS) data, according to any one of claims 1 to 14.

Images