Software for microfluidic systems interfaced with mass spectrometry

Abstract

Methods, apparatuses, and systems for improving the quality of electrospray ionization mass spectrometer (ESI-MS) data are described as methods, apparatuses, and systems for enabling improved correlation between chemical separation data and mass spectrometry data.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 078,856, filed September 15, 2020; U.S. Provisional Patent Application No. 63 / 021,020, filed May 6, 2020; U.S. Provisional Patent Application No. 62 / 966,372, filed January 27, 2020; and U.S. Provisional Patent Application No. 62 / 940,071, filed November 25, 2019, each of which is incorporated herein by reference in its entirety for all intents and purposes. Background Technology

[0003] This disclosure relates to the field of chemical analysis, and more specifically to the separation of analytes from mixtures and their subsequent analysis by mass spectrometry (MS). Separating analyte components from more complex mixtures of analytes based on their inherent mass and providing a collection of fractions enriched to that mass is a key part of analytical chemistry. Simplifying complex mixtures in this way reduces the complexity of downstream analysis. However, complications arise when attempting to interface known enrichment methods and / or equipment with analytical apparatus and / or techniques.

[0004] Various methods have been used, such as interfacing protein sample preparation techniques with downstream detection systems (e.g., mass spectrometry). A common approach is to prepare samples using liquid chromatography and collect fractions for mass spectrometry (LC-MS). The drawback of this is the requirement to digest the protein sample into peptide fragments, resulting in a large number of sample fractions that must be analyzed and complex data reconstruction after the run. While certain forms of liquid chromatography can be coupled to mass spectrometry, such as peptide mapping reversed-phase chromatography, these known techniques are limited to using peptide fragments rather than whole proteins, which restricts their practicality.

[0005] Another method for introducing samples into a mass spectrometer is electrospray ionization (ESI). In ESI, small droplets of sample and solution are ejected from the distal end of a capillary or microfluidic device, which includes electrospray features such as emitter tips or orifices, by applying an electric field between the capillary tip or emitter tip and the mass spectrometer source plate. The droplets stretch and expand in this induced electric field to form a cone-shaped emission (i.e., a “Taylor cone”), which consists of increasingly smaller droplets that evaporate and produce gaseous ions that are introduced into the mass spectrometer for further separation and detection. Typically, the emitter tip is formed by a capillary, which provides a convenient droplet volume for ESI. However, capillaries are limited to linear flow paths, which does not allow for multi-step sample processing. ESI also depends on the voltage at the ESI tip remaining constant throughout the analysis, which can be challenging in many assays because the internal fluid resistance changes over time, altering the voltage drop in different parts of the circuit and thus the voltage at the ESI tip.

[0006] Further work has been done on microfluidic devices. Microfluidic devices can be produced using various known techniques and provide fluid channels of defined dimensions, which can form a network of channels designed to perform different fluid manipulations. Compared to capillaries, these devices offer an additional level of control and complexity, making them a better choice for sample preparation. However, like capillaries, these tools often provide limited characterization of separated analyte fractions before being introduced into a mass spectrometer (if available). Furthermore, systems with capillary or microfluidic devices generally do not provide tools for calibrating the system to re-establish the Taylor cone during operation.

[0007] Methods, apparatus, systems, and software for improving the quality of electrospray ionization mass spectrometry (ESI-MS) data are described, as well as methods, apparatus, systems, and software for achieving more quantitative characterization and improved correlation between chemical separation data and mass spectrometry data. Summary of the Invention

[0008] In one aspect, this paper discloses a computer-implemented method comprising: (a) receiving a time-series imaging dataset using a processor, the dataset comprising multiple images separated by isoelectric focusing in a separation channel, wherein each of the multiple images corresponds to a different time point; (b) converting each of the multiple images into an intensity or absorbance measurement based on the position along the length of the separation channel at the corresponding time point using the processor; and (c) generating a thermal map or 3D map based on the position along the length of the separation channel and the intensity or absorbance measurement based on time using the processor.

[0009] In some embodiments, the time-series imaging dataset further includes multiple images of the movement of analyte peaks separated in the separation channel. In some embodiments, the time-series imaging dataset includes multiple ultraviolet (UV) absorbance images. In some embodiments, the time-series imaging dataset includes multiple fluorescence images. In some embodiments, the fluorescence images include natural fluorescence images. In some embodiments, the time-series imaging dataset includes multiple images acquired at a frame rate of at least one image per minute. In some embodiments, the time-series imaging dataset includes multiple images acquired at a frame rate of at least one image per 30 seconds. In some embodiments, the time-series imaging dataset includes multiple images acquired at a frame rate of at least one image per 10 seconds. In some embodiments, the method further includes imaging the separation channel while performing isoelectric focusing separation, wherein (a)-(c) are performed iteratively as images are acquired. In some embodiments, a heatmap or 3D map is used to perform one or more tasks selected from: comparing isoelectric focusing separation with additional isoelectric focusing separation, comparing the moving reaction with isoelectric focusing separation, comparing the moving reaction with additional moving reaction, determining the completion of isoelectric focusing separation, monitoring the progress of the moving reaction, determining the presence of electroosmotic flow, and determining separation performance parameters. In some embodiments, the separation performance parameter is the separation resolution.

[0010] On the other hand, this paper provides a computer-implemented method comprising: (a) receiving, using a processor,: (i) a first dataset comprising multiple intensity or absorbance measurements from isoelectric focusing separation performed in a separation channel based on the length of the separation channel;

[0011] (ii) a second dataset comprising multiple mass spectrometer total ion measurements over time; (b) using a processor to convert the second dataset into a third dataset comprising ion count measurements based on mass; and (c) using a processor to overlay a graph of the first and third datasets.

[0012] In some embodiments, (c) further includes using a processor to overlay a graph of the second dataset with graphs of the first and third datasets. In some embodiments, (c) includes deconvolving the second dataset to generate the third dataset. In some embodiments, in (c), a first peak of intensity or absorbance of the first dataset is mapped to a set of peaks of the third dataset. In some embodiments, the second dataset is used to map the first dataset to the set of peaks of the third dataset. In some embodiments, the computer-implemented method further includes using a processor to correlate the first peak with the set of peaks to determine the mass distribution and isoelectric point of at least one analyte species of the first peak. In some embodiments, the first peak corresponds to an analyte peak and generates information about the isoelectric points of one or more analyte species among the analyte peaks. In some embodiments, the set of peaks corresponds to the mass distribution of one or more analyte species among the analyte peaks. In some embodiments, the computer-implemented method further includes using a processor to determine the identity of one or more analyte species among the analyte peaks for a given isoelectric point. In some embodiments, one or more analyte species include different protein isotypes. In some embodiments, protein isotypes include different post-translational modifications of proteins. In some embodiments, the overlay diagram illustrates (i) a time series of intensity or absorbance measurements based on multiple intensity or absorbance measurements along the length of the separation channel and (ii) a time series of ion count measurements based on mass. In some embodiments, the computer-implemented method further includes performing isoelectric focusing separation, movement, and electrospray ionization using a single integrated microfluidic device coupled to the mass spectrometer to obtain a first dataset and a second dataset. In some embodiments, (b) and (c) are performed within 1 minute of ESI-MS or concurrently with ESI-MS. In some embodiments, (b) or (c) is performed automatically as part of a software package for acquiring or processing electrospray ionization-mass spectrometry (ESI-MS) data. In some embodiments, (b) and (c) are performed automatically as part of a software package for acquiring or processing electrospray ionization-mass spectrometry (ESI-MS) data.

[0013] On the other hand, this paper provides a method comprising: assigning post-translational modifications to one or more analyte species using (i) mass spectrometry data for one or more analyte species and (ii) isoelectric focusing data for one or more analyte species.

[0014] In some embodiments, post-translational modifications are selected from: hydroxylation, methylation, esterification, acetylation, disulfide bonding, threonylation, ubiquitination, glycosylation, glycation, amino acid addition or removal, amidation, deamidation, isomerization, oxidation, fucosylation, sialylation, and phosphorylation. In some embodiments, the method further includes performing isoelectric focusing separation on a mixture of analytes comprising one or more analyte species to generate isoelectric focusing data, moving one or more analyte species, and performing electrospray ionization mass spectrometry (ESI-MS) to generate mass spectrometry data. In some embodiments, isoelectric focusing separation and moving are performed using a single microfluidic device comprising a separation channel and an integrated electrospray tip. In some embodiments, the isoelectric focusing data includes one or more intensity or absorbance measurements based on a distance along the separation channel, and wherein peaks in the intensity or absorbance measurements correspond to analyte peaks comprising one or more analyte species having the same given isoelectric point. In some embodiments, the method further includes using mass spectrometry data to distinguish at least one post-translational modification of one or more analyte species for a given isoelectric point. In some embodiments, isoelectric focusing data includes information about isoelectric points of one or more analyte species, and mass spectrometry data includes information about the mass of one or more analyte species. In some embodiments, the method further includes assigning post-translational modifications (PGMs) to at least one of the one or more analyte species using known values ​​of isoelectric point shifts and mass shifts of a plurality of PGMs. In some embodiments, the assignment of PGMs occurs within one minute of acquiring the ESI-MS data. In some embodiments, isoelectric focusing data includes information about one or more isoelectric points of one or more analyte species, wherein the mass spectrometry data includes information about one or more masses of one or more analyte species, and wherein the PGMs are assigned by matching one or more isoelectric points and one or more masses to a reference comprising a plurality of known isoelectric points and a plurality of known mass values ​​for PGMs. In some embodiments, the reference comprises publicly available data.

[0015] On the other hand, this document provides 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 a proximal end of a separation channel, wherein a distal end of the separation channel is in fluid and electrical communication with the ESI tip; (b) applying a second voltage to a proximal end of an auxiliary fluid channel, wherein a distal end of the auxiliary fluid channel is in fluid and electrical communication with the distal end of the separation channel; (c) performing a separation reaction to separate a mixture of analytes, wherein the separation reaction occurs within the separation channel; and (d) monitoring changes in the resistance of the separation channel or changes in the voltage at the ESI tip in a feedback loop, the feedback loop adjusting a third voltage applied to the mass spectrometer inlet to maintain a constant voltage difference between the ESI tip and the mass spectrometer inlet. In some embodiments, the separation channel is the lumen of a capillary. In some embodiments, the capillary includes a micro-tube nozzle. In some embodiments, the separation channel is a fluid channel within a microfluidic device. In some embodiments, the separation reaction includes an isoelectric focusing reaction. In some embodiments, the separation reaction includes an electrophoretic separation reaction. In some embodiments, a first voltage is applied at a cathode coupled to the separation channel and a second voltage is applied at an anode coupled to the separation channel. In some embodiments, the voltage at the ESI tip or mass spectrometer inlet is kept grounded. In some embodiments, the voltage at the mass spectrometer inlet is maintained 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 supply. In some embodiments, the power supply is coupled to a separation channel. In some embodiments, the power supply is coupled to another channel, which is coupled to the separation channel. In some embodiments, the power supply is set to 0 microamps. In some embodiments, the voltage at the ESI tip is measured using an electrode deployed at the ESI tip, wherein the electrode is configured to output a current of 0 microamps. 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.

[0016] On the other hand, this paper discloses a method for maintaining a constant voltage difference (ΔV) between the electrospray ionization (ESI) tip and the mass spectrometer inlet. TIP-MS The method includes: (a) ΔV TIP-MS Set the target value (ΔV)TARGET (b) Periodically or continuously monitor the first voltage at the ESI tip, wherein the ESI tip is in fluid and electrical communication with the separation channel; (c) Calculate ΔV TIP-MS The instantaneous value of ΔV; and (d) using a feedback loop to periodically or continuously adjust the second voltage at the mass spectrometer inlet so that ΔV TIP-MS =ΔV TARGET In some embodiments, the separation channel is the inner cavity of a capillary. In some embodiments, the capillary includes a micro-specimen nozzle. In some embodiments, the separation channel is a fluid channel within a microfluidic device. In some embodiments, the separation reaction performed in the separation channel includes an isoelectric focusing reaction. In some embodiments, the separation reaction performed in the separation channel includes an electrophoretic separation reaction. In some embodiments, a first voltage at the ESI tip or a second voltage at the mass spectrometer inlet is kept grounded. In some embodiments, the first voltage at the ESI tip is monitored using an electrode deployed at the ESI tip. In some embodiments, the electrode deployed at the ESI tip is configured to output a 0 μA current. In some embodiments, the first voltage at the ESI tip is monitored using a power supply electrically connected to the fluid channel and configured to output a 0 μA current, the fluid channel intersecting the separation channel near the ESI tip. 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 transfers ΔV TIP-MS Maintain at ΔV TARGET Within ±10%. In some embodiments, the feedback loop will ΔV TIP-MS Maintain at ΔV TARGET Within ±1%.

[0017] On the other hand, this document discloses a computer-implemented method for maintaining a constant voltage difference between an electrospray ionization (ESI) tip and a mass spectrometer inlet, the method comprising: (a) receiving a measurement of a first voltage at the ESI tip using a processor, wherein the ESI tip is in fluid and electrical communication with a separation channel; (b) receiving a measurement of a second voltage at the mass spectrometer inlet using the processor; and (c) comparing the first voltage with the second voltage using the processor, wherein if the second voltage differs from the first voltage, the processor adjusts the voltage at the mass spectrometer inlet or the ESI tip such that the difference between the voltage at the ESI tip and the voltage at the mass spectrometer inlet remains constant. In some embodiments, the method further comprises (d) repeating steps (a) through (c) at a specified frequency using a feedback loop. In some embodiments, the separation channel comprises (i) the lumen of a capillary or (ii) a fluid channel within a microfluidic device. In some embodiments, the capillary comprises a micro-tube nozzle. In some embodiments, a separation reaction is performed in the separation channel, and wherein the separation reaction comprises an isoelectric focusing reaction.

[0018] In some embodiments, the voltage at the ESI tip or the voltage at the mass spectrometer inlet remains grounded. In some embodiments, the voltage at the ESI tip is measured using electrodes placed at the ESI tip. In some embodiments, the voltage at the ESI tip is monitored using a power supply electrically connected to a fluid channel intersecting the separation channel near the ESI tip and configured to output 0 microamps of current. 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.

[0019] This document also discloses a method for maintaining an electrospray ionization (ESI) tip at a constant voltage relative to ground during the execution of a separation reaction, the method comprising: a) applying a first voltage to a proximal end of a separation channel, wherein a distal end of the separation channel is in fluid and electrical communication with the ESI tip; b) applying a second voltage to a proximal end of an auxiliary fluid channel, wherein a distal end of the auxiliary fluid channel is in fluid and electrical communication with the distal end of the separation channel; c) performing a separation reaction to separate a mixture of analytes, wherein the separation reaction occurs within the separation channel; and d) monitoring changes in the resistance of the separation channel or voltage changes at the ESI tip in a feedback loop, the feedback loop adjusting the first and second voltages to maintain 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 includes a micro-tube nozzle. In some embodiments, the separation channel is a fluid channel within a microfluidic device. In some embodiments, the separation reaction includes an isoelectric focusing reaction. In some embodiments, the separation reaction includes an electrophoretic separation reaction. In some embodiments, the first voltage is applied at the cathode and the second voltage is applied at the anode. In some embodiments, the voltage at the ESI tip is kept grounded. In some embodiments, the voltage at the ESI tip is maintained at a second voltage. In some embodiments, adjusting the first and second voltages includes subtracting a 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 supply providing 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 separation channel within ±10% of a preset value. In some embodiments, the feedback loop maintains the voltage drop across the separation channel within ±1% of a preset value.

[0020] This document also discloses a method comprising: a) providing a sample comprising a mixture of two or more analytes; b) performing separation within a fluid channel containing the sample to resolve individual analyte peaks from the mixture of two or more analytes; c) calculating the velocity of the analyte peaks as the contents of the fluid channel move toward the fluid channel outlet; and d) using the velocity of the analyte peaks to determine the time it takes for the analyte peaks to reach the fluid channel outlet. In some embodiments, the fluid channel is the lumen of a capillary. In some embodiments, the fluid channel is part of a microfluidic device. In some embodiments, the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isovelocity electrophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, the velocity of the analyte peaks is calculated based on the time interval required for the analyte peaks to move from a first position to a second position. In some embodiments, the first position, the second position, and the time interval are determined by a series of two or more images of the fluid channel. In some embodiments, the series of two or more images includes ultraviolet absorbance images, visible absorbance images, or fluorescence images. In some embodiments, the fluid channel outlet includes an electrospray interface with the mass spectrometer. In some embodiments, the time it takes for the analyte peak to reach the fluid channel outlet is used to correlate mass spectrometer data with the analyte peak. In some embodiments, movement of the contents of the fluid channel includes the use of electroosmotic movement, chemical movement, hydrodynamic movement, or any combination thereof. In some embodiments, two or more analytes include proteins, protein-drug conjugates, 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 from samples of a biopharmaceutical candidate and a reference drug is used to determine biosimilarity. In some embodiments, the velocity of the analyte peak is used in a feedback loop to adjust control parameters for the separation or movement 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.

[0021] The methods disclosed herein include: a) providing a sample comprising a mixture of two or more analytes; b) performing separation within a fluid channel containing the sample to resolve individual analyte peaks from the mixture of two or more analytes; and c) collecting mass spectrometer data of two or more individual analyte peaks emitted from the fluid channel via an electrospray interface with a mass spectrometer; wherein the data collection mode for the mass spectrometer alternates between high-quality scans and low-quality scans. In some embodiments, the mass spectrometer switches between high-quality scan and low-quality scan data collection modes at a frequency of at least 0.5 Hz. In some embodiments, the fluid channel is the lumen of a capillary. In some embodiments, the fluid channel is part of a microfluidic device. In some embodiments, the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isovelocity electrophoresis (CITP), or micelle electrokinetic chromatography (MEKC). In some embodiments, high-quality scans capture mass spectrometry data for biomacromolecules. In some embodiments, biomacromolecules include proteins, protein-drug conjugates, nucleic acid molecules, reduced proteins, fusion proteins, protein complexes, or any combination thereof. In some embodiments, the m / z ratio of high-quality scans ranges from 1500 to 6000. In some embodiments, low-quality scans capture mass spectrometry data of solution-phase amphoteric electrolytes for performing isoelectric focusing separation. In some embodiments, the m / z ratio of low-quality scans ranges from 150 to 1500. In some embodiments, mass spectrometry of one or more solution-phase amphoteric electrolytes is used to calibrate the isoelectric point (pI) of biomacromolecules identified in high-quality scans.

[0022] The method disclosed herein includes: a) performing separation within a fluid channel containing a sample, wherein the sample comprises a mixture of two or more analytes, and wherein the separation resolves individual analyte peaks from the mixture of the two or more analytes; b) moving the contents of the fluid channel toward a fluid channel outlet, wherein the fluid channel outlet includes an electrospray interface with a mass spectrometer; and c) simultaneously or alternately imaging: (i) at least a portion of the fluid channel to monitor the position of the analyte peaks during (a) and (b), and (ii) a Taylor cone present between the fluid channel outlet and the mass spectrometer inlet to monitor 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 outlet. In some embodiments, the time it takes for the analyte peak to reach the fluid channel outlet is used to correlate mass spectrometer data with the analyte peak. In some embodiments, data obtained from imaging 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. 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 isovelocity electrophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, imaging includes ultraviolet absorbance imaging, visible absorbance imaging, or fluorescence imaging. In some embodiments, movement of the contents of the fluid channel includes the use of electroosmotic movement techniques, chemical movement techniques, hydrodynamic movement techniques, or any combination thereof. In some embodiments, two or more analytes include proteins, protein-drug conjugates, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, small organic compounds, or any combination thereof.

[0023] This document discloses a computer-implemented method for maintaining an electrospray ionization (ESI) tip at a constant voltage relative to ground during the execution of a separation reaction. The method includes: a) receiving a first measurement of the voltage at the ESI tip using a processor, wherein a distal end of a separation channel is fluidly and electrically connected to the ESI tip; b) receiving a second measurement of the voltage at the ESI tip using the processor; c) comparing the second measurement with the first measurement using the processor, wherein if the second measurement differs from the first measurement, the processor causes the voltage at the proximal end of the separation channel and the voltage at the proximal end of an auxiliary fluid channel, the auxiliary fluid channel including a distal end fluidly and electrically connected to the distal end of the separation channel, such that the voltage at the ESI tip returns to the first measured value; and d) repeating steps (a) through (c) at a specified frequency. In some embodiments, the separation channel includes the lumen of a capillary or a fluid channel within a microfluidic device. In some embodiments, the separation reaction includes an isoelectric focusing reaction. In some embodiments, the separation reaction includes an electrophoretic separation reaction. In some embodiments, the voltage at the ESI tip is kept grounded. 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 a specified value.

[0024] This document also discloses a computer-implemented method comprising: a) receiving image data using a processor, the image data including two or more images acquired using a detector configured to image all or part of a separation channel in a capillary or microfluidic device; b) processing the image data using the same or different processors to determine the position of an analyte peak within the separation channel in the two or more images; c) calculating the velocity of the analyte peak based on the position of the analyte peak in the two or more images and a known time interval between the acquisition of the two or more images, using the same or different processors; and d) determining the time it takes for the analyte peak to reach the outlet of the separation channel using the same or different processors. In some embodiments, the separation reaction performed within the separation channel includes isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isovelocity electrophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, the two or more images include ultraviolet absorbance images, visible absorbance images, or fluorescence images. In some embodiments, the outlet of the separation channel is in fluid communication with a mass spectrometer or includes an electrospray interface with the mass spectrometer. In some embodiments, the time it takes for the analyte peak to reach the separation channel exit is used to correlate mass spectrometry data with the analyte peak. In some embodiments, the analyte is separated from the mixture and includes proteins, protein-drug conjugates, peptides, nucleic acid molecules, carbohydrate molecules, lipid molecules, metabolite molecules, or small organic compounds. In some embodiments, comparison of mass spectrometry data collected for samples of a biopharmaceutical 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 control parameters for the separation reaction performed 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.

[0025] By incorporating references

[0026] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually incorporated by reference in their entirety. In the event of any conflict between terminology used herein and terminology found in the incorporated references, the terminology used herein shall prevail. Attached Figure Description

[0027] The novel features of the invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments, which utilize the principles of the invention, and the accompanying drawings:

[0028] Figure 1A-B provides a schematic diagram of an isoelectric focusing (IEF) and electrospray ionization (ESI) apparatus for automatically loading samples according to an embodiment of the present disclosure. Figure 1A A schematic diagram of the device is shown. Figure 1B Another schematic diagram of the device is shown.

[0029] Figure 2 A sample flowchart of a computer-implemented method for calculating the isoelectric point of a separated analyte band is provided.

[0030] Figure 3 Another example flowchart is provided for a computer-implemented method for determining the velocity of one or more separated analyte bands and calculating the exit time.

[0031] Figure 4 Another example flowchart is provided for a computer-implemented method of implementing feedback and control of one or more operating parameters of an imaging-based ESI-MS analysis system.

[0032] Figure 5 A schematic block diagram of hardware components for one embodiment of the disclosed system is provided.

[0033] Figure 6 A schematic block diagram of software components for one embodiment of the disclosed system is provided.

[0034] Figure 7A -B illustrates a microfluidic device used in some embodiments of the present invention. Figure 7A A schematic diagram of a fluid channel network for an exemplary microfluidic device is provided. Figure 7B Computer-aided design (CAD) drawings of the assembled microfluidic device are provided. Figure 7A The fluid channel layer shown is sandwiched between two transparent layers to seal the fluid channel.

[0035] Figure 8 Images of the Taylor cone and electrospray ionization (ESI) plume during the movement of the separated samples are provided.

[0036] Figure 9A -F provides a non-limiting example of data on sample movement after separating analytes in a mixture using isoelectric focusing. Figure 9A The absorbance curve is shown at t=0 minutes (when isoelectric focusing is completed). Figure 9B The absorbance curve at t=1 minute is shown. Figure 9C The absorbance curve at t=2 minutes is shown. Figure 9D The absorbance curve at t=3 minutes is shown. Figure 9E The absorbance curve at t=4 minutes is shown. Figure 9FThe absorbance curve at t=5 minutes is shown.

[0037] Figure 10A -B provides a representative circuit diagram of a microfluidic device designed to perform isoelectric focusing to separate analytes and the subsequent movement of the separated analyte mixture. Figure 10A Provided that the ESI tip will remain grounded or close to ground. Figure 7A The diagram shows a representative circuit of the microfluidic device during isoelectric focusing. Figure 10B It shows Figure 7A The diagram shows a representative circuit of a microfluidic device during the chemical movement of a separated analyte mixture. In this example, it is assumed that channel 114 (in...) Figure 7A The resistance shown in the figure is negligible.

[0038] Figure 11A -B provides representative data for the movement while keeping the ESI tip at 0V. Figure 11A The curve showing the change of voltage over time is shown. Figure 11B The curve showing the change of current over time is shown.

[0039] Figure 12 An example flowchart of the voltage feedback loop is provided, in which the ESI tip is kept at +3000V.

[0040] Figure 13A -E provides an example of a representative circuit diagram of the microfluidic device disclosed herein. Figure 13A Provided Figure 7A The diagram shows a representative circuit of a microfluidic device during chemical movement, where the ESI tip will be held at a positive voltage using an additional resistor to draw current to ground. Figure 13B It shows Figure 7A The diagram shows a representative circuit of a microfluidic device during chemical movement, where the ESI tip will use an additional resistor to maintain the current at a positive voltage in order to draw the current to a third power source. Figure 13C It shows Figure 7A The diagram shows a representative circuit of a microfluidic device during chemical movement, where the ESI tip will be held at a positive voltage using a field-effect transistor (FET) to absorb current. Figure 13D It shows Figure 7A The diagram shows a representative circuit of a microfluidic device during chemical movement, where the ESI tip will be held at a positive voltage using a bipolar junction transistor (BJT) to absorb current. Figure 13E Provided Figure 7A The diagram shows a representative circuit of a microfluidic device during the chemical flow of a separated analyte mixture, where the ESI tip will remain grounded or close to ground.

[0041] Figure 14A-B provides a schematic diagram of a capillary connector sprayer. Figure 14A A representative diagram of a capillary connector sprayer is provided. Figure 14B It shows Figure 14A A representative resistor circuit diagram for a capillary connector sprayer.

[0042] Figure 15 An exemplary flowchart of a computer-controlled voltage feedback loop is provided, in which the ESI tip is held at 0V.

[0043] Figure 16 The AE panel provides examples of analyte separation data and the corresponding mass spectrometry data for the separated analytes. Figure 16 Panel A shows an electrophoretic graph of the separated analyte mixture. Figure 16 Panel B shows the mass spectrum of the acid peaks of the separated species. Figure 16 Panel C shows Figure 16 The mass spectrum of the main peak present in the electrophoresis image of panel A. Figure 16 Panel D and Figure 16 Panel E shows Figure 16 The mass spectrum of the two basic peaks in the electrophoresis pattern shown in panel A.

[0044] Figure 17A -B provides an example of separating data. Figure 17A A representative example of an isoelectric focusing electrophoresis image is shown. Figure 17B Representative examples of dynamic heatmap displays showing separation and movement within the separation channel are provided.

[0045] Figure 18 Representative examples of multiaxial curves are provided, showing combined isoelectric focusing electrophoresis, total ion chromatograms from mass spectrometry, and individual mass spectra.

[0046] Figure 19 Representative examples of dynamic heatmaps of separation and movement data are provided, displayed as three-axis graphs, where the x-axis plots distance, the y-axis plots time, and the z-axis plots absorbance (in arbitrary units).

[0047] Figure 20 Panel AB provides representative examples of monoclonal antibody data in isoelectric focusing and mass spectrometry. Figure 20 Panel A provides isoelectric focusing data and mass spectrometry chromatograms of the charge variants. Figure 20 Panel B shows an example of understanding the quality of convolution data.

[0048] Figure 21 Panel AB provides examples of post-translational modifications and tables showing the expected changes in protein quality and charge. Figure 21Panel A provides a representative table listing post-translational modifications and the expected changes in protein quality and charge due to these modifications. Figure 21 Panel B provides representative examples of modifications that can lead to the same mass change but have different effects on protein charge.

[0049] Figure 22 Panel AB provides an example of mass spectrometry. Figure 22 Panel A provides a representative example of deconvolution mass spectra obtained by analyzing charge variants separated by isoelectric focusing. Figure 22 Panel B provides another representative example of deconvolution mass spectra obtained from the analysis of charge variants separated by isoelectric focusing.

[0050] Figure 23 Panel AB provides representative examples comparing the quality of deconvolution of major protein charge variants with acidic and basic variants. Figure 23 Panel A provides an example of the quality of deconvolution between major protein charge variants and acid variants. Figure 23 Panel B provides an example of the quality of unconvolution between major protein charge variants and basic variants. Detailed Implementation

[0051] Some embodiments described herein relate to innovative software and systems for analyzing data from and guiding the operation of capillary and microfluidic-based separation systems integrated with mass spectrometry detection. In some embodiments, the analyte is imaged in a capillary or microfluidic device during separation and its molecular weight or mass-to-charge ratio is measured in a mass spectrometer after separation. The disclosed methods, apparatus, systems, and software provide more accurate characterization of separated analyte peaks and are used to achieve improved correlation between chemical separation data and mass spectrometry (MS) data. Methods, apparatus, systems, and software for improving the quality of electrospray ionization mass spectrometry (ESI-MS) data are also disclosed. The disclosed methods, apparatus, systems, and software have potential applications in a variety of fields, including but not limited to proteomics research, drug discovery and development, and clinical diagnostics. For example, in some embodiments, the disclosed methods, apparatus, systems, and software can be used to characterize biopharmaceuticals 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 virus vaccines, human plasma-derived proteins, cell-derived drugs, naturally derived proteins, antibody-drug conjugates, protein-drug conjugates, and other protein drugs.

[0052] The description describes a microfluidic device designed to perform any of a variety of chemical separation techniques and also includes an electrospray ionization interface for performing downstream mass spectrometry-based analysis. In a preferred embodiment, the disclosed device is designed to perform isoelectric focusing on proteins or other biomacromolecules. In another preferred embodiment, the disclosed device is designed for use with imaging techniques. Devices and methods for integrating imaging microfluidic separation with mass spectrometry have previously been described, for example, in published PCT patent application publication No. WO 2017 / 095813 and U.S. patent application publication No. US 2017 / 0176386, which are incorporated herein by reference for all purposes. Among other things, these applications particularly describe systems that perform imaging separation in conjunction with MS analysis. Such microfluidic systems represent a significant advance in the characterization of bioproducts. However, to maximize the benefits of such systems, innovative software and systems to assist in the operation of these systems and the downstream integration of imaging and MS data, as disclosed herein, would be beneficial.

[0053] Therefore, in a preferred embodiment, the disclosed microfluidic device can be used in conjunction with imaging techniques, for example, to accurately determine the isoelectric point (pI) of one or more analytes that have been isoelectrically separated from a mixture of analytes in a separation channel to form a series of enriched fractions comprising 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 location of two or more pI standards (or pI markers) injected along with the sample to be separated, thereby allowing for the calculation of a more accurate pI for each separated analyte peak by extrapolation to determine the local pH. In some embodiments, imaging of the analyte mixture within the separation channel is performed concurrently with separation, and optionally, the isoelectric point of one or more of the analytes being separated is determined and iteratively updated concurrently with separation. In some embodiments, the isoelectric point of one or more analytes that have been isoelectrically focused is determined based on imaging after separation is complete. In some embodiments, the isoelectric point of one or more analytes that have been isoelectrically focused is determined based on imaging after separation is complete and before the separated analyte mixture has moved toward the electrospray tip. In some embodiments, the imaging-based methods disclosed herein may be used with capillary-based ESI-MS systems rather than with microfluidic device-based ESI-MS systems. In some embodiments, the determination of the isoelectric point of one or more analyte peaks may be performed using computer-implemented methods.

[0054] In another preferred embodiment, the disclosed microfluidic device can be used in conjunction with imaging techniques to image the separated analyte peaks after they have moved, i.e., as they exit the separation channel and move toward the electrospray tip. In some embodiments, the moving step of imaging is the same as the separation step of imaging, such as when performing a separation step including capillary gel electrophoresis, capillary zone electrophoresis, isotachymetry, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, or any other separation technique by differential separation of the analyte mixture components. In some embodiments, the moving step of analytical imaging is used to correlate the enriched fraction in the imaged separation with mass spectrometry. Imaging of the moving analyte peaks can be used, for example, to determine the velocity of one or more analyte peaks based on their positions in a series of moving images, which can then be used to determine the time point at which the analyte peak(s) will exit the separation channel or be emitted by the electrospray tip, and thus can be used to correlate mass spectrometry data with a specific analyte peak. In some cases, the velocities of one or more analyte peaks are calculated based on 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, imaging of the moving analyte peaks can allow direct monitoring of the peaks as they pass through the fluid channel and are emitted by the electrospray tip, and can therefore be used to directly correlate mass spectrometry data with a specific analyte peak. In some embodiments, the imaging-based methods disclosed herein can be used with capillary-based ESI-MS systems rather than with microfluidic device-based ESI-MS systems. In some embodiments, the determination of the velocities of one or more analyte peaks, their actual or predicted separation channel exit times, and / or their electrospray emission times can be performed using computer-implemented methods.

[0055] In some embodiments, the movement of separated analyte peaks can be initiated by changing the electric field or flow parameters in the microfluidic device. In some embodiments, one or more electrodes connected to a power source for the microfluidic device are connected or disconnected to initiate movement via a computer-implemented method. In some embodiments, the Taylor cone formed at the electrospray tip can be imaged during the movement step. In some embodiments, computer-implemented image analysis can be used to identify stable electrospray operating conditions. In some embodiments, image analysis can be performed by an operator. In some embodiments, automated image processing software can be used to perform image analysis. In some embodiments, one or more operating parameters known to affect electrospray performance are adjusted to regain stable electrospray operating conditions. Examples of operating parameters that can be adjusted include, but are not limited to, electrophoresis voltage, flow rate, distance from the electrospray tip to the MS inlet, MS voltage, etc. In some embodiments, computer-implemented methods can be used to adjust electrospray parameters.

[0056] In some embodiments, more than one power source may be used to generate the electrophoretic electric field. In some embodiments, two power sources with positive polarity may be used. In some embodiments, one or more power sources may have negative polarity. In some embodiments, the voltage settings on the power sources may be consistently varied to maintain the same voltage gradient in the separation channel used for electrophoretic separation. In some embodiments, the voltage settings on the power sources may be varied to maintain a constant voltage at the electrospray tip. In some embodiments, the multiple power sources may be different channels in a single multichannel 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 movement may be performed in the separation channel, and the resistance in the channel may decrease over time. In some embodiments, pressure-driven movement 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 grounded. In some embodiments, the electrospray tip may be maintained 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-implemented method may adjust the voltage to maintain a constant electric field strength (or a constant voltage drop between the anode and cathode) in the separation channel and a constant voltage at the electrospray tip. In some embodiments, a voltmeter may be used to measure the voltage at the tip. In some embodiments, the voltage at the tip can be measured using electrodes located at or inside the tip. In some embodiments, an additional power supply can be set to 0 μA using current control and used as a voltmeter to read the tip voltage. In some embodiments, a computer-implemented method reads the voltage value at the tip and adjusts the voltage to maintain a constant electric field strength (or a constant voltage drop between the anode and cathode) in the separation channel and maintain a constant voltage at the tip. In some embodiments, a computer-implemented method calculates the voltage at the ESI tip based on the current flow through the separation electric field circuit. In some embodiments, the voltage drop across the separation channel is adjusted such that a constant or maximum power is applied in the separation channel, wherein the power applied in the separation channel is calculated as follows:

[0057] Power = Voltage across the separation channel x Current in the separation channel, where the current can be measured continuously or periodically during separation, and the current measurement can be used to adjust the voltage across the separation channel. This method of controlling the power in the separation channel can be useful for managing temperature effects in the separation channel.

[0058] In some embodiments, the separation path is a linear, coated or uncoated capillary, tube, or line of a certain length, with its inlet inserted into a vial containing an acidic anolyte and a positive electrode, or an alkaline catholyte and a negative electrode. In some embodiments, the outlet of the separation path is inserted into a connector sprayer. In some embodiments, the connector sprayer houses a tee for an auxiliary tube, line, or capillary that can introduce another conductive supplemental solution into the capillary outlet, thereby providing liquid-to-liquid electrical contact and liquid flow to support electrospraying and transport of analytes emerging from the separation channel to the tip for introduction into the mass spectrometer via electrospray ionization. In some embodiments, the system may be configured with an anolyte and a positive electrode at the separation path inlet, and the connector or distal portion of the separation path may be loaded with a catholyte prior to focusing. After focusing is complete, a moving agent with competing anions may be introduced into the connector by hydrodynamic or electroosmotic forces. In some embodiments, the separation path inlet may be immersed in a vial containing a catholyte and a negative electrode, and the connector or distal portion of the capillary may be loaded with an anolyte prior to focusing. After focusing is complete, a mobile agent with competing cations can be introduced into the connector using hydrodynamics or electroosmosis. In some embodiments, the separation channel is a linear capillary, one end inserted into an anolyte reservoir connecting the capillary to the positive electrode, and the other end inserted into a catholyte reservoir connecting the capillary to the negative electrode for isoelectric focusing. In some embodiments, after focusing, the catholyte end of the capillary is removed from the catholyte and inserted into a connector sprayer (e.g., a micro-canister sprayer) near the mass spectrometer, such as... Figure 14A As shown in the diagram. In some embodiments, the connector sprayer can provide a volume of moving agent to charge and move the focused analyte in the ESI. In some embodiments, the connector sprayer can provide an electrical connection to complete the moving circuit. In some embodiments, the voltage at the anolyte and the connector sprayer is adjusted such that changes in the voltage (ΔV) or electric field between the anolyte and the connector sprayer remain constant, and the voltage at the ESI tip remains constant. In some embodiments, the ΔV or electric field between the anolyte and the connector sprayer can fluctuate or change over time, and the voltage at the ESI tip can change. In such cases, the voltage or potential applied to the mass spectrometer inlet can be adjusted to maintain the voltage difference (ΔV) between the ESI tip and the mass spectrometer inlet. TIP-MS () Remain constant.

[0059] In some embodiments, the separation channel (e.g., a capillary) includes a micro-vibration device that facilitates the transfer of flowing effluent to the ESI. The micro-vibration device may be part of the capillary or may be attached to and / or fused to the separation channel. The micro-vibration device may be part of the ESI tip. In some cases, the micro-vibration device may include a connector atomizer or part of a connector atomizer. The micro-vibration device may provide a fluid flow path (e.g., for sheath fluid) within a portion of the channel or at the ESI tip.

[0060] In some embodiments, a power supply may be connected to a resistor to generate a current sink. In some embodiments, the resistor may sink current by grounding the electrophoresis circuit. 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 ladder resistor, or any other resistive element capable of providing a path for the sinking current. In some embodiments, the current sink may be a FET, wherein the FET is controlled to provide a constant current flowing through the FET or can be controlled to act as an open circuit or short circuit when needed. In some embodiments, a bipolar junction transistor (BJT) may be used for the current sinking function. In some embodiments, the resistor may sink current by connecting the electrophoresis circuit to a current sinking power supply. In some embodiments, the voltage setting of the current sinking power supply will be adjusted as the resistance in the discrete channel changes over time. In some embodiments, the voltage on the current sinking power supply will be adjusted to maintain a constant current across the resistor. In some embodiments, a resistor or a collection of resistors, a resistive circuit, etc., may be used as a current sink.

[0061] In some embodiments, the scanned mass-to-charge ratio (m / z) range can be changed during the migration / ESI step. In some embodiments, a computer-implemented method can be used to switch between high and low m / z ranges. In some embodiments, a mass spectrum within one m / z range can be used as an internal standard for separating analytes in different mass ranges. This spectrum may include data for isoelectric amphoteric electrolytes in free solution, which can be used as a standard for isoelectric point (pI), or this spectrum may include data for electrophoretic mobility standards that can be used as a standard for electrophoresis (e.g., capillary zone electrophoresis). In some cases, this spectrum may include data for any molecule that can be resolved in the separation step, for example, by pI, mass-to-charge ratio, gel reputation, electrophoretic mobility, etc., whose mass range differs from that of the analyte of interest.

[0062] In some embodiments, the correlation between charge variant peaks and mass spectrometry data can allow for the confirmation of post-translational modifications or other protein or peptide modifications. For example, mass differences between two molecules can be detected during mass spectrometry analysis. In some cases, multiple modifications may be present that could lead to a detected mass difference. In some cases, it may be known that certain modifications cause specific charge shifts (or changes in isoelectric point) in a molecule. In some cases, knowing the charge shift associated with the mass difference can allow for the exclusion of a specific modification or set of modifications. In some embodiments, knowing the charge shift and detecting the same or similar mass (within the mass spectrometer's mass accuracy limits) can allow for the exclusion of a specific modification or set of modifications. In some cases, knowing the charge shift and detecting the same or similar mass (within the mass spectrometer's mass accuracy range) can allow for the assignment of a specific modification or set of modifications to a molecule. In some cases, knowing the charge shift associated with the mass difference can allow for the assignment of a specific modification or set of modifications to a molecule.

[0063] The system disclosed herein may include one or more of the following: (i) a capillary or microfluidic device designed to perform analyte separation, such as isoelectric focusing-based separation, which provides 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 analyte separation operations and image acquisition based on the capillary or microfluidic device; (vi) software for processing images and determining the position of one or more pI standards or analyte peaks in the separation channel during separation, after separation is completed, or after moving pI standards and analyte peaks toward the electrospray tip; (vii) software for processing images and determining the velocity, exit time, and / or electrospray emission time of one or more pI standards or analyte peaks; and (viii) software for simultaneously or alternately acquiring images of the separation channel to monitor the position of analyte peaks and their presence in the electrospray. Software for monitoring electrospray performance using a Taylor cone between the spray tip and the mass spectrometer inlet; (ix) software for processing images of the Taylor cone and adjusting one or more of, or any combination thereof, the position of the electrospray tip relative to the mass spectrometer inlet, the fluid flowing through the electrospray tip, and the voltage between the electrospray tip and the mass spectrometer to influence changes in the mass spectrometer data quality; (x) software for controlling the collection of mass spectrometer data for individual analyte peaks emitted from the electrospray interface, wherein the mass spectrometer data collection mode alternates between high-quality scans and low-quality scans; (xi) software for reading the voltage at the electrospray tip and / or the mass spectrometer inlet and adjusting (a) the separation channel voltage to maintain a constant 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) the voltage applied to the mass spectrometer inlet to maintain a constant voltage between the tip and the mass spectrometer inlet; or 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 capillary or microfluidic devices, are replaceable or disposable components.

[0064] It should be understood that the foregoing general overview and the following description are exemplary and illustrative only, and do not limit the methods and apparatus described herein.

[0065] Definitions: Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0066] As used in this specification and the appended claims, the singular forms “a” and “the” include plural references unless the context clearly specifies otherwise. Unless otherwise stated, any reference to “or” herein is intended to cover “and / or”. Similarly, the phrases “comprising” and “including” are not intended to be limiting.

[0067] As used herein, the term "approximately" for a number means that number plus or minus 10% of that number. In the context of a range, the term "approximately" means the range minus 10% of its lowest value and plus 10% of its highest value.

[0068] Analytes: As described above, the disclosed methods, apparatus, systems, and software enable more accurate characterization of separated analyte peaks and improve the correlation between chemical separation data and mass spectrometry data. In some cases, these analytes can be, for example, released glycans, carbohydrates, lipids or derivatives thereof (e.g., extracellular vesicles, liposomes, etc.), DNA, RNA, intact proteins, digested proteins, protein complexes, antibody-drug conjugates, antibodies, antibody fragments, protein-drug conjugates, peptides, metabolites, organic compounds or other biologically relevant molecules, or any combination thereof. In some cases, these analytes can be small molecule drugs. In some cases, these analytes can be protein molecules in a protein mixture, such as biological protein drugs and / or lysates collected from cultured or in vivo isolated cells.

[0069] Samples: The disclosed methods, apparatus, systems, and software can 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, arterial, or capillary blood samples), plasma, serum, saliva, interstitial fluid, urine, sweat, tears, protein samples derived from industrial enzyme or biopharmaceutical manufacturing processes, environmental samples (e.g., air samples, water samples, soil samples, surface swab samples), etc. In some embodiments, any of a variety of techniques known to those skilled in the art can be used for integrated chemical separation and mass spectrometry characterization prior to analysis using the disclosed methods and apparatus. For example, in some embodiments, samples can be processed to extract proteins or nucleic acids. Samples can be collected from any of a variety of sources or subjects, such as bacteria, viruses, plants, animals, or humans.

[0070] Sample volume: In some embodiments of the disclosed methods and apparatus, miniaturization achieved using microfabrication techniques enables the processing of very small sample volumes. In some embodiments, the sample volume for analysis can range from approximately 0.1 μl to approximately 1 ml. In some embodiments, the sample volume for analysis can 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 lower and upper limits described in this paragraph may be combined to form ranges 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, such as about 10 μl.

[0071] Separation techniques: The disclosed methods, apparatus, systems, and software can utilize any of a variety of analyte separation techniques known to those skilled in the art. For example, in some embodiments, the imaging separation can be electrophoretic separation that produces one or more separated analyte fractions from an analyte mixture, such as isoelectric focusing, capillary gel electrophoresis, capillary zonal electrophoresis, isovelocity electrophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, electric field gradient focusing, dynamic field gradient focusing, etc.

[0072] Capillary Isoelectric Focusing (CIEF): In some embodiments, separation techniques may include isoelectric focusing (IEF), such as capillary isoelectric focusing (CIEF). Isoelectric focusing (or “electro-focusing”) is a technique for separating molecules by the difference in their isoelectric points (pI) (i.e., the pH at which they have a net zero charge). CIEF involves adding a solution of an amphoteric electrolyte (ampholyte) between a reagent container containing an anode or cathode to generate a pH gradient within the separation channel (i.e., a fluid channel connected to a trap containing electrodes) across which a separation voltage is applied. The amphoteric electrolyte may be a solution phase or immobilized on a surface of the channel walls. Negatively charged molecules migrate toward the positive electrode through the pH gradient in the medium, while positively charged molecules move toward the negative electrode. Proteins (or other molecules) in a pH range below their isoelectric point (pI) will become positively charged and will therefore migrate toward the cathode (i.e., the negatively charged electrode). The total net charge of a protein decreases 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 a pH region corresponding to its pI, at which point it has no net charge and migration ceases. Thus, a mixture of proteins is separated based on the relative abundance of their acidic and basic residues and focused into sharp, fixed bands, with each protein positioned at a point in the pH gradient corresponding to its pI. This technique offers extremely high resolution for proteins, the difference being that individual charges are separated into distinct bands. In some embodiments, isoelectric focusing can be performed in separation channels that have been 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 modifiers, hydroxypropyl cellulose (HPC), and polyvinyl alcohol (PVA). (Alcor Bioseparations), linear polyacrylamide, polyacrylamide, dimethacrylamide, polyvinylpyrrolidone (PVP), methylcellulose, hydroxyethylcellulose (HEC), hydroxypropyl methylcellulose (HPMC), triethylamine, propylamine, morpholine, diethanolamine, triethanolamine, diaminopropane, ethylenediamine, chitosan, polyethyleneimine, cadaverine, putrescine, spermidine, diethylenetriamine, tetraethylenepentamine, cellulose, dextran, polyethylene oxide (PE) O), cellulose acetate, amylopectin, ethylpyrrolidine methacrylate, dimethyl methacrylate, docosyl dimethyl ammonium bromide, Brij35, sulfobetaine, 1,2-dilauroyl n-phosphatidylcholine, 1,4-decyl-1,4-diazobicyclo[2,2,2]octane dibromide, agarose, poly(N-hydroxyethylacrylamide), pole-323, hyperbranched polyurethane, pullulan, glycerol, adsorption coatings, covalent coatings, dynamic coatings, etc. In some embodiments, isoelectric focusing (e.g., in uncoated separation channels) can be performed using additives (such as methylcellulose, glycerol, urea, formamide, surfactants (e.g., Triton-X 100, CHAPS, digitalis saponins)) in the separation medium to significantly reduce electroosmotic flow, allow better protein dissolution, and limit diffusion within the capillary of the fluid channels by increasing the viscosity of the electrolyte.

[0073] As described above, the pH gradient used in capillary isoelectric focusing is generated using an amphoteric electrolyte (i.e., an amphoteric molecule containing both acidic and basic groups and existing primarily as amphoteric ions within a certain pH range). The portion of the electrolyte solution located on the anodic side of the separation channel is referred to as the "anolyte." The portion of the electrolyte solution located on the cathode side of the separation channel is referred to as the "catholyte." Various electrolytes can be used in the disclosed methods and apparatus, including but not limited to phosphoric acid, sodium hydroxide, ammonium hydroxide, glutamic acid, lysine, formic acid, dimethylamine, triethylamine, acetic acid, piperidine, diethylamine, and / or any combination thereof. The electrolyte can be used at any suitable concentration, such as 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc. The concentration of the electrolyte can be at least 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The maximum concentration can be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, 0.01%, 0.001%, or 0.0001%. A range of electrolyte concentrations can be used, for example, from 0.1% to 2%. Amphoteric electrolytes can be selected from any commercial or non-commercial carrier amphoteric electrolyte mixture (e.g., Servalyt pH 4–9 (Serva, Heildelberg, Germany), Beckman pH 3–10 (Beckman Instruments, Fullerton, CA, USA), Ampholine 3.5–9.5 and Pharmalyte 3–10 (both from General Electric Healthcare in Orsay, France), AESlytes (AES), FLUKA amphoteric electrolyte (Thomas Scientific, Swedesboro, NJ), Biolyte (Bio-Rad, Hercules, CA)), etc. Carrier amphoteric electrolyte mixtures can comprise a mixture of small molecules (approximately 300–1000 Da) containing multiple aliphatic amino and carboxylic acid ester groups, which have closely spaced pI values ​​and good buffering capacity. In the presence of an applied electric field, the carrier amphoteric electrolyte splits into a smooth linear or non-linear pH gradient that gradually increases from the anode to the cathode.

[0074] Any of the various pI standards can be used in the disclosed methods and apparatus to calculate the isoelectric point of a separated analyte peak. For example, pI tags commonly used in CIEF applications, such as protein pI tags and synthetic small molecule pI tags, can be used. In some cases, the protein pI tag may be a specific protein with a generally accepted pI value. In some cases, the pI tag may be detectable, for example, via imaging. Multiple or combinations of commercially available protein pI tags or synthetic small molecule pI tags can be used, such as small molecule pI tags available from Advanced Electrophoresis Solutions (Cambridge, Ontario, Canada), ProteinSimple, peptide libraries designed by Shimura, and Slais dyes (Alcor Biosepartions).

[0075] Mobility Techniques: In some embodiments, such as those employing isoelectric focusing, the separated analyte bands may move toward the end of a separation channel that interfaces with a downstream analytical device (e.g., electrospray ionization interfaced with a mass spectrometer). In some embodiments, such as those employing capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, or any other separation technique that separates components of an analyte mixture by differential velocity, the separation step may be considered a mobility step.

[0076] In some embodiments, movement of the analyte band can be achieved by applying hydrodynamic pressure to one end of the separation channel. In some embodiments, movement of the analyte band can be achieved by orienting the separation channel in a vertical position, thereby employing gravity. In some embodiments, movement of the analyte band can be achieved using EOF-assisted movement. In some embodiments, movement of the analyte band can be achieved using chemical movement. In some embodiments, any combination of these movement techniques can be employed.

[0077] In one embodiment, the movement step of the analyte band for isoelectric focusing includes chemical movement. Compared to pressure-based movement, chemical movement has the advantage of exhibiting minimal band broadening by overcoming the hydrodynamic parabolic flow profile caused by the pressure used. Chemical movement can be achieved by introducing the inlet or outlet of a separation path containing a fully or partially focused pH gradient into a conductive solution having ions that compete with hydrated hydrogen ions or hydroxyl groups for electrophoretic entry into the separation path. This results in a gradual electrodynamic displacement of the pH gradient components by disrupting the near-zero net charge state. In the case of cathodic chemical movement, the supplied hydroxyl groups, i.e., the cathodic electrolyte, can be replaced with a moving solution containing competing anions. The competing anions can cause a pH decrease in the separation path, thereby generating a positive charge on the pH gradient components, allowing them to migrate toward the cathode. Correspondingly, in anodic movement supplying hydrated hydrogen ions, the anolyte solution is replaced with a moving solution containing competing cations, which increases the pH in the separation, thereby negatively charging the pH gradient components and allowing them to migrate toward the anode. In some embodiments, an acidic electrolyte (such as formic acid, acetic acid, carbonic acid, phosphoric acid, etc.) at any suitable concentration can be used to initiate cathode migration. In some embodiments, an alkaline electrolyte (such as ammonium hydroxide, dimethylamine, diethylamine, piperidine, sodium hydroxide, etc.) can be used to initiate anodic migration. In some embodiments, chemical migration can be initiated by adding a salt (such as sodium chloride or any other salt) to the anolyte or catholyte.

[0078] In a preferred embodiment, the chemical migration step can be initiated within a microfluidic device designed to integrate CIEF with ESI-MS by altering the electric field within the device to electrophoretically transfer migrating electrolytes into separation channels. In some embodiments, the alteration of the electric field can be achieved by connecting or disconnecting one or more electrodes attached to one or more power sources, wherein the electrodes are positioned in reagent traps within the device or integrated with the device's fluid channels. In some embodiments, the connection or disconnection of one or more electrodes can be controlled using computer-implemented methods and programmable switches, such that the timing and duration of the migration step can be coordinated with separation steps, electrospray ionization steps, and / or mass spectrometry data collection. In some embodiments, disconnection of one or more electrodes from the separation circuitry can be achieved using current control and setting the current to 0 μA.

[0079] Capillary zone electrophoresis (CZE): In some embodiments, the separation technique may include capillary zone electrophoresis, a method for separating charged analytes in solution under an applied electric field. The net velocity of the charged analyte molecules is influenced by the electroosmotic flow (EOF) mobility μ exhibited by the separation system. EOF and the electrophoretic mobility μ of a single analyte EPThe influence of (depending on the size, shape, and charge of the molecule) causes analyte molecules of different sizes, shapes, or charges to exhibit different migration rates and separate into bands.

[0080] Capillary gel electrophoresis (CGE): In some embodiments, separation techniques may include capillary gel electrophoresis, a method for separating and analyzing macromolecules (e.g., DNA, RNA, and proteins) and their fragments based on their size and charge. This method involves using separation channels filled with a gel, where the gel acts as an anti-convection and / or sieving medium during the electrophoretic movement of charged analyte molecules in an applied electric field. The gel serves to suppress thermal convection caused by the applied electric field and also acts as a sieving medium that impedes the passage of molecules, resulting in different migration rates for molecules of different sizes or charges.

[0081] Capillary isotachophoresis (CITP): In some embodiments, the separation technique may include capillary isotachophoresis, a method for separating charged analytes using a discontinuous system of two electrolytes (called a leader electrolyte and a stop electrolyte) within a capillary of suitable dimensions or fluid channels. The leader electrolyte may contain ions with the highest electrophoretic mobility, while the stop electrolyte may contain ions with the lowest electrophoretic mobility. The mixture of analytes to be separated (i.e., the sample) may be sandwiched between these two electrolytes, and an applied electric field causes the charged analyte molecules within the capillary or fluid channel to partition into closely adjacent bands to reduce their electrophoretic mobility. These bands move at a constant velocity in the applied electric field, allowing their passage along the separation channel to be recorded using detectors (e.g., conductivity detectors, photodetectors, or imaging devices). Unlike capillary zonation, it is not feasible to simultaneously identify or detect anionic and cationic analytes in a single analysis performed using capillary isotachophoresis.

[0082] Capillary electrokinetic chromatography (CEC): In some embodiments, the separation technique may include capillary electrokinetic chromatography, a method for separating mixtures of analytes based on a combination of liquid chromatography and electrophoresis. CEC offers the efficiency of capillary electrophoresis (CE) as well as the selectivity and sample capacity of packed capillary high-performance liquid chromatography (HPLC). Because the capillaries used in CEC are packed with HPLC packing material, the selectivity of a wide range of analytes available in HPLC is also provided in CEC. The high surface area of ​​these packing materials allows CEC capillaries to accommodate relatively large amounts of sample, thus simplifying the detection of subsequently eluted analytes compared to the detection task in capillary zone electrophoresis (CZE).

[0083] Micelle electrokinetic chromatography (MEKC): In some embodiments, the separation technique may include micellar electrokinetic chromatography, a method for separating analyte mixtures based on differential partitioning between surfactant micelles (pseudo-stationary phase) and the surrounding aqueous buffer solution (mobile phase). In MEKC, the buffer solution may contain a surfactant at a concentration greater than the critical micelle concentration (CMC) such that the surfactant monomers are equilibrated with the micelles. MEKC can be performed using alkaline conditions in open capillaries or fluid channels to generate strong electroosmotic flow. Various surfactants can be used in MEKC applications, such as sodium dodecyl sulfate (SDS). For example, the anionic sulfate groups of SDS cause the surfactant and micelles to have electrophoretic mobilities opposite to the direction of strong electroosmotic flow. Therefore, the surfactant monomers and micelles migrate slowly, although their net movement is still in the direction of electroosmotic flow, i.e., towards the cathode. During MEKC separation, the analyte can be distributed between the hydrophobic interior of the micelles and the hydrophilic buffer solution. The hydrophilic analyte insoluble in the interior of the micelles moves at an electroosmotic flow rate u. o Migration, and will occur during the retention time t in the buffer. M The hydrophobic analyte, completely dissolved within the micelles, was detected at a micelle velocity u. c Migration, and at the final elution time t c Wash off.

[0084] Flow-resistance balanced capillary electrophoresis (FCCE): In some embodiments, the separation technique may include flow-resistance balanced capillary electrophoresis, a method for improving the efficiency and resolution of capillary electrophoresis that utilizes pressure-induced countercurrent to actively delay, stop, or reverse the electrokinetic migration of analytes through a capillary. By delaying, 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 longer period than under normal separation conditions, thereby improving separation efficiency and resolution.

[0085] Separation Time and Separation Resolution: Generally, the separation time required to achieve complete separation will 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 software will determine when separation is complete based on imaging-based analysis of the analyte peaks, as described in co-pending U.S. Patent Application No. 16 / 261,382. In some embodiments, the separation time may range from approximately 0.1 minutes to approximately 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 at most 30 minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, at most 10 minutes, at most 5 minutes, at most 1 minute, at most 0.5 minutes, or at most 0.1 minutes. Any of the lower and upper limits described in this paragraph can be combined to form a range included in this disclosure. For example, in some embodiments, the separation time can range from about 1 minute to about 20 minutes. The separation time can have any value within this range, for example, about 7 minutes.

[0086] Similarly, the separation efficiency and resolution achieved using the disclosed methods and apparatus can vary depending on the specific separation techniques 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., the number of theoretical plates) can be in the range of approximately 1,000 to 1,000,000. In some cases, the separation efficiency can be at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 600,000, at least 700,000, at least 800,000, at least 900,000, or at least 1,000,000. The separation resolution of the efficiency can vary depending on one or more properties of the analyte in the mixture (e.g., molecular weight, diffusivity, electrophoretic or isoelectric mobility, etc.).

[0087] Microfluidic device design and fabrication: In some embodiments of the disclosed methods, devices and systems, the separation of analytes from mixtures and, optionally, their subsequent analysis using ESI-MS, can be performed using microfluidic devices designed to integrate one or more sample preparation steps (e.g., filtration, pre-concentration or extraction steps, etc.) and / or separation steps (e.g., as described above) with an electrospray ionization step.

[0088] In some embodiments, the disclosed microfluidic device may include one or more sample or reagent ports (also referred to as inlet ports, sample traps, or reagent traps), one or more waste ports (also referred to as outlet ports), one or more fluid channels connecting the inlets and outlets to each other or to an intermediate fluid channel (e.g., a separation channel), or any combination thereof. In some embodiments, the disclosed microfluidic device may also include one or more reaction chambers or mixing chambers, one or more microfabricated valves, one or more microfabricated pumps, one or more venting structures, one or more membranes (e.g., filter membranes), one or more microcolumn structures (e.g., fluid channels filled with chromatographic separation media or modified fluid channels), or any combination thereof.

[0089] 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. A non-limiting example of such an interface is described in co-pending U.S. Patent Application Publications Nos. US2017 / 0176386A1 and US2018 / 0003674A1. Figure 1A and 1B The illustration shows a non-limiting example of a microfluidic device designed to perform isoelectric focusing followed by ESI-MS characterization. Figure 1A and 1BThe fluid channel network shown is made of a single piece of soda-lime glass, etched using standard photolithography, and has a very low transmittance of 280 nm light. The device includes a sample inlet channel 414 connected to inlet 412, an enrichment channel 418, and a movement channel 438. An anode 416 is electrically contacted with an anolyte trap 426. The depth of the separation (or enrichment) channel 418 is the same as the thickness of the glass layer 402; that is, the enrichment channel 418 extends from the top to the bottom of the glass plate 402. The device 400 can be illuminated by a light source deployed on one side of the device 400 and imaged by a detector deployed on the opposite side of the device 400. Because the substrate 402 is opaque, but the enrichment channel 418 defines an optical slit, the substrate 402 can block light that does not pass through the enrichment channel 418, thereby blocking stray light and improving the resolution of the imaging process. The glass layer 402 is sandwiched between two molten quartz plates that are transmissive (e.g., transparent) to 280 nm light. The top plate contains through-holes for instrument and user interfaces 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 channels are made of diced capillaries (100 μm ID, Polymicro) bonded to the channel network. The operation of this device in performing isoelectric focusing and subsequent mass spectrometry characterization of proteins will be described in Example 1 below.

[0090] Any of a variety of fluid actuation mechanisms known to those skilled in the art can be used to control the fluid flow of samples and reagents through a device. Examples of suitable fluid actuation mechanisms for the disclosed methods, devices, and systems include, but are not limited to, applying positive or negative pressure, gravity or centrifugal force, electrodynamic force, electrowetting force, or any combination thereof to one or more inlet or outlet ports. In some embodiments, positive or negative pressure may be applied directly, for example, by using a mechanical actuator or piston coupled to the inlet and / or outlet ports to actuate the flow of samples or reagents through a fluid channel. In some embodiments, the mechanical actuator or piston may apply force to a flexible membrane or diaphragm used to seal the inlet and / or outlet ports. In some embodiments, positive or negative pressure may be applied indirectly, for example, by using a pressurized gas line or vacuum line connected to one or more inlets and / or outlets. In some embodiments, a pump (e.g., a programmable syringe pump, HPLC pump, or peristaltic pump) connected to one or more inlet and / or outlet ports may be used to drive the fluid flow. In some embodiments, electrodynamic force and / or electrowetting force may be applied by using an electric field and controlling surface properties within the device. An electric field can be applied by means of electrodes inserted into one or more inlet and / or outlet ports, or by means of electrodes integrated into one or more fluid channels within the device. The electrodes can be connected to one or more DC or AC power sources to control the voltage and / or current within the device.

[0091] Generally, the inlet ports, outlet ports, fluid channels, or other components of the disclosed microfluidic devices, including the device body, can be manufactured using any of a variety of materials, including but not limited to glass, fused silica, silicon, polycarbonate, polymethyl methacrylate, cyclic olefin copolymers (COC) or cyclic olefin polymers (COP), polydimethylsiloxane (PDMS), or other elastomeric materials. Suitable manufacturing techniques will generally depend on the choice of materials, and vice versa. Examples include, but are not limited to, CNC machining, photolithography and chemical etching, laser ablation, injection molding, thermoforming, die cutting, 3D printing, etc. In some embodiments, the microfluidic device may include a layered structure, for example, a fluid layer including fluid channels sandwiched between an upper and / or lower layer to seal the channels. The upper and / or lower layers may include openings aligned with the fluid channels in the jet layer to create inlet and / or outlet ports, etc. Two or more device layers may be sandwiched together to form a device that can be detached or permanently bonded. Suitable bonding techniques generally depend on the choice of materials used to manufacture the layers. Examples include, but are not limited to, anodic bonding, thermal bonding, laser welding, or the use of curable adhesives (e.g., heat- or light-curable adhesives).

[0092] In some embodiments, all or a portion of the inlet port, outlet port, or fluid channel within the microfluidic device may include a surface coating (e.g., an HPC or PVA coating) for modifying electroosmotic properties and / or hydrophobic / hydrophilic properties (e.g., a polyethylene glycol (PEG) coating) of the inlet port, outlet port, or fluid channel wall.

[0093] The inlet and / or outlet ports of the disclosed equipment can be manufactured in various shapes and sizes. Suitable inlet and / or outlet geometries include, but are not limited to, spheres, cylinders, ellipses, cubes, cones, hemispheres, rectangles, or polyhedra (e.g., three-dimensional geometries consisting of several planes, such as 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.

[0094] The dimensions of the inlet and / or outlet ports can be characterized by their 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 within a planar cross-section of the geometry of the inlet and / or outlet port. In some embodiments of this disclosure, the average diameter of the inlet and / or outlet ports can range from about 0.1 mm to about 10 mm. In some embodiments, the average diameter of the inlet and / or outlet ports can 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 can be at most 10 mm, at most 8 mm, at most 6 mm, at most 4 mm, at most 2 mm, at most 1 mm, or at most 0.5 mm. Any of the lower and upper limits described in this paragraph can be combined to form a range included within this disclosure; for example, in some embodiments, the average diameter can 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 has any value within this range, for example, about 5.5 mm.

[0095] In some embodiments, the depth of the inlet and / or outlet port (e.g., a sample or reagent trap) may range from about 5 μm to about 500 μm. In some embodiments, the depth may be at least 5 μm, at least 10 μm, at least 25 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, or at least 500 μm. In some embodiments, the depth may be at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, at most 25 μm, at most 10 μm, or at most 5 μm. Any of the lower and upper limits described in this paragraph may be combined to form the range included in this disclosure; for example, in some embodiments, the depth of the inlet and / or outlet port may range from about 50 μm to about 200 μm. 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 port (e.g., a sample or reagent trap) may range from about 500 μm to about 50 mm. In some embodiments, the depth may be at least 1 mm, at least 5 mm, at least 10 mm, at least 15 mm, at least 20 mm, or at least 50 mm. In some embodiments, the depth may be at most 50 mm, at most 20 mm, at most 15 mm, at most 10 mm, at most 5 mm, or at most 1 mm. Any of the lower and upper limits described in this paragraph may be combined to form the ranges included in this disclosure; for example, in some embodiments, the depth of the inlet and / or outlet port may range from about 50 μm to about 5 mm.

[0096] In some embodiments, the fluid channels of the disclosed device can have any of a variety of cross-sectional geometries, such as square, rectangular, circular, etc. Generally, the cross-sectional geometry of the fluid channels will depend on the manufacturing technology used to create them, and vice versa. In some embodiments, the cross-sectional dimensions of the fluid channels (e.g., the height, width, or average diameter of a fluid channel with a non-rectangular cross-section, wherein the average diameter is defined as the diameter of the largest circle that can be inscribed within the cross-sectional geometry of the fluid channel) can range from about 5 μm to about 500 μm. In some embodiments, the dimensions of the fluid channels can be at least 5 μm, at least 10 μm, at least 25 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, or at least 1000 μm. In some embodiments, the size of the fluid channel can be up to 1000 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 can be combined to form the ranges included in this disclosure; for example, in some embodiments, the dimension of the fluid channel can range from about 75 μm to about 300 μm. Those skilled in the art will recognize that this dimension can have any value within this range, for example, about 95 μm. In some embodiments, the depth of the fluid channel can be equal to the depth of the inlet and / or outlet ports of the device.

[0097] Imaging techniques: In some embodiments of the disclosed methods and apparatus, imaging of the analyte separation and / or movement steps may be performed using optical detection techniques, such as ultraviolet (UV) absorbance, visible absorbance, fluorescence, Fourier transform infrared spectroscopy, Fourier transform near-infrared spectroscopy, Raman spectroscopy, optical spectroscopy, etc. In some embodiments, all or a portion of the separation (or enrichment) channel, the end of the separation channel and downstream analytical instrument or electrospray orifice or tip, the connector or connecting channel of the electrospray orifice or tip itself, or any combination thereof, may be imaged. In some embodiments, the separation (or enrichment) channel may be the lumen of a capillary. In some embodiments, the separation (or enrichment) channel may be a fluid channel within a microfluidic device.

[0098] The wavelength range(s) used to detect the separated analyte bands will generally depend on the choice of imaging technique and the materials(s) used to manufacture the device or parts thereof. For example, in cases where UV 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 natural absorbance of peptide bonds) and / or approximately 280 nm (due to the natural absorbance of aromatic amino acid residues) can allow visualization of protein bands during separation and / or movement, provided that at least a portion of the device (e.g., the separation channel) is transparent to light at these wavelengths. In some embodiments, analytes to be separated and characterized by ESI-MS can be tagged prior to separation with, for example, fluorophores, chemiluminescent tags, or other suitable tags, so that they can be imaged using fluorescence imaging or other suitable imaging techniques. In some embodiments, for example, where the analytes comprise proteins produced by commercial manufacturing processes, the proteins can be genetically engineered to incorporate green fluorescent protein (GFP) domains or variants thereof, thereby enabling them to be imaged using fluorescence. In some embodiments, proteins can be labeled or tagged. The tagged proteins can be configured so that the tags do not interfere with or disrupt the analyte properties on which the selected separation technique is based. In some embodiments, no changes are required for imaging, and fluorescence for UV imaging can be performed on native proteins, peptides, or other analytes.

[0099] To achieve the objectives of the disclosed methods, apparatus, and systems, any of a variety of imaging system components can be used. Examples include, but are not limited to, one or more light sources (e.g., light-emitting diodes (LEDs), diode lasers, fiber lasers, gas lasers, halogen lamps, arc lamps, etc.), condenser lenses, objectives, 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 any combination thereof. Depending on the imaging mode used, the light source and image sensor may be positioned on opposite sides of the microfluidic device, for example, so that an absorbance-based image can be acquired. In some embodiments, for example, the light source and image sensor may be positioned on the same side of the microfluidic device, thereby enabling the acquisition of epifluorescence images.

[0100] Images may be acquired continuously during the separation, movement, and / or electrospraying steps, or at random or specified time intervals. In some embodiments, a series of one or more images may be acquired continuously, at random time intervals, or at specified time intervals. In some embodiments, a series of one or more images may include video images.

[0101] Imaging of pI markers for determining the isoelectric point of proteins prior to electrospraying: In some embodiments, as described above, the positions of two or more pI markers in an image of a separation channel including a CIEF-separated mixture of analytes can 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 for 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) describing the relationship between local pH and position along the separation channel. In some embodiments, the isoelectric point for one or more analytes is calculated based on the positions of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more pI criteria determined from an image of the separation channel.

[0102] In some embodiments, images for determining the positions of two or more pI markers are acquired during the separation of the analyte mixture, and the calculation of the pI for each analyte band is iteratively updated as separation continues. In some embodiments, images for determining the positions of two or more pI markers are acquired after separation is complete and before the movement step is initiated. In some embodiments, images for determining the positions of two or more pI markers are acquired as the separated mixture is moved and discharged through an electrospray tip or orifice. In some embodiments, images for determining the positions of two or more pI markers are acquired as the separated mixture is moved and discharged through a fluid channel connected to a downstream analytical instrument.

[0103] In some embodiments, the images used to determine the positions of two or more pI markers and(one or more) analyte bands in the separated mixture are acquired using a computer-implemented method (e.g., software package). In some embodiments, the positions of the two or more pI markers and(one or more) analyte bands are determined using a computer-implemented method including automated image processing. In some embodiments, the computer-implemented method further includes performing an isoelectric point calculation for one or more analyte bands based on position data derived from automated image processing.

[0104] Figure 2An example process flowchart of a computer-implemented method is provided, which acquires one or more images of a separation channel (or other part of a microfluidic device), determines the locations of pI markers and analyte bands in the images (i.e., where the separation step includes CIEF), and calculates the pI of one or more analyte bands in the mixture of separated analytes. In some embodiments, the computer-implemented method may include controlling the acquisition of a series of one or more images and then processing these images to identify the locations of pI markers and separated analyte bands. Examples of suitable automated image processing algorithms will be discussed in more detail below. In some embodiments, predetermined knowledge about the predicted locations of pI markers (e.g., the locations of pI markers determined from images of a “control” sample containing only pI markers) can be used to distinguish bands corresponding to pI markers from bands corresponding to separated analytes. In some embodiments, images of pI markers may be acquired at different wavelengths or using imaging modes different from the imaging modes used to acquire images of separated analyte bands. Figure 2 As shown, if the image processing step cannot determine the location of a known number of pI markers and / or separated analyte bands, the system can be instructed to acquire one or more new images so that the image processing step can be repeated. Once the locations of the pI markers and separated analyte bands are determined, the pI marker location data is fitted to a user-selected pH gradient model (e.g., a linear or nonlinear model), and the resulting fitted relationship between the local pH and the location along the separation channel is then used to calculate the isoelectric point of one or more analyte bands.

[0105] In some embodiments, the computer-implemented method may be an iterative process in which the steps of detecting pI markers and analyte band positions, fitting position data to a pH gradient model, and calculating the isoelectric point of one or more analyte bands are repeated so that the latter are continuously updated and refined (e.g., through several determined averagings). In some embodiments, the steps including image acquisition and processing, detecting pI markers and analyte band positions, fitting pI marker position data to a pH gradient model, and calculating the isoelectric point of one or more analyte bands can be completed in a sufficiently short time. The calculation of the isoelectric point may be updated and refined at least at 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 1000 Hz or any other relevant rate (e.g., at least a Nyquist rate).

[0106] Imaging Separation and Movement: In some embodiments, the movement of the peak through the separation channel can be monitored (e.g., during separation, during movement, etc.). Imaging can be UV imaging, fluorescence imaging, transmitted light imaging, or another imaging modality. In some embodiments, images of the separation and movement can be recorded at a defined rate. For example, the imaging rate can 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 can be combined into a single frame in a "movie" to show peak formation and movement. Figure 9A -F indicates a subset of images that can be combined to generate a movie. In some embodiments, the movie can be saved as GIF, AVI, MOV, MP4, or any other digital format capable of storing digital video data. In some embodiments, imaging can be performed in real time, for example, during separation, during movement, during electrospraying, etc.

[0107] In some embodiments, such as Figure 17B The dynamic heatmap shown can be used to display a series of images (e.g., a time-series imaging dataset of separation and / or movement performed in a separation channel, where each image in the series corresponds to a different time point). As an example, in Figure 17B In this approach, instead of displaying peaks together as a graphical representation (e.g., superimposing multiple intensity or absorbance curves along the length of the separate channels (i.e., pixel locations) on a single curve), peaks are represented as analyte bands (each containing intensity or absorbance measurements) along the length of the imaging channels and plotted over time. Figure 17B Each row of the medium image (heatmap) shows the position of the analyte band at a single time point during focusing and movement. For example, line 1704 shows an example row of pixels in the dynamic heatmap; each row corresponds to an image of the separated channels and can be used to generate (or can be generated from) an electrophoresis map at a given time point (e.g., as shown in the image). Figure 17A (as shown in the image). Figure 17A Analyte peak 1702 in the sample is composed of Figure 17B The bright pixels (also labeled 1702) are represented in the image. For Figure 17A Each analyte peak in the sample, Figure 17B The time process of analyte peak migration is shown. For example, during IEF separation, an analyte (or multiple analytes) can migrate from both ends of the channel to one or more isoelectric points of the analyte (or multiple analytes). At these isoelectric points, when focusing is complete (e.g., at the time corresponding to line 1704), the analyte can be enriched, resulting in a bright peak (pixel 1702). Additionally, at the start of the migration (in this case, ...), Figure 17BThe portion above the midline 1704 (where each peak can migrate rapidly toward the chip aperture and the mass spectrometer) allows for accelerated migration of each peak. Successive time-series images are stacked vertically, therefore... Figure 17B The column axis (Y-axis) represents time, while the X-axis represents the spatial resolution of the band at each time point (e.g., the position of the analyte based on its position along the length of the separation channel). In some embodiments, the relative intensity of the band can be represented by grayscale or color scale. In some embodiments, increasing or decreasing the color scale or grayscale can be correlated with an increase in signal, intensity, or absorbance. In some embodiments, the velocity of the band can be determined by measuring the slope of the band over time in a modified thermal map. In some embodiments, the modified thermal map can be used to display imaging data from focusing, movement, or both. This display of separation data can be particularly useful in comparing or characterizing the round-by-round variability of separation and / or movement, comparing separation and movement responses (e.g., timescales of completion of separation and movement responses, separation resolution achieved during separation, separation resolution maintained during movement, etc.), determining when separation is completed, monitoring or detecting faults 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.).

[0108] In some embodiments, time-series imaging data can be plotted on a three-dimensional or triaxial graph, such as... Figure 19 As shown in the diagram. One axis of the graph can represent distance (e.g., physical distance or pixel location along the length of the separating channel), and another axis can represent time. In some cases, a third axis can be used to represent signal strength, intensity, or absorbance, which can alternatively or additionally be represented by color levels or grayscale. In some embodiments, the x-axis can be used to represent distance, the y-axis to represent time, and the z-axis to represent signal or absorbance. It will be appreciated that the axes can be used to represent any parameter (e.g., distance or location along the channel, pI, intensity or absorbance, time, etc.).

[0109] Imaging data and data plotting (or other image processing) can be performed after separation and mass spectrometry analysis are completed, or in some cases, concurrently with separation, movement, and mass spectrometry analysis. For example, computer-implemented methods or software can be configured to receive the acquired imaging data, process the imaging data (e.g., to obtain an intensity map based on channel length), and plot the IEF data (e.g., iteratively or incrementally plotting in a 3D map or heatmap).

[0110] As described herein, computer-implemented methods or software can be used to collect mass spectra at a specified scan rate. In some embodiments, computer-implemented methods can be used to summarize mass spectrometry data in the form of chromatograms. For example, curves can be generated where the X-axis represents time and the Y-axis represents the sum of signals in the mass spectrometry data (e.g., total ion count), such as... Figure 18 The line trace is 1834. The Y-axis can represent the sum of all signals in an individual mass spectrometer (total ion chromatogram), the sum of signals from a specific base peak or extracted ions (base peak chromatogram, extracted ion chromatogram), or any other subset of mass spectrometry data.

[0111] Imaging of analyte bands to determine velocity: In some embodiments, as described above, the position of one or more analyte bands can be determined from a series of two or more images of the separation channel (or other part of the microfluidic device), such that the velocity for one or more analyte bands can be calculated based on the difference in 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, two or more images of at least a portion of the separation channel can be acquired simultaneously with the separation step. In some embodiments, two or more images can be acquired during the movement step. In some embodiments, two or more images can be acquired as the separated sample is discharged through a fluid channel connected to the end of the separation channel to a downstream analytical instrument. In some embodiments, two or more images can be acquired simultaneously as the separated sample is discharged through an electrospray tip or orifice to form a Taylor cone. In some embodiments, the velocity determined for one or more analyte bands can be used to calculate the time it takes for a given analyte band to leave the separation channel. In some embodiments, for example, when one or more interconnecting fluid connectors or fluid channels are present connecting the end of the separation channel to an outlet port (e.g., an electrospray orifice or tip), the velocity determined for one or more analyte bands can be used to calculate the time it takes for a given analyte band to reach the outlet port and leave the device. In some embodiments, the velocity determined for one or more analyte bands can be used to calculate the time it takes for a given analyte band to leave 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.

[0112] In some embodiments, the image sequence used to determine the velocity of one or more analyte bands can be acquired using a computer-implemented method (e.g., software package). In some embodiments, the velocity of one or more analyte bands is determined using a computer-implemented method including automated image processing. In some embodiments, the computer-implemented method further includes calculating the time it takes for a given analyte band to exit the separation channel. In some embodiments, the computer-implemented method further includes calculating the time it takes for a given analyte band to reach the exit port and exit the device. In some embodiments, the computer-implemented method further includes calculating the time it takes for a given analyte band to exit the electrospray tip or orifice and enter the Taylor cone formed between the electrospray tip or orifice and the mass spectrometer inlet. In some embodiments, the exit times(s) determined for one or more analyte bands are used to correlate specific analyte bands with mass spectrometry data or data collected using other analytical instruments.

[0113] Figure 3 Another example process flowchart of a computer-implemented method is provided for acquiring one or more images of a separation channel (or other part of a microfluidic device), determining the velocity of one or more analyte bands, and the time it takes for a given analyte band to reach a specified point in the device (e.g., the end of the separation channel, the junction point between the separation channel and a second fluid channel, the device's outlet port, or an electrospray tip or orifice). In some embodiments, the computer-implemented method may include controlling the acquisition of a series of one or more images, and then processing these images to identify the location of the separated analyte bands. Examples of suitable automatic image processing algorithms will be discussed in more detail below. Figure 3 As shown, if the image processing step fails to determine the location of the separated analyte bands, the system can be instructed to acquire one or more new images, allowing the image processing step to be repeated. Once the locations of the separated analyte bands in a series of two or more images are determined, the velocities of one or more analyte peaks are calculated based on their relative positions in the two or more images and one or more known time intervals 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 can be used to distinguish several separated analyte bands and refine the velocity calculations (e.g., by averaging velocity values ​​calculated from several pairs of images in the series). In some embodiments, pI markers or other internal standards detected using a selected imaging mode can be used as “velocity standards.” The analyte band velocities thus determined can be used to calculate the time it will take for a given band to reach a user-specified point in the device (e.g., the outlet port of a separation channel, a specific fluid connector within the device, the device's outlet port, the analyte entering the electrospray ionization tip or orifice of the Taylor cone, etc.).

[0114] In some embodiments, the computer-implemented method may be an iterative process in which the steps of detecting the analyte band position, determining the analyte band velocity, and calculating the exit time are repeated, such that the exit time prediction is continuously updated and further improves the correlation between chemical separation data and mass spectrometry data (or other types of downstream analysis data). In some embodiments, the cycle including image acquisition and processing, velocity calculation, and exit time prediction(s) can be completed in a sufficiently short time, such that the exit time prediction(s) can be updated at 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 1000 Hz or any other relevant rate (e.g., at a rate of at least Nyquist).

[0115] In some embodiments (e.g., embodiments including the CIEF step), the computer-implemented method of this disclosure can perform image-based determination of precise isoelectric points and image-based determination of the velocity of separated analyte bands.

[0116] Correlation between separation data and mass spectrometry data: In some embodiments, the computer-implemented method described above for performing imaging-based determination of the accurate isoelectric point of an analyte band using isoelectric focusing enables the correlation of isoelectric point data with specific m / z peaks in mass spectrometry data (or other analytical data), thereby improving the informative content of the dataset (even for a single run experiment) and allowing for more quantitative characterization of the analyte sample. The computer-implemented method can be configured to receive (e.g., using a processor) IEF data (e.g., based on multiple intensity or absorbance measurements along the length of the separation channel, or pI data for a specific peak) and MS data (e.g., total ion chromatograms, multiple ion measurements based on mass, etc.).

[0117] In some embodiments, the imaged analyte peaks can be correlated with mass spectrometry data to generate information about the mass and charge (or isoelectric point) of one or more analytes in the peaks. For example, during separation, one or more images of the separation channel can be acquired at any useful imaging rate to generate a time-series imaging dataset. The time-series imaging dataset can include multiple images of the separation channel, each corresponding to a different time point. In some cases, the IEF data (e.g., each image in the time series) can be plotted as an electrophoresis diagram, which can show the signal, intensity, or absorbance according to its position along the length of the separation channel. In some cases, as described elsewhere herein, the IEF data can be used to generate heatmaps or 3D maps (see, for example...). Figure 17A -B and Figure 19 ).

[0118] IEF data can be plotted together with MS data (e.g., using one or more processors). For example, Figure 18 Line 1832 in the image shows an IEF electrophoresis pattern of NIST monoclonal antibody separation. The X-axis of line 1832 indicates the spatial resolution or position along the length of the separation channel (which can be displayed in units of distance, number of pixels, or isoelectric points), and the Y-axis of line 1832 indicates the relative signal intensity (e.g., absorbance, intensity, etc.). Figure 18 As shown, an IEF electrophoresis plot is drawn on one or more chromatograms representing MS data. For example, one or more MS scans, averaged scans, processed data, or deconvoluted data can be plotted using an IEF electrophoresis plot by aligning the time axis of the MS data (e.g., total ion chromatogram 1834) with the isoelectric focusing peak in trace 1832. In one such example, trace 1836 represents an example of a deconvoluted mass spectrum collected by a mass spectrometer at equal time intervals, corresponding to time segments of total ion chromatogram 1834 and pI segments of IEF electrophoresis plot 1832. In some cases, the deconvoluted mass spectrum can be deconvoluted from total ion chromatogram 1834 or otherwise generated. Each trace 1836 is aligned with the x-axis of the total ion chromatogram 1834 and IEF electrophoresis plot 1832. Traces 1836 show the mass (represented by the vertical Y-axis) and signal intensity (e.g., ion count) of each deconvoluted mass spectrum (along the X-axis). Each mass spectral line trace 1836 can be shown using two reference scales—the darker line is normalized to the highest signal across all mass spectra, while the lighter trace is normalized for each individual mass spectrum. This curve provides a display of signal intensity but also allows for the display of low signals within the line trace 1836.

[0119] In some embodiments, correlating IEF and MS data can be particularly useful in identifying or distinguishing one or more analyte species that have similar properties (e.g., the same charge or isoelectric point, or the same mass) and / or different properties. For example, two molecules with different masses can be identified in mass spectrometry data. The two molecules may have different isoelectric points (pI) and focus in different regions of the pH gradient in the IEF, or the two molecules may have the same isoelectric point (pI) and focus in the same region of the pH gradient in the IEF. In the case where two molecules have the same pI and different masses, the correlation between IEF and MS data can be used to distinguish the two molecules (e.g., identify them as different species or isotypes).

[0120] For example, two molecules with the same pI and different masses (or alternatively, the same mass but different pIs) can be two protein isotypes, for example, isotypes where the difference in pI or mass is due to post-translational modifications, translation errors (e.g., incorrect amino acid additions, folding, disulfide rearrangements, or other translational modifications), transcription, or encoding in a DNA sequence. In this example, the correct post-translational modification can be identified by examining the differences (or similarities) in mass and charge or pI. For example, analyte peaks within a separation channel (e.g., IEF analyte peaks) can include one or more analyte species with the same isoelectric point but different masses. As described herein, different masses can be identified by performing MS on the analyte peaks. By using both pI and mass information, certain isotypes with known or expected pIs and masses that deviate from a particular isotype can be eliminated. Thus, differences in mass can be used to identify one or more analyte species even if they have the same isoelectric point. Alternatively or in combination, differences in pI can be used to identify one or more analyte species even if they have the same mass.

[0121] Coverage of IEF and MS data (e.g., total ion chromatograms and time-series ion measurements based on mass) can be useful for identifying protein isotypes by mapping pI to the mass of one or more analyte species. For example, reference Figure 18 IEF data (e.g., IEF electrophoresis plot 1832) can be mapped to a total ion chromatogram 1834. Each point (e.g., time interval) in the total ion chromatogram 1834 can be deconvolved to generate a line trace 1836, which shows the mass distribution and relative intensity. Thus, for each time interval in the total ion chromatogram 1834, the corresponding deconvolved mass distribution data and isoelectric point can be determined. Similarly, for each isoelectric point, the corresponding mass distribution can be obtained.

[0122] For example, Figure 20 Example isoelectric focusing and mass spectrometry data from NIST monoclonal antibody analysis are shown. Figure 20 Panel A shows isoelectric focusing data for the charge variants in the inset (labeled as acid 1, acid 2, major, basic 1, and basic 2), while the larger plot shows the mass spectrometric base chromatograms of the charge variants introduced into the mass spectrometer corresponding to the charge variants in the inset. Figure 20 Panel B shows the convolution quality data, which displays the quality assignment of the base peak chromatographic sample for each time interval, each time interval can be mapped back to the position along the length of the separation channel (e.g., from the acid end to the basic end). Figure 20 The differences in peak distribution in panel B demonstrate the differences in the quality and relative abundance of different charge variant peaks separated during isoelectric focusing.

[0123] IEF and MS data can be used to assign post-translation editing. Figure 21 Panel A shows a list of examples of post-translational modifications and the expected changes in charge and mass resulting from these modifications. These values ​​can be obtained from publicly available sources (e.g., published data, protein databases, etc.) or derived empirically. In the examples, in Figure 21 In panel A, both glycosylation and galactose (glycosylation) modifications result in a 162 Dalton increase in molecular weight. However, glycosylation events can be distinguished from glycosylation events because glycosylation makes the molecule more acidic and shifts it to a lower isoelectric point in the IEF or a different elution time in the CZE. Figure 21 Panel B outlines this example and others, but many other combinations of modifications may exist in protein analytes. For example, post-translational modifications can include hydroxylation, methylation, lipidation, acetylation, disulfide bonding, threoylation, 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 characteristics (e.g., charge, mass, pI changes, etc.) are available from publicly available sources (e.g., UniProt, BLAST, or other protein databases).

[0124] Figure 22 Panel A shows the unconvolutional mass calculated from mass spectrometry analysis of Basic 2, Basic 1, and the main peak (analyte peak separated by IEF). The peaks in Basic 1 and the main peak show a continuous increase of 162 Daltons per molecule, indicating that successive glycosylation steps resulted in mass differences but no difference in charge (or pI). Figure 23 As shown in panel B, the relative abundance of molecules of different masses did not change between basicity 1 and majority, only... Figure 22 The 128 Dalton shift shown in panel A indicates the presence of an additional lysine residue on the molecule in the basic 1 peak. Similarly, in Figure 22 In panel B, when comparing the deconvolution mass calculated from the mass spectrometry analysis of the main, acid 1, and acid 2 peaks (analyte peaks separated by IEF), we can see a consistent 162 Dalton shift series in the molecule within each charge variant peak. However, when the main acid 1 and acid 2 profiles cover... Figure 23 In panel A, a relative increase in the mass of the larger peaks in the acid charge variant can be observed, indicating glycosylation in addition to the glycosylation sequences seen in basic 1 and the main peak.

[0125] Computer-based methods: As described herein, one or more data presentation and analysis algorithms can be implemented using computer-based methods. Computer algorithms can be used to perform a variety of functions, including but not limited to: receiving IEF and MS data, transforming or processing the data, generating plots or curves of the data, overlaying IEF and MS data, and analyzing IEF data, such as evaluating charge and mass changes to correctly assign post-translational modifications. In some embodiments, neural networks or other artificial intelligence algorithms can be used to evaluate charge and mass changes or similarities to correctly assign post-translational modifications. In some cases, computer-based methods can 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 ​​can then be used by one or more processors to match IEF and MS data to determine or identify post-translational modifications in the analyte peaks.

[0126] One or more computer-implemented methods can be configured to automatically perform one or more functions (e.g., without human interaction). For example, one or more computer-implemented methods may be part of a software package for acquiring data (e.g., performing imaging), receiving data (e.g., separating, moving, and / or MS data), processing data, displaying data, etc. Data processing or presentation may be performed substantially simultaneously with imaging, or may occur after imaging is complete. Similarly, data processing or presentation may be performed during or after ESI-MS. For example, the determination or assignment of post-translation modifications may be performed within 1 second, 1 minute, 10 minutes, 1 hour, etc., of acquiring ESI-MS data.

[0127] In some embodiments, the computer-implemented method described above for using image-derived data to calculate the velocity and predict the exit time of separated analyte bands (using any of a variety of different separation techniques) enables the improvement of the temporal correlation between chemical separation data (e.g., retention time, electrophoretic mobility, isoelectric point, etc.) and specific m / z peaks in mass spectrometry data (or other analytical data), thereby improving the information content of the dataset (even for a single run experiment) and allowing for more quantitative comparisons of data collected from different sample runs, different samples, or data collected on different instruments, because it is possible to correct for inter-experimental or inter-instrumental differences in separation time.

[0128] Therefore, the disclosed methods, apparatus, and systems can be particularly advantageous for a wide range of metabolomics, proteomics, and drug development or manufacturing applications. It will be appreciated that while the examples above involve isoelectric focusing coupled to a mass spectrometer, the separations performed prior to the introduction of the mass spectrometer can be electrophoresis, chromatography, or other separations, as described elsewhere herein.

[0129] Mass spectrometry and electrospray ionization: In some embodiments, the methods, apparatus, and systems of this disclosure can be configured to perform electrospray ionization of a mixture of analytes for separation and inject it into a mass spectrometer. Mass spectrometry (MS) is an analytical technique that measures the “mass” of analyte molecules in a sample by ionizing and classifying the generated ions based on their mass-to-charge ratio (m / z). Combined with preceding liquid or gas phase sample separation systems, mass spectrometry provides one of the most efficient means of analyzing complex samples containing a variety of low-abundance analytes, such as those common in biological samples.

[0130] All mass spectrometers require ions to be in the gas phase before being introduced into the mass analyzer. Various sample ionization modes have been developed, including but not limited to matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI). In MALDI, 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 cyano-4-hydroxycinnamic acid) and crystallized onto a metal plate. Surface-enhanced laser desorption / ionization (SELDI) is a common variant of this technique, which incorporates additional surface chemicals on a metal plate to promote the specific binding of certain classes of proteins. The plate is inserted into a vacuum chamber, and then the matrix crystals are struck with pulses of light from a nitrogen laser. The energy absorbed by the matrix molecules is transferred to the proteins, causing them to desorb, ionize, and generate a cluster of ions in the gas phase. These ions are accelerated in the presence of an electric field and drawn into a flight tube, where they drift until they collide with a detector that records the time of flight. The time of flight can then be used to calculate the m / z ratio of the ionized species. In some embodiments of the disclosed device, the device's outlet port may include a capillary or other features for depositing separated analyte bands (or portions thereof) onto a MALDI plate to prepare for mass spectrometry analysis, such as correlating the isoelectric point of a particular analyte band with MALDI mass spectrometer data.

[0131] Electrospray ionization (ESI; also referred to herein as “espray”) can also be used due to its inherent compatibility with mass spectrometer-connected liquid chromatography or electrochromatographic separation techniques. As described above, in electrospray ionization, small droplets of sample and solution are emitted from the distal end of a capillary or microfluidic device, which includes 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 in this induced electric field to form a cone emission (i.e., a “Taylor cone”), which comprises increasingly smaller droplets that evaporate and generate gaseous ions that are introduced into the mass spectrometer for further separation and detection. The emitter tip can be formed from a capillary or corner or an ESI tip built into a microfluidic chip design, which provides a convenient droplet volume for ESI. The emitter tip can be sharpened to provide a small surface area and droplet volume using grinding wheels, files, machining tools, CNC machining tools, waterjet cutting, or other tools or processes to shape the ESI tip to provide a small surface volume, etc. In some embodiments, the tip can be drawn by heating and stretching the tip portion of the chip. In some embodiments, the tip can then be cut to the desired length or diameter. In some embodiments, the electrospray tip can be coated with a hydrophobic coating that minimizes the size of droplets formed on the tip. In some embodiments, when no analyte is eluted from the device, the system can electrospray a moving agent, cathode electrolyte, or any other liquid during the separation step.

[0132] In some embodiments of the disclosed methods, apparatus and systems, other ionization methods are used, such as inductively coupled laser ionization, fast atomic bombardment, soft laser desorption / resorption, atmospheric pressure chemical ionization, secondary ion mass spectrometry, spark ionization, thermal ionization, etc.

[0133] Regarding electrospray ionization, in some embodiments, the disclosed microfluidic devices include features designed to facilitate efficient electrospray ionization and facilitate interface with downstream mass spectrometry analysis, as shown in Figure 1. The mass-to-charge ratio (or “mass”) of the analyte expelled from the microfluidic device (e.g., a biological or bioanalyte) and introduced into the mass spectrometer can be measured using any 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 orbital trap mass spectrometry, distance-of-flight mass spectrometry, Fourier transform ion cyclotron resonance, resonance mass measurement, and nanomechanical mass spectrometry.

[0134] 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 at a right angle or a mid-angle relative to the separation channel. In some embodiments of the disclosed method, substantially all separated and / or enriched analyte fractions from the final separation or enrichment step performed in the capillary or microfluidic device are discharged in a continuous flow from the electrospray tip or feature. 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 enrich at least a portion of a sample). Another portion of the analyte mixture (e.g., containing fractions other than the fraction of interest) may be discharged via a waste channel.

[0135] In some embodiments, pressure, electricity, ionization, or any combination thereof are used to perform discharge from the capillary or microfluidic device. In some embodiments, discharge is consistent with the movement step described above. In some embodiments, the sheath fluid used for electrospray ionization is used as the electrolyte for electrophoretic separation. In some embodiments, an atomizing gas is provided to reduce the analyte fraction to a fine spray.

[0136] Imaging-Based Feedback for Electrospray Ionization Performance: Conventional ESI-MS systems using capillary or microfluidic devices generally do not provide 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 channels in the microfluidic device or capillary. Changes in reagent conductivity between or during runs can alter the voltage potential at the interface with the mass spectrometer. Changes in the interface potential can adversely affect the Taylor cone and lead to a loss of electrospray ionization efficiency. This document discloses methods and systems for improving electrospray ionization performance and thus enhancing the quality of mass spectrometry data collected for capillary or microfluidic device-based ESI-MS systems. In some embodiments, such as imaging of the Taylor cone in an electrospray ionization device, the image can be used in a computer-implemented method to provide feedback control of one or more operating parameters such that the shape, density, or other characteristics of the Taylor cone are maintained within specified ranges. In some embodiments, the operating parameters that can be controlled by such a feedback process include, but are not limited to, the alignment of the electrospray tip or orifice with 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 including an integrated electrospray feature on a programmable precision XYZ translation stage), the flow rate of the analyte sample through the electrospray tip (e.g., by adjusting the pressure, electric field strength, or a combination thereof used to drive the discharge of the analyte sample), such as the voltage applied to the proximal end of the channel (e.g., between the electrospray tip or orifice and the mass spectrometer inlet), the volumetric flow rate of the sheath fluid, or the sheath gas surrounding the discharged analyte sample, or any combination thereof.

[0137] Figure 4 An example process flowchart of a computer-implemented method is provided, which involves: (i) acquiring an image of the Taylor cone (using any of a variety of image sensors, such as a CCD image sensor or a CMOS image sensor); (ii) processing the image to determine the shape, density, or other characteristics of the Taylor cone; (iii) comparing the shape, density, or other characteristics of the Taylor cone with a specified or target set of values; and (iv) based on the comparison, using a mathematical algorithm that associates the shape, density, or other characteristics of the Taylor cone with one or more operating parameters to determine appropriate adjustments to one or more operating parameters to restore the Taylor cone to the specified or target values. In some embodiments, in addition to data derived from the Taylor cone image, data acquired from a mass spectrometer (e.g., total ion current data) may also be used to monitor system performance and adjust one or more operating parameters.

[0138] In some embodiments, Figure 4The illustrated cyclic process includes the following steps: image acquisition and processing, identification of Taylor cone features, comparison of the Taylor cone features with a set of target values, and calculation of adjustments required for one or more ESI-MS system operating parameters, which can be completed in a sufficiently short time so that one or more operating parameters can be updated at a rate of at least 0.01Hz, 0.1Hz, 0.2Hz, 0.3Hz, 0.4Hz, 0.5Hz, 0.6Hz, 0.7Hz, 0.8Hz, 0.9Hz, 1Hz, 10Hz, 100Hz, or 1000Hz or any other relevant rate (e.g., at least the Nyquist rate).

[0139] Alternating high-quality / low-quality scans: In some embodiments of the disclosed methods, apparatus, and systems, the mass spectrometer may be configured to alternate between a high-quality scan range (e.g., approximately 1500–6000 m / z) or “high-quality scan” and a low-quality scan range (e.g., approximately 150–1500 m / z) or “low-quality scan”, such that the low-quality scan can be used to identify low-quality markers, such as free-solution amphoteric electrolytes in the case of performing an isoelectric focusing separation step, which can be identified in the mass spectrometry data and used to calibrate the spectrometer regarding the properties indicated by the low-quality markers (e.g., isoelectric points within a specific range in the case of detecting free-solution amphoteric electrolytes, peptides, small molecule markers). The switching between high-quality and low-quality scans, as well as the scan rate, should be rapid relative to the outflow of the analyte sample from the electrospray interface. In some cases, the switching rate between high-quality and low-quality scans may range from approximately 0.5 Hz to approximately 50 Hz. In some cases, the switching rate can be at least 0.5 Hz, at least 1 Hz, at least 5 Hz, at least 10 Hz, at least 20 Hz, at least 30 Hz, at least 40 Hz, or at least 50 Hz.

[0140] Changing the high and low separation / movement voltages to maintain a constant ESI tip voltage: In some embodiments, the ESI ion source on the mass spectrometer will have an adjustable power supply capable of setting a negative voltage on the mass spectrometer. In some embodiments, the ESI ion source on the mass spectrometer will have an adjustable power supply capable of setting a positive voltage on the mass spectrometer. In some embodiments, the ESI ion source on the mass spectrometer will be kept grounded. In some embodiments, the ESI tip on the capillary or microfluidic device will be kept grounded or close to ground to generate an electric field between the ESI tip and the charged ESI ion source on the mass spectrometer. In some embodiments, the ESI tip on the capillary or microfluidic device will be maintained at a positive or negative voltage to generate an electric field between the ESI tip and the grounded ESI ion source on the mass spectrometer.

[0141] Figure 15An exemplary flowchart of a computer-controlled feedback loop is provided to maintain a constant voltage drop of 3000V between the anode and cathode during movement, while keeping the ESI tip voltage at 0V. In some embodiments, this feedback loop can be implemented when the mass spectrometer ESI ion source is set to a positive or negative voltage (e.g., -3500V) relative to ground. In this example, by... Figure 7A In this configuration, the anolyte port 108 is initially set to +3000V and the moving agent port 104 is set to 0V, with the ΔV between the anolyte port 108 and the moving agent port 104 maintained at 3000V. In some embodiments, different ΔV can be set by setting the anolyte port 108 to different values. In some embodiments, anolyte movement can be used, and port 108 will be the catholyte port set to, for example, -3000V. Figure 15 In the example outlined below, during the transition, the resistance in separation channel 112 decreases as the analyte and amphoteric electrolyte regain charge during separation. This causes a decrease in the voltage drop across channel 112, resulting in an increase in the voltage at the ESI tip 116, according to Equation 1:

[0142] V 116 =(△V) 108-104 )*(R 105 ) / (R 109 +R 112 +R 105 )

[0143] However, by measuring or calculating the ESI tip voltage 116, the voltage settings at the anolyte port 108 and the mobility agent port 104 can be adjusted. By subtracting the ESI tip voltage 116 from the settings at the anolyte port 108 and the mobility agent port 104, ΔV 108-104 Maintaining 3000V, movement is unaffected, but according to Equation 2, the voltage at the ESI tip 116 is set to 0:

[0144] V 116 =(△V) 108-104 )*(R 105 ) / (R 109 +R 112 +R 105 )+V 104

[0145] This feedback loop continues to operate until the movement is complete, adjusting the voltage at the ESI tip 116 to 0 at a regular frequency (e.g., the Nyquist rate or approximately 0.2 Hz). In some cases, the voltage at the ESI tip 116 can 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 1000 Hz. Maintaining a constant, stable voltage at the ESI tip 116 is crucial for maintaining stable electrospray during the movement process.

[0146] In some cases, the feedback loop operates to maintain the voltage at the ESI tip within a specified percentage range 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 the preset value. In other cases, the feedback loop operates to maintain the voltage at the ESI tip within 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V of the preset value.

[0147] In some embodiments, the mass spectrometer ESI ion source remains grounded, and the ESI tip 116 will need to be maintained 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 +5000V, approximately +4000V, approximately +3500V, approximately +3000V, approximately +2500V, approximately +2000V, approximately +1500V, approximately +1000V, approximately +500V, or approximately -5000V, approximately -4000V, approximately -3500V, approximately -3000V, approximately -2500V, approximately -2000V, approximately -1500V, approximately -1000V, or approximately -500V. Figure 12 An example flowchart of a computer-controlled feedback loop is provided to maintain a constant voltage drop of 3000V between the anode and cathode during migration, while keeping the ESI tip voltage at 3000V. The operation of the computer-controlled feedback loop is similar to, except that the voltage at the anode electrolyte port 108 and the migration agent port 104 is offset by +3000V (which offsets the voltage at the ESI tip 116 to +3000V, still conforming to Equation 2). Figure 15The same applies in the above. In some embodiments, analog circuitry can be used to control the electric field strength. In some embodiments, the voltage at one or more electrodes in contact with a capillary-based or microfluidic-based separation system can be controlled by using one, two, three, four, or more independent high-voltage power supplies. In some cases, the voltage at one or more electrodes in contact with a capillary-based or microfluidic-based separation system can be controlled, for example, by using a single multiplexed high-voltage power supply.

[0148] In some cases, the feedback loop operates to maintain the electric field strength within the separation 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 within the separation 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 other cases, the feedback loop operates to maintain the electric field strength within the separation channel or the voltage drop between the anode and cathode within a range of 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V.

[0149] Changing the mass spectrometer inlet potential 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 supply coupled thereto, which is capable of setting a negative voltage on the mass spectrometer (e.g., at the inlet). In some embodiments, the mass spectrometer may have an adjustable power supply coupled thereto, which is capable of setting a positive voltage on the mass spectrometer (e.g., at the inlet). In various embodiments, the mass spectrometer or the mass spectrometer inlet may be kept grounded. In some cases, the ESI tip on a capillary or microfluidic device may be kept grounded or close to ground to generate an electric field between the ESI tip and the charged ESI ion source on the mass spectrometer. In some cases, the ESI tip on a capillary or microfluidic device may be maintained 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 may be adjusted, for example, in a feedback loop, to maintain a constant voltage difference (ΔV) between the ESI tip and the mass spectrometer inlet. TIP-MS In some cases, the voltage difference (ΔV) TIP-MS ) can be set as the target value (ΔV) TARGET (or target value range). In some cases, the potential of the ESI tip and the voltage or potential of the mass spectrometer (e.g., at the inlet) can be adjusted, for example, to keep the voltage drop between the ESI tip and the mass spectrometer constant or within the target value range.

[0150] In various situations, 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 can be implemented when the mass spectrometer ESI ion source is set to a positive or negative voltage relative to ground (e.g., -3500V). In this example, reference... Figure 7A By setting the anolyte port 108 to +3000V and the migration agent port 104 to 0V, the ΔV between the anolyte port 108 and the migration agent port 104 can be set to an initial voltage of 3000V. In some embodiments, different ΔV can be set by setting the anolyte port 108 to different values. In some embodiments, anolyte migration can be used, and port 108 will be the catholyte port, set to, for example, -3000V. In some cases, during migration, the resistance in the separation channel 112 can decrease due to the analyte and amphoteric electrolyte regaining charge in the separation channel. This causes the voltage across channel 112 to decrease according to the following equation, resulting in an increase in the voltage at the ESI tip 116:

[0151] V 116 =(△V) 108-104 )*(R 105 ) / (R 109 +R 112 +R 105 )

[0152] The mass spectrometer voltage can be adjusted by measuring or calculating the voltage at the ESI tip 116. For example, an increase in the voltage at the ESI tip 116 can be added to the voltage applied to the mass spectrometer, such that the voltage difference between the mass spectrometer inlet and the ESI tip 116, i.e., ΔV, is increased. TIP-MS The voltage difference between the ESI tip 116 and the mass spectrometer can be adjusted in some cases to match the voltage of the ESI tip 116. The feedback loop can continue operating until the movement is complete, thereby adjusting the mass spectrometer voltage (e.g., at the inlet) at a regular frequency (e.g., the Nyquist rate or approximately 0.2 Hz) to match the voltage of the ESI tip 116. In some cases, the voltage applied to the mass spectrometer can 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 1000 Hz. Maintaining a constant, stable voltage difference between the ESI tip 116 and the mass spectrometer inlet can maintain a stable electrospray during the analyte movement to the mass spectrometer.

[0153] In some cases, the feedback loop can 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 can operate to maintain the voltage at the mass spectrometer at a preset value (e.g., ΔV). TARGET The voltage drop between the ESI tip and the mass spectrometer may be within at least 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. In another example, in some cases, the feedback loop may operate to maintain the voltage drop between the ESI tip and the mass spectrometer at a preset value (e.g., ΔV). TARGET The voltage can be maintained at at least 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 can operate to maintain the mass spectrometer voltage at at least 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V (e.g., ΔV). TARGET Within a certain range. In some cases, the feedback loop can be operated to maintain the voltage difference between the mass spectrometer and the ESI tip at a preset value (e.g., ΔV). TARGET The voltage rating is at least 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V or 1V.

[0154] In some embodiments, the ESI tip 116 may remain grounded, and the mass spectrometer or mass spectrometer inlet may be maintained at a constant positive or negative voltage to generate 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 +5000V, approximately +4000V, approximately +3500V, approximately +3000V, approximately +2500V, approximately +2000V, approximately +1500V, approximately +1000V, approximately +500V, or approximately -5000V, approximately -4000V, approximately -3500V, approximately -3000V, approximately -2500V, approximately -2000V, approximately -1500V, approximately -1000V, or approximately -500V. Although Figure 12The illustration shows an example flowchart of a computer-controlled feedback loop to maintain a constant voltage drop of 3000V between the anode and cathode during movement, while keeping the ESI tip voltage at 3000V. However, it will be appreciated that similar computer-controlled feedback loops can be used to maintain a constant voltage drop, such as a 3000V difference, between the ESI tip and the mass spectrometer inlet during movement. In such cases, the voltage at the ESI tip can be measured (e.g., via measuring resistance, current, or voltage on the chip or the ESI tip), and the voltage at the mass spectrometer inlet can be adjusted to match the voltage change at the ESI tip. In some embodiments, the potential at the tip can be measured and used to predict potential changes during subsequent operation. The mass spectrometer and / or chip potential can then be adjusted based on the predicted changes to maintain a constant voltage between the ESI tip and the mass spectrometer. In some embodiments, measurements of voltage and current in channels within the chip can be used to calculate resistance, and these resistances can be used in subsequent operation to calculate the voltage at the tip or in a specific channel.

[0155] The voltage at the ESI tip can be measured using various methods or mechanisms, including the use of a power source or electrodes. For example, microfluidic devices may include additional channels that may intersect with or be fluidly or electrically connected to the separation channel (e.g., near the ESI tip). For example, the location where the additional channel intersects with the separation channel can be 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, or 10 mm from the ESI tip. An additional power supply can be connected to this additional channel and set to a 0 microamp current. This additional power supply can be used to measure the potential at the ESI tip without introducing additional circuitry into the microfluidic device. Alternatively or additionally, the electrodes can be placed near the ESI tip. For example, the electrode can be placed at a distance 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, or 10 mm from the ESI tip. The electrode can also be configured to not be supplied with power or current, thereby allowing measurement of the ESI tip without the addition of an additional circuit system. This allows adjustment of the potential applied to the mass spectrometer inlet, the ESI tip (e.g., via potential application to the anolyte and catholyte ports, or the anolyte and flow ports), or both, to maintain a constant ΔV. TIP-MS .

[0156] In some embodiments, analog circuitry can be used to control the electric field strength. In some embodiments, voltage control at one or more electrodes in contact with a capillary-based or microfluidic-based separation system can be provided by using one, two, three, four, or more independent high-voltage power supplies. In some cases, the voltage at one or more electrodes in contact with a capillary-based or microfluidic-based separation system can be controlled, for example, by using a single multiplexed high-voltage power supply.

[0157] In some cases, the feedback loop can operate to maintain the electric field strength within the separation 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 can operate to maintain the electric field strength within the separation 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 other cases, the feedback loop can operate to maintain the electric field strength within the separation 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 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V of a preset value.

[0158] System hardware: Figure 5A schematic diagram of a system hardware block diagram for one embodiment of the disclosed methods, apparatus, and systems is provided. As shown, the system of this disclosure may include 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, such as isoelectric focusing-based separation, and one or more high-voltage power supplies), (ii) an electrospray interface for a mass spectrometer, which in some cases may be directly integrated with the separation system (as indicated by the dashed lines), (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 operate for capillary or microfluidic devices, additional light 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., including graphical user interfaces), and digital communication devices (e.g., intranets, the Internet, WiFi, etc.). Maintain a specified temperature for all or part of (or other hardwired or wireless communication hardware).

[0159] In some embodiments, the system may include an integrated system in which the selection of functional hardware components is packaged in a fixed configuration. In some embodiments, the system may include a modular system in which the selection of functional hardware components can be changed to reconfigure the system for new applications. In some embodiments, some of these functional system components (e.g., capillary or microfluidic devices) are replaceable or disposable components.

[0160] As described above, any of a variety of different mass spectrometers can 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 orbital trap mass spectrometers, distance-of-flight mass spectrometers, Fourier transform ion cyclotron resonance spectrometers, resonance mass measurement spectrometers, and nanomechanical mass spectrometers.

[0161] System and application software: such as Figure 6As shown, the system disclosed herein may include multiple software modules. For example, the system may include a system control software module, a data acquisition software module, a data processing software module, or any combination thereof. Generally, these software modules will be configured to operate within an operating system or environment hosted by a computer processor, and can communicate with and / or share data with the operating system.

[0162] In some embodiments, the system control software module may include software for:

[0163] (i) Coordinate the operation of an analyte separation system based on a capillary or microfluidic device with image acquisition via an imaging system; (ii) Coordinate the data acquisition of an analyte separation system based on a capillary or microfluidic device with a mass spectrometer system; (iii) Coordinate the image acquisition via an imaging system with the operation of an analyte separation system based on a capillary or microfluidic device and / or a mass spectrometer system; (iv) Provide feedback control of one or more operating parameters of the electrospray ionization device and / or the mass spectrometer based on data derived from imaging of the separation channel and / or Taylor cone; (v) Control data acquisition via the mass spectrometer while switching alternately between high-quality and low-quality scan ranges; (vi) Monitor the voltage at the ESI tip and adjust the separation circuit voltage to maintain a constant separation electric field strength (or voltage drop between the anode and cathode) and a constant voltage at the ESI tip; (vii) Monitor the voltage at the ESI tip and adjust the separation circuit voltage and / or the 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 inlet), or any combination thereof.

[0164] In some embodiments, the data acquisition module may include software for: (i) controlling image acquisition through 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 through 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, or any combination thereof.

[0165] In some embodiments, the data processing module may include software for: (i) processing images and determining the position of one or more pI standard or analyte peaks in the separation channel while performing separation, after separation is complete, or after the pI standard and analyte peaks have moved toward the separation channel outlet or electrospray tip; (ii) processing images and determining the velocity, exit time, and / or electrospray emission time of one or more pI standard or analyte peaks; (iii) processing images of the separation channel to monitor the position of the analyte peaks and images of the Taylor cone to monitor electrospray performance, wherein the images of the separation channel and the Taylor cone are acquired either simultaneously or alternately; (iv) processing images of the Taylor cone to determine the shape, density, or other characteristics of the Taylor cone and calculating adjustments to one or more operating parameters, including the position of the electrospray tip or orifice relative to the mass spectrometer inlet (i.e., alignment 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, to affect changes in the quality of the mass spectrometer data; or any combination thereof.

[0166] The disclosed system and application software can be implemented using any of a variety of programming languages ​​and environments known to those skilled in the art. Examples include, but are not limited to, C, C++, C#, PL / I, PL / S, PL / 8, PL-6, SYMPL, Python, Java, LabVIEW, Visual Basic, .NET, etc.

[0167] Image processing software: In some embodiments, as described above, the data processing module may include image processing software for determining the location of pI markers or separated analyte bands, for characterizing the shape, density, or other visual indicators of the Taylor cone, etc. Any of a variety of image processing algorithms known to those skilled in the art can be used for image preprocessing or image processing to implement the disclosed methods and systems. Examples include, but are not limited to, Canny edge detection methods, Canny-Deriche edge detection methods, first-order gradient edge detection methods (e.g., Sobel operator), second-order differential edge detection methods, phase coherence 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 for detecting arbitrary shapes, circular Hough transform, etc.), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation, Savitzky-Golay smoothing, feature analysis, etc.) or any combination thereof.

[0168] Processors and Computer Systems: One or more processors or computers may be used to implement the methods disclosed herein. One or more processors may include 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 consist of any of a variety of suitable integrated circuits (e.g., application-specific integrated circuits (ASICs) or field-programmable gate arrays (FPGAs) specifically designed for implementing deep learning network architectures to accelerate computation time and / or facilitate deployment), microprocessors, emerging next-generation microprocessor designs (e.g., memristor-based processors), logic devices, etc. While this disclosure is described with reference to processors, other types of integrated circuits and logic devices may also be applicable. Processors may have any suitable data manipulation capabilities. For example, a processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations. One or more processors may be single-core or multi-core processors, or multiple processors configured for parallel processing.

[0169] One or more processors or computers used to implement the disclosed methods may be part of a larger computer system and / or may be operatively coupled to a computer network (“network”) with the aid of a communication interface to facilitate the transmission and sharing of data. The network may be a local area network (LAN), an intranet and / or extranet, an intranet and / or extranet communicating with the Internet, or the Internet itself. In some cases, the network is a telecommunications and / or data network. The network may include one or more computer servers, which in some cases enables distributed computing, such as cloud computing. In some cases, with the aid of the computer system, the network may implement a peer-to-peer network, which enables devices coupled to the computer system to act as clients or servers.

[0170] Computer systems may also include memory or memory locations (e.g., random access memory, read-only memory, flash memory, etc.). Optane TM The memory, storage units (e.g., hard disks), and communication interfaces (e.g., network adapters) are used to communicate with one or more other systems and peripherals (such as caches, other memories, data storage devices, and / or electronic display adapters). The memory, storage units, interfaces, and peripherals can communicate with one or more processors (e.g., CPUs) via communication buses, for example, found on the motherboard. The storage units (one or more) can be data storage units (or data repositories) for storing data.

[0171] One or more processors (e.g., a CPU) execute a series of machine-readable instructions implemented in a program (or software). The instructions are stored in a memory location. The instructions are directed to the CPU and subsequently program or otherwise configure the CPU to implement the methods of this disclosure. Examples of 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).

[0172] Storage units store files, such as drivers, libraries, and saved programs. Storage units also store user data, such as user-specified preferences and user-specified programs. In some cases, a computer system may include one or more additional data storage units located outside the computer system, such as those located on a remote server that communicates with the computer system via an intranet or the Internet.

[0173] Some aspects of the methods and systems provided herein are implemented through machine-executable code (e.g., a processor) stored in electronic storage locations within a computer system, such as, for example, memory or electronic storage units. The machine-executable or machine-readable code is provided in the form of software. During use, the code is executed by one or more processors. In some cases, the code is retrieved from a storage unit and stored in memory for access by one or more processors at any time. In some cases, electronic storage units are excluded, and machine-executable instructions are stored in memory. The code can be pre-compiled and configured for use with a machine having one or more processors adapted to execute the code, or it can be compiled at runtime. The code can be supplied in a programming language selected to enable execution in a pre-compiled or compiled manner.

[0174] Various aspects of the disclosed methods and apparatus may be considered "products" or "articles of art," such as "computer programs or software products," typically in the form of machine (or processor) executable code and / or associated data stored in a type of machine-readable medium, wherein the executable code includes multiple instructions for controlling a computer or computer system to perform one or more of the methods disclosed herein. The machine-executable code may be stored in an optical storage unit, including optically readable media such as optical discs, CD-ROMs, DVDs, or Blu-ray discs. The machine-executable code may also be stored in an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media includes any or all tangible memory of computers, processors, etc., or associated modules thereof, such as various semiconductor memory chips, optical drives, tape drives, disk drives, etc., which can provide non-transitory storage at any time for software encoding the methods and algorithms disclosed herein.

[0175] Software code, in whole or in part, can sometimes be communicated via the Internet or various other telecommunications networks. For example, such communication enables the loading of software from one computer or processor into another, such as from a management server or host computer into a computer platform for an application server. Therefore, other types of media used for transmitting software-coded instructions include light waves, radio waves, and electromagnetic waves, such as those used through physical interfaces between local devices, wired and optical terrestrial networks, and various atmospheric links. Physical elements carrying such waves (such as wired or wireless links, optical links, etc.) are also considered as media for transmitting software-coded instructions for performing the methods disclosed herein. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as "computer or machine-readable medium" refer to any medium involved in providing instructions to a processor for execution.

[0176] Computer systems typically include, or can communicate with, electronic displays to provide, for example, images captured by a machine vision system. Displays often also provide user interfaces (UIs). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs), web-based user interfaces, etc.

[0177] Applications: As described above, the disclosed methods, apparatus, systems, and software have potential applications in a variety of fields, including but not limited to proteomics research, drug discovery and development, and clinical diagnostics. For example, separation-based ESI-MS analysis of analyte samples using the disclosed methods can achieve improved information content and data quality, which can be highly beneficial for characterizing biological and biosimilar drugs during development and / or manufacturing. Other applications may include, but are not limited to, the analysis of environmental pollutants, pesticides, small molecules, metabolites, peptides, post-translational modifications, glycoforms, antibody-drug conjugates, fusion proteins, viruses, allergens, single-cell organisms, and other applications.

[0178] Biologics and biosimilars are a class of drugs that include, for example, recombinant proteins, antibodies, live viral vaccines, human plasma-derived proteins, cell-derived drugs, naturally derived proteins, antibody-drug conjugates, protein-drug conjugates, and other protein drugs. The FDA and other regulatory agencies require a step-by-step approach to demonstrating biosimilarity, which may include comparisons of the proposed product and a reference product in terms of structure, function, animal toxicity, human pharmacokinetics (PK), and pharmacodynamics (PD), as well as clinical immunogenicity and clinical safety and efficacy (see “Scientific Considerations in Demonstrating Biosimilarity to a Reference Product: Guidance for Industry,” FDA, U.S. Department of Health and Human Services, April 2015). Examples of structural characterization data that may be required for protein products include primary structure (i.e., the amino acid sequence), secondary structure (i.e., the degree of folding to form α-helices or β-sheets), tertiary structure (i.e., the three-dimensional shape of the protein resulting from the folding of the polypeptide backbone and secondary domains), and quaternary structure (e.g., the number of subunits required to form an active protein complex, or the aggregation state of the protein)). In many cases, this information may not be available without the use of laborious, time-consuming, and costly techniques (such as X-ray crystallography). Therefore, experimental techniques that allow for convenient, real-time, and relatively high-throughput characterization of protein structures are needed to establish biosimilarity between candidate biopharmaceuticals and reference drugs.

[0179] In some embodiments, the disclosed methods, apparatus, and systems can be used to provide structural comparison data for biopharmaceutical candidates (e.g., monoclonal antibodies (mAbs)) and reference biopharmaceuticals to establish biosimilarity. For example, in some cases, isoelectric point data and / or mass spectrometry data of the candidate and reference drugs can provide important evidence to support proof of biosimilarity. In some embodiments, isoelectric point data and / or mass spectrometry data of the candidate and reference drugs, which have been treated with site-specific proteases under the same reaction conditions, can provide important evidence to support proof of biosimilarity. In some embodiments, the disclosed methods, apparatus, and systems can be used to monitor the biopharmaceutical manufacturing process to ensure product quality and consistency by analyzing samples taken at different points in the production process or from different production runs. In some embodiments, the disclosed methods, apparatus, and systems can be used to evaluate the stability of formulation buffers. In some embodiments, the disclosed methods, apparatus, and systems can be used to evaluate the production and quality of biopharmaceutical candidates from clonal cell lines.

[0180] Example

[0181] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims presented herein.

[0182] Example 1 – Characterization of protein charge on the chip prior to performing mass spectrometry analysis

[0183] The fabrication of the microfluidic device shown in Figure 1 has been described above. In operation, the device is mounted on an instrument comprising a nitrogen source, heater, positive pressure pump (e.g., Parker, T5-1IC-03-1EEP), an electrophoresis power supply (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 automated sampler for loading samples onto the device. The power supply shares a common ground with the mass spectrometer. The instrument is controlled via software (e.g., LabVIEW).

[0184] Protein samples are premixed with an amphoteric electrolyte pH gradient and pI markers, then placed in vials and loaded into an autosampler. They are continuously loaded from the autosampler through inlet 412 onto a microfluidic device 400 via enrichment channel 418, and discharged from the device to waste 430 through outlet 434.

[0185] Sheath liquid / cathode liquid (50% MeOH, N4OH / H2O) was loaded onto two cathodic liquid traps 404 and 436, anodic liquid (10mM H3PO4) was loaded onto anodic liquid trap 426, and a heated nitrogen source was connected to two gas traps 408 and 440.

[0186] After loading all reagents, an electric field of +600 V / cm is applied from the anolyte trap 426 to the cathode electrolyte traps 404 and 436 by connecting electrodes to the anolyte trap 426 and the cathode electrolyte traps 404 and 436 to initiate isoelectric focusing. A UV light source is aligned below the enrichment channel 418, and a camera is placed above the enrichment channel 418 to measure the light passing through the enrichment channel 418, thereby detecting the focusing protein by means of their absorbance. A glass plate 402 made of soda-lime glass is used to block any stray light from the camera, thus suppressing light that does not pass through the enrichment channel 418 from reaching the camera, which increases the sensitivity of the measurement.

[0187] Images of the focused proteins can be captured continuously and / or periodically during IEF. When focusing is complete, a low pressure is applied from inlet 412, causing the pH gradient to move towards orifice 424. An electric field can be maintained at this time to sustain high-resolution IEF separation. Continued imaging of enrichment channel 418 during the ESI process can be used to determine the pI at which each protein exits orifice 424.

[0188] As the enriched protein fraction moves from enrichment channel 418 to confluence 420, it mixes with sheath fluid, which flows from cathodic liquid traps 404 and 436 through sheath / cathode liquid channels 406 and 438 to confluence 420. Mixing the enriched protein fraction with sheath fluid allows the protein fraction to be placed in a mass spectrometry-compatible solution and restores the charge of the focused protein (IEF drives the protein to an uncharged state), thereby improving ionization.

[0189] The enriched protein fraction then continues to orifice 424, which can be defined by the countersunk surface 422 of glass plate 402. Once trapped in the electric field between the bottom of the sheath trap and the negative electrode of the mass spectrometer, the enriched protein fraction can generate a Taylor cone.

[0190] As the solution continues to push the Taylor cone from enrichment channel 418, small droplets will exit from the Taylor cone and fly towards the mass spectrometer inlet. Nitrogen gas (e.g., at 150°C) can descend from gas traps 408, 440, along gas channels 410, 432, and form a nitrogen jet located on the side of the Taylor cone. This jet can transform the droplets emanating from the Taylor cone into finer droplets to form a fine mist before leaving the microfluidic device, which facilitates detection in the mass spectrometer. Adjusting the pressure from inlet 412 can adjust the Taylor cone size as needed to improve detection in the mass spectrometer.

[0191] Example 2 - Tracking speed of analyte peaks as they leave the microfluidic chip and enter the mass spectrometer

[0192] For this example, Figure 7A The microfluidic channel network 100 is fabricated within a 250-micrometer-thick opaque cyclic olefin polymer layer. Channel 112 has a depth of 250 micrometers, thus extending through the 250-micrometer layer. All other channels have a depth of 50 micrometers. Figure 7B As shown, a channel layer is sandwiched between two transparent cyclic olefin polymer layers to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, and 110 provide access to the channel network for introducing reagents from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel, thus enabling the filling of other reagents through the channel network to be "wasted". Acid (1% formic acid) is filled through port 108 into channels 109, 112, 114, and 103 and flows out through 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)) was loaded through port 106 into channels 107, 112, 114, and 103 and exited through port 102. This left channel 112 containing the sample analyte. The base (1% dimethylamine) was loaded through port 104 into channels 105, 114, and 103 and exited through port 102. The transfer agent (1% formic acid, 49% methanol) was loaded through port 110 into channels 111, 114, and 103 and exited through channel 103 to port 102.

[0193] Electrophoresis of the analyte sample in channel 112 was performed by applying a 4000V voltage to port 108 and grounding port 110. The amphoteric electrolytes in the analyte sample established a pH gradient across channel 112. Separation absorbance imaging was performed using a 280nm light source aligned with channel 112, and the transmittance of 280nm light through channel 112 was measured using a CCD camera. The software calculated absorbance by comparing the light transmittance during separation or movement with a “blank” reference measurement taken before analyte operation without focusing, and then displayed the absorbance of each pixel along the length of channel 112. The location where the standard or analyte was focused was displayed as a peak, such as... Figure 9A As indicated in –9F.

[0194] Once the analyte is focused, the final focused absorbance image is captured. The software identifies the spatial location of the pI markers and interpolates between the markers to calculate the pI of the focused analyte fraction peak. At this point, the control software triggers a relay to disconnect the ground at port 110 and connect port 104 to ground. Pressure is then set on the mobile agent reservoir connected to port 104 to establish a flow rate of 100 nL / min of the mobile agent solution entering channels 105 and 114 through port 104 and exiting the chip at orifice 116. Orifice 116 is positioned 2 mm from the ESI inlet of the mass spectrometer, with an inlet voltage of -3500V to -4500V.

[0195] As the pressure-driven flow guides the migrating agent from port 104 to orifice 116, some formic acid in the migrating agent will be electrophoresed to the anode at port 108 in the form of formate from channel 105 through channel 112. As the formate passes through channel 112, it disrupts the isoelectric pH gradient, causing the amphoteric electrolytes, standards, and analyte samples to gain charge and migrate electrophoretically from channel 112 to channel 114, where the pressure-driven flow from port 104 carries them into the ESI at orifice 116.

[0196] As the migration occurs, the software continues to capture absorbance images and identify peaks, tracking their migration from imaging channel 112 to channel 114. By tracking the time each peak takes to leave imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time it takes for the peak to pass through channel 114 and be introduced into the mass spectrometer through orifice 116, thus allowing a direct correlation between the originally focused peak and the resulting mass spectrum.

[0197] Figure 9A -F provides a series of examples of absorbance traces, spaced 1 minute apart, showing the movement of the isoelectric point (pI) standard determined from the images of the separated channels. Figure 9AThe graph shows the absorbance 910 as a function of channel distance 905 after isoelectric focusing of five pI standards (peaks 915, 920, 925, 930, 935) was completed, before movement. Figure 9B As shown, after 1 minute of movement, peak 915, corresponding to the pI=9.99 standard, is located at the edge of the imaging system's field of view. Figure 9C As shown, after 2 minutes of movement, peak 915 (pI = 9.99 standard) had exited the portion of the channel being imaged. Figure 9D As shown, after 3 minutes of movement, peak 920 (pI = 8.40 standard) had exited the portion of the channel being imaged. Figure 9E As shown, after 4 minutes of movement, peak 925 (pI = 7.00 standard) had exited the portion of the channel being imaged. Figure 9F As shown, after 5 minutes of movement, peak 930 (pI = 4.05 standard) had exited the portion of the channel being imaged.

[0198] Example 3 - Using feedback to adjust MS and ESI parameters

[0199] In Example 3, the chip, instruments, and software perform all the same processes as in Example 2. Furthermore, a second CCD camera is used to image the Taylor cone during ESI, such as... Figure 8 As shown in the image. These images were used to assess the quality and consistency of the Taylor cone. Evaluating the images and / or total counts on the mass spectrometer allows for the identification of ESI Taylor cone malfunctions and diagnosis of their causes.

[0200] The formation of the Taylor cone in ESI depends on the input flow rate into the cone that matches the fluid rate at which evaporation and ESI losses occur. 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.

[0201] Imaging the Taylor cone allows for the diagnosis of ESI malfunctions. For example, a loss in the Taylor cone indicates insufficient flow, and the software can increase the flow of the moving agent into the microfluidic device. Similarly, corona discharge indicates excessively high voltage, and the software can reduce the voltage. Expansion of the ESI cloud indicates excessively high voltage, while droplet formation instead of a Taylor cone indicates excessively low voltage. These differences, along with any other visual discrepancies, can be identified in the image, and the software can automatically compensate to reconstruct the Taylor cone.

[0202] Example 4 - Low-quality scan as a marker for separation

[0203] In Example 4, the chip, instrument, and software perform all the same procedures as in Example 2. Furthermore, once a shift occurs and the analyte peaks begin to migrate to the MS, the MS is set to alternate between m / z ranges of 1500–6000 and 150–1500. The 1500–6000 range is used to identify NIST antibody analyte fraction peaks as they are introduced into the MS. The 150–1500 m / z range scan is used to identify free-solution phatidylcholine (Pharmalytes) introduced into the MS. Pharmalytes can be identified in mass scans and used to calibrate the total ion chromatograph from the MS, as the presence of a specific phatidylcholine defines a portion of the isoelectric pH gradient analyzed in the MS at any given time point.

[0204] Example 5 – Changing high and low voltages to maintain electric field strength and constant voltage at the tip

[0205] For this example, Figure 7A The microfluidic channel network 100 is fabricated within a 250-micrometer-thick opaque cyclic olefin polymer layer. Channel 112 has a depth of 250 micrometers, thus extending through the 250-micrometer layer. All other channels have a depth of 50 micrometers. Figure 7B As shown, a channel layer is sandwiched between two transparent cyclic olefin polymer layers to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, and 110 provide access to the channel network for introducing reagents from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel, thus enabling the filling of other reagents through the channel network to be "wasted". Acid (1% formic acid) is filled through port 108 into channels 109, 112, 114, and 103 and flows out of 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)) was loaded through port 106 into channels 107, 112, and 114 and exited through port 102. This left channel 112 containing the sample analyte. Base (1% dimethylamine) was loaded through port 104 into channels 105, 114, and 103 and exited through port 102. Mobilizer (1% formic acid, 49% methanol) was loaded through port 110 into channels 110, 114, and 103 and then exited through channel 102 back to port 102. Pressure is applied to the alkali reservoir to generate a flow rate of 100 nL / min through port 104 into channels 105 and 114 and out through orifice 116.

[0206] Isoelectric focusing of the analyte sample in channel 112 is initiated by applying 2000V to port 108 using power supply 1005 and connecting port 110 to high-voltage power supply 1010 and applying -2000V. This establishes... Figure 10A The circuit shown includes high-voltage power supplies 1005 and 1010 (in some cases, power supplies 1005 and 1010 may include two channels of a single multiplexed high-voltage power supply) to generate a 4000V voltage drop between the anode and cathode. The resistance of the channel depends on the channel dimension and the conductivity of the reagent. In this example, with channel 109 (see...) Figure 7A The resistance R109 of the corresponding acid channel is 10 megohms, which is the same as that of channel 112 (see [link]). Figure 7A The resistance R112 of the corresponding sample channel starts at 40 megohms and is the same as that of channel 111 (see [link]). Figure 7A The resistance R111 of the alkali electrode in the corresponding channel is 50 megohms. Orifice 116 (see...) Figure 7A The resistance R113 at the electrospray ionization (ESI) interface between the mass spectrometer 1015 and the mass spectrometer 1015 is 2 gigahertz. Channels 109, 112, and 111 (see...) Figure 7A The total voltage drop is 4000V, and since these channels represent three series resistors, the voltage at the tips (V) is... 116 Calculate according to Equation 1:

[0207] V 116 =△V 108-110 *(R 111 ) / (R 109 +R 112 +R 111 (Voltage setting of high voltage power supply 1010) At the start of isoelectric focusing, V 116 =0 volts. Orifice 116 (see...) Figure 7A Position it 2 mm from the ESI inlet of the mass spectrometer, with an inlet voltage of -3500V to -4500V to form a Taylor cone. Figure 10B It shows Figure 10A Another embodiment of the circuit shown in the figure includes a resistor R105 for channel 105.

[0208] The amphoteric electrolytes in the analyte sample established a pH gradient across channel 112. Separation absorbance imaging was performed using a 280 nm light source aligned with channel 112, and the transmittance of 280 nm light through channel 112 was measured using a CCD camera. The software calculated absorbance by comparing the light transmittance during separation or movement with a “blank” reference measurement taken before analyte operation without focusing, and then displayed the absorbance for each pixel along the length of channel 112. The location where the standard or analyte was focused was displayed as a peak, such as... Figures 9A-9F As shown in the image.

[0209] As the sample focuses, the resistance of sample channel 112 increases as the amphoteric electrolyte, antibody isomers, and standards reach their isoelectric points and lose charge, while the resistance of channels 109 and 111, as well as the ESI interface, remains constant. A computer-implemented method monitors the current at power supply 1005 and can calculate the resistance in channel 112 at any given time. This information is used to adjust power supplies 1005 and 1010. For example, when the resistance in channel 112 rises to 140 megohms, without adjusting the power supplies, the voltage at orifice 116 would be -1000V, which would damage the Taylor cone. However, by adjusting power supply 1005 to +3000V and power supply 1010 to -1000V, the tip will remain at 0V, and the total voltage drop across channels 109, 112, and 111 will remain at 4000V. These adjustments are made instantaneously as the resistance in channel 112 changes.

[0210] Once the analyte is focused, the final focused absorbance image is captured. The software identifies the spatial location of the pI markers and interpolates between the markers to calculate the pI of the focused analyte fraction peak. At this point, the control software triggers a relay, disconnecting power supply 1010 at port 110 and connecting port 104 to power supply 1010. Pressure is then set on the mobile agent reservoir connected to port 104 to establish a flow rate of 100 nL / min for the mobile agent solution entering channels 105 and 114 through port 104 and exiting the chip at orifice 116 (see [link to documentation]). Figure 7A Chip diagram and Figure 10B (Circuit shown). The aperture 116 is positioned 2 mm from the ESI inlet of the mass spectrometer, and the inlet voltage is -3500V to -4500V.

[0211] As the pressure-driven flow guides the mobile agent from port 104 to orifice 116, some formic acid in the mobile agent reagent will be electrophoresed to the anode at port 108 in the form of formate from channel 105 through channel 112. As the formate passes through channel 112, it disrupts the isoelectric pH gradient, causing the amphoteric electrolytes, standards, and analyte samples to gain charge and migrate electrophoretically from channel 112 to channel 114. The pressure-driven flow from port 104 carries them into the ESI ejector orifice 116.

[0212] When movement occurs, the resistance of channel 112 will decrease. Figure 11A -B shows an example of the voltage and current data for channel 112, which can be used to derive the channel's resistance. Figure 11A The curve showing the change of voltage over time is shown. Figure 11BThe curve showing the current changing over time is displayed. The software monitors the changes in current and adjusts the power supply to maintain a voltage drop of 3000V between the anode and cathode and 0V at the peak 116, as shown. Figure 15 As described above, voltage changes can be instantaneous or stable.

[0213] As movement occurs, the software continues to capture absorbance images and identify peaks, tracking their migration from imaging channel 112 to channel 114. By tracking the time each peak takes to leave imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time it takes for a peak to pass through channel 114 and be introduced into the mass spectrometer through aperture 116, thus allowing a direct correlation between the originally focused peak and the resulting mass spectrum.

[0214] Figure 13A Provided Figure 7A The diagram shows a representative circuit diagram of the microfluidic device during chemical migration, where the ESI tip will be held at a positive voltage using an additional resistor R120 to draw current to ground. The circuit may include a high-voltage power supply 1305, which may be substantially similar to 1005, and a high-voltage power supply 1310, which may be substantially similar to 1010, to generate a specified voltage drop (e.g., 4000V) between the anode and cathode. The circuit may also include a third high-voltage power supply 1307. The resistance of the channel depends on the channel dimension and the conductivity of the reagent. Channel 109 (see [link to circuit diagram]) is also integrated into the circuit. Figure 7A The resistance of the corresponding acid channel R109, and the resistance of channel 112 (see...) Figure 7A The resistance of the sample channel R112 corresponding to ) and the resistance of channel 111 (see Figure 7A The resistance corresponding to the alkali channel R111, and the resistance of orifice 116 (see...) Figure 7A The circuit includes a resistor at the electrospray ionization (ESI) interface between channel 105 and a voltage source that can be substantially similar to the voltage source of mass spectrometer 1315 (see 1015). The circuit may also include a resistor R105 for channel 105. A power supply 1307 can be connected to channel 111 (see 1315). Figure 7A It uses a current control set to 0μA during movement. This power supply can read the voltage at the tip and use it to implement a computer-controlled feedback loop to maintain a constant voltage at the tip.

[0215] Figure 13B It shows Figure 7A The diagram shows a representative circuit of a microfluidic device during chemical movement, where the ESI tip will be held at a positive voltage using resistor R120 to draw current to power supply 1320. Figure 13C It shows Figure 7AThe circuit diagram shown represents a microfluidic device during chemical migration, where the ESI tip is held at a positive voltage using a field-effect transistor (FET) 1325 to absorb current. The circuit may additionally include an amplifier 1330, a voltage reference 1335, and an additional resistor R200. Figure 13D It shows Figure 7A The circuit diagram shown represents a microfluidic device during chemical migration, where the ESI tip will be held at a positive voltage using a bipolar junction transistor (BJT) 1340 to absorb current. Power supply 1307 can be connected to channel 111 (see [link to circuit diagram]). Figure 7A It uses a current control set to 0μA. This power supply can read the voltage at the tip and be used to implement a computer-controlled feedback loop to maintain a constant voltage at the tip. Figure 13E Provided Figure 7A The diagram shows a representative circuit of the microfluidic device during the chemical movement of a separated analyte mixture, where the ESI tip will remain grounded or close to ground. Power supply 1307 can be connected to channel 111 (see [link to circuit diagram]). Figure 7A It uses a current control set to 0μA. This power supply can read the voltage at the tip and be used to implement a computer-controlled feedback loop to maintain a constant voltage at the tip.

[0216] Example 6 – Adjusting high and low voltages based on measured tip voltage to maintain electric field strength and constant voltage at the tip.

[0217] For this example, Figure 7A The microfluidic channel network 100 is fabricated within a 250-micrometer-thick opaque cyclic olefin polymer layer. Channel 112 has a depth of 250 micrometers, thus extending through the 250-micrometer layer. All other channels have a depth of 50 micrometers. Figure 7BAs shown, a channel layer is sandwiched between two transparent cyclic olefin polymer layers to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, and 110 provide access to the channel network for introducing reagents from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel, thus enabling the filling of other reagents through the channel network to be "wasted". Acid (1% formic acid) is filled through port 108 into channels 109, 112, 114, and 103 and flows out through 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)) was loaded through port 106 into channels 107, 112, and 114, and exited through port 102. This left channel 112 containing the sample analyte. The base (1% dimethylamine) was loaded through port 104 into channels 105, 114, and 103, and exited through port 102. The moving agent (1% formic acid, 49% methanol) was loaded through port 110 into channels 110, 114, and 103, and exited through channel 102 back to port 102 (see [link to product description]). Figure 7A Chip schematic diagram and Figure 13E (The circuit shown).

[0218] Electrophoresis of the analyte sample in channel 112 is initiated by applying 1500V to port 108 using power supply 1305 and connecting port 110 to power supply 1307 (set to 0V). After 5 minutes, power supply 1305 is increased to 3000V and held for 3 minutes to complete focusing.

[0219] The amphoteric electrolytes in the analyte sample established a pH gradient across channel 112. Separation absorbance imaging was performed using a 280 nm light source aligned with channel 112, and the transmittance of 280 nm light through channel 112 was measured using a CCD camera. The software calculated absorbance by comparing the light transmittance during separation or movement with a "blank" reference measurement taken before analyte operation without focusing, and then displayed the absorbance for each pixel along the length of channel 112. The location where the standard or analyte was focused was displayed as a peak, such as... Figure 9A As shown in –9F.

[0220] Once the analyte is focused, the final focused absorbance image is captured. The software identifies the spatial location of the pI markers and interpolates between the markers to calculate the pI of the focused analyte fraction peak. At this point, the control software triggers a relay to connect port 104 to power supply 1310 and sets pressure on the mobile agent reservoir connected to port 104 to establish a flow rate of 100 nL / min of the mobile agent solution entering channels 105 and 114 through port 104 and exiting the chip at orifice 116. Orifice 116 is positioned 2 mm from the mass spectrometer ESI inlet 1315, with an inlet voltage of -3500V to -4500V. Power supply 1307 is set to 0 μA using current control, power supply 1305 is set to 3000V, and power supply 1310 is set to 0V, with the MS ESI ion source set between -3500V and -4500V.

[0221] As the pressure-driven flow guides the migrating agent from port 104 to orifice 116, some formic acid in the migrating agent will be electrophoretically transferred from channel 105 through channel 112 to the anode at port 108 in the form of formate. As the formate passes through channel 112, it disrupts the isoelectric pH gradient, causing the amphoteric electrolytes, standards, and analyte samples to gain charge and migrate electrophoretically from channel 112 to channel 114, where the pressure-driven flow from port 104 carries them into the ESI at orifice 116.

[0222] When movement occurs, the resistance of channel 112 will decrease. The power supply 1307, set to 0μA, will be equal to... Figure 13E The voltage at V116 is zero because the voltage drop across channel 111 is now 0 (ΔV = IR = 0 * R111 = 0V). Figure 11A As shown in -B, 8 minutes (480 seconds) after focusing is complete, the software monitors changes in current and adjusts the power supply to maintain a constant voltage drop of 3000V between the anode and cathode and 0V at the tip 116, as... Figure 15 As described in [the text]. The voltage (V116) at the tip is described by Equation 2:

[0223] V 116 =△V 108-110 *(R 111 ) / (R 109 +R 112 +R 105 (Power supply 1310 voltage setting)

[0224] As the migration occurs, the software continues to capture absorbance images and identify peaks, tracking their migration from imaging channel 112 to channel 114. By tracking the time each peak takes to leave imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time it takes for the peak to pass through channel 114 and be introduced into the mass spectrometer through orifice 116, thus allowing a direct correlation between the originally focused peak and the resulting mass spectrum.

[0225] Example 7 - Based on measuring the tip voltage and changing the high and low voltages with a resistor, to maintain the electric field strength and constant voltage at the tip.

[0226] For this example, Figure 7A The microfluidic channel network 100 is fabricated within a 250-micrometer-thick opaque cyclic olefin polymer layer. Channel 112 has a depth of 250 micrometers, thus extending through the 250-micrometer layer. All other channels have a depth of 50 micrometers. Figure 7B As shown, a channel layer is sandwiched between two transparent cyclic olefin polymer layers to fabricate a planar microfluidic device. Ports 102, 104, 106, 108, and 110 provide access to the channel network for introducing reagents from external reservoirs and electrical contacts. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel, thus enabling the filling of other reagents through the channel network to be "wasted". Acid (1% formic acid) is filled through port 108 into channels 109, 112, 114, and 103 and flows out of port 102. The sample (4% Pharmalyte 3-10, 12.5mM pI standard 5.52 (purified peptide, sequence: Trp-Glu-His), 12.5mM pI standard 8.4 (purified peptide, sequence: Trp-Tyr-Lys), infliximab biosimilar monoclonal antibody standard (part number MCA6090, Bio-Rad)) is loaded through port 106 into channels 107, 112, and 114, and exits through port 102. This leaves channel 112 containing the sample analyte. The base (1% dimethylamine) is loaded through port 104 into inlet channels 105, 114, and 103, and exits through port 102. The moving agent (1% formic acid, 49% methanol) is loaded through port 110 into channels 110, 114, and 103, and exits through channel 102 to port 102 (see [link to product description]). Figure 7A Chip schematic diagram and Figure 13B (The circuit shown).

[0227] Electrophoresis of the analyte sample in channel 112 is initiated by applying 1500V to port 108 using power supply 1305 and connecting port 110 to power supply 1307 (set to 0V). After 5 minutes, power supply 1305 is increased to 3000V.

[0228] The amphoteric electrolytes in the analyte sample established a pH gradient across channel 112. Separation absorbance imaging was performed using a 280 nm light source aligned with channel 112, and the transmittance of 280 nm light through channel 112 was measured using a CCD camera. The software calculated absorbance by comparing the light transmittance during separation or movement with a "blank" reference measurement taken before analyte operation without focusing, and then displayed the absorbance for each pixel along the length of channel 112. The location where the standard or analyte was focused was displayed as a peak, such as... Figure 9A As shown in –9F.

[0229] Once the analyte is focused, the charge variants of infliximab are separated, such as... Figure 16 As shown in panel A, the final focused absorbance image is captured. The software identifies the spatial location of the pI markers and interpolates between the markers to calculate the pI of the focused analyte fraction peak. At this time, the control software triggers a relay to connect port 104 to power supply 1310 and sets pressure on the mobile agent reservoir connected to port 104 to establish a flow of 100 nL / min of mobile agent solution entering channels 105 and 114 through port 104 and exiting the chip at orifice 116. Orifice 116 is positioned 2 mm from the mass spectrometer ESI inlet 1315. Power supply 1307 is set to 0 μA using current control, power supply 1305 is set to 7000 V, power supply 1310 is set to 4000 V, and the MS ESI ion source 1315 remains grounded. An additional resistor R120 is connected to the system between power supply 1310 and channel 105 (R current absorber), and the other side of resistor R120 is connected to power supply 1320, as shown. Figure 13B As shown in the diagram. Power supply 1320 will be set at least 4000V lower than power supply 1310 to act as a current sink. Resistor R120 can be replaced as shown in the diagram. Figure 13A Grounding the circuit as described in the example can be done as follows: Figure 13C The field-effect transistor (FET) shown can be as follows: Figure 13D The bipolar junction transistor (BJT) shown can be any other resistive element that can draw current from power supply 1310 to create a properly functioning electrophoresis circuit.

[0230] As the pressure-driven flow guides the migrating agent from port 104 to orifice 116, some formic acid in the migrating agent will be electrophoresed to the anode at port 108 in the form of formate from channel 105 through channel 112. As the formate passes through channel 112, it disrupts the isoelectric pH gradient, causing the amphoteric electrolytes, standards, and analyte samples to gain charge and migrate electrophoretically from channel 112 to channel 114, where the pressure-driven flow from port 104 carries them into the ESI at orifice 116.

[0231] When the movement occurs, the resistance of channel 112 will decrease. The power supply 1307, set to 0 μA, will be equal to the voltage at V116 because the voltage drop across channel 111 is now 0 (ΔV = IR = 0 * R111 = 0V). Figure 11A and 11B The data shows that the software monitors changes in current and adjusts the power supply to maintain a constant voltage drop of 3000V between the anode and cathode, and 3000V at tip 116, as shown. Figure 12 As described in [the text]. The voltage (V116) at the tip is described by Equation 2:

[0232] V 116 =△V 108-110 *(R 111 ) / (R 109 +R 112 +R 105 (Power supply 1310 voltage setting)

[0233] As the migration occurs, the software continues to capture absorbance images and identify peaks, tracking their migration from imaging channel 112 to channel 114. By tracking the time each peak takes to leave imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time it takes for the peak to pass through channel 114 and be introduced into the mass spectrometer through orifice 116, thus allowing a direct correlation between the originally focused peak and the resulting mass spectrum. For example, Figure 16 Panel B shows the mass of glycoforms electrosprayed into the mass spectrometer, which contain... Figure 16 The acidic peaks are shown in the electrophoresis pattern on panel A. Figure 16 Panel C shows from Figure 16 Quality of glycoform in the infliximab main peak of panel A. Figure 16 Panel D and Figure 16 Panel E shows from Figure 16 The mass of the alkaline peak in the electrophoresis diagram shown in panel A.

[0234] Example 8 – Changing high and low voltages in a 2-step capillary IEF to maintain electric field strength and constant voltage

[0235] In Example 8, the two-step IEF (isoelectric focusing followed by movement) is performed in a 60cm capillary and moved to the ESI-MS via a connector atomizer, as shown. Figure 14AAs outlined in the diagram, the separating capillary 1808 is immersed in the anolyte bottle 1806. A high-voltage power supply 1802 is connected to the anolyte bottle 1806 via electrode 1804. The other end of the capillary 1808 is connected to the connector sprayer 1814 via a tee 1812. The capillary 1808 is inserted into the connector sprayer 1814, so that the capillary outlet is very close to the ESI tip 1824. The third arm of the tee 1812 is connected to the moving agent capillary 1816, which is immersed in a pressurized moving agent bottle 1818. The pressurized moving agent bottle 1818 is also grounded via electrode 1817, thus acting as a current absorber. Furthermore, the connector sprayer 1814 is connected to the power supply 1810 via wire 1820, which is connected to the outside of the sprayer 1814. In this example, the mass spectrometer ion source remains grounded.

[0236] The reagents were prepared as follows: Anodic solution bottle 1806 contained 1% formic acid aqueous solution; separating capillary 1808 contained aqueous samples (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); connector spray chamber 1826 and moving agent capillary 1816 contained 1% diethylamine aqueous solution; and pressurized moving agent bottle 1818 contained 1% formic acid, 50% acetonitrile, and 49% water.

[0237] In this example, the mass spectrometer ion source remains grounded. For initiating focusing, power supply 1802 is set to +30 kV and power supply 1810 is set to 4 kV. A pressure-driven flow from the mobile agent vial 1818 is initiated at a rate of 100 nL / min. In this manner, ESI is initiated using diethylamine in the connector atomizer chamber 1826, and diethylamine also serves as the catholyte for the isoelectric focusing step.

[0238] As focusing proceeds within capillary 1808, the sample loses its charge-carrying capacity, and the resistance within capillary 1808 increases. This is because the ESI tip is electrically positioned between the diethylamine in capillary 1808 and chamber 1826 (see [link to ESI tip]). Figure 14B Therefore, the ESI tip voltage (V) 1824 The value will decrease according to Equation 3:

[0239] V 1824 =△V 1806-1814 *R 1826 / (R 1808 +R 1826 )+V 1814

[0240] Furthermore, as the resistance in capillary 1808 increases, the current through the capillary will decrease, which can be measured at power supply 1802. According to Equation 4, the increased current will be directly related to the change in resistance in capillary 1808:

[0241] I 1806 =△V 1806-1814 / (R 1808 +R 1826 )

[0242] Use such as Figure 12 The computer-controlled feedback loop described herein allows the system to calculate changes in the resistance in capillary 1808 (and thus the change in the voltage drop across capillary 1808 that defines the voltage at ESI tip 1824), and the system can adjust power supplies 1802 and 1810 to maintain a ΔV of 26 kV and sustain an ESI tip voltage of 4000 kV.

[0243] After focusing is complete (approximately 30 minutes), the moving agent solution in pressurized moving agent bottle 1818 replaces the diethylamine in connector atomizer chamber 1826, thereby initiating the movement of the NIST mAb protein isotype in capillary 1808. In a similar but reverse manner to isoelectric focusing, as movement proceeds, the resistance in capillary 1808 decreases, thus affecting the voltage at ESI tip 1824. Again, a computer-controlled feedback loop uses Equations 3 and 4 to calculate the necessary changes to power supplies 1802 and 1810 to maintain a 26 kV electric field while keeping the voltage at ESI tip 1824 at 4 kV.

[0244] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in any combination in practice. The following claims are intended to define the scope of the invention, and the methods and structures within the scope of these claims and their equivalents are thereby covered.

Claims

1. A computer-implemented method, comprising: (a) Receive using a processor: (i) A first dataset comprising multiple intensity or absorbance measurements from isoelectric focusing separation performed in the separation channel, based on the length of the separation channel; as well as (ii) A second dataset, comprising total ion measurements from multiple mass spectrometers over time; (b) Using a processor, the second dataset is converted into a third dataset, which includes ion count measurements based on mass; as well as (c) Use the processor to cover the graphs of the first and third datasets; (d) Using a processor to map the first peak of intensity or absorbance of the first dataset to the set of peaks of the third dataset and / or using the second dataset to map the set of peaks of the first dataset to the set of peaks of the third dataset; (e) Using a processor, correlate the first peak with a set of peaks to determine the mass distribution and isoelectric point of at least one analyte species in the first peak, wherein the set of peaks corresponds to the mass distribution of one or more analyte species in the analyte peaks; and (f) Use the processor to determine the identity of one or more analyte species in the analyte peak.

2. The computer-implemented method of claim 1, wherein (c) further comprises using a processor to overlay a graph of the second dataset with a graph of the first dataset and a graph of the third dataset.

3. The computer-implemented method of claim 1 or 2, wherein (c) includes deconvolving the second dataset to generate the third dataset.

4. The computer-implemented method according to any one of claims 1 to 3, wherein the one or more analyte species include different protein isotypes.

5. The computer-implemented method of claim 4, wherein protein isotypes include different post-translational modifications of proteins.

6. The computer-implemented method of claim 5, wherein the post-translational modification is selected from the group consisting of: hydroxylation, methylation, esterification, acetylation, disulfide bond, threonylation, ubiquitination, glycosylation, glycation, amino acid addition or removal, amidation, deamidation, isomerization, oxidation, sialylation, and phosphorylation.

7. The computer-implemented method of claim 5, wherein the post-translational modification is selected from the group consisting of: hydroxylation, methylation, esterification, acetylation, disulfide bond, threonylation, ubiquitination, glycosylation, amino acid addition or removal, amidation, deamidation, isomerization, oxidation, fucosylation, sialylation, and phosphorylation.

8. The computer-implemented method of any one of claims 1-7, wherein the overlay plot illustrates a time series of (i) the plurality of intensity or absorbance measurements along the length of the separation channel and (ii) the ion count measurements based on mass.

9. The computer-implemented method of any one of claims 1-8, further comprising performing isoelectric focusing separation, movement, and electrospray ionization using a single integrated microfluidic device coupled to the mass spectrometer to obtain a first dataset and a second dataset.

10. The computer-implemented method of claim 9, wherein (b) and (c) are executed within 1 minute of ESI-MS or concurrently with ESI-MS.

11. The computer-implemented method according to any one of claims 1-10, wherein (b), (c), (d), (e) and / or (f) are executed automatically as part of a software package for acquiring or processing electrospray ionization-mass spectrometry (ESI-MS) data.

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