Software for coupling microfluidics systems to mass spectrometry

Abstract

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

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 678,265, filed May 31, 2018, and U.S. Provisional Application No. 62 / 684,090, filed June 12, 2018, both of which are incorporated herein by reference. Background Art

[0003] The present disclosure relates to the field of chemical analysis, and more particularly to the separation of analytes in a mixture and subsequent analysis thereof by mass spectrometry (MS). Separating analyte components from a more complex analyte mixture based on the intrinsic mass of the analyte, and providing a grade grouping rich in the mass state, is a key part of analytical chemistry. Simplifying complex mixtures in this way reduces the complexity of downstream analysis. However, when attempting to combine known enrichment methods and / or devices with analytical equipment and / or technology, complications may arise.

[0004] For example, a variety of methods have been used to combine protein sample preparation techniques with downstream detection systems such as mass spectrometers. One common method is to use liquid chromatography to prepare samples and collect fractions for mass spectrometry (LC-MS). This method has the disadvantage of requiring protein samples to be digested into peptide fragments, resulting in a large number of sample fractions that must be analyzed and complex data reconstruction after the run. Although some forms of liquid chromatography such as peptide mapping reverse phase chromatography can be coupled to a mass spectrometer, these known techniques are limited to using peptide fragments rather than intact proteins, which limits their practicality.

[0005] Another method for introducing a sample into a mass spectrometer is electrospray ionization (ESI). In ESI, a small droplet of sample and solution is emitted from a capillary or microfluidic device distal end including an electrospray feature (such as an emitter tip or orifice) by applying an electric field between the capillary tip or emitter tip and the mass spectrometer source plate. The droplets are stretched and expanded in this induced electric field to form a conical emission (i.e., a "Taylor cone"), which includes smaller and smaller droplets that evaporate and produce gas phase ions, which are then 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, the capillary is limited to a linear flow path that does not allow multi-step sample processing. ESI also depends on the voltage at the ESI tip to remain constant throughout the analysis, which can be a challenge in many assays, where the internal fluid resistance can change over time, thereby changing the voltage drop in different parts of the circuit, and thereby changing the voltage at the ESI tip.

[0006] Microfluidic devices have been used to perform other work. Microfluidic devices can be produced by various known techniques and provide fluid channels of defined size that can form a channel network designed to perform different fluid operations. These devices provide a higher level of control and complexity than capillaries, making them a better choice for sample preparation. However, like capillaries, these tools usually provide limited characterization of the separated analyte fractions before introduction into a mass spectrometer (if any). Moreover, systems with capillaries or microfluidic devices generally do not provide tools for calibrating the system during operation to reestablish the Taylor cone.

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

[0008] Disclosed herein is a method for maintaining an electrospray ionization (ESI) tip at a constant voltage relative to ground while performing a separation reaction, the method comprising: a) applying a first voltage to the proximal end of a separation channel, wherein the distal end of the separation channel is fluidically and electrically connected to the ESI tip; b) applying a second voltage to the proximal end of an auxiliary fluid channel, wherein the distal end of the auxiliary fluid channel is fluidically and electrically connected to the distal end of the separation channel; c) performing the separation reaction to separate a mixture of analytes, wherein the separation reaction is performed within the separation channel; and d) monitoring a change in resistance of the separation channel or a change in voltage at the ESI tip in a feedback loop, the feedback back adjusting the first voltage and the second voltage 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 comprises a microvial spray tip. In some embodiments, the separation channel is a fluid channel within a microfluidic device. In some embodiments, the separation reaction comprises an isoelectric focusing reaction. In some embodiments, the separation reaction comprises an electrophoretic separation reaction. In some embodiments, the first voltage is applied to the cathode and the second voltage is applied to the anode. In some embodiments, the voltage at the ESI tip is maintained at ground. In some embodiments, the voltage at the ESI tip is maintained at the second voltage. In some embodiments, the adjustment of the first voltage and the second voltage includes subtracting a transient voltage change measured at the ESI tip from the first voltage and the second voltage. In some embodiments, the voltage at the ESI tip is measured using a power supply that provides the first voltage or the 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 ±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.

[0009] Also disclosed herein is a method comprising: a) providing a sample comprising a mixture of two or more analytes; b) performing separation in a fluid channel containing the sample to resolve individual analyte peaks from the mixture of two or more analytes; c) calculating the velocity of the analyte peak as the contents of the fluid channel migrate toward the fluid channel outlet; and d) using the velocity of the analyte peak to determine the time when the analyte peak arrives at 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 isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, the velocity of the analyte peak is calculated based on the time interval required for the analyte peak to move from a first position to a second position. In some embodiments, the first position, the second position, and the time interval are determined based on a series of two or more images of the fluid channel. In some embodiments, the series of two or more images includes an ultraviolet absorbance image, a visible light absorbance image, or a fluorescence image. In some embodiments, the fluid channel outlet includes an electrospray interface docked with a mass spectrometer. In some embodiments, the time at which the analyte peak arrives at the fluid channel outlet is used to associate mass spectrometer data with the analyte peak. In some embodiments, the migration of the fluid channel contents includes the use of electroosmotic migration technology, chemical migration technology, fluid dynamic migration technology, or any combination thereof. In some embodiments, the 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 for samples of biological candidate drugs and reference drugs is used to determine biological similarity. In some embodiments, the speed of the analyte peak is used in a feedback loop to adjust the control parameters of the separation or migration 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.

[0010] The method disclosed herein comprises: a) providing a sample comprising a mixture of two or more analytes; b) performing separation in a fluid channel containing the sample to distinguish individual analyte peaks from the mixture of two or more analytes; and c) collecting mass spectrometer data of the two or more individual analyte peaks emitted from the fluid channel through an electrospray interface docked with a mass spectrometer; wherein the data collection mode for the mass spectrometer alternates between high-quality scanning and low-quality scanning. In some embodiments, the mass spectrometer switches between the high-quality scanning and low-quality scanning 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 isotachophoresis (CITP) or micellar electrokinetic chromatography (MEKC). In some embodiments, the high-quality scan captures mass spectrometry data of biomacromolecules. In some embodiments, the biomacromolecule comprises a protein, a protein-drug conjugate, a nucleic acid molecule, a reduced protein, a fusion protein, a protein complex or any combination thereof. In some embodiments, the m / z ratio of the high mass scan is in the range of 1500 to 6000. In some embodiments, the low mass scan captures mass spectral data of a solution phase ampholyte for isoelectric focusing separation. In some embodiments, the m / z ratio of the low mass scan is in the range of 150 to 1500. In some embodiments, the mass spectrum of one or more solution phase ampholytes is used to calibrate the isoelectric point (pI) of the biomacromolecule identified in the high mass scan.

[0011] The methods disclosed herein include: a) performing a separation in a fluid channel containing a sample, wherein the sample includes a mixture of two or more analytes, and wherein the separation resolves individual analyte peaks from the mixture of two or more analytes; b) causing the contents of the fluid channel to migrate toward a fluid channel outlet, wherein the fluid channel outlet includes an electrospray interface docked 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 peak during (a) and (b), and (ii) a Taylor cone present between the fluid channel outlet and the inlet of the mass spectrometer 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 when the analyte peak will arrive at the fluid channel outlet. In some embodiments, the time when the analyte peak arrives at the fluid channel outlet is used to associate mass spectrometer data with the analyte peak. In some embodiments, the data derived from the imaging of the Taylor cone is used in a feedback loop to adjust electrospray performance. In some embodiments, the feedback loop operates at a frequency of at least 0.1 Hz. In some embodiments, the fluid channel is the lumen of a capillary. In some embodiments, the fluid channel is part of a microfluidic device. In some embodiments, the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP) or micellar electrokinetic chromatography (MEKC). In some embodiments, the imaging includes ultraviolet absorbance imaging, visible light absorbance imaging or fluorescence imaging. In some embodiments, the migration of the contents of the fluid channel includes the use of electroosmotic migration technology, chemical migration technology, fluid dynamic migration technology or any combination thereof. In some embodiments, the 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.

[0012] Disclosed herein is a computer-implemented method for maintaining an electrospray ionization (ESI) tip at a constant voltage relative to ground while performing a separation reaction, the method comprising: a) receiving, using a processor, a first measurement of a voltage at the ESI tip, wherein the distal end of the separation channel is in fluidic and electrical communication with the ESI tip; b) receiving, using the processor, a second measurement of the voltage at the ESI tip; c) using the processor, comparing the second measurement with the first measurement, wherein if the second measurement is different 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 including the distal end that is in fluidic and electrical communication with the distal end of the separation channel to be adjusted so that the voltage at the ESI tip returns to the first measurement; and d) repeating steps (a) to (c) at a specified frequency. In some embodiments, the separation channel comprises a lumen of a capillary or a fluid channel within a microfluidic device. In some embodiments, the separation reaction comprises an isoelectric focusing reaction. In some embodiments, the separation reaction comprises an electrophoretic separation reaction. In some embodiments, the voltage at the ESI tip is maintained at ground. In some embodiments, the specified frequency is at least 1 Hz. In some embodiments, the voltage at the ESI tip is maintained within ±5% of a specified value.

[0013] Also disclosed herein is a computer-implemented method comprising: a) receiving image data using a processor, the image data comprising two or more images acquired using a detector, the detector being 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 processor to determine the position of an analyte peak in the separation channel in the two or more images; c) using the same or different processor to calculate the velocity of the analyte peak based on the position of the analyte peak in the two or more images, and the known time interval between the acquisition of the two or more images; and d) using the same or different processor to determine the time when the analyte peak will reach the exit of the separation channel. In some embodiments, the separation reaction performed in the separation channel comprises isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP), or micellar electrokinetic chromatography (MEKC). In some embodiments, the two or more images comprise ultraviolet absorbance images, visible light absorbance images, or fluorescence images. In some embodiments, the separation channel outlet is fluidically connected to or includes an electrospray interface docked with a mass spectrometer. In some embodiments, the time at which the analyte peak arrives at the separation channel outlet is used to associate mass spectrometer data with the analyte peak. In some embodiments, the analyte is separated from a 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 spectrometer data collected for samples of biological candidate drugs and reference drugs is used to determine biosimilarity. In some embodiments, the speed of the analyte peak is used in a feedback loop to adjust the control parameters of 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.

[0014] Incorporation by reference

[0015] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term in this document and a term in an incorporated reference, the term in this document controls. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention may be better understood by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the present invention are utilized, wherein:

[0017] Figure 1 A schematic diagram of an apparatus for isoelectric focusing (IEF) and electrospray ionization (ESI) of automatically loaded samples is provided according to one embodiment of the present disclosure.

[0018] Figure 2 An example flow chart of a computer-implemented method for calculating the isoelectric point of separated analyte bands is provided.

[0019] Figure 3 Another example flow chart of a computer-implemented method for determining the velocity of one or more separated analyte bands and calculating the exit time is provided.

[0020] Figure 4 Another example flow chart of a computer-implemented method for implementing imaging-based feedback and controlling one or more operating parameters of an ESI-MS analysis system is provided.

[0021] Figure 5 A schematic block diagram of the hardware components of one embodiment of the disclosed system is provided.

[0022] Figure 6 A schematic block diagram of the software components of one embodiment of the disclosed system is provided.

[0023] Figure 7A-7B A microfluidic device useful in some embodiments of the present invention is shown. Fig. 7A A schematic diagram of the fluidic channel network of an exemplary microfluidic device is provided. Figure 7B Computer-aided design (CAD) drawings of the assembled microfluidic devices are provided. Fig. 7A The fluid channel layer is shown sandwiched between two transparent layers to seal the fluid channel.

[0024] Figure 8 Images of the Taylor cone and electrospray ionization (ESI) plume during the separation sample migration process are provided.

[0025] Figure 9A-9F Non-limiting examples of sample migration data following separation of analytes in an analyte mixture using isoelectric focusing are provided. Fig. 9A The absorbance trace at t = 0 min (completion of isoelectric focusing) is shown. Fig. 9B The absorbance trace at t = 1 minute is shown. Fig. 9C The absorbance trace at t = 2 minutes is shown. Fig.9DThe absorbance trace at t = 3 minutes is shown. Fig.9E The absorbance trace at t = 4 minutes is shown. Fig.9F The absorbance trace at t = 5 minutes is shown.

[0026] Figure 10A-10B Representative circuit diagrams of microfluidic devices designed to perform isoelectric focusing to separate analytes and subsequently migrate the separated analyte mixtures are provided. Fig. 10A Provided that the ESI tip will remain grounded or nearly grounded during isoelectric focusing, Fig. 7A Representative circuit diagram of the microfluidic device shown. Fig. 10B shows that during the chemical migration of a separated analyte mixture, Fig. 7A A representative circuit diagram of a microfluidic device is shown in FIG. 1 . In this example, it is assumed that channel 114 (eg Fig. 7A The resistance of the resistor (shown) can be ignored.

[0027] Figure 11A-11B Representative data for migration while holding the ESI tip at 0 V are provided. Fig.11A A graph of voltage as a function of time is shown. Fig. 11B A graph of current as a function of time is shown.

[0028] Fig.12 An example flow diagram for a voltage feedback loop is provided, where the ESI tip is held at +3000V.

[0029] Figure 13A-13E Examples of representative circuit diagrams of microfluidic devices of the present disclosure are provided. Fig.13A Provides that during chemical migration, Fig. 7A Representative circuit diagram of a microfluidic device shown, where the ESI tip will be held at a positive voltage, with an additional resistor used to sink current to ground. Fig. 13B shows that during chemical migration, Fig. 7A Representative circuit diagram of a microfluidic device shown, where the ESI tip will be held at a positive voltage, using an additional resistor to sink current into a third power supply. Fig. 13C shows that during chemical migration, Fig. 7A A representative circuit diagram of a microfluidic device is shown, where the ESI tip is held at a positive voltage and a field effect transistor (FET) is used to sink current. Fig.13D shows that during chemical migration, Fig. 7A A representative circuit diagram of a microfluidic device is shown, where the ESI tip is held at a positive voltage and a bipolar junction transistor (BJT) is used to sink current. Fig.13E Provided during chemical migration of a separated analyte mixture, Fig. 7ARepresentative circuit diagram of a microfluidic device shown, where the ESI tip would be kept at or near ground.

[0030] Fig.14A A representative diagram of a capillary junction nebulizer is provided. Fig. 14B Shows Fig.14A Representative resistor circuit diagram for the capillary junction nebulizer diagram.

[0031] Fig.15 An exemplary flow chart of a computer-controlled voltage feedback loop is provided, in which the ESI tip is held at 0 V.

[0032] Fig.16 A- Fig.16 E provides an example of analyte separation data and the corresponding mass spectrometry data of the separated analyte species. Fig.16 A shows the electropherogram of the separated analyte mixture. Fig.16 B shows the mass spectrum of the acidic peak of the separated species. Fig.16 C shows that Fig.16 Mass spectrum of the main peak present in the electropherogram of A. Fig.16 D and Fig.16 E shows Fig.16 Mass spectrum of the two basic peaks of the electropherogram shown in A. DETAILED DESCRIPTION

[0033] Some embodiments described herein relate to innovative software and systems, which are used to analyze data from capillary-based and microfluidic-based separation systems integrated with mass spectrometry detection and guide their operation. In some embodiments, analytes are imaged during the separation process on a capillary or microfluidic device, and molecular weight or mass-to-charge ratio is measured in a mass spectrometer after separation. The disclosed methods, devices, systems and software provide more accurate characterization of the analyte peaks separated, and achieve improved correlation between chemical separation data and mass spectrometry (MS) data. Methods, devices, systems and software for improving the quality of electrospray ionization mass spectrometry (ESI-MS) data are also disclosed. The disclosed methods, devices, systems and software have potential applications in various fields, including but not limited to proteomics research, drug discovery and development and clinical diagnosis. For example, in some embodiments, the disclosed methods, devices, systems and software can be used to characterize biological and biosimilar drugs during development and / or manufacturing, which will be discussed in more detail below. Biologics and biosimilars are a class of drugs that include, for example, recombinant proteins, antibodies, live viral vaccines, proteins derived from human plasma, cell-based drugs, proteins from natural sources, antibody-drug conjugates, protein-drug conjugates, and other protein drugs.

[0034] A microfluidic device designed to perform any of a variety of chemical separation techniques and also including an electrospray ionization interface for performing downstream mass spectrometry analysis is described. 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 to be used with imaging techniques. Devices and methods for combining imaging microfluidic separation with mass spectrometry have been previously described in, for example, disclosed PCT patent application publication number WO 2017 / 095813 and U.S. patent application publication number US 2017 / 0176386, which are hereby incorporated by reference herein for all purposes. Among them, these applications describe systems for imaging separation in conjunction with MS analysis. This microfluidic system represents a major advance in the characterization of biological agents. However, in order for such a system to provide the greatest benefit, as disclosed herein, it would be beneficial to have innovative software and systems to assist the operation of these systems and the downstream integration of imaging and MS data.

[0035] Therefore, in a preferred embodiment, the disclosed microfluidic device can be used in combination with imaging techniques, so as to, for example, accurately determine the isoelectric point (pI) of one or more analytes that have been isoelectrically separated from the analyte mixture in the separation channel to form a series of enriched fractions (also referred to herein as "peaks" or "bands") containing substantially pure single analyte components. Imaging all or part of the separation channel allows the position of two or more pI standards (or pI markers) that have been injected with the sample to be separated to determine the local pH value by extrapolation, allowing a more accurate pI to be calculated for each separated analyte peak. In some embodiments, while performing separation, the analyte mixture in the separation channel is imaged, and optionally, one or more of the analytes being separated are subjected to isoelectric point determination, and iteratively updated while performing separation. In some embodiments, after the separation is completed, the isoelectric point determination based on imaging is performed on one or more analytes that have been isoelectrically focused. In some embodiments, after the separation is completed and before the separated analyte mixture has migrated toward the electrospray tip, the isoelectric point determination based on imaging is performed on one or more analytes that have been isoelectrically focused. In some embodiments, the imaging-based methods disclosed herein can be used with a capillary-based ESI-MS system rather than a microfluidic device-based ESI-MS system. In some embodiments, isoelectric point determination of one or more analyte peaks can be performed by a computer-implemented method.

[0036] In another preferred embodiment, the disclosed microfluidic device can be used in conjunction with an imaging technique to image the separated analyte peaks after the separated analyte mixture migrates, i.e., when the peaks move out of the separation channel and toward the electrospray tip. In some embodiments, the imaged migration step is the same step as the imaged separation step, such as when implementing a separation step comprising capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, or any other separation technique that separates the components of the analyte mixture by differential separation. In some embodiments, the imaged migration step is analyzed to associate the enriched fractions in the imaged separation with a mass spectrum. The imaging of the migrated analyte peaks can be used, for example, to determine their velocities based on the positions of one or more analyte peaks in a series of migration images, which can then be used to determine the time point at which the analyte peak will leave the separation channel or be emitted by the electrospray tip, thereby being used to associate mass spectrometer data with specific analyte peaks. In some cases, the speed of the analyte peak is calculated according to the time interval required for the analyte peak to move a specific displacement value (e.g., from a first position to a second position). In some embodiments, imaging of the migrated analyte peak can allow direct monitoring of the peak when they pass through a fluid channel and are emitted by an electrospray tip, so it can be used to directly associate mass spectrometer data with a specific analyte peak. In some embodiments, the imaging-based method disclosed herein can be used with a capillary-based ESI-MS system rather than with an ESI-MS system based on a microfluidic device. In some embodiments, the speed of one or more analyte peaks, its actual or predicted departure time from the separation channel and / or its electrospray emission time can be determined by a computer-implemented method.

[0037] In some embodiments, the migration of the separated analyte peaks can be initiated by changes in the electric field or flow parameters in the microfluidic device. In some embodiments, one or more electrodes connecting the power supply to the microfluidic device will be connected or disconnected to initiate migration by a computer-implemented method. In some embodiments, the Taylor cone formed at the electrospray tip can be imaged during the migration step. In some embodiments, computer-implemented image analysis can be used to identify stable electrospray operating conditions. In some embodiments, the image analysis can be performed by an operator. In some embodiments, the image analysis can be performed using automated image processing software. In some embodiments, one or more operating parameters known to affect the performance of the electrospray will be adjusted to restore stable electrospray operating conditions. Examples of adjustable operating parameters 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, a computer-implemented method can be used to adjust the electrospray parameters.

[0038] In some embodiments, more than one power supply may be used to generate the electrophoretic electric field. In some embodiments, two power supplies with positive polarity may be used. In some embodiments, one or more power supplies may have negative polarity. In some embodiments, the voltage setting on the power supply may be changed in unison to maintain the same voltage gradient in the separation channel of the electrophoretic separation. In some embodiments, the voltage setting on the power supply may be changed to maintain a constant voltage at the electrospray tip. In some embodiments, the multiple power supplies may be different channels in a single multi-channel power supply. 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 migration may be performed in the separation channel, and the resistance in the channel may decrease over time. In some embodiments, pressure-driven migration may be performed, and the resistance in the channel may change over time as new reagents are pushed into the channel. In some embodiments, the electrospray tip may remain 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 grounding. In some embodiments, the computer-implemented method can adjust the voltage to maintain a constant electric field strength in the separation channel (or a constant voltage drop between the anode and the cathode) and a constant voltage at the electrospray tip. In some embodiments, the voltage at the tip can be measured using a voltmeter. In some embodiments, the voltage at the tip can be measured using an electrode located at the tip or inside the tip. In some embodiments, the 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, the computer-implemented method will read the voltage value at the tip and adjust the voltage to maintain a constant electric field strength in the separation channel (or a constant voltage drop between the anode and the cathode) and a constant voltage at the tip. In some embodiments, the computer-implemented method will calculate the voltage at the ESI tip based on the current flowing through the separation electric field circuit. In some embodiments, the voltage drop across the separation channel will be adjusted so that a constant power or maximum power is applied in the separation channel, wherein the power applied in the separation channel is calculated as:

[0039] Power = Voltage across separation channel x Current in separation channel Wherein the current may be measured continuously or periodically during the separation process, and the current measurement may be used to adjust the voltage of the separation channel. This method of controlling power in a separation channel may be useful for managing temperature effects in the separation channel.

[0040] In some embodiments, the separation path will be a linear coated or uncoated capillary, tube or pipeline, wherein the inlet is inserted into a vial containing an acidic anolyte and a positive electrode or an alkaline cathode electrolyte and a negative electrode. In some embodiments, the outlet of the separation path will be inserted into a joint sprayer. In some embodiments, the joint sprayer accommodates a T-piece for an auxiliary tube, pipeline or capillary, which can introduce another conductive supplementary solution into the capillary outlet, thereby providing liquid-liquid electrical contact; and a liquid flow, which is used to support electrospray and transport the analytes that will flow out of the separation channel to the tip for introduction into a mass spectrometer by electrospray ionization. In some embodiments, the system can be configured with an anolyte and a positive electrode at the entrance of the separation path, and a cathode electrolyte can be installed at the junction or distal portion of the separation path just before focusing. After focusing is completed, a migration agent with a competitive anion can be introduced into the junction by fluid dynamics or electroosmotic forces. In some embodiments, the inlet of the separation path can be immersed in a vial having a catholyte and a negative electrode, and the junction or distal portion of the capillary can be filled with an anolyte just before focusing. After focusing is completed, a migration agent with a competing cation can be introduced into the junction by fluid dynamics or electroosmotic forces. In some embodiments, the separation channel will be a section of linear capillary, one end of which is inserted into an anolyte container connecting the capillary to the positive electrode, and the other end is inserted into a catholyte container connecting the capillary to the negative electrode for isoelectric focusing. In some embodiments, after focusing, the catholyte end of the capillary will be removed from the catholyte and inserted into a junction sprayer (e.g., a micro-bottle sprayer) near the mass spectrometer, such as Fig.14A As shown. In some embodiments, the junction sprayer can provide a certain amount of migration agent to charge the analyte in the ESI and migrate the focused analyte. In some embodiments, the junction sprayer can provide an electrical connection to complete the migration circuit. In some embodiments, the voltage of the anolyte and the junction sprayer will be adjusted so that the voltage (ΔV) or the change in the electric field between the anolyte and the junction sprayer remains constant, and the voltage at the ESI tip remains constant.

[0041] In some embodiments, the separation channel (e.g., a capillary) includes a micro-bottle, which can facilitate the transfer of the migrated effluent to the ESI. The micro-bottle can be a part of the capillary, or can be attached and / or fused to the separation channel. The micro-bottle can be a part of the ESI tip. In some cases, the micro-bottle can include or be a part of the joint sprayer. The micro-bottle can provide a fluid flow path (e.g., for a sheath fluid) in a part of the channel or at the ESI tip.

[0042] In some embodiments, a power supply can be connected to a resistor to produce a current sink. In some embodiments, the resistor can absorb current by grounding the electrophoresis circuit. In some embodiments, the resistor is a field effect transistor (FET) adjustable resistor. In some embodiments, the resistor can be a precision variable resistor, a relay resistor network, a resistor ladder, or any other resistor element capable of providing a path for absorbing current. In some embodiments, the current sink can be a FET, wherein the FET is controlled so that it provides a constant current flowing through the FET, or it can be controlled to be used as an open circuit or a short circuit when needed. In some embodiments, a bipolar junction transistor (BJT) can be used for the current sink function. In some embodiments, the resistor can absorb current by connecting the electrophoresis circuit to a current absorbing power supply. In some embodiments, the voltage setting of the current absorbing power supply will be adjusted as the resistance in the separation channel changes over time. In some embodiments, the voltage on the current absorbing power supply will be adjusted to maintain a constant current on the resistor. In some embodiments, a resistor or a group of resistors, a resistor circuit, etc. can be used as a current sink.

[0043] In some embodiments, the mass-to-charge (m / z) range scanned can be changed during the migration / ESI step. In some embodiments, the computer-implemented method can be used to switch between a high m / z range and a low m / z range. In some embodiments, a mass spectrum in an m / z range can be used to separate the internal standard of the analyte in different mass ranges. The mass spectrum can include data for free solution isoelectric gradient ampholytes, which can be used as a standard for isoelectric point (pI), or the mass spectrum can include standard data for electrophoretic mobility, which can be used as a standard for electrophoresis (e.g., capillary zone electrophoresis). In some cases, the mass spectrum can include data for any molecule that can be distinguished, such as by pI, charge-to-mass ratio, by the credibility (reputation) of gel, electrophoretic mobility, etc., in a separation step, which is in different mass ranges from the analyte of interest.

[0044] The systems of the present disclosure may include one or more of the following: (i) a capillary or microfluidic device designed to perform analyte separations (e.g., separations based on isoelectric focusing) that provides an electrospray interface for interfacing with a mass spectrometer, (ii) a mass spectrometer, (iii) an imaging device or system, (iv) a processor or computer, (v) software for coordinating the operation of the capillary or microfluidic device-based analyte separation with image acquisition, (vi) software for processing the image and determining the location of one or more pI standards or analyte peaks in the separation channel while the separation is in progress, after the separation is complete, or after the pI standards and analyte peaks migrate toward the electrospray tip; (vii) software for processing the image and determining the velocity, departure time, and / or electrospray emission time of one or more pI standards or analyte peaks, (viii) software for simultaneously or alternately performing the image acquisition and / or image acquisition of the pI standards or analyte peaks; (ix) software for processing the image of the Taylor cone and adjusting the position of the electrospray tip relative to the mass spectrometer inlet, the fluid flowing through the electrospray tip, the voltage between the electrospray tip and the mass spectrometer, or any combination thereof, to affect changes in mass spectrometer data quality; (x) software for controlling mass spectrometer data collection for each analyte peak emitted from the electrospray interface, wherein the data collection mode of the mass spectrometer alternates between high-quality scans and low-quality scans; (xi) software for reading the voltage at the electrospray tip and adjusting 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; or any combination thereof. In some embodiments, the system may include an integrated system in which the selection of these functional components is packaged as 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 a new application. In some embodiments, some of these functional system components, such as capillaries or microfluidic devices, are replaceable or disposable components.

[0045] It is to be understood that both the foregoing general summary and the following description are exemplary and explanatory only and are not restrictive of the methods and apparatus described herein.

[0046] 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 belongs.

[0047] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference to "or" herein is intended to include "and / or" unless otherwise stated. Similarly, the phrases "including" and "comprising" are not intended to be limiting.

[0048] As used herein, the term "about" a numerical value refers to the numerical value plus or minus 10% of the numerical value. "About" a range is used in the context of the range minus 10% of its minimum value and plus 10% of its maximum value.

[0049] Analytes: As described above, the disclosed methods, devices, systems, and software can more accurately characterize 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, 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 a biological protein drug and / or a lysate collected from cells isolated from culture or in vivo.

[0050] Sample: The disclosed methods, apparatus, systems, and software can be used to separate and characterize 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, tissue fluid, urine, sweat, tears, protein samples from industrial enzyme or biopharmaceutical production processes, environmental samples (e.g., air samples, water samples, soil samples, surface wipe samples), etc. In some embodiments, the sample can be processed using any of a variety of techniques known to those skilled in the art prior to analysis using the disclosed methods and apparatus for integrated chemical separation and mass spectrometry characterization. For example, in some embodiments, the sample can be processed to extract proteins or nucleic acids. The sample can be collected from any of a variety of sources or subjects, such as bacteria, viruses, plants, animals, or humans.

[0051] Sample volume: In some embodiments of the disclosed methods and devices, miniaturization achieved through the use of microfabrication techniques can enable processing of very small sample volumes. In some embodiments, the sample volume for analysis can be in the range of about 0.1 μl to about 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 for analysis can be at most 1ml, at most 750μl, at most 500μl, at most 250μl, at most 100μl, at most 75μl, at most 50μl, at most 25μl, at most 10μl, at most 7.5μl, at most 5μl, at most 2.5μl, at most 1μl or at most 0.1μl. Any of the lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure, for example, in some embodiments, the sample volume for analysis can range from about 5μl to about 500μl. Those skilled in the art will recognize that the sample volume for analysis can have any value within this range, for example, about 10μl.

[0052] Separation Techniques: The methods, devices, systems and software of the present disclosure can utilize any of a variety of analyte separation techniques known to those skilled in the art. For example, in some embodiments, the imaged separation can be an electrophoretic separation that can produce one or more separated analyte fractions from an analyte mixture, such as isoelectric focusing, capillary gel electrophoresis, capillary zone electrophoresis, isotachophoresis, capillary electrokinetic chromatography, micellar electrokinetic chromatography, flow equilibrium capillary electrophoresis, electric field gradient focusing electrophoresis, dynamic field gradient focusing electrophoresis, etc.

[0053] Capillary isoelectric focusing (CIEF): In some embodiments, the separation technique may include isoelectric focusing (IEF), for example, capillary isoelectric focusing (CIEF). Isoelectric focusing (or "electric focusing") is a technique for separating molecules by differences in their isoelectric points (pI), i.e., the pH at which they have a net zero charge. CIEF involves adding an amphoteric electrolyte (amphoteric electrolyte) solution to a sample channel between reagent containers containing an anode or cathode to create a pH gradient within a separation channel (i.e., a fluid channel connecting a hole containing an electrode), across which a separation voltage is applied. The amphoteric electrolyte may be in solution phase or immobilized on the surface of the channel wall. 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 region below their isoelectric point (pI) will be positively charged and will therefore migrate toward the cathode (i.e., the negatively charged electrode). The overall net charge of a protein decreases as it migrates through an increasing pH gradient (e.g., due to protonation of carboxyl or other negatively charged functional groups) until it reaches the pH region corresponding to its pI, at which point it has no net charge and migration ceases. As a result, a mixture of proteins is separated according to their relative content of acidic and basic residues and focused into sharp, fixed bands, with each protein located at the point in the pH gradient corresponding to its pI. The technique has extremely high resolution, with proteins differing in that individual charges are fractionated into separate bands. In some embodiments, isoelectric focusing can be performed in a separation channel that has been permanently or dynamically coated with, for example, 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, dimethylacrylamide, polyvinylpyrrolidone (PVP), methylcellulose, hydroxyethylcellulose (HEC), hydroxypropylmethylcellulose (HPMC), triethylamine, n-propylamine (proylamine), morpholine, diethanolamine, triethanolamine, diaminopropane, ethylenediamine, chitosan, polyethyleneimine, cadaverine, putrescine, spermidine, diethylenetriamine, tetraethylenepentamine, cellulose, dextran, polyethylene oxide (PEO), cellulose acetate, pullulan, ethylpyrrolidone methacrylate, dimethyl methacrylate, didodecyldimethylammonium bromide, Brij 35, sulfobetaine, 1,2-dilauryloyl sn-phosphatidylcholine (1,2-dilauryloyl sn-phosphatidylcholine), 1,4-didecyl-1,4-diazabicyclo[2,2,2]octane dibromide, agarose, poly(N-hydroxyethylacrylamide), pole-323, hyperbranched polyaminoesters, pullalan, glycerol, adsorption coatings, covalent coatings, dynamic coatings, etc. In some embodiments, additives such as methylcellulose, glycerol, urea, formamide, surfactants (e.g., Triton-X 100, CHAPS, digitonin) can be used in the separation medium for isoelectric focusing (e.g., in an uncoated separation channel) to significantly reduce electroosmotic flow, allow better protein solubilization, and limit diffusion inside the fluid channel capillary by increasing the viscosity of the electrolyte.

[0054] As described above, the pH gradient used for capillary isoelectric focusing technology is generated by using amphoteric electrolytes (i.e., amphipathic molecules that contain both acidic and basic groups and exist mainly in the form of zwitterions within a certain pH range). The portion of the electrolyte solution on the anode side of the separation channel is called the "anolyte". The portion of the electrolyte solution on the cathode side of the separation channel is called the "catholyte". A variety of electrolytes can be used in the disclosed methods and devices, 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 in 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%, 90%. The concentration of the electrolyte can be up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%. A range of electrolyte concentrations can be used, for example, 0.1%-2%. The ampholytes may be selected from any commercial or non-commercial carrier ampholyte 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, Orsay, France), AESlytes (AES), FLUKA ampholytes (Thomas Scientific, Swedesboro, NJ), Biolyte (Bio-Rad, Hercules, CA), etc. The carrier ampholyte mixture may contain a mixture of small molecules (about 300-1,000 Da) containing multiple aliphatic amino and carboxylate groups, with closely spaced pI values ​​and good buffering capacity. In the presence of an applied electric field, the carrier ampholytes partition into a smooth linear or non-linear pH gradient that increases gradually from the anode to the cathode.

[0055] Any of the various pI standards can be used in the disclosed method and apparatus to calculate the isoelectric point of the analyte peak separated. For example, the pI markers commonly used in CIEF applications can be used, for example, protein pI markers and synthetic small molecule pI markers. In some cases, the protein pI marker can be a specific protein with a generally accepted pI value. In some cases, the pI marker can be detectable, for example, by imaging. Commercially available multiple protein pI markers or synthetic small molecule pI markers or combinations thereof can be used, for example, small molecule pI markers obtained from Advanced Electrophoresis Solutions, Ltd. (Cambridge, Ontario, Canada), Protein Simple, peptide libraries designed by Shimura, and Slais dyes (Alcor Biosepartions).

[0056] Migration Techniques: In some embodiments, such as where isoelectric focusing is employed, the separated analyte bands can be moved to one end of a separation channel that interfaces with a downstream analytical device (e.g., electrospray ionization interfaces 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 can be considered a migration step.

[0057] In some embodiments, the migration of the analyte band can be achieved by applying a hydrodynamic pressure to one end of the separation channel. In some embodiments, the migration of the analyte band can be achieved by orienting the separation channel in a vertical position so that gravity can be used to achieve the migration of the analyte band. In some embodiments, the migration of the analyte band can be achieved using EOF-assisted migration. In some embodiments, the migration of the analyte band can be achieved using chemical migration. In some embodiments, any combination of these migration techniques can be used.

[0058] In one embodiment, the migration step of the isoelectric focusing analyte band includes chemical migration. Compared with pressure-based migration, the advantage of chemical migration is to show minimum band broadening by overcoming the hydrodynamic parabolic flow curve caused by the use of pressure. Chemical migration can be achieved by introducing the inlet or outlet of the separation path containing a fully or partially focused pH gradient into a conductive solution, wherein ions compete with hydronium or hydroxyl groups for electrophoresis and enter the separation path. This causes the stepwise electrokinetic displacement of the pH gradient components by destroying the approximate zero net charge state. In the case of cathode chemical migration, the supply of hydroxyl groups can be replaced by a migration solution containing competitive anions, i.e., a cathode electrolyte solution. Competitive anions can cause the pH in the separation path to drop, generating positive charges on the pH gradient components, thereby causing them to migrate to the cathode. Accordingly, in anode migration, the anolyte solution is replaced by a migration solution containing competitive cations, which increases the pH value in the separation, causing the pH gradient components to generate negative charges, thereby causing them to migrate to the anode. In some embodiments, any suitable concentration of acidic electrolytes such as formic acid, acetic acid, carbonic acid, phosphoric acid, etc. can be used to trigger cathode migration. In some embodiments, anodic migration can be initiated using alkaline electrolytes such as ammonium hydroxide, dimethylamine, diethylamine, piperidine, sodium hydroxide, etc. In some embodiments, chemical migration can be initiated by adding salts such as sodium chloride or any other salt to the anolyte solution or the catholyte solution.

[0059] In a preferred embodiment, the chemical migration step can be started in a microfluidic device, which is designed to combine CIEF with ESI-MS by changing the electric field in the device to electrophoresing the migrated electrolyte into the separation channel. In some embodiments, the change of the electric field can be achieved by connecting or disconnecting one or more electrodes connected to one or more power supplies, wherein the one or more electrodes are located in the reagent wells on the device or integrated with the fluid channel of the device. In some embodiments, the connection or disconnection of one or more electrodes can be controlled using computer-implemented methods and programmable switches to coordinate the time and duration of the migration step with the separation step, the electrospray ionization step, and / or the mass spectrometry data collection. In some embodiments, the disconnection of one or more electrodes from the separation circuit can be achieved by using current control and setting the current to 0 μA.

[0060] Capillary Zone Electrophoresis (CZE): In some embodiments, the separation technique may include capillary zone electrophoresis, which is a method for separating charged analytes in solution under conditions of an applied electric field. The net velocity of the charged analyte molecules is governed by the electroosmotic flow (EOF) mobility μ displayed by the separation system. EOF and the electrophoretic mobility μ of individual analytes EPThe influence of the molecular size, shape and charge causes analyte molecules of different sizes, shapes or charges to exhibit different migration speeds and separate into spectral bands.

[0061] Capillary Gel Electrophoresis (CGE): In some embodiments, the separation technique may include capillary gel electrophoresis, a method for separation and analysis of macromolecules (e.g., DNA, RNA, and proteins) and their fragments based on molecular size and charge. The method involves the use of a gel-filled separation channel in which the gel acts as an opposing current and / or sieving medium during the electrophoretic movement of the charged analyte molecules in an applied electric field. The gel acts to inhibit thermal convection caused by the applied electric field and also acts as a sieving medium that blocks the passage of molecules, resulting in different migration rates for molecules of different sizes or charges.

[0062] Capillary isotachophoresis (CITP): In some embodiments, the separation technique may include capillary isotachophoresis, a method for separating charged analytes that uses a discontinuous system of two electrolytes (called a leading electrolyte and a trailing electrolyte) in a capillary or fluid channel of suitable size. The leading electrolyte may contain ions with the highest electrophoretic mobility, while the trailing electrolyte may contain ions with the lowest electrophoretic mobility. The analyte mixture to be separated (i.e., the sample) may be sandwiched between the two electrolytes, and the application of an electric field causes the charged analyte molecules within the capillary or fluid channel to be distributed into closely continuous regions in order of decreasing electrophoretic mobility. The regions move at a constant speed in the applied electric field, making it possible to record their passage along the separation channel using a detector such as a conductivity detector, a photodetector, or an imaging device. Unlike capillary zone electrophoresis, it is not feasible to simultaneously determine or detect anionic and cationic analytes in a single analysis performed using capillary isotachophoresis.

[0063] Capillary electrokinetic chromatography (CEC): In some embodiments, the separation technique may include capillary electrokinetic chromatography, a method for separating analyte mixtures based on a combination of liquid chromatography and electrophoretic separation. CEC offers both the efficiency of capillary electrophoresis (CE) and the selectivity and sample capacity of packed capillary high performance liquid chromatography (HPLC). Because the capillaries used in CEC are packed with HPLC packing materials, the wide range of analyte selectivities available in HPLC can also be used in CEC. The high surface area of ​​these packing materials enables CEC capillaries to accommodate relatively large amounts of sample, making the detection of subsequently eluted analytes simpler than their detection in capillary zone electrophoresis (CZE).

[0064] Micellar electrokinetic chromatography (MEKC): In some embodiments, the separation technique may include micellar electrokinetic chromatography, a method for separating analyte mixtures based on differential partitioning of surfactant micelles (pseudo-stationary phase) and a surrounding aqueous buffer solution (mobile phase). In MEKC, the concentration of surfactant contained in the buffer solution can be greater than the critical micelle concentration (CMC), so that the surfactant monomers are in equilibrium with the micelles. MEKC can be performed in an open capillary or fluid channel using alkaline conditions to generate a strong electroosmotic flow. A variety of surfactants can be used in MEKC applications, such as sodium dodecyl sulfate (SDS). For example, the anionic sulfate groups of SDS give the surfactant and micelles an electrophoretic mobility that is opposite to the direction of the strong electroosmotic flow. As a result, the migration of the surfactant monomers and micelles is slow, although their net movement is still in the direction of the electroosmotic flow, i.e., toward the cathode. During the MEKC separation process, the analyte can be distributed between the hydrophobic interior of the micelle and the hydrophilic buffer solution. Hydrophilic analytes that are insoluble in the interior of the micelle move at an electroosmotic flow rate u. o Migrate and will be retained in the buffer at the retention time t M The hydrophobic analyte completely dissolved in the micelle moves at the micelle velocity u c migration, and at the final elution time t c Elution.

[0065] Flow Balance Capillary Electrophoresis (FCCE): In some embodiments, the separation technique may include flow balance capillary electrophoresis, a method that uses pressure-induced countercurrent to actively retard, stop, or reverse the migration of analytes through a capillary to increase the efficiency and resolution of capillary electrophoresis. By retarding, stopping, or moving analytes back and forth in the detection window, the target analytes can be effectively confined to the separation channel for a much longer period of time than under normal separation conditions, thereby improving separation efficiency and resolution.

[0066] Separation time and separation resolution: Generally, the separation time required for complete separation will vary according to the specific separation technology and the operating parameters used (e.g., separation channel length, microfluidic device design, buffer composition, applied voltage, etc.). In some embodiments, as described in the same U.S. Patent Application No. 16 / 261,382, the software will determine when the separation is completed based on the imaging-based analysis of the analyte peak. In some embodiments, the separation time can be in the range of about 0.1 minute to about 30 minutes. In some embodiments, the separation time can be at least 0.1 minute, at least 0.5 minute, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes or at least 30 minutes. In some embodiments, the separation time is 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 the present disclosure, for example, in some embodiments, the separation time can be in the range of about 1 minute to about 20 minutes. The separation time can have any value within this range, for example, about 7 minutes.

[0067] Similarly, the separation efficiency and resolution achieved using the disclosed methods and devices may vary depending on the specific separation technique and the operating parameters used (e.g., separation channel length, microfluidic device design, buffer composition, applied voltage, etc.). In some embodiments, the separation efficiency achieved (e.g., the number of theoretical plates) may be in the range of about 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 efficiency can vary, depending on one or more properties of the analytes in the mixture (e.g., molecular weight, diffusivity, electrophoretic or isoelectric mobility, etc.).

[0068] Design and fabrication of microfluidic devices: In some embodiments of the disclosed methods, devices, and systems, separation of analytes from a mixture and, optionally, subsequent analysis thereof using ESI-MS can be performed using a microfluidic device designed to integrate one or more sample preparation steps (e.g., filtration, preconcentration or extraction steps, etc.) and / or separation steps (e.g., as described above) with an electrospray ionization step.

[0069] In some embodiments, the disclosed microfluidic device may include one or more sample or reagent ports (also referred to as inlets, sample wells, or reagent wells); one or more waste ports (also referred to as outlets); one or more fluid channels, the fluid channels being connected to each other or to an intermediate fluid channel (e.g., a separation channel) with the inlet port and the outlet port; 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 exhaust 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.

[0070] In a preferred embodiment, the disclosed microfluidic device is combined with 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-existing U.S. Patent Application Publication Nos. US2017 / 0176386 A1 and US2018 / 0003674 A1. Figure 1 A non-limiting example of a microfluidic device designed to perform isoelectric focusing followed by ESI-MS characterization is shown. Figure 1The fluid channel network shown in is made of a soda-lime glass plate using standard photolithography etching technology, which has a very low transmittance to 280nm light. 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 runs from the top to the bottom of the glass plate 402. The device 400 can be illuminated by a light source arranged on one side of the device 400 and imaged by a detector arranged on the opposite side of the device 400. Since the substrate 402 is opaque, but the enrichment channel 418 defines an optical gap, the substrate 402 can block the 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 fused silica plates that are transmissive to 280nm light (e.g., transparent to it). The top plate contains through holes for instruments and users to interface with the channel network, while the bottom plate is solid. The three plates are bonded at 520°C for 30 minutes. Inlet and outlet tubing were made from cut capillaries (100 μm inner diameter, Polymicro) and bonded to the channel network.The operation of this device for isoelectric focusing of proteins and subsequent mass spectrometric characterization is described in Example 1 below.

[0071] 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 the device. Examples of suitable fluid actuation mechanisms for disclosed methods, devices, and systems include, but are not limited to, applying positive pressure or negative pressure, gravity or centrifugal force, electromotive force, electrowetting force, or any combination thereof to one or more inlet ports or outlet ports. In some embodiments, positive pressure or negative pressure can be directly applied, such as by using a mechanical actuator or piston, which is coupled to the inlet port and / or outlet port to actuate sample or reagent to flow through the fluid channel. In some embodiments, a mechanical actuator or piston can apply force on a flexible film or diaphragm for sealing the inlet port and / or outlet port. In some embodiments, positive pressure or negative pressure can be indirectly applied, such as by using a pressurized gas line or a vacuum line connected to one or more inlet ports and / or outlet ports. In some embodiments, a pump connected to one or more inlet ports and / or outlet ports, such as a programmable syringe pump, HPLC pump, or a peristaltic pump can be used to drive fluid flow. In some embodiments, electromotive force and / or electrowetting force can be applied by using an electric field and the surface properties in the control device. The electric field can be applied by electrodes inserted into one or more inlet ports and / or outlet ports or by 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.

[0072] Typically, the inlet port, outlet port, fluid channel or other components of the disclosed microfluidic device, including the body of the device, can be made using any of a variety of materials, including but not limited to glass, fused silica, silicon, polycarbonate, polymethyl methacrylate, cyclic olefin copolymer (COC) or cyclic olefin polymer (COP), polydimethylsiloxane (PDMS) or other elastic materials. Suitable manufacturing techniques generally depend on the choice of material, and vice versa. Examples include but are not limited to CNC machining, photolithography and chemical etching, laser ablation, injection molding, hot embossing, die cutting, 3D printing, etc. In some embodiments, the microfluidic device may include a layered structure, wherein, for example, a fluid layer including a fluid channel is sandwiched between an upper layer and / or a lower layer to seal the channel. The upper layer and / or the lower layer may include an opening aligned with the fluid channel in the fluid layer to form an inlet port and / or an outlet port, etc. Two or more device layers may be sandwiched together to form a device that can be disassembled or permanently combined. Suitable bonding techniques generally depend on the selection of materials used to manufacture the layer. Examples include, but are not limited to, anodic bonding, thermal bonding, laser welding, or using a curable adhesive (eg, a heat-curable or light-curable adhesive).

[0073] In some embodiments, all or a portion of an inlet port, outlet port, or fluid channel within a microfluidic device may include a surface coating (e.g., a polyethylene glycol (PEG) coating) for altering the electroosmotic flow properties (e.g., an HPC or PVA coating) and / or the hydrophobic / hydrophilic properties of the inlet port, outlet port, or fluid channel wall.

[0074] The inlet port and / or outlet port of the disclosed device can be made into various shapes and sizes. Suitable inlet port and / or outlet port geometries include, but are not limited to, spherical, cylindrical, elliptical, cubic, conical, hemispherical, rectangular or polyhedral (e.g., a three-dimensional geometric shape composed of multiple planes, such as a rectangular cuboid, a hexagonal prism, an octagonal prism, an inverted triangular pyramid, an inverted quadrangular pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid or an inverted truncated pyramid) or any combination thereof.

[0075] Inlet port and / or outlet port size can be characterized by mean diameter and depth. As used herein, the mean diameter of an inlet port or an outlet port refers to the largest circle that can be inscribed in the plane cross section of the inlet port and / or outlet port geometry. In some embodiments of the present disclosure, the mean diameter of an inlet port and / or an outlet port can be in the range of about 0.1mm to about 10mm. In some embodiments, the mean diameter of an inlet port and / or an outlet port can be at least 0.5mm, at least 1mm, at least 2mm, at least 4mm, at least 8mm or at least 10mm. In some embodiments, the mean diameter can be at most 10mm, at most 8mm, at most 6mm, at most 4mm, at most 2mm, at most 1mm or at most 0.5mm. Any one of the lower limit and the upper limit described in this paragraph can be combined to form a scope included in the present disclosure, for example, in some embodiments, the mean diameter can be in the range of about 2mm to about 8mm. Those skilled in the art will recognize that the mean diameter of an inlet port and / or an outlet port has any value within the range, for example, about 5.5mm.

[0076] In some embodiments, the depth of the inlet port and / or outlet port (e.g., sample or reagent hole) can be in the range of about 5 μm to about 500 μm. In some embodiments, the depth 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 or at least 500 μm. In some embodiments, the depth can 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 can be combined to form a range included in the present disclosure, for example, in some embodiments, the depth of the inlet port and / or outlet port can be in the range of about 50 μm to about 200 μm. Those skilled in the art will recognize that the depth can have any value within this range, for example, about 130 μm. In some embodiments, the depth of the inlet port and / or outlet port (e.g., sample or reagent well) can be in the range of about 500 μm to about 50 mm. In some embodiments, the depth can 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 can 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 can be combined to form a range included in the present disclosure, for example, in some embodiments, the depth of the inlet port and / or outlet port can be in the range of about 50 μm to about 5 mm.

[0077] In some embodiments, the fluid channels of the disclosed devices may have any of a variety of cross-sectional geometries, such as square, rectangular, circular, and the like. Typically, the cross-sectional geometry of the fluid channels will depend on the manufacturing technology used to make 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 of a non-rectangular cross-section, where the average diameter is defined as the diameter of the largest circle that can be inscribed within the cross-sectional geometry of the fluid channel) can range from about 5 μm to about 500 μm. In some embodiments, the size of the fluid channel 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 at most 1000 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 50 μm, at most 25 μm, at most 10 μm or at most 5 μm. Any of the lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure, for example, in some embodiments, the size of the fluid channel can be in the range of about 75 μm to about 300 μm. Those skilled in the art will recognize that the size 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 port and / or outlet port of the device.

[0078] Imaging Techniques: In some embodiments of the disclosed methods and devices, imaging of the analyte separation step and / or migration step can be performed using optical detection techniques, such as ultraviolet (UV) light absorption, visible light absorption, fluorescence, Fourier transform infrared spectroscopy, Fourier transform near infrared spectroscopy, Raman spectroscopy, optical spectroscopy, etc. In some embodiments, all or part of the separation (or enrichment) channel, the junction or connecting channel connecting the end of the separation channel and the downstream analytical instrument or electrospray orifice or tip, the electrospray orifice or tip itself, or any combination thereof can be imaged. In some embodiments, the separation (or enrichment) channel can be the lumen of a capillary. In some embodiments, the separation (or enrichment) channel can be a fluid channel within a microfluidic device.

[0079] The wavelength range used to detect the separated analyte bands will generally depend on the choice of imaging technique and the materials used to manufacture the device or a portion thereof. For example, where ultraviolet light absorbance is used to image all or part of a separation channel or other portion of a microfluidic device, detection at about 220 nm (due to the intrinsic absorbance of peptide bonds) and / or at about 280 nm (due to the intrinsic absorbance of aromatic amino acid residues) may allow one to visualize protein bands during separation and / or migration, provided that at least a portion of the device, such as the separation channel, is transparent to these wavelengths of light. In some embodiments, the analyte to be separated and characterized by ESI-MS may be labeled with, for example, a fluorophore, a chemiluminescent tag, or other suitable marker prior to separation so that it may be imaged using fluorescence imaging or other suitable imaging techniques. In some embodiments, for example, where the analyte includes a protein produced by a commercial manufacturing process, the protein may be genetically engineered to incorporate a green fluorescent protein (GFP) domain or a variant thereof so that they may be imaged using fluorescence. In some embodiments, the protein may be tagged or labeled. The labeled protein can be configured so that the label does not interfere with or perturb the properties of the analyte upon which the chosen separation technique is based.

[0080] Any of a variety of imaging system components may be used to achieve the purposes of the disclosed methods, apparatuses, and systems. 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.), focusing lenses, objective lenses, reflectors, 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.), etc., or any combination thereof. Depending on the imaging mode used, the light source and image sensor may be positioned, for example, on opposite sides of the microfluidic device so that an absorbance-based image may be acquired. In some embodiments, the light source and image sensor may be positioned on the same side of the microfluidic device, for example, so that an epifluorescence image may be acquired.

[0081] Images can be acquired continuously during the separation, migration and / or electrospraying steps, or can be acquired randomly or at specified time intervals. In some embodiments, a series of one or more images are 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.

[0082] Imaging of pI markers for determining protein isoelectric points prior to electrospray: In some embodiments, as described above, the positions of two or more pI markers in an image of a separation channel comprising a separated analyte mixture that has been subjected to CIEF can be used to determine the isoelectric points of one or more individual analyte peaks (e.g., protein analyte peaks). In some embodiments, the isoelectric points of one or more analyte peaks are 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 points of one or more analyte peaks are calculated from the positions of three or more pI markers based on a nonlinear fit function (e.g., a nonlinear polynomial) that describes the relationship between local pH and position along the separation channel. In some embodiments, the isoelectric points of one or more analytes are calculated based on the positions of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more pI standards determined from an image of a separation channel.

[0083] In some embodiments, when separating the analyte mixture, an image for determining the position of two or more pI markers is obtained, and as the separation continues, the calculation of the pI of each analyte band is iteratively updated. In some embodiments, an image for determining the position of two or more pI markers is obtained after the separation is complete and before the migration step begins. In some embodiments, when the separated mixture migrates and is discharged through an electrospray tip or orifice, an image for determining the position of two or more pI markers is obtained. In some embodiments, when the separated mixture migrates and is discharged through a fluid channel that connects the separation channel to a downstream analytical instrument, an image for determining the position of two or more pI markers is obtained.

[0084] In some embodiments, a computer-implemented method (e.g., a software package) is used to obtain an image for determining the position of two or more pI markers and analyte bands in a separation mixture. In some embodiments, a computer-implemented method comprising automatic image processing is used to determine the position of two or more pI markers and analyte bands. In some embodiments, the computer-implemented method further comprises calculating the isoelectric point of one or more analyte bands based on the position data derived from the automatic image processing.

[0085] Figure 2An example process flow diagram is provided for a computer-implemented method for acquiring an image of a separation channel (or other portion of a microfluidic device), determining the locations of PI markers and analyte bands in the image (i.e., where the separation step includes CIEF), and calculating the pI of one or more analyte bands in a separated mixture of analytes. In some embodiments, the computer-implemented method may include controlling the acquisition of a series of one or more images, which are then processed to identify the locations of the pI marker and the separated analyte bands. Examples of suitable automatic image processing algorithms are discussed in more detail below. In some embodiments, predetermined knowledge of the predicted location of a PI marker, for example, the location of the PI marker determined from an image of a "control" sample that includes only the pI marker, can be used to distinguish between bands corresponding to the pI marker and bands corresponding to the separated analyte. In some embodiments, images of the pI marker can be acquired at a different wavelength or using a different imaging mode than that used to acquire images of the separated analyte bands. As Figure 2 As shown, if the image processing step fails to determine the position of a known number of pI markers and / or separated analyte bands, the system can be instructed to acquire a new image so that the image processing step can be repeated. Once the positions of the pI markers and separated analyte bands are determined, the data of the pI marker positions can be fit to a user-selected pH gradient model (e.g., a linear or nonlinear model), and the resulting fitted relationship between local pH and position along the separation channel is then used to calculate the isoelectric point of one or more analyte bands.

[0086] In some embodiments, the computer-implemented method can be an iterative process in which the following steps are repeated: detecting pI markers and analyte band positions, fitting the position data to a pH gradient model, and calculating the isoelectric point of one or more analyte bands so as to continuously update and improve the latter (e.g., by averaging multiple determinations). In some embodiments, a cycle comprising the steps of image acquisition and processing, detecting pI markers and analyte band positions, fitting the 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 so that the calculation of the isoelectric point can be updated and improved at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz, or 1,000 Hz, or any other relevant rate, e.g., at a rate of at least the Nyquist rate.

[0087] 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 a separation channel (or other portion of a microfluidic device) so that the velocity of the one or more analyte bands can be calculated based on the difference in their relative position in the two or more images and the 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 while the separation step is being performed. In some embodiments, two or more images can be acquired during the migration step. In some embodiments, two or more images can be acquired when the separated sample is discharged through a fluid channel connecting one end of the separation channel with a downstream analytical instrument. In some embodiments, two or more images can be acquired when 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 when a given analyte band leaves the separation channel. In some embodiments, for example, when there are one or more interconnecting fluid junctions or fluid channels connecting one end of the separation channel with 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 when a given analyte band reaches the outlet port and leaves the device. In some embodiments, the velocities determined for one or more analyte bands can be used to calculate the time it takes for a given analyte band to exit the electrospray tip or electrospray orifice and enter the Taylor cone formed between the electrospray tip or orifice and the mass spectrometer inlet.

[0088] In some embodiments, a computer-implemented method (e.g., a software package) can be used to obtain an image sequence for determining the speed of one or more analyte bands. In some embodiments, the speed of one or more analyte bands is determined using a computer-implemented method including automatic image processing. In some embodiments, the computer-implemented method also includes calculating the time for a given analyte band to leave the separation channel. In some embodiments, the computer-implemented method also includes calculating the time for a given analyte band to arrive at the outlet port and leave the device. In some embodiments, the computer-implemented method also includes calculating the time 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 mass spectrometer inlet. In some embodiments, the departure time determined for one or more analyte bands is used to associate a specific analyte band with mass spectrometry data or data collected using other analytical instruments.

[0089] Figure 3Another example process flow diagram of a computer-implemented method for acquiring an image of a separation channel (or other portion of a microfluidic device), determining the velocity of one or more analyte bands, and calculating the time it takes for a given analyte band to arrive at a specified point in the device, such as the end of a separation channel, a junction between a separation channel and an auxiliary fluid channel, an outlet port of the device, or an outlet of an electrospray tip or orifice is provided. In some embodiments, the computer-implemented method can include controlling the acquisition of a series of one or more images, which are then processed to identify the location of separated analyte bands. Examples of suitable automatic image processing algorithms are discussed in more detail below. Figure 3 As shown, if the image processing step fails to determine the position of the separated analyte band, the system can be instructed to acquire a new image, so that the image processing step can be repeated. Once the position of the separated analyte band is determined for a series of two or more images, the speed is calculated for one or more analyte peaks based on its relative position in the two or more images and the known time interval between the acquisition time 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 multiple separated analyte bands and used to improve speed calculations (e.g., by averaging the speed values ​​calculated from multiple pairs of images in the series). In some embodiments, pI markers or other internal standards that can be detected using a selected imaging mode can be used as "speed standards". The analyte band speed so determined can be used to calculate the time when a given band arrives at a user-specified point in the device, the user-specified point, for example, the outlet end of the separation channel, a specific fluid junction in the device, the outlet port of the device, the tip of the electrospray ionization or the orifice of the analyte entering the Taylor cone, etc.

[0090] In some embodiments, the computer-implemented method can be an iterative process in which the following steps are repeated: detecting analyte band position, determining analyte band velocity, and calculating departure time, so that the prediction of departure time is continuously updated and the correlation of chemical separation data with mass spectrometry data (or other types of downstream analytical data) is further improved. In some embodiments, the cycle of steps including image acquisition and processing, velocity calculation, and departure time prediction can be completed in a sufficiently short time that the departure time prediction can be updated at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz, or 1,000 Hz, or any other relevant rate, for example, at a rate of at least the Nyquist rate.

[0091] Correlation of separation data with mass spectrometry data: In some embodiments, the above-described computer-implemented method for accurate imaging-based isoelectric point determination of isoelectric focused analyte bands enables correlation of isoelectric point data with specific m / z peaks in mass spectrometry data (or other analytical data), thereby improving the information content of the data set (even for a single run experiment) and allowing for more quantitative characterization of the analyte sample.

[0092] In some embodiments, the above-described computer-implemented methods for calculating velocities and predicting the departure times of separated analyte bands (using any of a variety of different separation techniques) using image-derived data enable improved temporal correlations 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 data set (even for a single run experiment), and enabling more quantitative comparisons of data collected from different sample runs, different samples, or on different instruments due to the ability to correct for experiment-to-experiment or instrument-to-instrument differences in separation times. Thus, the disclosed methods, apparatus, and systems may be particularly advantageous for various metabolomics, proteomics, and drug development or manufacturing applications.

[0093] In some embodiments (eg, those including a CIEF step), the computer-implemented methods of the present disclosure can perform both imaging-based determination of the precise isoelectric point and imaging-based determination of the velocity of the separated analyte bands.

[0094] Mass Spectrometry and Electrospray Ionization: In some embodiments, the methods, apparatuses, and systems of the present disclosure may be configured to perform electrospray ionization of a separated analyte mixture 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 the analyte molecules and classifying the resulting ions according to their mass-to-charge ratio (m / z). Combined with the previous liquid or gas phase sample separation systems, mass spectrometry provides one of the most effective means for analyzing complex samples that include multiple low-abundance analytes, such as are commonly found in biological samples.

[0095] All mass spectrometers require that ions must be in the gas phase before being introduced into the mass analyzer. A variety of sample ionization modes have been developed, including, but not limited to, matrix-assisted laser desorption and ionization (MALDI) and electrospray ionization (ESI). In the MALDI technique, a sample (e.g., a biological sample including a mixture of proteins) is mixed with an energy absorbing matrix (EAM) such as sinapinic acid or α-cyano-4-hydroxycinnamic acid and crystallized onto a metal plate. Surface-enhanced laser desorption and ionization (SELDI) is a common variant of this technique that incorporates additional surface chemistry on a metal plate to promote specific binding of a particular type of protein. The plate is inserted into a vacuum chamber and hits the matrix crystals with light pulses from a nitrogen laser. The energy absorbed by the matrix molecules is transferred to the proteins, causing them to desorb, ionize, and generate ion plumes in the gas phase, which are accelerated and drawn into a flight tube in the presence of an electric field, where they drift until they hit a detector that records the time of flight. The time of flight can in turn be used to calculate the m / z ratio of the ionized species. In some embodiments of the disclosed device, the outlet port of the device may include a capillary or other feature for depositing separated analyte bands (or fractions thereof) onto a MALDI plate in preparation for mass spectrometry analysis, for example, correlating the isoelectric point of a particular analyte band with MALDI mass spectrometer data.

[0096] Electrospray ionization (ESI; also referred to herein as "electrospray") is another widely used technique because of its inherent compatibility for docking liquid chromatography or electrokinetic chromatography separation techniques with mass spectrometers. As described above, in electrospray ionization, small droplets of sample and solution are emitted from the far end of a capillary or microfluidic device including an electrospray feature (e.g., an emitter tip or orifice) by applying an electric field between the tip or orifice and the mass spectrometer source plate. The droplets are then stretched and expanded in the induced electric field to form a conical emission (i.e., a "Taylor cone") that includes smaller and smaller droplets that evaporate and produce gas-phase ions that are introduced into a mass spectrometer for further separation and detection. The emitter tip can be formed by a capillary or corner or ESI tip built into the microfluidic chip design, which provides a convenient droplet volume for ESI. The emitter tip can be sharpened using a grinding wheel, file, machining tool, CNC machining tool, water jet cutting, or other tool or process to provide a smaller surface and droplet volume, to shape the ESI tip to provide a smaller surface volume, and the like. In some embodiments, the tip can be pulled out by heating and stretching the tip portion of the chip. In some embodiments, the tip can then be cut to a desired length or diameter. In some embodiments, the electrospray tip can be coated with a hydrophobic coating that can minimize the size of droplets formed on the tip. In some embodiments, the system can electrospray a mobile agent, catholyte, or any other liquid during the separation step when no analyte is eluted from the device.

[0097] In some embodiments of the disclosed methods, devices, and systems, other ionization methods are used, such as inductively coupled laser ionization, fast atom bombardment, soft laser desorption, atmospheric pressure chemical ionization, secondary ion mass spectrometry, spark ionization, thermal ionization, etc.

[0098] With respect to electrospray ionization, in some embodiments, the disclosed microfluidic devices include features designed to facilitate efficient electrospray ionization and convenient interfacing with downstream mass spectrometry analysis, such as Figure 1 As shown. The mass-to-charge ratio (or "mass") of an analyte (e.g., a biological or biosimilar) discharged from a microfluidic device and introduced into a 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.

[0099] In some embodiments, the electrospray feature of the microfluidic device can be in line with the separation channel. In some embodiments, the electrospray feature of the microfluidic device can be oriented at right angles or at an intermediate angle relative to the separation channel. In some embodiments of the disclosed method, substantially all separations and / or enriched analyte fractions from the final separation or enrichment steps carried out in the capillary or microfluidic device are discharged from the electrospray tip or feature in the form of a continuous flow. In some embodiments, a portion of the analyte mixture (e.g., a fraction of interest) can be discharged from the microfluidic device via an outlet configured to be connected to an analytical instrument (e.g., a mass spectrometer) or another device configured for classification and / or enrichment of at least a portion of the sample. Another portion of the analyte mixture (e.g., a fraction containing except the fraction of interest) can be discharged via a waste channel.

[0100] In some embodiments, the expulsion from the capillary or microfluidic device is performed using pressure, electricity, ionization, or any combination of these. In some embodiments, the expulsion is consistent with the migration step as described above. In some embodiments, the sheath fluid used for electrospray ionization is used as the electrolyte for the electrophoretic separation. In some embodiments, a nebulizing gas is provided to reduce the analyte fraction to a fine spray.

[0101] Feedback of Imaging-Based Electrospray Ionization Performance: Conventional ESI-MS systems using capillaries or microfluidic devices do not typically provide tools for calibrating the system to reconstruct the Taylor cone during operation. Maintaining a stable Taylor cone can be complicated by the electrophoretic electric field applied across the separation channel in the microfluidic device or capillary. Changes in reagent conductivity between runs or during runs can change the voltage potential at the interface with the mass spectrometer. Changes in the potential at the interface can adversely affect the Taylor cone and can result in a loss of electrospray ionization efficiency. Disclosed herein are methods and systems for improving electrospray ionization performance, thereby improving the quality of mass spectral data collected by a capillary-based or microfluidic device-based ESI-MS system. In some embodiments, for example, imaging of the Taylor cone in an electrospray ionization device can be used in a computer-implemented method to provide feedback control of one or more operating parameters so that the shape, density, or other characteristics of the Taylor cone remain within a specified range. In some embodiments, operating parameters that can be controlled by such a feedback process include, but are not limited to, 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 regulating pressure, electric field strength, or a combination thereof for expelling the analyte sample), such as a voltage applied at the proximal end of the channel (e.g., a voltage between the electrospray tip or orifice and the mass spectrometer inlet), the volumetric flow rate of sheath liquid or sheath gas surrounding the expelled analyte sample, or any combination thereof.

[0102] Figure 4 An example process flow diagram of a computer-implemented method is provided for: (i) acquiring an image of a Taylor cone (using various image sensors, such as a CCD image sensor or a CMOS image sensor), (ii) processing the image to determine the shape, density, or other characteristic of the Taylor cone, (iii) comparing the shape, density, or other characteristic of the Taylor cone to a set of specified or target values, and (iv) determining, based on the comparison, an appropriate adjustment to one or more operating parameters using a mathematical algorithm that relates the shape, density, or other characteristic of the Taylor cone to one or more operating parameters to restore the Taylor cone to the specified or target value. In some embodiments, in addition to data derived from the image of the Taylor cone, data acquired from a mass spectrometer (e.g., total ion current data) can be used to monitor system performance and make adjustments to one or more operating parameters.

[0103] In some embodiments, this can be accomplished in a sufficiently short time Figure 4The cyclic process shown includes the steps of: image acquisition and processing, identifying Taylor cone characteristics, comparing the Taylor cone characteristics to a set of target values, and calculating the required adjustments to one or more ESI-MS system operating parameters, so that one or more operating parameters can be updated at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz or 1,000 Hz or any other relevant rate, for example, at a rate of at least the Nyquist rate.

[0104] Alternating high mass / low mass scans: In some embodiments of the disclosed methods, apparatus, and systems, the mass spectrometer can be configured to alternate between a high mass scan range (e.g., an m / z range of about 1500-6000) or "high mass scan," and a low mass scan range (e.g., an m / z range of about 150-1500) or "low mass scan," such that the low mass scan can be used to identify low mass markers that can be identified in the mass spectrometry data (e.g., free solution ampholytes in the case of an isoelectric focusing separation step) and to calibrate the low mass markers with respect to the properties indicated by them (e.g., isoelectric point ranges, peptides, small molecule markers in the case of free solution ampholytes detected). The switching between high mass scans and low mass scans and 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 mass scans and low mass scans can be in the range of about 0.5 Hz to about 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.

[0105] Varying the high and low separation / migration voltages to keep the ESI tip voltage constant: 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 remain grounded. In some embodiments, the ESI tip on the capillary or microfluidic device will remain grounded or nearly grounded to create 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 create an electric field between the ESI tip and the grounded ESI ion source on the mass spectrometer.

[0106] Fig.15An exemplary flow chart of a computer-controlled feedback loop for maintaining a constant voltage drop between the anode and cathode at 3000 V during migration while maintaining the ESI tip voltage at 0 V is provided. 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., -3500 V) relative to ground. In this example, by first setting Fig. 7A In the embodiment, the anolyte port 110 is set to +3000V and the migration agent port 104 is set to 0V, and the ΔV between the anolyte port 110 and the migration agent port 104 is maintained at 3000V. In some embodiments, different ΔVs can be set by setting the anolyte port 110 to different values. In some embodiments, anodic migration can be used, and port 110 can be a catholyte port, which is set to, for example, -3000V. Fig.15 In the example outlined, during migration, the resistance in the separation channel 112 decreases as the separating analytes and ampholytes regain charge. This results in a decrease in the voltage drop across the channel 112, which in turn results in an increase in the voltage at the ESI tip 116, according to Equation 1:

[0107] V 116 =(ΔV 110-104 )*(R 105 ) / (R 109 +R 112 +R 105 )

[0108] However, by measuring or calculating the ESI tip voltage 116, the voltage settings at the anolyte port 110 and the migrater port 104 can be adjusted. By subtracting the ESI tip voltage 116 from the anolyte port 110 and migrater port 104 settings, ΔV 110-104 The voltage is kept at 3000 V so that migration is not affected, but the ESI tip 116 voltage is set to 0 according to Equation 2:

[0109] V 116 =(ΔV 110-104 )*(R 105 ) / (R 109 +R 112 +R 105 )+V 104

[0110] This feedback loop continues to operate until the migration is complete, adjusting the voltage at the ESI tip 116 to 0 at a regular frequency (e.g., the Nyquist rate or about 0.2 Hz). In some cases, the voltage at the ESI tip 116 can be adjusted to 0 at a rate of at least 0.01 Hz, 0.1 Hz, 0.2 Hz, 0.3 Hz, 0.4 Hz, 0.5 Hz, 0.6 Hz, 0.7 Hz, 0.8 Hz, 0.9 Hz, 1 Hz, 10 Hz, 100 Hz, or 1,000 Hz. Maintaining a constant, stable voltage at the ESI tip 116 is critical to maintaining a stable electrospray during the migration process.

[0111] In some cases, the feedback loop operates to maintain the voltage at the ESI tip within a specified percentage of a preset value. For example, in some cases, the feedback loop operates to maintain the voltage at the ESI tip within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the preset value. In some cases, the feedback loop operates to maintain the ESI tip voltage within 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V of the preset value.

[0112] 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 in order to generate an electric field between the ESI tip 116 and the mass spectrometer. In some embodiments, the ESI tip voltage (e.g., a preset value) can be about +5000V, about +4000V, about +3500V, about +3000V, about +2500V, about +2000V, about +1500V, about +1000V, about +500V, or about -5000V, about -4000V, about -3500V, about -3000V, about -2500V, about -2000V, about -1500V, about -1000V, or about -500V. Fig.12 An example flow chart of a computer-controlled feedback loop for maintaining a constant voltage drop between the anode and cathode at 3000 V during migration while maintaining the ESI tip voltage at 3000 V is provided. The operation of the computer-controlled feedback loop is similar to Fig.15, except that the voltage at the anolyte port 110 and the migration agent port 104 is offset by +3000V, which causes the voltage at the ESI tip 116 to be offset to +3000V, still following equation 2. In some embodiments, analog circuits can be used to achieve control of the electric field strength. In some embodiments, control of the voltage at one or more electrodes in contact with a capillary-based or microfluidic device-based separation system can be provided by using one, two, three, or four or more independent high-voltage power supplies. In some cases, for example, control of the voltage at one or more electrodes in contact with a capillary-based or microfluidic device-based separation system can be provided by using a single multiplexed high-voltage power supply.

[0113] 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 the 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 the cathode within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01% of the preset value. 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 the cathode within 1000V, 500V, 100V, 75V, 50V, 25V, 10V, 5V, or 1V of the preset value.

[0114] System Hardware: Figure 5A schematic diagram of a system hardware block diagram of one embodiment of the disclosed method, apparatus, and system is provided. As shown, the system of the present 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 separation based on isoelectric focusing, 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 shown by the dashed line), (iii) a mass spectrometer, (iv) an imaging device or system, (v) a processor or computer, and (vi) a computer storage device or any combination thereof. In some embodiments, the system may also include one or more capillary or microfluidic device flow controllers (e.g., programmable syringe pumps, peristaltic pumps, HPLC pumps, etc.); temperature controllers configured to maintain a specified temperature of all or part of the capillary or microfluidic device; additional light sensors 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 storage devices; computer display devices (e.g., including graphical user interfaces); digital communication devices (e.g., intranet, Internet, WiFi, or other hardwired or wireless communication hardware), etc.

[0115] In some embodiments, the system may include an integrated system in which a selection of functional hardware components is packaged as 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 a new application. In some embodiments, some of these functional system components, such as capillaries or microfluidic devices, are replaceable or disposable components.

[0116] As described above, any of a variety of different mass spectrometers may be utilized in different embodiments of the disclosed system, including, but not limited to, time-of-flight mass spectrometers, quadrupole mass spectrometers, ion 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.

[0117] System and application software: such as Figure 6As shown, the system of the present disclosure 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. Typically, these software modules will be configured to run in an operating system or environment controlled by a computer processor, and may communicate and share data with each other and / or with the operating system.

[0118] In some embodiments, the system control software module may include software for: (i) coordinating the operation of a capillary or microfluidic device-based analyte separation system with images acquired by an imaging system, (ii) coordinating the operation of a capillary or microfluidic device-based analyte separation system with data acquisition by a mass spectrometer system, (iii) coordinating image acquisition by an imaging system with the operation of a capillary or microfluidic device-based analyte separation system and / or a mass spectrometer system, (iv) providing feedback control of one or more operating parameters of an electrospray ionization device and / or a mass spectrometer based on data obtained from imaging of a separation channel and / or Taylor cone, (v) controlling data acquisition by a mass spectrometer while switching between a high mass range and a low mass scan range in an alternating manner, (vi) monitoring the voltage at the ESI tip and adjusting 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, or any combination thereof.

[0119] 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 system control and / or data processing software modules, and (ii) controlling data acquisition of one or more mass spectrometer systems, storing the mass spectrometer data (or other downstream analytical instruments), and providing a software interface with system control and / or data processing software, or any combination thereof.

[0120] In some embodiments, the data processing module can include software for: (i) processing images and determining the position of one or more pI standards or analyte peaks in the separation channel as separation is occurring, after separation is complete, or after migration of the pI standards and analyte peaks toward the separation channel exit port or electrospray tip, (ii) processing images and determining the velocity, exit time, and / or electrospray emission time of one or more pI standards or analyte peaks, (iii) processing images of the separation channel to monitor the position of analyte peaks, and processing images of Taylor cones to monitor electrospray performance, wherein images of the separation channel and Taylor cones are acquired simultaneously or alternately, (iv) processing images of the Taylor cones, determining the shape, density, or other characteristics of the Taylor cones, and calculating adjustments to be made 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, etc., or any combination thereof) to affect changes in mass spectrometer data quality; or any combination thereof.

[0121] 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.

[0122] Image processing software: As described above, in some embodiments, 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. In implementing the disclosed methods and systems, any of a variety of image processing algorithms known to those skilled in the art may be used for image preprocessing or image processing. Examples include, but are not limited to, Canny edge detection, Canny-Deriche edge detection, first-order gradient edge detection (e.g., Sobel operator), second-order differential edge detection, phase-consistent (phase-coherent) edge detection, 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 transforms for detecting arbitrary shapes, circular Hough transforms, etc.), and mathematical analysis algorithms (e.g., Fourier transforms, fast Fourier transforms, wavelet analysis, autocorrelation, Savitzky-Golay smoothing, eigenanalysis, etc.), or any combination thereof.

[0123] Processor and computer system: 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 processing unit, or a computing platform. One or more processors may be composed of any of a variety of suitable integrated circuits (e.g., an application-specific integrated circuit (ASIC) designed specifically to implement a deep learning network architecture; or a field programmable gate array (FPGA) for accelerating computing time, etc., and / or facilitating deployment), a microprocessor, an emerging next-generation microprocessor design (e.g., a memristor-based processor), a logic device, etc. Although the present disclosure is described with reference to a processor, other types of integrated circuits and logic devices may also be applicable. The processor may have any suitable data manipulation capabilities. For example, the 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.

[0124] One or more processors or computers for implementing the disclosed method can be part of a larger computer system and / or can be operably coupled to a computer network ("network") by means of a communication interface to facilitate the transmission and sharing of data. The network can be a local area network, an intranet and / or an extranet, an intranet and / or an extranet that communicates with the Internet, or the Internet. In some cases, the network is a telecommunications and / or data network. The network can include one or more computer servers, which in some cases can implement distributed computing, such as cloud computing. In some cases, with the help of a computer system, the network can implement a peer-to-peer network that can enable a device coupled to the computer system to act as a client or server.

[0125] A computer system may also include memory or memory locations (e.g., random access memory, read-only memory, flash memory, Optane TM Technology), electronic storage units (e.g., hard disks), communication interfaces (e.g., network adapters) for communicating with one or more other systems, and peripheral devices (e.g., cache, other memory, data storage, and / or electronic display adapters). The memories, storage units, interfaces, and peripheral devices may communicate with one or more processors, such as a CPU, via a communication bus (e.g., present on a motherboard). The storage unit may be a data storage unit (or data repository) for storing data.

[0126] One or more processors, e.g., CPUs, execute a series of machine-readable instructions that are implemented in a program (or software). Instructions are stored in a memory location. The instructions are directed to the CPU, which then programs or otherwise configures the CPU to implement the methods of the present disclosure. Examples of operations performed by the CPU include fetching, decoding, executing, and writing back. The CPU may be a circuit, such as part of an integrated circuit. The circuit may include one or more other components of the system. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0127] The storage unit stores files, such as drivers, libraries, and saved programs. The storage unit stores user data, such as user-specified preferences and user-specified programs. In some cases, the computer system may include one or more additional data storage units external to the computer system, such as located on a remote server that communicates with the computer system via an intranet or the Internet.

[0128] Some aspects of the methods and systems provided herein are implemented by machine (e.g., processor) executable code stored in an electronic storage location (e.g., memory or electronic storage unit) of a computer system. 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 the storage unit and stored in the memory for access at any time by one or more processors. In some cases, the electronic storage unit is excluded and the machine executable instructions are stored in the memory. The code can be precompiled and configured for use with a machine having one or more processors suitable for executing the code, or can be compiled at runtime. The code can be provided in a selected programming language so that the code can be executed in a precompiled or real-time compiled manner.

[0129] Various aspects of the disclosed methods and apparatus may be considered to be a "product" or "article of manufacture," e.g., a "computer program or software product," typically in the form of machine (or processor) executable code and / or associated data stored in some type of machine-readable medium, wherein the executable code includes a plurality of instructions for controlling a computer or computer system when performing one or more of the methods disclosed herein. The machine executable code may be stored in an optical storage unit including an optically readable medium such as a compact disc, CD-ROM, DVD, or Blu-ray disc. The machine executable code may be stored in an electronic storage unit such as a memory (e.g., read-only memory, random access memory, flash memory) or on a hard disk. "Storage" type media include any or all tangible memories of a computer, processor, etc., or related modules thereof, such as various semiconductor memory chips, optical disk drives, tape drives, disk drives, etc., which may readily provide non-temporary storage for software encoding the methods and algorithms disclosed herein.

[0130] All or part of the software code may sometimes be communicated over the Internet or various other telecommunication networks. Such communication, for example, enables software to be loaded from one computer or processor to another, for example, from a management server or host to a computer platform of an application server. Therefore, other types of media for transmitting software coding instructions include optical waves, radio waves, and electromagnetic waves, such as those used over wired and optical landline networks and via various atmospheric links at physical interfaces between local devices. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., are also considered to be media for transmitting software coding instructions for executing the methods disclosed herein. As used herein, unless limited to non-temporary, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0131] The computer system typically includes an electronic display, or can communicate with an electronic display to provide, for example, images captured by a machine vision system. The display can also typically provide a user interface (UI). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs), web-based user interfaces, and the like.

[0132] 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, the improved information content and data quality that can be achieved for separation-based ESI-MS analysis of analyte samples using the disclosed methods can be very beneficial for the characterization of biologics and biosimilars during development and / or manufacturing. Other applications may include, but are not limited to, 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.

[0133] Biologics and biosimilars are a class of drugs that include, for example, recombinant proteins, antibodies, live viral vaccines, proteins derived from human plasma, cell-based drugs, proteins from natural sources, antibody-drug conjugates, protein-drug conjugates, and other protein drugs. The FDA and other regulatory agencies require a stepwise approach to demonstrate biosimilarity, which may include comparisons of the proposed product to a reference product in terms of structure, function, animal toxicity, human pharmacokinetics (PK) and pharmacodynamics (PD), clinical immunogenicity, and clinical safety and effectiveness (see "Scientific Considerations in Demonstrating Biosimilarity to a Reference Product: Guidance for Industry," U.S. Department of Health and Human Services, Food and Drug Administration, April 2015). Examples of structural characterization data that may be required for protein products include primary structure (i.e., amino acid sequence), secondary structure (i.e., degree of folding to form alpha helical or beta pleated structures), tertiary structure (i.e., the three-dimensional shape of the protein resulting from 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 expensive techniques such as X-ray crystallography. Therefore, there is a need for an experimental technique that allows for convenient, real-time, and relatively high-throughput characterization of protein structure for use in establishing biosimilarity between candidate biopharmaceuticals and reference drugs.

[0134] In some embodiments, for the purpose of establishing biosimilarity, the disclosed methods, apparatuses and systems can be used to provide structural comparison data of biological candidate drugs (e.g., monoclonal antibodies (mAbs)) and reference biological drugs. For example, in some cases, the isoelectric point data and / or mass spectrometry data of the candidate drugs and the reference drugs can provide important evidence to support the proof of biosimilarity. In some embodiments, the isoelectric point data and / or mass spectrometry data of the candidate drugs and the reference drugs that have been treated with site-specific proteases under the same reaction conditions can provide important evidence to support the proof of biosimilarity. In some embodiments, the disclosed methods, apparatuses and systems can be used to monitor the biopharmaceutical production process to ensure the quality and consistency of the product by analyzing samples extracted at different points in the production process or samples extracted from different production runs. In some embodiments, the disclosed methods, apparatuses and systems can be used to evaluate the stability of formulation buffers. In some embodiments, the disclosed methods, apparatuses and systems can be used to evaluate the production and quality of biopharmaceutical candidates of clonal cell lines.

[0135] Example

[0136] These examples are provided for illustrative purposes only, and do not limit the scope of the claims provided herein.

[0137] Example 1 - Characterization of protein charge on chip prior to mass spectrometry analysis

[0138] Figure 1 The manufacture of the microfluidic device shown in has been described above. For operation, the device is installed on an instrument containing a nitrogen source, a heater, a 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 automatic sampler for loading samples onto the device. The power supply shares a common ground with the mass spectrometer. The instrument is controlled by software (e.g., LabView).

[0139] The protein sample is premixed with an ampholyte pH gradient and pI markers, then placed in a vial and loaded onto the autosampler. The mixture is continuously loaded from the autosampler onto the microfluidic device 400 via inlet 412, passes through the enrichment channel 418 and leaves the device through outlet 434 to the waste channel 430.

[0140] Sheath fluid / catholyte fluid (50% MeOH, N4OH / H2O) was loaded onto the two catholyte holes 404, 436, anolyte (10 mM H3PO4) was loaded onto the anolyte hole 426, and a heated nitrogen source was attached to the two gas holes 408, 440.

[0141] After all reagents are loaded, isoelectric focusing is initiated by applying a +600 V / cm electric field from the anolyte well 426 to the catholyte wells 404, 436 by connecting electrodes to the anolyte well 426 and the catholyte wells 404, 436. A UV light source is directed below the enrichment channel 418, and a camera is placed above the enrichment channel 418 to measure the light that passes through the enrichment channel 418, thereby detecting the focused proteins by their absorbance. A glass plate 402 made of soda-lime glass is used to block any stray light for the camera, so that light that does not pass through the enrichment channel 418 is blocked from reaching the camera, increasing the sensitivity of the measurement.

[0142] 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 to move the pH gradient toward orifice 424. The electric field can be maintained at this point to maintain high-resolution IEF separation. Continuing to image the enrichment channel 418 during the ESI process can be used to determine the pI of each protein as it exits orifice 424.

[0143] As the enriched protein fraction moves from the enrichment channel 418 into the junction 420, it will mix with the sheath fluid, which can flow from the catholyte wells 404, 436 to the junction 420 via the sheath fluid / catholyte fluid channels 406, 438. Mixing the enriched protein fraction with the sheath fluid can place the protein fraction in a solution compatible with mass spectrometry and restore the charge of the focused proteins (IEF puts the proteins into an uncharged state), thereby improving ionization.

[0144] The enriched protein fraction then continues to the orifice 424, which may be defined by the countersunk surface 422 of the glass plate 402. The enriched protein fraction may generate a Taylor cone once trapped in the electric field between the sheath fluid orifice ground and the mass spectrometer cathode.

[0145] As the solution continues to push the Taylor cone from the enrichment channel 418, small droplets of fluid will be discharged from the Taylor cone and fly toward the mass spectrometer inlet. Nitrogen (e.g., at 150°C) can flow downward from the gas holes 408, 440 through the gas channels 410, 432 and form a nitrogen jet on the side of the Taylor cone, which can convert the droplets emanating from the Taylor cone into a fine mist before leaving the microfluidic device, which can facilitate detection in the mass spectrometer. Adjusting the pressure from the inlet 412 can adjust the Taylor cone size as needed to improve detection in the mass spectrometer.

[0146] Example 2 - Tracking the velocity of an analyte peak as it leaves a microfluidic chip and enters a mass spectrometer

[0147] For this embodiment, Fig. 7A The microfluidic channel network 100 in FIG. 1 is fabricated in a 250 micron thick opaque cyclic olefin polymer layer. Channel 112 has a depth of 250 microns and therefore cuts through the 250 micron layer. All other channels have a depth of 50 microns. Figure 7BAs shown, the channel layer is sandwiched between two transparent layers of cyclic olefin polymer to make a planar microfluidic device. Ports 102, 104, 106, 108 and 110 provide access to the channel network for introducing reagents from external containers and making electrical contacts. Port 102 is connected to a vacuum source, making channel 103 a waste channel, so that other reagents are perfused through the channel network as "waste". Acid (1% formic acid) is perfused into channels 109, 112, 114 and 103 through port 108 and comes out to port 102. The sample (4% Pharmalyte 3-10, 12.5 mM pI standard 3.38 (purified peptide, sequence: Trp-Asp-Asp-Asp), 12.5 mM pI standard 10.17 (purified peptide, sequence: Trp-Tyr-Lys-Arg), NIST monoclonal antibody standard (Part No. 8671, NIST)) was poured into channels 107, 112, 114, and 103 through port 106 and out to port 102. This leaves channel 112 containing the sample analyte. Base (1% dimethylamine) was poured into channels 105, 114, and 103 through port 104 and out to port 102. Migrator (1% formic acid, 49% methanol) was poured into channels 111, 114, and 103 through port 110 and out to port 102 through channel 103.

[0148] The analyte sample in channel 112 is electrophoresed by applying 4000V to port 108 and grounding port 110. The ampholytes in the analyte sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed as follows: a 280 nm light source is aligned with channel 112, and the transmittance of the 280 light through channel 112 is measured using a CCD camera. The software calculates the absorbance by comparing the light transmittance during the separation or migration to a "blank" reference measurement made before the analyte run without the analyte in focus, and then displays the absorbance for each pixel over the entire length of channel 112. The location of the standard or analyte being focused is displayed as a peak, such as Figure 9A-9F shown.

[0149] Once the analyte is focused, the final focused absorbance image is captured. The software will identify the spatial location of the pI markers and interpolate between the markers to calculate the pI of the focused analyte fraction peak. At this point, the control software will trigger a relay to disconnect the ground of port 110, ground port 104, and set the pressure on the migration agent container connected to port 104 to establish a 100nL / min flow of the migration agent solution, which enters channels 105 and 114 through hole 104 and exits the chip at orifice 116. Orifice 116 is located 2mm from the ESI inlet of the mass spectrometer, where the inlet voltage is -3500V to -4500V.

[0150] As the pressure-driven flow directs the mobilizing agent from port 104 to orifice 116, some of the formic acid in the mobilizing agent will electrophoresce as formate from channel 105 through channel 112 to the anode at port 108. The formate will disrupt the isoelectric pH gradient as it passes through channel 112, causing the ampholytes, standards, and analyte samples to increase in charge and migrate electrophoretically from channel 112 to channel 114, where the pressure-driven flow from port 110 out of orifice 116 carries it into the ESI spray.

[0151] As migration occurs, the software continues to capture absorbance images and identifies peaks, tracking their migration from imaging channel 112 into channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time it takes for the peak to travel through channel 114 and be introduced into the mass spectrometer through orifice 116, allowing a direct correlation between the original focused peak and the resulting mass spectrum.

[0152] Figure 9A-9F An example of a series of absorbance traces, at 1 minute intervals, showing the migration of an isoelectric point (pI) standard determined from images of a separation channel is provided. Fig. 9A A graph of absorbance 910 as a function of channel distance 905 is shown after isoelectric focusing of five pI standards (peaks 915, 920, 925, 930, 935) prior to migration. Fig. 9B As shown, after 1 minute of migration, the peak 915 corresponding to the pI=9.99 standard is located at the edge of the imaging system's field of view. Fig. 9C As shown, after 2 minutes of migration, peak 915 (pI=9.99 standard) has left the portion of the channel being imaged. Fig.9D As shown, after 3 minutes of migration, peak 920 (pI=8.40 standard) has left the portion of the channel being imaged. Fig.9E As shown, after 4 minutes of migration, peak 925 (pI=7.00 standard) has left the portion of the channel being imaged. Fig.9F As shown, after 5 minutes of migration, peak 930 (pI=4.05 standard) has left the portion of the channel being imaged.

[0153] Example 3 - Using feedback to adjust MS and ESI parameters

[0154] In Example 3, the chip, instrument and software perform all the same procedures as in Example 2. In addition, Figure 8 As shown, a second CCD camera is used to image the Taylor cones during ESI. These images are used to evaluate the quality and consistency of the Taylor cones. Evaluation of the images and / or totals on the mass spectrometer allows identification of ESI Taylor cone failures and diagnosis of the cause.

[0155] The formation of a Taylor cone in ESI depends on maintaining an input flow into the cone that matches the rate of fluid loss to evaporation and ESI. The size of the Taylor cone depends on the flow rate, the voltage gradient between the microfluidic device and the MS, the distance between the microfluidic device and the MS, and subtle variations in the microfluidic device ESI tip and the local environment.

[0156] Imaging of the Taylor cone allows diagnosis of the cause of ESI failure. For example, loss of the Taylor cone indicates insufficient flow and the software can increase the flow of the transporter into the microfluidic device. Similarly, a coronal discharge indicates that the voltage is too high and the software can reduce the voltage. Expansion of the ESI cloud indicates that the voltage is too high, while droplet formation without a Taylor cone indicates that the voltage is too low. These differences, as well as any other visual differences, can be identified in the image and the software can automatically compensate to reconstruct the Taylor cone.

[0157] Example 4 - Low-mass scanning as a separation marker

[0158] In Example 4, the chip, instrument and software perform all the same procedures as in Example 2. In addition, once migration occurs and the analyte peak begins to migrate to the MS, the MS is set to alternate between the m / z ranges of 1500-6000 and 150-1500. The 1500-6000 range is used to identify the NIST antibody analyte fraction peak when it is introduced into the MS. The 150-1500 m / z range scan is used to identify free solution ampholytes (Pharmalytes) when they are introduced into the mass spectrometer. Ampholytes can be identified in mass spectrometry scans and used to calibrate the total ion chromatographic analysis from the MS because the presence of specific ampholytes defines the portion of the isoelectric pH gradient analyzed in the MS at any time point.

[0159] Example 5 - Varying high and low voltages to keep the electric field strength and tip voltage constant

[0160] For this embodiment, Fig. 7A The microfluidic channel network 100 in FIG. 1 is fabricated in a 250 micron thick opaque cyclic olefin polymer layer. Channel 112 has a depth of 250 microns and therefore cuts through the 250 micron layer. All other channels have a depth of 50 microns. Figure 7BAs shown, the channel layer is sandwiched between two transparent layers of cyclic olefin polymer to make a planar microfluidic device. Ports 102, 104, 106, 108 and 110 provide access to the channel network for introducing reagents from external containers and making electrical contacts. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel, thereby allowing other reagents to be perfused through the channel network as "waste". Acid (1% formic acid) is perfused into channels 109, 112, 114 and 103 through port 108 and out to port 102. The sample (4% Pharmalyte 3-10, 12.5 mM pI standard 3.38 (purified peptide, sequence: Trp-Asp-Asp-Asp), 12.5 mM pI standard 10.17 (purified peptide, sequence: Trp-Tyr-Lys-Arg), NIST monoclonal antibody standard (Part No. 8671, NIST)) was poured into channels 107, 112, and 114 through port 106 and out to port 102. This leaves channel 112 containing the sample analyte. Base (1% dimethylamine) was poured into channels 105, 114, and 103 through port 104 and out to port 102. Migrator (1% formic acid, 49% methanol) was poured into channels 110, 114, and 103 through port 110 and out through channel 102 to port 102. Pressure is applied to the base container to produce a flow of 100 nL / min through port 104 into channels 105 and 114 and out orifice 116 .

[0161] Isoelectric focusing of the analyte sample in channel 112 is initiated by applying 2000 V to port 108 using power supply 1005 and connecting port 110 to high voltage power supply 1010 and applying -2000 V. This establishes Fig. 10A The circuit shown includes a high voltage power supply 1005 and a high voltage power supply 1010 (in some cases, power supplies 1005 and 1010 may include two channels of a single multi-channel high voltage power supply) to produce a voltage drop of 4000V between the anode and the cathode. The resistance of the channel depends on the size of the channel and the conductivity of the reagent. In this embodiment, the corresponding channel 109 (see Fig. 7A ) of the acid channel has a resistance R109 of 10 megohms, corresponding to channel 112 (see Fig. 7A ) of the sample channel starts at 40 megohms and corresponds to channel 111 (see Fig. 7A ) has a resistance R111 of 50 megohms for the base channel. Fig. 7A ) and mass spectrometer 1015. The resistance R113 of the electrospray ionization (ESI) interface between channels 109, 112, and 111 (see Fig. 7A) is 4000V, and since these channels represent three resistors in series, the voltage at the tip (V 116 ):

[0162] V 116 =ΔV 108-110 *(R 111 ) / (R 109 +R 112 +R 111 )+(voltage setting of high voltage power supply 1010). At the beginning of isoelectric focusing, V 116 = 0 volts. Orifice 116 (see Fig. 7A ) was located 2 mm from the ESI inlet of the mass spectrometer, where the inlet voltage was -3500 V to -4500 V to form a Taylor cone. Fig. 10B Shows Fig. 10A Another embodiment of a circuit is presented, which includes a resistor R105 of the channel 105 .

[0163] Amphoteric electrolytes in the analyte sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed using a 280 nm light source aligned with channel 112 and measuring the transmission of the 280 nm light through channel 112 with a CCD camera. The software calculates the absorbance by comparing the light transmission during the separation or migration to a "blank" reference measurement made without the analyte in focus before the analyte run, and then displays the absorbance for each pixel across the length of channel 112. The position of the standard or the analyte being focused is displayed as a peak, such as Figure 9A-9F shown.

[0164] As the sample is focused, the resistance of sample channel 112 increases as ampholytes, antibody isoforms, and standards reach their isoelectric points and lose their charge, while the resistance in channels 109 and 111 and at the ESI interface remains constant. The computer-implemented method monitors the current at power supply 1005 and can calculate the resistance at any point in time in channel 112. The computer-implemented method uses this information to adjust power supplies 1005 and 1010. For example, when the resistance in channel 112 has climbed to 140 megohms, if the power supply is not adjusted, the voltage at orifice 116 will be -1000V, which will destroy 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 dynamically when the resistance in channel 112 changes.

[0165] Once the analyte is focused, the final focused absorbance image is captured. The software will identify the spatial location of the pI markers and interpolate between the markers to calculate the pI of the focused analyte fraction peak. At this point, the control software will trigger a relay to disconnect the power supply 1010 at port 110 and connect port 104 to the power supply 1010, as well as set the pressure on the migration agent container connected to port 104 to establish a 100nL / min flow of the migration agent solution, which enters channels 105 and 114 through hole 104 and exits the chip at orifice 116 (see Fig. 7A Chip schematic and Fig. 10B The orifice 116 is located 2 mm from the ESI inlet of the mass spectrometer, where the inlet voltage is -3500 V to -4500 V.

[0166] As the pressure driven flow directs the mobilizing agent from port 104 to orifice 116, some of the formic acid in the mobilizing agent will electrophoresce as formate from channel 105 through channel 112 to the anode at port 108. As the formate passes through channel 112, it will disrupt the isoelectric pH gradient, causing the ampholytes, standards, and analyte samples to increase in charge and migrate by electrophoresis from channel 112 to channel 114, where the pressure driven flow from port 110 out of orifice 116 carries it into the ESI spray.

[0167] As migration occurs, the resistance of channel 112 will decrease. Figure 11A-11B An example of voltage and current data for channel 112 is shown, which can be used to derive the resistance of the channel. Fig.11A A graph of voltage as a function of time is shown. Fig. 11B A graph of the current as a function of time is shown. The software monitors the change in current and adjusts the power supply to maintain a voltage drop of 3000 V between the anode and cathode and to maintain 0 V at the tip 116, as shown in FIG. Fig.15 The voltage change can be transient or steady.

[0168] As migration occurs, the software continues to capture absorbance images and identifies peaks, tracking their migration from imaging channel 112 into channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time it takes for the peak to travel through channel 114 and be introduced into the mass spectrometer through orifice 116, allowing a direct correlation between the original focused peak and the resulting mass spectrum.

[0169] Fig.13A Provides protection during chemical migration Fig. 7AA representative circuit diagram of a microfluidic device is shown, in which an additional resistor R120 is used to maintain the ESI tip at a positive voltage to sink current to ground. The circuit can include a high voltage power supply 1305 that can be substantially similar to 1005 and a high voltage power supply 1310 that can be substantially similar to 1010 to produce a specified voltage drop (e.g., 4000V) between the anode and cathode. The circuit can also include a third high voltage power supply 1307. The resistance of the channel depends on the size of the channel and the conductivity of the reagent. Also integrated in the circuit is a resistor corresponding to channel 109 (see Fig. 7A ) of the acid channel, corresponding to the resistance R109 of channel 112 (see Fig. 7A ) of the sample channel and the resistor R112 corresponding to channel 111 (see Fig. 7A ) of the base channel resistor R111, and the orifice 116 (see Fig. 7A ) and a power source of a mass spectrometer 1315 (which may be substantially similar to 1015). Circuit 105 may also include a resistor R105 for channel 105. Power source 1307 may be connected to channel 111 (see Fig. 7A ) and used a current control set to 0 μA during migration. This power supply can read the voltage at the tip and is used to implement a computer-controlled feedback loop to maintain a constant voltage at the tip.

[0170] Fig. 13B It shows that during chemical migration Fig. 7A A representative circuit diagram of a microfluidic device is shown, where the ESI tip is held at a positive voltage using resistor R120 to sink current to power supply 1320. Fig. 13C It shows that during chemical migration Fig. 7A A representative circuit diagram of a microfluidic device is shown, where a field effect transistor (FET) 1325 is used to hold the ESI tip at a positive voltage to sink current. The circuit may also include an amplifier 1330, a voltage reference 1335, and an additional resistor R200. Fig.13D It shows that during chemical migration Fig. 7A A representative circuit diagram of a microfluidic device is shown, in which a bipolar junction transistor (BJT) 1340 is used to hold the ESI tip at a positive voltage to sink current. A power supply 1307 can be connected to channel 111 (see Fig. 7A ) and uses the current control set to 0 μA. This power supply can read the voltage at the tip and is used to implement a computer-controlled feedback loop to maintain a constant voltage at the tip. Fig.13E Provides a chemical migration profile during separation of analyte mixtures Fig. 7AA representative circuit diagram of a microfluidic device is shown, in which the ESI tip will be held at or near ground. A power source 1307 can be connected to channel 111 (see Fig. 7A ) and uses the current control set to 0 μA. This power supply can read the voltage at the tip and is used to implement a computer-controlled feedback loop to maintain a constant voltage at the tip.

[0171] Example 6 - Based on measuring the tip voltage, varying the high and low voltages to maintain the electric field strength and constant voltage at the tip

[0172] For this embodiment, Fig. 7A The microfluidic channel network 100 in FIG. 1 is fabricated in a 250 micron thick opaque cyclic olefin polymer layer. Channel 112 has a depth of 250 microns and therefore cuts through the 250 micron layer. All other channels have a depth of 50 microns. Figure 7B As shown, the channel layer is sandwiched between two transparent layers of cyclic olefin polymer to make a planar microfluidic device. Ports 102, 104, 106, 108 and 110 provide access to the channel network for introducing reagents from external containers and making electrical contacts. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel, thereby allowing other reagents to be perfused through the channel network as "waste". Acid (1% formic acid) is perfused into channels 109, 112, 114 and 103 through port 108 and out to port 102. 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 No. 8671, NIST)) was poured into channels 107, 112, and 114 through port 106 and out to port 102. This leaves channel 112 containing the sample analyte. Base (1% dimethylamine) was poured into channels 105, 114, and 103 through port 104 and out to port 102. Migrator (1% formic acid, 49% methanol) was poured into channels 110, 114, and 103 through port 110 and out to port 102 through channel 102 (see Fig. 7A Chip schematic and Fig.13E circuit shown).

[0173] Electrophoresis of the analyte sample in channel 112 was initiated by applying 1500 V to port 108 using power supply 1305 and connecting port 110 to power supply 1307 (set to 0 V). After 5 minutes, power supply 1305 was increased to 3000 V for 3 minutes to complete focusing.

[0174] Amphoteric electrolytes in the analyte sample establish a pH gradient across channel 112. Absorbance imaging of the separation is performed using a 280 nm light source aligned with channel 112 and measuring the transmission of the 280 light through channel 112 with a CCD camera. The software calculates the absorbance by comparing the light transmission during the separation or migration to a "blank" reference measurement made before the analyte run without the analyte in focus, and then displays the absorbance for each pixel across the length of channel 112. The location of the standard or focused analyte is displayed as a peak, such as Figure 9A-9F shown.

[0175] Once the analyte is focused, the final focused absorbance image is captured. The software will identify the spatial position of the pI markers and interpolate between the markers to calculate the pI of the focused analyte fraction peak. At this point, the control software will trigger a relay, connect port 104 to a power supply 1310, and set the pressure on the migration agent container connected to port 104 to establish a 100nL / min flow of the migration agent solution, which enters channels 105 and 114 through hole 104 and exits the chip at orifice 116. Orifice 116 is located 2mm from the mass spectrometer ESI inlet 1315, where the inlet voltage is -3500V to -4500V. Use current control to set power supply 1307 to 0μA, power supply 1305 to 3000V, and power supply 1310 to 0V, and the MS ESI ion source to -3500V to -4500V.

[0176] As the pressure-driven flow directs the mobilizing agent from port 104 to orifice 116, some of the formic acid in the mobilizing agent will electrophoresce as formate from channel 105 through channel 112 to the anode at port 108. The formate will disrupt the isoelectric pH gradient as it passes through channel 112, causing the ampholytes, standards, and analyte samples to increase in charge and migrate electrophoretically from channel 112 to channel 114, where the pressure-driven flow from port 110 out of orifice 116 carries it into the ESI spray.

[0177] When migration occurs, the resistance of channel 112 will decrease. Power supply 1307 set to 0 μA will be equal to Fig.13E The voltage of V116 in the circuit is 0 because the voltage drop across channel 111 is now 0 (ΔV=IR=0*R111=0V). Figure 11A-11B As shown in the data, 8 minutes (480 seconds) after focusing is completed, the software monitors the current changes and adjusts the power supply to maintain a constant voltage drop of 3000 V between the anode and cathode and maintain 0 V at the tip 116, as shown in FIG. Fig.15 The voltage at the tip (V116) is expressed by Equation 2:

[0178] V116 =ΔV 108-110 *(R 111 ) / (R 109 +R 112 +R 105 )+(power supply 1310 voltage setting).

[0179] As migration occurs, the software continues to capture absorbance images and identifies peaks, tracking their migration from imaging channel 112 into channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time it takes for the peak to travel through channel 114 and be introduced into the mass spectrometer through orifice 116, allowing a direct correlation between the original focused peak and the resulting mass spectrum.

[0180] Example 7 - Based on the measured tip voltage and resistance, varying the high and low voltages to maintain the electric field strength and constant voltage at the tip

[0181] For this embodiment, Fig. 7A The microfluidic channel network 100 in FIG. 1 is fabricated in a 250 micron thick opaque cyclic olefin polymer layer. Channel 112 has a depth of 250 microns and therefore cuts through the 250 micron layer. All other channels have a depth of 50 microns. Figure 7B As shown, the channel layer is sandwiched between two transparent layers of cyclic olefin polymer to make a planar microfluidic device. Ports 102, 104, 106, 108 and 110 provide access to the channel network for introducing reagents from external containers and making electrical contacts. Port 102 is connected to a vacuum source, allowing channel 103 to act as a waste channel, thereby allowing other reagents to be perfused through the channel network as "waste". Acid (1% formic acid) is perfused into channels 109, 112, 114 and 103 through port 108 and out to port 102. Sample (4% Pharmalyte 3-10, 12.5 mM pI standard 5.52 (purified peptide, sequence: Trp-Glu-His), 12.5 mM pI standard 8.4 (purified peptide, sequence: Trp-Tyr-Lys), Infliximab biosimilar monoclonal antibody standard (Part No. MCA6090, Bio-rad) was perfused through port 106 into channels 107, 112, and 114 and out to port 102. This leaves channel 112 containing the sample analyte. Base (1% dimethylamine) was perfused through port 104 into channels 105, 114, 103 and out to port 102. Migrator (1% formic acid, 49% methanol) was perfused through port 110 into channels 110, 114, and 103 and out through channel 102 to port 102 (see Fig. 7A Chip schematic and Fig. 13B circuit shown).

[0182] Electrophoresis of the analyte sample in channel 112 was 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 was increased to 3000V.

[0183] The ampholytes in the analyte sample establish a pH gradient across the channel 112. Absorbance imaging of the separation is performed using a 280 nm light source aligned with the channel 112 and measuring the transmission of the 280 nm light through the channel 112 with a CCD camera. The software calculates the absorbance by comparing the light transmission during the separation or migration to a "blank" reference measurement made before the analyte run without the analyte in focus, and then displays the absorbance for each pixel over the entire length of the channel 112. The location of the standard or analyte that has been focused is displayed as a peak, such as Figure 9A-9F shown.

[0184] Once the analytes have been focused, the charge variants of infliximab are separated, e.g. Fig.16 A, and captures the final focused absorbance image. The software will identify the spatial position of the pI markers and interpolate between the markers to calculate the pI of the focused analyte fraction peak. At this point, the control software will trigger a relay to connect port 104 to a power supply 1310, and set a pressure on the migration agent container connected to port 104 to establish a 100nL / min flow of the migration agent solution, which enters channels 105 and 114 through hole 104 and exits the chip at orifice 116. Orifice 116 is located 2mm from the mass spectrometer ESI inlet 1315. Using current control, power supply 1307 is set to 0μA, power supply 1305 is set to 7000V, power supply 1310 is set to 4000V, and the MS ESI ion source 1315 is kept grounded. An additional resistor R120 is connected to the system between the power supply 1310 and channel 105 (R current sink), and the other end of resistor R120 is connected to the power supply 1320, as shown in FIG. Fig. 13B To act as a current sink, power supply 1320 would be set to at least 4000V less than power supply 1310. Resistor R120 could alternatively connect the circuit to ground (as in Fig.13A ), can be Fig. 13C The field effect transistor (FET) shown may be Fig.13D A bipolar junction transistor (BJT) as shown, or any other resistive element that can sink current from power supply 1310 to create a functional electrophoretic circuit.

[0185] As the pressure-driven flow directs the mobilizing agent from port 104 to orifice 116, some of the formic acid in the mobilizing agent will electrophoresce as formate from channel 105 through channel 112 to the anode at port 108. The formate will disrupt the isoelectric pH gradient as it passes through channel 112, causing the ampholytes, standards, and analyte samples to increase in charge and migrate electrophoretically from channel 112 to channel 114, where the pressure-driven flow from port 110 out of orifice 116 carries it into the ESI spray.

[0186] As migration occurs, the resistance of channel 112 will decrease. Power supply 1307, set to 0 μA, will equal the voltage of V116 because the voltage drop across channel 111 is now zero (ΔV=IR=0*R111=0V). Fig.11A and Fig. 11B As shown in the data, the software monitors the change in current and adjusts the power supply to maintain a constant voltage drop of 3000 V between the anode and cathode and maintain 3000 V at the tip 116, as shown in the data. Fig.12 The voltage at the tip (V116) is expressed by Equation 2:

[0187] V 116 =ΔV 108-110 *(R 111 ) / (R 109 +R 112 +R 105 )+(power supply 1310 voltage setting).

[0188] As migration occurs, the software continues to capture absorbance images and identifies peaks, tracking their migration from imaging channel 112 into channel 114. By tracking the time each peak leaves imaging channel 112, its velocity, and the flow rate in channel 114, the software can calculate the time it takes for the peak to traverse channel 114 and be introduced into the mass spectrometer through orifice 116, allowing a direct correlation between the original focused peak and the resulting mass spectrum. For example, Fig.16 B shows the masses of glycoforms electrosprayed into the mass spectrometer, which are contained in Fig.16 In the acidic peak of the electropherogram shown in A. Fig.16 C shows the Fig.16 A. Glycoform masses in the major infliximab peak. Fig.16 D and Fig.16 E shows Fig.16 A shows the mass of the basic peak in the electropherogram.

[0189] Example 8 - Varying high and low pressures to maintain electric field strength and constant voltage in two-step capillary IEF

[0190] In Example 8, two-step IEF (isoelectric focusing followed by migration) was performed in a 60 cm capillary and migrated to ESI-MS by coupling a nebulizer, as shown in FIG. Fig.14A As shown in the schematic. The separation capillary 1808 is immersed in the anolyte vial 1806. The high voltage power supply 1802 is connected to the anolyte vial 1806 via the electrode 1804. The other end of the capillary 1808 is connected to the junction sprayer 1814 via a three-way pipe joint 1812. The capillary 1808 is inserted into the junction sprayer 1814, so that the capillary outlet is close to the ESI tip 1824. The third arm of the three-way pipe joint 1812 is connected to the migration capillary 1816 immersed in the pressurized migration agent vial 1818. The pressurized migration agent vial 1818 is also grounded via the electrode 1817, so it can be used as a current absorber. In addition, the junction sprayer 1814 is connected to the power supply 1810 via the wire 1820, and the wire 1820 is connected to the outside of the sprayer 1814. In this embodiment, the mass spectrometer ion source remains grounded.

[0191] Reagents were prepared as follows. Anolyte vial 1806 was filled with 1% formic acid in water, separation capillary 1808 was filled with aqueous sample (250 μg / mL NIST mAb, 1.5% Pharmalyte 5-8 ampholytes, 1.5% Pharmalyte 8-10.5, 5 mg / mL pI standards 7.00 and 10.17), junction nebulizer chamber 1826 and migration agent capillary 1816 were filled with 1% diethylamine in water, and pressurized migration agent vial 1818 was filled with 1% formic acid, 50% acetonitrile and 49% water.

[0192] In this embodiment, the mass spectrometer ion source was kept grounded. To initiate focusing, power supply 1802 was set to +30 kV and power supply 1810 was set to 4 kV. Pressure driven flow from mobility agent vial 1818 was initiated at 100 nL / min. In this way, diethylamine was used in the junction nebulizer chamber 1826 to initiate ESI, and diethylamine was also used as the catholyte for the isoelectric focusing step.

[0193] As focusing progresses in capillary 1808, the sample loses its charge carrying capacity and increases in resistance in capillary 1808. Since the ESI tip is electrically positioned between capillary 1808 and the diethylamine in chamber 1826 (see Fig. 14B ), so the ESI tip voltage (V 1824 ) will drop according to Equation 3:

[0194] V 1824 =ΔV 1806-1814 *R 1826 / (R 1808 +R 1826)+V 1814

[0195] Additionally, as the resistance in the capillary 1808 increases, the current through the capillary will decrease, which can be measured at the power supply 1802. The increased current will be directly related to the change in resistance of the capillary 1808 via Equation 4:

[0196] I 1806 =ΔV 1806-1814 / (R 1808 +R 1826 )

[0197] Use as Fig.12 Using the computer-controlled feedback loop described in , the system can calculate the change in resistance in capillary 1808 (and therefore, the change in voltage drop across capillary 1808, which defines the voltage at ESI tip 1824), and the system can adjust power supplies 1802 and 1810 to maintain ΔV at 26 kV and maintain the ESI tip voltage at 4000 kV.

[0198] After focusing is complete (~30 minutes), the mobilizing agent solution in the pressurized mobilizing agent vial 1818 will have replaced the diethylamine in the junction nebulizer chamber 1826, thereby initiating migration of the NIST mAb protein isoforms in the capillary 1808. In a similar manner, but in contrast to isoelectric focusing, as migration proceeds, the resistance in the capillary 1808 will drop, affecting the voltage at the ESI tip 1824. The computer controlled feedback loop will again use Equations 3 and 4 to calculate the necessary changes in power supplies 1802 and 1810 to maintain an electric field of 26 kV while maintaining the ESI tip 1824 voltage at 4 kV.

[0199] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate many variations, changes and substitutions. It should be understood that in practicing the present invention, various alternatives of the embodiments of the present invention described herein may be used in any combination. The appended claims are intended to define the scope of the present invention, and thus encompass methods and structures within the scope of these claims and their equivalents.

[0200] The present invention also provides the following items:

[0201] 1. A method for maintaining an electrospray ionization (ESI) tip at a constant voltage relative to ground while performing a separation reaction, the method comprising:

[0202] 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;

[0203] b) applying a second voltage to the proximal end of the auxiliary fluid channel, wherein the distal end of the auxiliary fluid channel is in fluid and electrical communication with the distal end of the separation channel;

[0204] c) performing the separation reaction to separate the mixture of analytes, wherein the separation reaction is performed in the separation channel; and

[0205] d) monitoring a change in resistance of the separation channel or a change in voltage at the ESI tip in a feedback loop that adjusts the first voltage and the second voltage to maintain a constant voltage drop across the separation channel and a constant voltage at the ESI tip.

[0206] 2. The method according to item 1, wherein the separation channel is the inner lumen of a capillary.

[0207] 3. The method of claim 2, wherein the capillary comprises a micro-bottle spray tip.

[0208] 4. The method according to item 1, wherein the separation channel is a fluid channel within a microfluidic device.

[0209] 5. The method according to any one of items 1 to 4, wherein the separation reaction comprises an isoelectric focusing reaction.

[0210] 6. The method according to any one of items 1 to 4, wherein the separation reaction comprises an electrophoretic separation reaction.

[0211] 7. The method according to any one of items 1 to 6, wherein the first voltage is applied to the cathode and the second voltage is applied to the anode.

[0212] 8. A method according to any one of items 1 to 7, wherein the voltage at the ESI tip is maintained at ground.

[0213] 9. The method of any one of items 1 to 7, wherein the voltage at the ESI tip is maintained at the second voltage.

[0214] 10. The method of any one of items 1 to 9, wherein the adjusting of the first voltage and the second voltage comprises subtracting a transient voltage change measured at the ESI tip from the first voltage and the second voltage.

[0215] 11. The method of any one of items 1 to 10, wherein the voltage at the ESI tip is measured using a power supply that provides the first voltage or the second voltage.

[0216] 12. A method according to any one of items 1 to 11, wherein the feedback loop operates at a frequency of at least 0.1 Hz.

[0217] 13. A method according to any one of items 1 to 11, wherein the feedback loop operates at a frequency of at least 10 Hz.

[0218] 14. A method according to any one of items 1 to 13, wherein the feedback loop maintains the voltage at the ESI tip within ±10% of a preset value.

[0219] 15. The method of any one of items 1 to 13, wherein the feedback loop maintains the voltage at the ESI tip within ±1% of a preset value.

[0220] 16. A method according to any one of items 1 to 15, wherein the feedback loop maintains the voltage drop across the separation channel within ±10% of a preset value.

[0221] 17. A method according to any one of items 1 to 15, wherein the feedback loop maintains the voltage drop across the separation channel within ±1% of a preset value.

[0222] 18. A method comprising:

[0223] a) providing a sample comprising a mixture of two or more analytes;

[0224] b) performing separation within a fluid channel containing said sample to resolve individual analyte peaks from said mixture of two or more analytes;

[0225] c) calculating the velocity of the analyte peak as the contents of the fluid channel migrate toward an outlet of the fluid channel; and

[0226] d) using the velocity of the analyte peak to determine the time at which the analyte peak arrives at the fluid channel outlet.

[0227] 19. The method according to item 18, wherein the fluid channel is the lumen of a capillary tube.

[0228] 20. The method according to item 18, wherein the fluid channel is part of a microfluidic device.

[0229] 21. A method according to any one of items 18 to 20, wherein the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP) or micellar electrokinetic chromatography (MEKC).

[0230] 22. The method according to any one of items 18 to 21, wherein the velocity of the analyte peak is calculated based on the time interval required for the analyte peak to move from a first position to a second position.

[0231] 23. A method according to item 22, wherein the first position, the second position and the time interval are determined based on a series of two or more images of the fluid channel.

[0232] 24. The method according to item 23, wherein the series of two or more images comprises an ultraviolet absorbance image, a visible light absorbance image or a fluorescence image.

[0233] 25. A method according to any one of items 18 to 24, wherein the fluid channel outlet comprises an electrospray interface docked with a mass spectrometer.

[0234] 26. A method according to any one of items 18 to 25, wherein the time at which the analyte peak arrives at the fluid channel outlet is used to correlate mass spectrometer data with the analyte peak.

[0235] 27. A method according to any one of items 18 to 26, wherein the migration of the contents of the fluid channel comprises the use of electroosmotic migration technology, chemical migration technology, hydrodynamic migration technology or any combination thereof.

[0236] 28. A method according to any one of items 18 to 27, wherein the 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.

[0237] 29. A method according to any one of items 26 to 28, wherein comparison of mass spectrometer data collected for samples of the biological candidate drug and the reference drug is used to determine biosimilarity.

[0238] 30. A method according to any one of items 18 to 29, wherein the velocity of the analyte peak is used in a feedback loop to adjust a control parameter of the separation or migration of the analyte peak.

[0239] 31. The method according to item 30, wherein the control parameter is voltage.

[0240] 32. A method according to item 30 or 31, wherein the feedback loop operates at a frequency of at least 0.1 Hz.

[0241] 33. A method comprising:

[0242] a) providing a sample comprising a mixture of two or more analytes;

[0243] b) performing separation within a fluid channel containing said sample to resolve individual analyte peaks from said mixture of two or more analytes; and

[0244] c) collecting mass spectrometer data of the two or more individual analyte peaks emitted from the fluid channel via an electrospray interface docked with a mass spectrometer; wherein a data collection mode for the mass spectrometer alternates between high mass scans and low mass scans.

[0245] 34. A method according to item 33, wherein the mass spectrometer switches between the high mass scan and low mass scan data collection modes at a frequency of at least 0.5 Hz.

[0246] 35. The method according to item 33 or 34, wherein the fluid channel is the lumen of a capillary tube.

[0247] 36. A method according to item 33 or 34, wherein the fluid channel is part of a microfluidic device.

[0248] 37. A method according to any one of items 33 to 36, wherein the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP) or micellar electrokinetic chromatography (MEKC).

[0249] 38. A method according to any one of items 33 to 37, wherein the high mass scan captures mass spectrometric data of biological macromolecules.

[0250] 39. The method according to item 38, wherein the biomacromolecule comprises a protein, a protein-drug conjugate, a nucleic acid molecule, a reduced protein, a fusion protein, a protein complex or any combination thereof.

[0251] 40. A method according to any one of items 33 to 39, wherein the m / z ratio of the high mass scan is in the range of 1500 to 6000.

[0252] 41. A method according to any one of items 33 to 40, wherein the low mass scan captures mass spectrometric data of solution phase ampholytes used for isoelectric focusing separation.

[0253] 42. A method according to any one of items 33 to 41, wherein the m / z ratio of the low mass scan is in the range of 150 to 1500.

[0254] 43. A method according to any one of items 41 to 42, wherein the mass spectrum of one or more solution-phase ampholytes is used to calibrate the isoelectric points (pIs) of biomacromolecules identified in the high mass scan.

[0255] 44. A method comprising:

[0256] a) performing a separation in 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 two or more analytes;

[0257] b) causing the contents of the fluid channel to migrate toward a fluid channel outlet, wherein the fluid channel outlet comprises an electrospray interface that interfaces with a mass spectrometer; and

[0258] c) simultaneously or alternately imaging: (i) at least a portion of the fluid channel to monitor the position of the analyte peak during (a) and (b), and (ii) a Taylor cone between the fluid channel outlet and the inlet of the mass spectrometer to monitor electrospray performance.

[0259] 45. A method according to item 44, wherein 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.

[0260] 46. ​​A method according to item 45, wherein the velocity of the analyte peak is used to determine the time when the analyte peak will reach the outlet of the fluid channel.

[0261] 47. A method according to item 46, wherein the time when the analyte peak arrives at the fluid channel outlet is used to associate mass spectrometer data with the analyte peak.

[0262] 48. A method according to any one of items 44 to 47, wherein data derived from the imaging of the Taylor cone is used in a feedback loop to adjust electrospray performance.

[0263] 49. A method according to item 48, wherein the feedback loop operates at a frequency of at least 0.1 Hz.

[0264] 50. The method according to any one of items 44 to 49, wherein the fluid channel is the lumen of a capillary.

[0265] 51. A method according to any one of items 44 to 49, wherein the fluid channel is part of a microfluidic device.

[0266] 52. A method according to any one of items 44 to 51, wherein the separation is based on isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP) or micellar electrokinetic chromatography (MEKC).

[0267] 53. The method according to any one of items 44 to 52, wherein the imaging comprises ultraviolet light absorbance imaging, visible light absorbance imaging or fluorescence imaging.

[0268] 54. A method according to any one of items 44 to 53, wherein the migration of the contents of the fluid channel comprises the use of electroosmotic migration technology, chemical migration technology, hydrodynamic migration technology or any combination thereof.

[0269] 55. A method according to any one of items 44 to 54, wherein the 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.

[0270] 56. A computer-implemented method for maintaining an electrospray ionization (ESI) tip at a constant voltage relative to ground while performing a separation reaction, the method comprising:

[0271] a) receiving, with a processor, a first measurement of a voltage at the ESI tip, wherein a distal end of a separation channel is in fluid and electrical communication with the ESI tip;

[0272] b) receiving, with the processor, a second measurement of the voltage at the ESI tip;

[0273] c) comparing, using the processor, the second measurement to the first measurement, wherein if the second measurement is different from the first measurement, the processor causes an adjustment to a voltage at a proximal end of the separation channel and a voltage at a proximal end of an auxiliary fluid channel such that the voltage at the ESI tip returns to the first measurement, wherein the auxiliary fluid channel includes a distal end in fluidic and electrical communication with the distal end of the separation channel; and

[0274] d) Repeat steps (a) to (c) at a specified frequency.

[0275] 57. A computer-implemented method according to item 56, wherein the separation channel comprises the inner cavity of a capillary or a fluid channel within a microfluidic device.

[0276] 58. A computer-implemented method according to item 56 or 57, wherein the separation reaction comprises an isoelectric focusing reaction.

[0277] 59. A computer-implemented method according to item 56 or 57, wherein the separation reaction comprises an electrophoretic separation reaction.

[0278] 60. A computer-implemented method according to any one of items 56 to 59, wherein the voltage at the ESI tip is maintained at ground.

[0279] 61. A computer-implemented method according to any one of items 56 to 60, wherein the specified frequency is at least 1 Hz.

[0280] 62. A computer-implemented method according to any of items 56 to 61, wherein the voltage at the ESI tip is maintained within ±5% of a specified value.

[0281] 63. A computer-implemented method comprising:

[0282] a) receiving, using a processor, image data comprising two or more images acquired using a detector configured to image all or a portion of a separation channel in a capillary or microfluidic device;

[0283] b) processing the image data using the same or a different processor to determine the position of an analyte peak within the separation channel in the two or more images;

[0284] c) using the same or a different processor, calculating a 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 acquisition of the two or more images; and

[0285] d) using the same or a different processor to determine when the analyte peak will arrive at the exit of the separation channel.

[0286] 64. A computer-implemented method according to item 63, wherein the separation reaction performed in the separation channel includes isoelectric focusing (IEF), capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isotachophoresis (CITP) or micellar electrokinetic chromatography (MEKC).

[0287] 65. A computer-implemented method according to item 63 or item 64, wherein the two or more images include ultraviolet absorbance images, visible light absorbance images, or fluorescence images.

[0288] 66. A computer-implemented method according to any one of items 63 to 65, wherein the separation channel outlet is fluidically connected to or includes an electrospray interface docked with a mass spectrometer.

[0289] 67. A computer-implemented method according to any one of items 63 to 66, wherein the time at which the analyte peak arrives at the separation channel outlet is used to associate mass spectrometer data with the analyte peak.

[0290] 68. A computer-implemented method according to any one of items 63 to 67, wherein the analyte is separated from a mixture and comprises a protein, a protein-drug conjugate, a peptide, a nucleic acid molecule, a carbohydrate molecule, a lipid molecule, a metabolite molecule or a small organic compound.

[0291] 69. A computer-implemented method according to any one of items 63 to 68, wherein comparison of mass spectrometer data collected for samples of the biological candidate drug and the reference drug is used to determine biosimilarity.

[0292] 70. A computer-implemented method according to any one of items 63 to 69, wherein the velocity of the analyte peak is used in a feedback loop to adjust control parameters of a separation reaction performed in the separation channel.

[0293] 71. The computer-implemented method of item 70, wherein the control parameter is voltage.

[0294] 72. A computer-implemented method according to item 70 or 71, wherein the feedback loop operates at a frequency of at least 0.1 Hz.

Claims

1. A method for maintaining an electrospray ionization tip at a constant voltage relative to ground while performing 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 electrospray ionization 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 the separation reaction to separate the mixture of analytes, wherein the separation reaction is performed in the separation channel; as well as d) monitoring a change in resistance of the separation channel or a change in voltage at the electrospray ionization tip in a feedback loop that adjusts the first voltage and the second voltage to maintain a constant voltage drop across the separation channel and a constant voltage at the electrospray ionization tip. The method according to claim 1 , wherein the separation channel is the lumen of a capillary tube.

3. The method of claim 2, wherein the capillary tube comprises a micro-vial spray tip. The method of claim 1 , wherein the separation channel is a fluid channel within a microfluidic device.

5. The method according to any one of claims 1 to 4, wherein the separation reaction comprises an isoelectric focusing reaction.

6. The method according to any one of claims 1 to 4, wherein the separation reaction comprises an electrophoretic separation reaction.

7. The method according to any one of claims 1 to 4, wherein the first voltage is applied to a cathode, and the second voltage is applied to an anode.

8. The method of any one of claims 1 to 4, wherein the voltage at the electrospray ionization tip is maintained at ground.

9. The method of any one of claims 1 to 4, wherein the voltage at the electrospray ionization tip is maintained at the second voltage.

10. The method of any one of claims 1 to 4, wherein the adjusting of the first voltage and the second voltage comprises subtracting a transient voltage change measured at the electrospray ionization tip from the first voltage and the second voltage.

11. The method according to any one of claims 1 to 4, wherein the voltage at the electrospray ionization tip is measured using a power supply that provides the first voltage or the second voltage.

12. The method of any one of claims 1 to 4, wherein the feedback loop operates at a frequency of at least 0.1 Hz.

13. The method of any one of claims 1 to 4, wherein the feedback loop operates at a frequency of at least 10 Hz.

14. The method of any one of claims 1 to 4, wherein the feedback loop maintains the voltage at the electrospray ionization tip within ±10% of a preset value.

15. The method of any one of claims 1 to 4, wherein the feedback loop maintains the voltage at the electrospray ionization tip within ±1% of a preset value.

16. The method of any one of claims 1 to 4, wherein the feedback loop maintains the voltage drop across the separation channel within ±10% of a preset value.

17. The method of any one of claims 1 to 4, wherein the feedback loop maintains the voltage drop across the separation channel within ±1% of a preset value.

18. A computer-implemented method for maintaining an electrospray ionization tip at a constant voltage relative to ground while performing a separation reaction, the method comprising: a) receiving, with a processor, a first measurement of a voltage at the electrospray ionization tip, wherein a distal end of a separation channel is in fluid and electrical communication with the electrospray ionization tip; b) receiving, with the processor, a second measurement of the voltage at the electrospray ionization tip; c) comparing the second measurement with the first measurement using the processor, wherein if the second measurement is different from the first measurement, the processor causes an adjustment to a voltage at a proximal end of the separation channel and a voltage at a proximal end of an auxiliary fluid channel so that the voltage at the electrospray ionization tip returns to the first measurement, wherein the auxiliary fluid channel includes a distal end in fluidic and electrical communication with the distal end of the separation channel; and d) Repeat steps (a) to (c) at a specified frequency.

19. The computer-implemented method of claim 18, wherein the separation channel comprises a lumen of a capillary or a fluid channel within a microfluidic device.

20. The computer-implemented method of claim 18 or 19, wherein the separation reaction comprises an isoelectric focusing reaction.

21. The computer-implemented method of claim 18 or 19, wherein the separation reaction comprises an electrophoretic separation reaction.

22. The computer-implemented method of claim 18 or 19, wherein the voltage at the electrospray ionization tip is maintained at ground.

23. The computer-implemented method of claim 18 or 19, wherein the specified frequency is at least 1 Hz.

24. The computer-implemented method of claim 18 or 19, wherein the voltage at the electrospray ionization tip is maintained within ±5% of a specified value.

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