EP-4396570-B1 - SYSTEMS AND METHODS FOR FRACTIONATION AND COLLECTION OF ANALYTES IN A SAMPLE

EP4396570B1EP 4396570 B1EP4396570 B1EP 4396570B1EP-4396570-B1

Inventors

ZHOU, CHENG
XU, HUI
Wang, Roland Y.
ROACH, DAVID J.
DERMODY, JESSICA
YANG, TOM WEISAN
WU, JIAQI

Dates

Publication Date: 20260506
Application Date: 20220822

Claims (18)

A method (800), comprising: introducing (871), at a first time, a sample containing a plurality of analytes in a conductive medium into a capillary, ionically coupling a first end of the capillary to a first running buffer having a first pH; ionically coupling (872) a second end of the capillary to a second running buffer having a second pH, such that a pH gradient forms along the capillary; separating (873), at a second time after the first time, at least a subset of the plurality analytes according to their isoelectric points by applying a voltage across the first running buffer and the second running buffer when the first end of the capillary is ionically coupled to the first running buffer and the second end of the capillary is ionically coupled to the second running buffer; the method being characterized by : placing (874) the second end of the capillary into a well including a chemical mobilizer at a third time after the first time to elute an analyte from the plurality of analytes from the capillary and into the well, the well being from a plurality of wells; and applying (875) a second voltage across the first running buffer and the chemical mobilizer at the third time and when the first end of the capillary is ionically coupled to the first running buffer and the second end of the capillary is disposed in the well, the second voltage and the chemical mobilizer collectively causing the analyte to be eluted into the well.
The method of claim 1, wherein a duration for which the second voltage is applied is based on a speed of mobilization associated with the analyte.
The method of claim 1, wherein the well is a first well, the chemical mobilizer is a first chemical mobilizer, and the analyte is a first analyte the method further comprising: placing the second end of the capillary into a second well of the plurality of wells including a second chemical mobilizer at a fourth time after the third time to elute an analyte from the plurality of analytes from the capillary and into the well; and applying a third voltage across the first running buffer and the second chemical mobilizer when the second end of the capillary is disposed in the second well, the third voltage and the second chemical mobilizer collectively causing the second analyte to be eluted into the second well.
The method of claim 3, wherein the first chemical mobilizer and the second chemical mobilizer are different.
The method of claim 3, wherein the first chemical mobilizer and the second chemical mobilizer the same chemical mobilizer.
The method of claim 1, wherein: the chemical mobilizer has a third pH different from the first pH and the second pH, such that when the second end of the capillary is disposed in the well the pH gradient is disrupted.
The method of claim 5, wherein the first running buffer has a first composition, the second running buffer has a second composition, and the chemical mobilizer has a third composition, the third pH is the same as the first pH or the second pH, and the third composition being different from at least one of the first composition or the second composition.
An apparatus, comprising: a capillary (106) configured to electrophoretically focus an analyte contained within a sample; a running buffer reservoir (105) configured to contain a first running buffer having a first pH in ionic communication with a first end of the capillary (106); a sample plate defining a plurality of wells, the sample plate and the capillary (106) collectively configured such that a second end of the capillary (106) can move between each well from the plurality of wells; a first well from the plurality of wells configured to contain the sample, the capillary and the sample plate collectively configured such that, when a second end of the capillary is disposed in the first well, a portion of the sample can be drawn into the capillary; a second well from the plurality of wells configured to contain a second running buffer having a second pH different from the first pH such that when the second end of the capillary is disposed in the second well, the buffer reservoir (105) containing the first running buffer and the second well containing the second running buffer are in ionic communication causing a pH gradient to be established across the capillary; an electrical power source configured to apply a first voltage across the running buffer reservoir (105) and the second running buffer such that, when the second end of the capillary (106) is disposed in the second well and the first voltage is applied, the analyte migrates to a portion of the capillary associated with its isoelectric point; the apparatus being characterized by a third well from the plurality of wells configured to contain a chemical mobilizer, and the electrical power source configured to apply a second voltage, such that when the second end of the capillary (106) is disposed in the third well and the second voltage is applied, the analyte migrates into the third well from the portion of the capillary (106) associated with its isoelectric point when the capillary (106) is disposed in the second well.
The apparatus of claim 8, further comprising: a metal tip disposed over the second end of the capillary and configured to be disposed in at least one of the first well, the second well, or the third well when the second end of the capillary is disposed in at least one of the first well, the second well, or the third well, respectively, the metal tip configured to provide electrical connectivity between the second end of the capillary and the first end of the capillary.
The apparatus of claim 8, further comprising: a first electrode electrically coupled the running buffer reservoir and configured to be electrically coupled to the first running buffer; and a second electrode electrically coupled to the metal tip such that the first electrode, the second electrode, and a portion of the sample drawn into the capillary define a portion of an electrical circuit.
The apparatus of claim 8, wherein the capillary has an inner diameter of 320 - 530 µm.
The apparatus of claim 8, wherein the capillary has an inner diameter of 200 - 500 µm.
The apparatus of claim 8, wherein the capillary has a length of 60-120 mm.
The apparatus of claim 8, wherein the capillary has a length of 20-30cm.
The apparatus of claim 8, wherein there is no porous membrane tubing disposed on the second end of the capillary.
The apparatus of claim 8, wherein: the electrical power source is further configured to apply a second voltage across the running buffer reservoir and the chemical mobilizer when the second end of the capillary is disposed in the third well such that the analyte migrates into the third well from the portion of the capillary associated with its isoelectric point.
The apparatus of claim 8, wherein: the analyte is included in a first portion of the sample and is focused at a first distance from the second end of the capillary; the sample includes a plurality of portions including the first portion and a second portion focused at a second distance from the second end of the capillary, the second distance being greater than the first distance such that when the first voltage is applied, the first portion migrates to the first distance and the second portion migrates to the second distance; and the electrical power source is further configured to apply a second voltage across the running buffer reservoir and the chemical mobilizer when the second end of the capillary is disposed in the third well such that the first portion migrates into the third well from the first distance, a magnitude of the second voltage being determined based on a measure of separation between the first distance and the second distance.
The apparatus of claim 17, wherein the magnitude of the second voltage and the chemical mobilizer collectively cause the first portion to migrate towards the third well at a rate 1mm/min to 2mm/min when the second voltage is applied.

Description

Cross Reference to Related Application This application claims priority to and benefit of U.S. Patent Application No. 17/463,326, filed on August 31, 2021. Background The embodiments described herein relate generally to systems and methods for separating, analyzing, and eluting fractions from sample mixtures. Some embodiments described herein relate to separation and/or fractionation of an analyte or analytes present in a sample based on a property. More particularly, the embodiments described herein generally relate to systems and methods configured to perform capillary-based fractionation using electrophoretic methods of separation followed by elution. Summary Systems and methods for capillary electrophoresis, analyte visualization and fraction collection are described herein. The method of the invention includes introducing, at a first time, a sample containing a plurality of analytes in a conductive medium into a capillary. The method includes ionically coupling a first end of the capillary to a first running buffer having a first pH, and ionically coupling a second end of the capillary to a second running buffer having a second pH, such that a pH gradient forms along the capillary. The method further includes separating, at a second time after the first time, at least a subset of the plurality analytes according to their isoelectric points by applying a voltage across the first running buffer and the second running buffer when the first end of the capillary is ionically coupled to the first running buffer and the second end of the capillary is ionically coupled to the second running buffer. The method further includes detecting an analyte from the plurality of analytes separated along the pH gradient. The method further includes identifying a peak of a distribution associated with an amount of separated analyte along the pH gradient. The method further includes placing the second end of the capillary into a well including a chemical mobilizer at a third time after the first time to mobilize and selectively elute an analyte from the plurality of analytes from the capillary and into the well, based on the identifying the peak of the distribution associated with an amount of separated analyte. The method optionally includes monitoring a migration of the peak of distribution associated with the amount of separated analyte during elution to collect an individual fraction of the separated analyte. Brief Description of the Drawings FIG. 1 is a schematic illustration of a fractionation system, a system configured to perform capillary electrophoresis and/or fractionate analytes separated by capillary electrophoresis, according to an embodiment.FIG. 2 is a schematic of a side view of an example capillary cartridge that can be used with a fractionation system, according to an embodiment.FIGS. 3 is a schematic of a side view of an example capillary cartridge that can be used with a fractionation system, according to an embodiment.FIG. 4 is an image of a side view of a capillary cartridge that can be used with a fractionation system, according to an embodiment.FIG. 5 is a schematic of a side view of a portion of an example capillary cartridge that can be used with a fractionation system, according to an embodiment.FIG. 6 is a perspective view of a fractionation system, according to an embodiment.FIGS. 7A and 7B are perspective views of sample plate assemblies configured to be used with a fractionation system, according to two example embodiments.FIG.8 is a flowchart of a method of using a fractionation system, according to one implementation.FIGS. 9A, 9B, and 9C are plots of experimental data indicating separation of an analyte using a standard separation system, a fractionation system with a single segment capillary, and a fractionation system using a multi-segment capillary, respectively.FIG. 10 depicts experimental data indicating a mobilization of an analyte using a fractionation system.FIG. 11 depicts experimental data indicating a mobilization of an analyte using a fractionation system.FIG. 12 depicts experimental data indicating a separation and mobilization of analytes using a fractionation system.FIG. 13 depicts experimental data indicating fractions of a sample mixture collected using a fractionation system.FIG. 14 depicts experimental data indicating fractions of a sample mixture collected using a fractionation system.FIG. 15 depicts experimental data indicating fractions of a sample mixture collected using a fractionation system.FIG. 16 depicts experimental data of mass spectra obtained from fractions of a sample mixture collected using a fractionation system.FIG. 17 depicts experimental data indicating fractions of a sample mixture collected using a fractionation system.FIG. 18 depicts experimental data indicating fractions of a sample mixture collected using a fractionation system. Detailed Description The method of the invention includes introducing, at a first time, a sample containing a plurality o