Method for single-channel free-flow electrophoresis with sequential pH adjustment

Abstract

The embodiments described herein relate to a single-channel free-flow electrophoresis device or apparatus, and a method of separating and collecting an analyte of interest from a sample by sequentially adjusting the pH of an electrolyte buffer and separating the analyte of interest according to the corresponding isoelectric point of the analyte of interest. The method includes flowing the sample through a single central channel, applying an electric field perpendicular to the direction of sample flow through an anolyte channel and a catholyte channel parallel to the central channel, and then collecting fractions of the analyte of interest according to their respective isoelectric points.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 912,963, filed October 9, 2019, entitled "Methods for Single-Channel Free-Flow Electrophoresis with Sequential pH Adjustment", the entire disclosure of which is hereby incorporated by reference. Technical Field

[0003] Some embodiments described herein relate to devices and methods for separating and collecting protein samples. Background Art

[0004] Electrophoresis, including isoelectric focusing (IEF), is a common technique for protein separation. IEF is an electrophoretic technique for separating proteins and other amphoteric solutes according to their isoelectric points (pI) in a pH gradient. Synthetic carrier ampholytes are small amphoteric molecules that rapidly establish a pH gradient after the application of an electric field. Once the pH gradient is established, slower-moving proteins and other amphoteric molecules will concentrate and condense at their pI. IEF can be performed at the preparative and analytical levels. Due to the inability of preparative-scale devices to effectively dissipate Joule heat and keep convective mixing to a minimum, preparative-scale IEF devices typically lag behind their analytical counterparts. Additionally, since the electrolyte solutions are incompatible with common downstream analyses such as mass spectrometry, subsequent purification steps may be required. As a result, commercially available preparative-scale IEF devices suffer from low throughput and suboptimal performance.

[0005] Free-flow electrophoresis (FFE) is a technique similar to capillary electrophoresis, with comparable resolution, where semi-preparative and preparative amounts of samples can be generated. Two typical separation modes of FFE are zone electrophoresis (ZE) and IEF. In an FFE system, sample separation and collection are continuous processes. Although FFE systems have some advantages in throughput and resolution over preparative-scale capillary IEF devices, such FFE systems are expensive to operate because they consume large amounts of reagents, such as ampholytes, during operation. In addition, known FFE instrument setups and maintenance are cumbersome, and bubbles are a source of non-reproducibility.

[0006] In view of the severe drawbacks of current commercial products, the present disclosure describes devices and methods for single-channel free-flow electrophoresis that maintain the continuous-flow characteristics for sample separation and collection. The devices described herein are operable to separate analytes with a specific pI and have medium to high throughput for sample preparation. Compared to known FFE devices, the single-channel devices described herein generally allow for much lower reagent consumption and have a simpler setup and operation. Additionally, unlike known FFE devices, some embodiments described herein do not require ampholytes. Summary of the Invention

[0007] Some embodiments described herein relate to devices and methods for collecting and separating samples containing biological materials or analytes such as proteins.

[0008] Some embodiments described herein relate to an instrument configured to electrophoretically fractionate a sample of an analyte-containing mixture as the sample flows hydrodynamically through a central channel. The device can be configured to receive the sample via an inlet and discharge at least a portion of the sample (e.g., the fractionated analyte of interest) via an outlet. The device can be configured to operate with a continuous flow of the sample such that the sample is electrophoretically fractionated while the sample moves hydrodynamically through the central channel. Anolyte channels and catholyte channels can be provided parallel to and on opposite sides of the central channel. The anolyte channels and catholyte channels can be configured to be filled with an electrolyte and connected to an anode and a cathode, respectively. A hydrodynamic barrier such as a porous membrane can be provided between the central channel and at least one of the anolyte channels and catholyte channels. When energized (i.e., when a potential is applied to the anode and cathode), the anolyte and catholyte channels can jointly induce an electric field oriented perpendicular to the central channel. As discussed further in detail below, analytes having a pI with a pH different from that of the sample buffer and / or electrolyte buffer can migrate into or through the hydrodynamic barrier and out of the central channel in the direction of the electric field (perpendicular to the direction of the hydrodynamic flow). Thus, sample fractions that do not have a pI matching the pH of the sample buffer and / or electrolyte buffer can be removed from the overall flow of the sample, and fractions containing enriched fractions (in some cases, substantially pure fractions) of one or more analytes having a pI matching the pH of the sample buffer and / or electrolyte buffer can exit the central channel via the outlet. As discussed further in detail herein, specific analytes of interest can be purified by controlling the pH of the sample and / or electrolyte buffer.

[0009] In some embodiments, the body of the device can define an inlet configured to receive a sample of an analyte-containing mixture. In some embodiments, the analyte-containing mixture can include proteins. The body of the device can define an outlet configured to discharge a fractionated portion of the sample (e.g., containing the enriched or substantially pure analyte of interest). The body of the device can define a catholyte channel configured to be coupled to a cathode and an anolyte channel configured to be coupled to an anode. The device can include a lid and a hydrodynamic barrier (e.g., made of cellulose, polyvinylidene fluoride, polyvinylidene difluoride, polytetrafluoroethylene, or any other suitable material) disposed between the lid and the body. The hydrodynamic barrier, the body, and the lid can jointly form a central channel between the inlet and the outlet that is parallel to the catholyte channel and the anolyte channel. In some embodiments, the central channel can be partially defined by a hollow space or opening in the hydrodynamic barrier.

[0010] In some embodiments, at least one of the catholyte channel or the anolyte channel can be fluidly connected to a reservoir configured to contain an electrolyte buffer. For example, the reservoir can contain a MES-BisTris buffer. In some cases, the electrolyte buffer can contain one or more polymers, such as methylcellulose (e.g., 0.1% to 0.5% by weight). Such reservoirs can have a volume of 100 mL to 500 mL. A pump can be configured to recirculate the electrolyte solution from the reservoir through the catholyte channel and / or the anolyte channel via a separate loop. In other embodiments, the pump can be configured to recirculate the electrolyte buffer from the reservoir first through the anolyte channel and then through the catholyte channel (or vice versa) before returning to the reservoir.

[0011] In some embodiments, the device can include an anode and a cathode. The anode and the cathode can be electrically coupled to the anolyte channel and the catholyte channel, respectively, such that when powered, the anolyte channel and the catholyte channel together apply an electric field across and perpendicular to the central channel.

[0012] In some embodiments, the body of the device as described herein can define an inlet and an outlet of the catholyte channel. In one embodiment, the body can be plastic and / or substantially waterproof.

[0013] In some embodiments, the analyte mixture can include peptides having an isoelectric point (pI) of 1 to 11. As discussed in further detail herein, the electrolyte buffer and / or the sample buffer can be configured to fractionate the analytes of the sample such that one or more analytes of interest are selectively enriched or purified based on their pI points. Thus, in some embodiments, the electrolyte buffer can have a pH value of 0.1 to 14 such that analytes having corresponding pI values within this range can be selectively enriched or purified.

[0014] In some embodiments, the pores of the hydrodynamic barrier can have a median characteristic length (e.g., diameter) of 25 nm to 800 nm. In some embodiments, the hydrodynamic barrier can have a thickness of 100 μm to 200 μm. In some embodiments, the central channel can have a width of 1 mm to 10 mm. In some embodiments, the central channel can have a length of 10 cm to 20 cm.

[0015] Some embodiments described herein relate to methods for fractionating an analyte mixture. A sample can flow through a central channel of a single-channel free-flow electrophoresis device. An electric field can be applied perpendicular to the flow direction of the sample via an anolyte channel and a catholyte channel containing an electrolyte buffer and parallel to the central channel. The central channel can be electrically coupled and / or ionically coupled, but is fluidically isolated from at least one of the anolyte channel and the catholyte channel by a hydrodynamic barrier. Analytes of interest can be separated from the sample based on the isoelectric point of the analyte of interest and the pH of the electrolyte buffer and / or sample buffer. A sample fraction containing the analyte of interest can be separated from the analyte mixture and collected. The fraction can contain an enriched or substantially pure analyte of interest.

[0016] In some embodiments, the method can include circulating the electrolyte buffer from a reservoir and through the anolyte channel and / or the catholyte channel. In some embodiments, the method can include applying a voltage across the anolyte channel and the catholyte channel to generate an electric field. In some embodiments, the method can include circulating the electrolyte buffer from a reservoir such that the electrolyte buffer can flow through the anolyte channel and the catholyte channel before returning to the reservoir. In some embodiments, the method as described herein can include circulating the electrolyte buffer from a reservoir such that the electrolyte buffer can flow through the anolyte channel and the catholyte channel in two separate loops.

[0017] In some embodiments, a sample fraction used in the method as described herein can contain an analyte of interest collected at a rate of 5 μL / minute to 15 μL / minute. In some embodiments, the pH value of the electrolyte buffer can be adjusted sequentially. In some embodiments, the pH value of the electrolyte buffer can be adjusted sequentially by modifying the ratio of MES and BisTris. In some embodiments, the pH value of the electrolyte buffer can be the same as the sample buffer contained in the sample.

[0018] In some embodiments, the pH value of the electrolyte buffer and / or the sample can be adjusted sequentially, for example, by a metering pump or a valve. Thus, multiple analytes having different isoelectric points can be sequentially separated based on the pH of the electrolyte buffer and / or the sample buffer during the time the fraction flows through the central channel and / or is collected. In some embodiments, the analyte of interest can be collected at a constant rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A and 1B Illustrates the assembly of a single-channel free-flow electrophoresis device according to an embodiment. Figure 1A Shows the assembled device. Figure 1BShows an exploded view of the device, which shows three components: a body typically made of plastic, an intermediate section made of a porous membrane material, and a cap located at the bottom of the device typically made of glass or metal.

[0020] Figure 2A and 2B Shows a recirculation scheme for the electrolyte buffer of a single-channel free-flow electrophoresis device according to an embodiment. Figure 2A Shows an embodiment of recirculation with a single loop. Figure 2B Shows an embodiment of recirculation with two separate loops.

[0021] Figures 3A - 3C Shows a fractionation example of a peptide mixture with a pI range of 3.4 to 10.1. In this example, the buffer system is based on MES-BisTris. The buffer pH is adjusted by changing the ratio of MES and BisTris. Figure 3A Shows the fractionation of a peptide mixture with a buffer pH of 5.8. Figure 3B Shows the fractionation of a peptide mixture with a buffer pH of 6.3. Figure 3C Shows the fractionation of a peptide mixture with a buffer pH of 6.7.

[0022] Figure 4A and 4B Shows a fractionation example of a peptide mixture containing acidic IgG molecules. In this example, the buffer system is based on MES-BisTris. Figure 4A Shows the fractionation of a peptide mixture with a buffer pH of 6.3. Figure 4B Shows the fractionation of a peptide mixture with a buffer pH of 6.5.

[0023] Figure 5 Shows a fractionation example of the basic protein Herceptin. 2-Amino-2-methyl-1,3-propanediol (AMPD) is used as the buffer, and the pH varies from 8.8 to 9.4.

[0024] Figure 6 Is a flowchart of a method for sequentially separating one or more proteins of interest and an analyte mixture according to their isoelectric points according to an embodiment.

[0025] Figure 7 Shows an exploded view of a device with at least six components according to an embodiment: a top cover, a spacer, a bottom cover, two buffer tanks and electrodes, and two diaphragms. Detailed Description

[0026] Although various embodiments of the present disclosure 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. Many variations, substitutions, and alternatives will be apparent to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed.

[0027] As used in this specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the term "member" is intended to mean a single member or a combination of members, and the term "material" is intended to mean one or more materials or combinations thereof.

[0028] As used herein, the term "protein" refers to proteins, oligopeptides, peptides, and analogs, including proteins and peptidomimetics containing non-naturally occurring amino acids and amino acid analogs. The term "protein" also refers to proteins, oligopeptides, peptides, and analogs having various isoelectric points.

[0029] As used herein, the term "analyte" refers to any molecule or compound to be detected or separated as described herein. Suitable analytes can include, but are not limited to, small chemical molecules such as, for example, environmental molecules, clinical molecules, chemicals, contaminants, and / or biomolecules. More specifically, such chemical molecules can include, but are not limited to, pesticides, insecticides, toxins, therapeutic and / or abused drugs, hormones, antibiotics, antibodies, organic materials, proteins (e.g., enzymes, immunoglobulins, and / or glycoproteins), nucleic acids (e.g., DNA and / or RNA), lipids, lectins, carbohydrates, whole cells (e.g., prokaryotic cells such as pathogenic bacteria and / or eukaryotic cells such as mammalian tumor cells), viruses, spores, polysaccharides, glycoproteins, metabolites, cofactors, nucleotides, polynucleotides, transition state analogs, inhibitors, nutrients, electrolytes, growth factors, and other biomolecules and / or non-biological molecules, as well as fragments and combinations thereof. Some analytes described herein can be proteins, such as enzymes, drugs, cells, antibodies, antigens, cell membrane antigens, and / or receptors or their ligands (e.g., neurotransmitter receptors or their ligands, hormone receptors or their ligands, nutrient receptors or their ligands, and / or cell surface receptors or their ligands).

[0030] As used herein, the term "catholyte" can refer to the electrolyte on the cathode side of an electrophoresis device. As used herein, the term "anolyte" can refer to the electrolyte on the anode side of an electrophoresis device. In some embodiments, a common electrolyte is used on both sides of the electrophoresis device.

[0031] As used herein, the term "sample" refers to a composition containing one or more analytes to be detected or separated. A sample can be heterogeneous, containing various components (e.g., different proteins) or homogeneous, containing one component. In some cases, a sample can be a naturally occurring biological material and / or an artificial material. Additionally, a sample can be in its native or denatured form. In some cases, a sample can be a single cell (or single cell contents) or multiple cells (or multiple cell contents), a blood sample, a tissue sample, a skin sample, a urine sample, a water sample, and / or a soil sample. In some cases, a sample can be from a living organism, such as a eukaryote, a prokaryote, a mammal, a human, a yeast, and / or a bacterium, or a sample can be from a virus. In some cases, a sample can be one or more stem cells (e.g., any cell that can divide for an indefinite period of time and give rise to a specific cell). Suitable examples of stem cells can include, but are not limited to, embryonic stem cells (e.g., human embryonic stem cells (hES)) and non-embryonic stem cells (e.g., mesenchymal, hematopoietic, induced pluripotent stem cells (iPS cells), or adult stem cells (MSC)).

[0032] The apparatuses and methods of the present disclosure generally relate to separating and collecting analytes of interest based on their isoelectric point (pI) contained in a sample. In some embodiments, various analytes of interest can be separated and collected sequentially. As described herein, a single-channel free-flow electrophoresis system allows a protein sample to be mixed with a pH-controlled buffer and continuously flow into a channel while an electric field is applied that is not parallel to the direction of flow. The electric field causes charged analytes to migrate along the direction of the electric field or in a direction opposite to the direction of the electric field, such that the charged analytes move away from the direction of the hydrodynamic flow and, in some cases, out of the central channel, separating them from uncharged and / or less charged analytes. In some embodiments, the electric field is oriented perpendicular to the direction of the hydrodynamic flow, causing non-target analytes to migrate vertically out of the channel. In other embodiments, the electric field can have any suitable orientation that is not parallel to the direction of the hydrodynamic flow such that at least one component (e.g., vector component) of the electric field causes charged analytes to move in a direction perpendicular to the direction of the hydrodynamic flow. As used herein, features (e.g., the electric field and the central channel) are "perpendicular" when they are substantially perpendicular. As used herein, substantially perpendicular means features that are oriented at 90 degrees (plus or minus less than 5 degrees) to each other.

[0033] In some embodiments, the porous membrane is configured to form at least a portion of the channel. Accordingly, non-target analytes having a velocity vector in a direction not parallel to the channel (e.g., as induced by a non-parallel electric field) can leave the channel of the sample and the overall hydrodynamic flow and enter the porous membrane. In some such embodiments, the channel is partially defined by a hollow space in the center of the porous membrane. In some embodiments, the sidewalls of the channel can be defined by the porous membrane material through which buffer ions and proteins can migrate.

[0034] The present disclosure provides that proteins having a positive or negative charge based on their pI will be driven out of the channel by an electric field applied to the device or apparatus as described herein, depending on the background buffer pH. The device or apparatus can separate neutral molecules (e.g., analytes having a pI matching the pH of the background buffer) from their charged counterparts. Such neutral molecules can remain in the channel and flow into a collection vessel located at the end of the channel. The present disclosure provides that by sequentially changing the pH of the background buffer (e.g., sample buffer and / or electrolyte buffer), proteins with different pI values can be collected one at a time, resulting in protein fractionation depending on their charge.

[0035] Figure 1A and 1B Depicted is a single-channel free-flow electrophoresis device or apparatus according to an embodiment. The apparatus includes: (1) a body 150 (i.e., a top cover), (2) a porous membrane 160 (also referred to as a spacer), and (3) a bottom cover 170. The body defines two buffer channels 140 that are parallel to each other and are configured to be filled with an electrolyte buffer. Typically, one channel is configured to contain anolyte and the other channel is configured to contain catholyte. The body 150, the porous membrane 160, and the bottom cover 170 together define a central channel that is parallel to the two buffer channels 140 and between the two buffer channels 140. An inlet 120 allows a sample (typically containing a mixture of analytes) to enter the central channel, and an outlet 130 allows for the collection of sample fractions at the opposite side of the device. As used herein, a channel (or other feature) is "parallel" to another channel (or other feature) when the channel (or other feature) is substantially parallel to the other channel (or other feature). As used herein, substantially parallel means that the features are offset by less than 30 degrees, less than 10 degrees, or 0 degrees, including all ranges and sub-ranges therebetween.

[0036] The body 150 is typically made of a waterproof and non-conductive material, such as plastic (e.g., acrylic, polycarbonate, cycloolefin copolymer (COC), cycloolefin polymer (COP), polyethylene, or polystyrene), but can be made of any suitable material.

[0037] The porous membrane 160 is disposed between the body 150 and the bottom cover 170, and the bottom cover 170 and the body 150 together define a central channel. The body 150 defines the top of the central channel, while the bottom cover 170 defines the bottom of the central channel. The porous membrane 160 acts as a spacer between the body 150 and the bottom cover 170 such that the thickness of the porous membrane defines the height of the central channel. As Figure 1A and 1B shown, the hollow space or opening from the porous membrane 160 defines the length and width of the central channel.

[0038] The porous membrane 160 is configured to be wetted on opposite sides by the sample as it flows through the central channel and the electrolyte buffer as it flows through the buffer channel 140. The porous membrane 160 is configured to electro- and / or ion-couple the sample to the electrolyte buffer while preventing or impeding hydrodynamic flow from the central channel into the buffer channel 140.

[0039] The porous membrane 160 can be made of cellulose, polyvinylidene fluoride or polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE), or any other suitable material. The porous membrane 160 is generally configured to allow ions and / or analytes to migrate into / through the porous membrane 160 while preventing hydrodynamic fluid flow. As disclosed herein, "preventing or impeding hydrodynamic flow" or "fluid isolation" means reducing the volumetric flow rate by at least 95%, at least 99%, at least 99.9%, or at least 100% relative to the central channel, including all ranges and sub-ranges therebetween, on a volume basis, relative to the flow through the central channel.

[0040] Although the embodiments are generally described as containing the porous membrane 160, it should be understood that any suitable object or structure can be disposed between the central channel and at least one of the anode buffer channel and / or the cathode buffer channel. For example, a hydrodynamic barrier can be configured to electro- and / or ion-couple the sample to the electrolyte buffer while preventing or impeding hydrodynamic flow from the central channel into the buffer channel 140. For example, a gel or other material suitable for electrophoresis, a microchannel network, a nanochannel network, the porous membrane 160, and / or any other suitable structure or material can act as a hydrodynamic barrier and be disposed between the central channel and at least one of the anode buffer channel and the cathode buffer channel.

[0041] The bottom cover 170 can be made of a non-porous material. In some embodiments, the non-porous material can be glass. In some embodiments, the non-porous material can be aluminum. In some embodiments, the non-porous material is electrically insulating. In some embodiments, the non-porous material is non-conductive. In some embodiments, made of PTFE, A film made of PVDF or any other suitable insulating and / or hydrophobic material can be applied to the bottom cover 170 to prevent protein adsorption to the bottom cover 170 and provide electrical isolation if needed. In some embodiments, the film of insulating material can reduce electroosmotic flow. In some embodiments, the film of insulating material can reduce the magnitude of the ζ potential of the bottom cover 170. In some embodiments, the film of insulating and / or hydrophobic material can reduce or prevent protein or other analytes from adhering to the bottom cover 170. In some embodiments, the film of insulating material can be positioned between the bottom cover 170 and the porous membrane 160. In some embodiments, the film made of insulating material can be positioned between the bottom of the s body 150 and the porous membrane 160. In some embodiments, the film made of insulating material has a thickness of about 50μm, about 55μm, about 60μm, about 65μm, about 70μm, about 75μm, about 80μm, about 85μm, including all ranges and subranges therebetween. In some embodiments, the bottom cover 170 of the assembly device can be placed on top of a thermoelectric cooler or cold block that regulates temperature by recirculating a cryogenic coolant.

[0042] The cathode buffer channel is configured to be coupled to the cathode, and the anode buffer channel is configured to be coupled to the anode. In some embodiments, the device or apparatus can include an anode and a cathode. In some embodiments, the electrodes (i.e., the cathode and / or anode) can be made of platinum. In some embodiments, the electrodes can be made of copper. In some embodiments, the electrodes can be made of graphite. In some embodiments, the electrodes can be made of titanium. In some embodiments, the electrodes can be made of brass. In some embodiments, the electrodes can be made of silver. In some embodiments, the electrodes can be made of carbon fiber material. In some embodiments, the electrodes can be made of gold. In some embodiments, the electrodes can be made of stainless steel or any material suitable for the electrophoresis process.

[0043] As Figure 1A and 1BAs shown, the electrolyte buffer can be stored in an electrolyte buffer tank, which is fluidly coupled to the anode electrolyte buffer channel and / or the cathode electrolyte buffer channel through port 110. In other embodiments, buffer channel 140 itself can be a buffer reservoir. The buffer reservoir can have a volume of from 10 mL to 1000 mL, from 20 mL to 900 mL, from 30 mL to 800 mL, from 40 mL to 700 mL, from 50 mL to 600 mL, from 60 mL to 500 mL, from 70 mL to 400 mL, from 80 mL to 300 mL, from 90 mL to 200 mL, from 100 mL to 150 mL, from 100 mL to 500 mL, from 100 mL to 400 mL, from 100 mL to 300 mL, from 100 mL to 200 mL, including all ranges and sub-ranges therebetween. In some embodiments, the buffer reservoir can have a volume of from 100 mL to 500 mL, from 100 mL to 400 mL, from 100 mL to 300 mL, from 100 mL to 200 mL, including all ranges and sub-ranges therebetween. In other embodiments, the buffer reservoir can have a volume of from 100 mL to 500 mL.

[0044] In some embodiments, buffer (i.e., anode electrolyte and cathode electrolyte) channels 140 can be located on either side of the body, parallel to the central channel. Embodiments having a single channel (e.g., a single "central" channel) for sample separation can be advantageous because reagent or buffer consumption with such designs tends to be lower than designs having multiple channels for sample separation, which can reduce the overall cost of separating and collecting the desired analyte compared to known devices. However, it should be understood that other designs having multiple channels for sample separation are possible.

[0045] In some embodiments, a device or apparatus as described herein can include only one inlet 120 and only one outlet 130. In some embodiments, a single inlet 120 and a single outlet 130 can be preferred because it avoids potential difficulties with unbalanced flow that can occur when using multiple inlets and / or outlets. However, it should be understood that in other embodiments, multiple inlets and / or outlets can be used, for example, to increase throughput. Another advantage of the device or apparatus as described herein is the reduction in the formation of air bubbles that can become trapped inside the channels. The sizes of the inlet 120, outlet 130, and / or channels included in the present device or apparatus can be narrow, which is beneficial for stable liquid filling and avoids turbulence similar to that of microfluidic devices. In some embodiments, inlet 120 and outlet 130 can be oriented perpendicular to buffer channel 140 and the central channel.

[0046] The porous membrane 160 can prevent or substantially impede hydrodynamic flow while allowing electrokinetic (and / or electrophoretic) transport of ions and analytes. By preventing or substantially impeding hydrodynamic flow while allowing electrokinetic transport of analytes through the porous membrane 160, the device or apparatus is configured to allow substantially only the target analyte (i.e., the analyte of interest) to be hydrodynamically transported downward along the central channel to the outlet 130.

[0047] For example, when the background pH value (e.g., the pH value of the central channel and / or the buffer channel 140) is set at 6.0, an analyte with a pI value of 6.0 is freely hydrodynamically transported downward along the central channel to the outlet 130, while an analyte with a pI value other than 6.0 moves in a direction not parallel to the hydrodynamic flow, toward and / or into the porous membrane 160. The analyte transferred into the porous membrane 160 leaves the hydrodynamic flow and does not move toward the outlet 130 with the hydrodynamic flow. Thus, the porous membrane 160 is operable to filter out non-target analytes.

[0048] In some embodiments, the central channel may have a width of from about 1 mm to about 10 mm, from about 1 mm to about 9 mm, from about 1 mm to about 8 mm, from about 1 mm to about 7 mm, from about 1 mm to about 6 mm, from about 1 mm to about 5 mm, from about 1 mm to about 4 mm, from about 1 mm to about 3 mm, including all ranges and sub-ranges therebetween. In some embodiments, the central channel may have a length of from about 1 cm to 30 cm, 5 cm to 25 cm, 10 cm to 30 cm, 10 cm to about 20 cm, about 10 cm to about 19 cm, about 10 cm to about 18 cm, about 10 cm to about 17 cm, about 10 cm to about 16 cm, about 10 cm to about 15 cm, including all ranges and sub-ranges therebetween. In some embodiments, the central channel has a length of from about 10 cm to about 20 cm.

[0049] In some embodiments, the pore size of the porous membrane 160 can range from about 25 nm to about 800 nm, about 30 nm to about 700 nm, about 40 nm to about 600 nm, about 50 nm to about 500 nm, about 60 nm to about 400 nm, about 70 nm to about 300 nm, about 80 nm to about 200 nm, about 90 nm to about 100 nm, about 35 nm to about 750 nm, about 45 nm to about 650 nm, about 55 nm to about 550 nm, about 65 nm to about 450 nm, including all ranges and sub-ranges therebetween. In some embodiments, the pore size of the porous membrane 160 can be any suitable size as long as it is compatible with the devices or apparatuses disclosed herein or at least allows the target analyte to permeate. In some embodiments, the thickness range of the porous membrane 160 can be from about 100 μm to about 200 μm, about 110 μm to about 190 μm, about 120 μm to about 180 μm, about 130 μm to about 170 μm, about 140 μm to about 150 μm, including all ranges and sub-ranges therebetween. In some embodiments, the thickness range of the porous membrane 160 is from about 100 μm to about 200 μm, about 90 nm to about 600 μm, 100 nm to 500 μm, 200 nm to 400 μm, 300 nm to 300 μm, 400 nm to 200 μm, 500 nm to 100 μm, 600 nm to 90 μm, 700 nm to 80 μm, 800 nm to 70 μm, 900 nm to 60 μm, 1 μm to 50 μm, 10 μm to 40 μm, 20 μm to 30 μm, including all ranges and sub-ranges therebetween.

[0050] Figure 7 An embodiment of a single-channel free-flow electrophoresis device or apparatus is depicted, which includes: (1) a top cover 750 that can be made of glass, or any other suitable material, (2) a bottom cover 770 that can be made of plastic or any other suitable material, (3) a spacer 764 positioned between the top cover and the bottom cover, (4) two parallel membranes 762, 764, (5) an inlet 720 positioned on top of the top cover 750, (6) an outlet 730 positioned at the bottom of the bottom cover 770, (7) an anodic electrolyte buffer tank 742, and (8) a cathodic electrolyte buffer tank 744. The anodic electrolyte buffer tank 742 and the cathodic electrolyte buffer tank 744 can act as parallel electrodes such that a voltage can be applied to the buffers within the anodic electrolyte buffer tank 742 and the cathodic electrolyte buffer tank 744. Figure 7 The embodiment of is different from the embodiment of FIG. 1 mainly because, unlike having a single porous membrane 160 that electrically and / or ionically couples the central channel to two electrolyte buffer channels 140, Figure 7An embodiment has two porous membranes 762, 764, one electrically and / or ionically coupling the central channel to the buffer reservoir 742, and the other electrically and / or ionically coupling the central channel to the cathode electrolyte buffer reservoir 744. Figure 7 The various components of can be structurally and / or functionally similar to those of FIG. 1. Additionally, Figure 7 The overall function of the device is similar to that of the device of FIG. 1.

[0051] The two porous membranes 762, 764 are positioned to define the sides of the central channel. The top and bottom of the central channel are defined by a top cover 750 and a bottom cover 770, respectively. A spacer 765 defines the height of the central channel. The two porous membranes 762, 764 can each be configured to be wetted on one side by a sample flowing through the central channel and on the other side by a buffer (e.g., from buffer reservoirs 742, 744). The porous membrane 760 can be configured such that buffer ions and / or proteins can migrate into / through the membrane while preventing hydrodynamic flow. As discussed in further detail herein, one or more analytes of interest can be separated from non-target analytes, which can migrate from the central channel into / through the porous membrane 760, and then one or more fractionated target analytes can be collected at the outlet 730.

[0052] Figure 2A Shows a single-loop electrolyte buffer recirculation scheme for a single-channel free-flow electrophoresis device according to an embodiment, where an inlet 220 allows a sample to enter a single central channel, and an outlet 230 allows collection of sample fractions at the opposite side of the device. Figure 2B Shows a double-loop electrolyte buffer recirculation scheme for a single-channel free-flow electrophoresis device according to an embodiment, where an inlet 220' allows a sample to enter a single central channel and an outlet 230' allows collection of sample fractions at the opposite side of the device 200'. In some embodiments, at least one of the cathode electrolyte buffer channel 244 or the anode electrolyte buffer channel 242 is fluidly connected to a reservoir 290 containing an electrolyte buffer. Figure 2A and 2B The schematic diagrams shown can be implemented using any suitable device, such as the devices of FIGS. 1 and / or 7.

[0053] In some cases, before the sample is introduced into the central channel via the inlet 220, the temperature of a cooler or cold block (e.g., coupled to the bottom plate) can be adjusted downward to 5°C to 15°C, including all ranges and sub-ranges therebetween. After the temperature stabilizes, an electrolyte buffer that can be stored in a buffer reservoir having a volume of 100 ml to 500 mL or other suitable volume as disclosed herein can be recirculated through the buffer channels of the device using a peristaltic pump or another suitable pump 280. The recirculation of the electrolyte buffer through each buffer channel can be accomplished with a single fluid loop, asFigure 2A as shown. For example, pump 280 can deliver the electrolyte buffer from buffer reservoir 290 down one buffer channel and back through another buffer channel.

[0054] In other cases, the electrolyte buffer can be recycled through two buffer channels using two fluid loops, as Figure 2B shown. For example, pump 280' can deliver the electrolyte buffer from buffer reservoir 290' to one end of each of the anode electrolyte buffer channel 242' and the cathode electrolyte buffer channel 244', and exit from the opposite ends of the anode electrolyte buffer channel and the cathode electrolyte buffer channel (e.g., return to the buffer reservoir).

[0055] In other cases ( Figure 2A or not shown in Figure 2B), the electrolyte buffer can be circulated through the buffer channels via a completely separate loop. For example, a pump can deliver the anode electrolyte buffer from a dedicated anode electrolyte buffer reservoir through the anode electrolyte buffer channel, and another separate pump can deliver the cathode electrolyte buffer from a cathode electrolyte buffer reservoir through the cathode electrolyte buffer channel.

[0056] The electrolyte buffer (e.g., the buffer contained in one or more buffer reservoirs) typically contains an electrolyte solution and a polymer. In some embodiments, the electrolyte buffer can be a MES buffer. In some embodiments, the electrolyte buffer can be a BisTris buffer. In some embodiments, the electrolyte buffer can include any buffer suitable for an electrophoresis process, such as Tris / borate / EDTA, Tris / acetate / EDTA, etc. In some embodiments, the electrolyte buffer can contain methylcellulose. In some embodiments, the reservoir can contain an electrolyte buffer having 0.01% to 1%, 0.05% to 1%, 0.5% to 1%, 0.1% to 0.5%, 0.1% to 0.4%, 0.1% to 0.3%, 0.1% to 0.2% methylcellulose, including all ranges and subranges therebetween.

[0057] In some embodiments, the cathode electrolyte and the anode electrolyte are remixed in a buffer tank or reservoir, thereby maintaining a constant pH and the buffer capacity during use. The resistance of the channels for recycling can be at least 50 times, 40 times, 30 times, 20 times, 10 times the resistance of the device passing through the central channel and one or more porous membranes, including all ranges and subranges therebetween. This effectively prevents a "short circuit" through the recycling reservoir or through the channel loop, as Figure 2A shown.

[0058] In some embodiments, a sample buffer can be used to prepare a sample containing an analyte of interest. In some embodiments, the electrolyte buffer is the same as the sample buffer with a matching pH such that the sample can be maintained at a constant pH in the presence of electroosmotic flow. To slow down the electroosmotic flow and allow for more effective control of the fractionation process, a polymer such as methylcellulose at a concentration of 0.1%-0.5% can be added to the electrolyte buffer. In some embodiments, the sample can be a buffer exchanged into a predetermined sample buffer and can be further diluted in real time in a pH control buffer before entering the device.

[0059] The present disclosure provides a method for separating an analyte of interest from a sample according to the pI value of the analyte by using single-channel free-flow electrophoresis. Figure 6 is a flowchart of a method for separating an analyte of interest according to an embodiment. At 610, a sample buffer can be combined with an analyte mixture to form a sample. At 620, the sample can be introduced through an inlet and flow through a central channel of a single-channel free-flow electrophoresis device. The sample can be pumped through the central channel such that the sample flows hydrodynamically from the inlet of the central channel to the outlet of the central channel. At 625, a buffer pump can recirculate an electrolyte buffer from a buffer reservoir and through an electrolyte channel that extends parallel to and on either side of the central channel. At 640, electrodes coupled to the electrolyte channel can be energized such that an electric field is applied perpendicular to the central channel and the flow direction of the sample. In some embodiments, the anolyte channel, the central channel, and the catholyte channel can be electrically and / or ionically coupled but fluidically isolated by a porous membrane.

[0060] At 640, by applying an electric field perpendicular to the central channel, analytes having a pI value different from the pI value of the analyte of interest will migrate away from the direction of the central channel, toward, into, and / or through one or more porous membranes. The flow rate of the sample through the central channel and / or the intensity of the electric field can control the purity of the fractionated sample exiting the outlet of the central channel. At 630, the analyte of interest can be selectively isolated by controlling the pH of the electrolyte buffer and / or the sample buffer such that analytes having a pI value different from the pH value of one or more of the buffers are selectively rejected from entering / through one or more porous membranes. However, it should be understood that any electric field not parallel to the central channel will have a vector component perpendicular to the central channel such that analytes having a pI value different from the pI value of the analyte of interest will migrate in a direction not parallel to the hydrodynamic flow direction and toward, into, and / or through one or more porous membranes.

[0061] At 650, multiple purified fractions of a variety of analytes and / or samples of interest can be collected by adjusting the pH of the electrolyte buffer and / or sample buffer at 630. In some cases, the sample can be divided into multiple aliquots, each aliquot being mixed with a sample buffer having a different pH. After running each aliquot, the pH of the electrolyte buffer can be adjusted to match the pH of the next aliquot. Blanks can be run between aliquots. In other cases, the sample can be run continuously, and when sufficient volume of each sample fraction has been collected, the sample buffer and / or electrolyte buffer can be adjusted during the run (e.g., using a metering pump or valve).

[0062] In some embodiments, the mixture of analytes in a sample as described herein can include peptides having different isoelectric points of 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, including all ranges and sub-ranges therebetween. In some embodiments, the reservoir can contain an electrolyte buffer having a pH value of 0.1 to 14, 0.5 to 13, 1 to 14, 2 to 13, 3 to 12, 4 to 11, 5 to 10, 6 to 9, 7 to 8, including all ranges and sub-ranges therebetween. In some embodiments, the pH value of the electrolyte buffer can be adjusted sequentially by modifying the ratio of MES and BisTris. In some embodiments, the pH value of the electrolyte buffer can be adjusted sequentially by changing the temperature of the electrolyte buffer. Without wishing to be bound by any theory, the pKa value of the buffer will change in response to a change in temperature and thereby change the pH value.

[0063] For example, when collecting sufficient fractions of a sample (e.g., an analyte of interest) having a pI value corresponding to pH 3.5, the pH values of the electrolyte buffer and the sample buffer can be increased from pH 3.5 to pH 5.5. In other instances, when collecting sufficient fractions of a sample (e.g., an analyte of interest) having a pI value corresponding to pH 6.0, the pH values of the electrolyte buffer and the sample buffer can be increased from pH 6.0 to pH 7.5. In other instances, when collecting sufficient fractions of a sample (e.g., an analyte of interest) having a pI value corresponding to pH 11.0, the pH values of the electrolyte buffer and the sample buffer can be decreased from pH 11.0 to pH 10.5. In other aspects of the present disclosure, the pH value of the electrolyte buffer does not need to be modified to collect the fractionated analyte of interest from the sample. For example, the fractionated analyte of interest can be collected from the sample by applying pressure or vacuum to the central channel in a controlled manner and pushing out the unwanted pI fragments (i.e., fragments that do not have the pI value of interest) from the channel, thereby guiding the target fragments into the collection container. In yet another example, the fractionated analyte of interest can be collected from the sample by applying electroosmotic flow through a hydrodynamic barrier to push out the unwanted pI fragments (i.e., fragments that do not have the pI value of interest) from the channel, thereby guiding the target fragments into the collection container.

[0064] In some embodiments, the fractionated analyte of interest exits the single-channel electrophoresis device via the outlet of the device or apparatus. In some embodiments, at 650, the sample can be collected from the outlet of the device. In some embodiments, the sample can be continuously collected from the outlet of the device. The device or apparatus as described herein can maintain the characteristics of continuous separation and collection, which allows for excellent flexibility in the throughput of sample fractions containing the analyte of interest. In some embodiments, sample fractions can be collected from the outlet of the device at a rate of 1 μL / minute to 50 μL / minute, 5 μL / minute to 15 μL / minute, 2 μL / minute to 40 μL / minute, 3 μL / minute to 30 μL / minute, 15 μL / minute to 45 μL / minute, including all ranges and sub-ranges therebetween.

[0065] Although Figure 6 not shown, in some embodiments, a blank sample can be run before introducing the sample and collecting subsequent samples (e.g., an analyte of interest) corresponding to proteins having a pI equal to the newly adjusted pH. This process can be repeated for any number of pH values to ensure collection accuracy. At 630, buffer pH adjustment can be automatically accomplished using a metering pump or a metering valve.

[0066] Although Figure 6Not shown, but in some embodiments, before electrophoresis begins, the device can be pre-wetted first by flowing a 25% ethanol solution into the central channel. In some embodiments, the device is pre-wetted by flowing an ethanol solution having any suitable concentration into the central channel. In some embodiments, the device is pre-wetted by flowing a 0.1% Tween 20 solution into the central channel. In some embodiments, the device is pre-wetted by flowing a Tween 20 solution having any suitable concentration into the central channel. Flowing the ethanol solution or the Tween 20 solution into the central channel or the membrane can minimize the formation of air bubbles in the channel. In some embodiments, incubating in the ethanol solution or the Tween 20 solution for 5 minutes to 10 minutes can ensure that one or more membranes are completely wetted.

[0067] In some embodiments, the sample fraction can contain the analyte of interest collected at a rate of: 1 μL / minute to 50 μL / minute, 5 μL / minute to 15 μL / minute, 2 μL / minute to 40 μL / minute, 3 μL / minute to 30 μL / minute, 15 μL / minute to 45 μL / minute, including all ranges and sub-ranges therebetween.

[0068] As an example, Figures 3A - 3C shows the fractionation of a peptide mixture with a pI value in the range of 3.4 to 10.1 using a MES-BisTris buffer, and the pI value is measured by commercially available IEF (such as an isoelectric focusing system or technique). In some embodiments, the pH value can be changed by adjusting the ratio of MES to BisTris. The sample is a mixture of five peptides with pI values of 3.4, 5.85, 6.15, 9.9, and 10.1. By sequentially changing the buffer pH from 5.8 to 6.7, after fractionation, peptides with pI values not within this range of pI values (e.g., pI values of 3.4, 9.9, and 10.1) are hardly detectable. When the buffer pH is increased from 5.8 to 6.7, the relative amounts of peptides with pI values of 5.85 and 6.15 change. Figure 3C shows that at pH 6.7, the peptide with a pI value of 5.85 becomes undetectable, and only the single peptide with a pI value of 6.15 can be collected.

[0069] As another example, Figure 4B shows the fractionation of IgG with a pI value in the range of 5.6 - 5.9 by using a MES-BisTris buffer and by using the device or equipment as described herein. In some embodiments, the pH value can be changed by adjusting the ratio of MES to BisTris. The sample is a mixture of four fragments of this IgG with pI values of 5.65, 5.72, 5.8, and 5.9. As Figure 4AAs shown, when the buffer pH is 6.3, after fractionation, the fragment with a pI value of 5.65 is undetectable, while the fragments with higher pI values increase their relative abundance. Figure 4B It is shown that as the buffer pH is increased to 6.5, after the fractionation process, both fragments with pIs of 5.65 and 5.72 are undetectable, while the relative amount of the fragment with a pI value of 5.9 increases from 3.5% to approximately 50%.

[0070] Figure 5 Examples of the fractionation of basic proteins using the apparatus or device as described herein are shown. As an example, Herceptin monoclonal antibody with four major fragments having pI values of 8.62, 8.73, 8.85, and 8.95 can be fractionated by using 2-amino-2-methyl-1,3-propanediol (AMPD) as the buffer. In some embodiments, the fractionation of Herceptin monoclonal antibody can be processed by using any other suitable buffer. Without wishing to be bound by any theory, AMPD has an effective pH range of 7.8 to 9.7. At a buffer pH of 8.8, the relatively minor peak with a pI of 8.63 can become the most abundant peak, which represents an increase in the percentage of the total peak area of this peak from approximately 13.4% to approximately 83.5%.

[0071] Figure 5 It is also shown that when the buffer pH is increased to 9.0, the second peak with a pI value of 8.73 can be enriched from 34.9% to 83.7%, while the abundances of the other peaks can be significantly reduced. At pH 9.2, the third peak, which is the main peak before fractionation, can be enriched from 36.1% to 68.3%, while the first peak with a pI value of 8.63 can no longer be detected. At pH 9.4, the pI fragment of 8.95 is 70.2%, compared with 14.7% before the fractionation process. At pH 9.4, the fragments with pI values of 8.63 and 8.73 are not detected. A slight mismatch between the pI values of the collected fragments and the buffer pH is observed, which may be due to the presence of EOF, as EOF may distort the collection of the fractionated analytes of interest. Another reasonable explanation is the measurement error caused by the pH buffer.

[0072] When the above-described apparatus and / or method indicate that certain events and / or procedures occur in a certain order, the order of certain events and / or procedures can be modified. Additionally, certain steps and / or procedures can be performed simultaneously in parallel processes where possible, as well as sequentially.

Claims

1. A method for electrophoretic fractionation of a sample, comprising: Flowing the sample through a central channel of a single-channel free-flow electrophoresis device; Applying an electric field that is not parallel to the flow direction of the sample via an anolyte channel and a catholyte channel parallel to the central channel, wherein the anolyte channel, the central channel, and the catholyte channel are fluidically coupled by hydrodynamic barrier ions but fluidically isolated; Separating a first analyte from the sample based on the isoelectric point of the first analyte and the first pH value of an electrolyte buffer, wherein the isoelectric point of the first analyte corresponds to the first pH value; and Collecting a first fraction of the sample containing the first analyte separated from the sample at an outlet and rejecting at least a portion of a second analyte having a second isoelectric point corresponding to a second pH value from entering the anolyte channel or the catholyte channel; After collecting the first fraction, adjusting the first pH value of the electrolyte buffer to a second pH value; and Collecting a second fraction of the sample containing the second analyte separated from the sample at the outlet and rejecting at least a portion of the first analyte from entering the anolyte channel or the catholyte channel.

2. The method according to claim 1, further comprising mixing the sample with a sample buffer before flowing the sample through the central channel, the sample buffer having a pH matching the first pH value of the electrolyte buffer.

3. The method according to claim 1, further comprising circulating the electrolyte buffer from a reservoir and through the anolyte channel and the catholyte channel.

4. The method according to claim 1, further comprising applying a voltage across the anolyte channel and the catholyte channel to generate the electric field.

5. The method according to claim 1, further comprising circulating the electrolyte buffer from a reservoir such that the electrolyte buffer flows through the anolyte channel and the catholyte channel before returning to the reservoir.

6. The method according to claim 1, further comprising circulating the electrolyte buffer from a reservoir such that the electrolyte buffer flows through the anolyte channel and the catholyte channel via two loops.

7. The method according to claim 2, wherein the sample buffer comprises MES-BisTris.

8. The method according to claim 1, wherein the first fraction of the sample containing the first analyte is collected at a rate of 5 µL / min to 15 µL / min.

9. The method according to claim 1, wherein the first fraction of the sample containing the first analyte is collected at a rate of 1 µL / min to 50 µL / min.

10. The method according to claim 1, wherein the first pH value of the electrolyte buffer is adjusted to the second pH value by modifying the ratio of MES and BisTris in the electrolyte buffer.

11. The method according to claim 1, wherein the first pH value of the electrolyte buffer is adjusted to the second pH value via a metering pump or a valve.

12. The method according to claim 1, wherein the first fraction and the second fraction of the sample are collected at a constant rate.

13. The method according to claim 1, wherein the first pH value of the electrolyte buffer is adjusted to a second pH value by changing the temperature of the electrolyte buffer.

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