Improved methods, devices, and kits for reducing or labeling carbohydrates

Abstract

The present invention provides methods, apparatus, and kits for improving the process of reducing carbohydrates, such as glycans released from glycoconjugates or labeling carbohydrates by reductive amination.

Description

[0001] This application is a divisional application of patent application No. 201880034596.3, filed on March 22, 2018, entitled "Improved Method, Apparatus and Reagent Kit for Reducing or Labeling Carbohydrates".

[0002] Cross-reference to related applications

[0003] This application claims the rights and priorities of the following applications: U.S. Provisional Application No. 62 / 475,815, filed March 23, 2017; U.S. Provisional Application No. 62 / 636,815, filed February 28, 2018; and U.S. Provisional Application No. 62 / 644,460, filed March 17, 2018, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0004] This invention relates to the field of improving the efficiency, convenience, and speed of labeling carbohydrates (such as oligosaccharides) by reductive amination. Background Technology

[0005] In various commercial and regulatory contexts, it is essential to determine the nature and quantity of carbohydrates present in a sample. This is particularly true for glycans attached to glycoproteins, especially those used as therapeutic agents. Since glycans attached to glycoproteins influence key properties of glycoprotein function, including their pharmacokinetics, stability, biological activity, and immunogenicity, identifying which glycans are present on glycoproteins is crucial. The Food and Drug Administration (“FDA”) requires the characterization of carbohydrates attached to biologics (such as therapeutic glycoproteins and vaccines) to demonstrate conformity between the composition and manufacturing process, necessitating extensive product characterization. Analyzing carbohydrate profiles is also important for quality control in the production of both therapeutic and non-therapeutic recombinant proteins, where variations in carbohydrate distribution can predict system pressures, signaling conditions, and potential discarding of contents from commercial-scale fermenters. Therefore, biochemists, clinical chemists, pharmaceutical manufacturers, and protein producers have considerable interest in characterizing the distribution of glycans in biological samples, such as therapeutic glycoproteins.

[0006] A common approach to analyzing carbohydrates, particularly glycans released from glycoproteins via enzymatic digestion, is to label them using reductive amination with appropriate dyes or markers. For example, reductive amination of N-glycans released from glycoproteins by the conventional deglycosylation enzyme PNGase F is typically achieved by conjugating the free reduced end of the glycan to the free amino group of a marker, such as a fluorescent dye or charged moiety. A common marker used for reductive amination is 2-aminobenzamide, or "2-AB". Reductive amination and labeling with 2-AB is disclosed in common U.S. Patent No. 5,747,347. The labeled glycans can then be analyzed using any of a variety of analytical methods, depending on the marker used, such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE, including capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, capillary isovelocity electrophoresis, and micellar electrokinetic chromatography), or microfluidic separation. The labeling and analysis of N-glycans are further described, for example, in common U.S. Patent Nos. 8,124,792 and 8,445,292. International Publication No. WO2015 / 166399 discloses the use of carboxyl-derived magnetic beads in deglycosylation and labeling processes.

[0007] There remains a need in the art for improved methods of labeling carbohydrates (such as glycans) at a faster and more efficient rate, particularly for methods of labeling carbohydrates (such as glycans) by reductive amination. Surprisingly, the present invention addresses these and other needs. Summary of the Invention

[0008] The present invention provides a method, apparatus and kit for improving the labeling of carbohydrates by reduction or by reductive amination.

[0009] In some embodiments, the present invention provides an in vitro method for reducing or by reducing amination of carbohydrates provided in an aqueous solution, and optionally, an in vitro method for analyzing the reduced or labeled carbohydrates, the method comprising the following steps in the following order: (a) mixing the carbohydrates and the aqueous solution with an organic solvent to form a mixture of the organic solvent and the aqueous solution of the carbohydrates, wherein the mixture has a ratio of about 80% or more of the organic solvent to about 20% or less of the aqueous solution; (b) transferring the mixture of carbohydrates to a reaction vessel having (1) a first opening allowing the introduction of reagents, and (2) a body having, in the following order: a first segment having a cross-section of a defined area, configured to extend throughout the first segment. The method comprises: (a) a first porous solid carrier having a cross-sectional area, a second segment having a cross-sectional area, and a second opening allowing reagent discharge when necessary; (b) passing a mixture through the first porous solid carrier, allowing carbohydrates to remain on the first porous solid carrier, thereby immobilizing the carbohydrates on the first porous solid carrier; (c) washing the carbohydrates immobilized on the first porous solid carrier with an organic solvent or a solution of organic solvent concentration greater than 95% to remove the mixture of organic solvent and aqueous solution and any compounds in the mixture not retained on the first porous solid carrier; and (d) reducing or labeling the carbohydrates immobilized on the first porous solid carrier by reductive amination, thereby reducing or labeling the carbohydrates by reductive amination. In some embodiments, the method further comprises: step e', after reduction or labeling, washing the first porous solid carrier with an organic solvent or a solution of organic solvent concentration greater than 95% to remove any excess reducing agent or labeling agent. In some embodiments, the method further includes: step e", after washing, eluting the washed and reduced or labeled carbohydrates in the first porous solid carrier using an aqueous solution containing up to 20% organic solvent. In some embodiments, the aqueous solution containing up to 20% organic solvent includes water or a buffer solution. In some embodiments, the aqueous solution does not contain any organic solvent. In some embodiments, the first porous solid carrier is made of a hydrophilic material or has a hydrophilic material on its surface, wherein the hydrophilic material does not contain carboxyl groups. In some embodiments, the first porous solid carrier is made of a hydrophilic material. In some embodiments, the hydrophilic material is cellulose; glass; alumina; a functionalized surface having diol, aminopropyl, amide, cyanopropyl, or ethylenediamine-N-propyl; silica; silica derivatized with diol, aminopropyl, or amide (carbamoyl); a porous hydrophilic material but without one or more carboxyl groups; or a combination of two or more of these. In some embodiments, the glass is glass fiber.In some embodiments, the first porous solid support is made of a non-hydrophilic material or has a surface of a non-hydrophilic material, and the mixture of the organic solvent and the aqueous solution of the carbohydrate in step (a) has an organic solvent concentration of more than 95%. In some embodiments, the first porous solid support is made of a non-hydrophilic material. In some embodiments, the non-hydrophilic material has pores or openings with a width of 10 micrometers or less. In some embodiments, the width of the pores or openings is 5 micrometers or less. In some embodiments, the width of the pores or openings is 1 micrometer or less. In some embodiments, the non-hydrophilic material is polyethylene, nylon, polyvinylidene fluoride, or polypropylene. In some embodiments, the non-hydrophilic material has pores or openings with a width of 10 micrometers or less. In some embodiments, the non-hydrophilic material is polyethylene. In some embodiments, (1): the reaction vessel in step (b) further comprises a hydrophilic second porous solid support disposed between the first porous solid support and the second opening; and (2): the reduction or labeling in step (e) is carried out by a reducing agent or labeling agent that is poorly soluble in organic solvents. In some embodiments, the method further includes: washing a reducing agent or labeling agent poorly soluble in the organic solvent with a washing solution containing about 80% to 90% organic solvent and the remainder being an aqueous solution, thereby releasing the reduced or labeled carbohydrates in the first porous solid carrier and capturing the reduced or labeled carbohydrates through a hydrophilic second porous solid carrier. In some embodiments, the concentration of the organic solvent in the washing solution is about 80%. In some embodiments, the method further includes: eluting the released labeled carbohydrates captured by the hydrophilic second porous solid carrier with an aqueous solution containing no more than 20% organic solvent. In some embodiments, the aqueous solution containing up to 20% organic solvent comprises water or a buffer solution. In some embodiments, the aqueous solution does not contain any organic solvent. In some embodiments, the hydrophilic second porous solid support is made of: (a) cellulose; (b) glass; (c) alumina; (d) silica; (e) a functionalized surface having diol, aminopropyl, amide (carbamoyl), cyanopropyl, ethylenediamine-N-propyl, or zwitterionic groups; (f) silica covalently bonded with one or more carbamoyl groups; or (g) a combination of two or more of these. In some embodiments, the second porous solid support is made of silica. In some embodiments, silica covalently bonded with one or more carbamoyl groups. In some embodiments, silica is in the form of beads or particles. In some embodiments, the silica beads or particles have a size of 3 to 60 micrometers. In some embodiments, the silica spheres or particles have a size of about 30 micrometers. In some embodiments, the silica beads or particles covalently bonded with carbamoyl groups are Amide-80.In some embodiments, the first porous solid support is in the form of a membrane. In some embodiments, the first porous solid support is in the form of a monolith. In some embodiments, in step (e), the carbohydrate is reduced by a reducing agent. In some of these embodiments, the reducing agent is sodium cyanoborohydride or methylpyridinium borane. In some embodiments, the reducing agent is in an organic solvent. In some embodiments, in step (e), the carbohydrate is labeled with a marker. In some of these embodiments, the marker is 2-aminobenzamide (2-AB), anthranilic acid (2-AA), 8-aminopyrene-1,3,6-trisulfonic acid (APTS), or procainamide hydrochloride. In some embodiments, the method further includes: step (f), eluting the reduced or labeled carbohydrate from the first porous solid support and separating or analyzing the reduced or labeled carbohydrate, or separating and analyzing the reduced or labeled carbohydrate. In some embodiments, the reduced or labeled carbohydrate is provided to a separation element. In some embodiments, the separation element is an apparatus for separating reduced or labeled carbohydrates by high-performance liquid chromatography, capillary electrophoresis, microfluidic separation, hydrophilic interaction liquid chromatography, or mass spectrometry, or a combination of two or more of these. In some embodiments, the reduced or labeled carbohydrate is a reduced carbohydrate, and the reduced carbohydrate is analyzed by mass spectrometry. In some embodiments, the reduced or labeled carbohydrate is a labeled carbohydrate, and the labeled carbohydrate is analyzed by detecting the fluorescence of a label. In some embodiments, the organic solvent is acetonitrile, anhydrous ethanol, anhydrous methanol, isopropanol, butanol, toluene, ethyl acetate, acetone, tetrahydrofuran, diethyl ether, dichloromethane, chloroform, tert-butyl methyl ether, benzene, carbon tetrachloride, isooctane, hexane, or any two or more combinations of these. In some embodiments, the organic solvent is acetonitrile. In some embodiments, the carbohydrate is an N-glycan. In some embodiments, the carbohydrate is an O-glycan. In some embodiments, the carbohydrate is a polysaccharide, oligosaccharide, disaccharide, or monosaccharide. In some embodiments, the reaction vessel is a well. In some embodiments, the reaction vessel is a chamber of a microfluidic device. In some embodiments, the well is located in a porous plate, and the first porous solid support is composed of polyethylene. In some embodiments, the polyethylene has pores of 10 micrometers or smaller. In some embodiments, the vessel further includes a hydrophilic second porous solid support disposed between the first porous solid support and the second opening. In some embodiments, the hydrophilic second porous solid support is composed of beads or particles of silica bonded with carbamoyl groups.

[0010] In another set of embodiments, the present invention provides an apparatus for reducing carbohydrates provided in an aqueous solution by reductive amination. The apparatus of the present invention includes a reaction vessel having a body having (a) a first opening, (b) a first section having a first chamber having a first cross-section over a defined area, wherein the first chamber is in fluid communication with the first opening, (c) a second section having a second chamber having a second cross-section over a defined area, the second chamber being in fluid communication with the first chamber of the first section, (d) a second opening in fluid communication with the second chamber, (e) a first porous solid carrier disposed between the first and second openings and filling the area of ​​the first cross-section of the first chamber, and (f) a second porous solid carrier composed of or having a surface of a hydrophilic material, the second porous solid carrier being disposed between the first porous solid carrier and the second opening and filling the area of ​​the first or second cross-section. In some embodiments, the diameter of the first section of the vessel body is larger than the second diameter of the second section. In some embodiments, a first porous solid support is disposed in a first section of a reaction vessel, and a second porous solid support is disposed in a second section of the reaction vessel. In some embodiments, the reaction vessel is a tube or cylinder. In some embodiments, the reaction vessel is a hole in a porous plate. In some of these embodiments, the second section of the hole is a nozzle protruding from the bottom of the hole. In some embodiments, the second porous solid support is disposed in the nozzle. In some embodiments, the reaction vessel is within a microfluidic device. In some embodiments, the first porous solid support is made of a non-hydrophilic material or has a non-hydrophilic material surface. In some embodiments, the non-hydrophilic material of the first porous solid support has pores or openings with a width of 10 micrometers or less. In some embodiments, the non-hydrophilic material is polyethylene and has pores or openings with a width of 10 micrometers or less. In some embodiments, the first porous solid support is composed of a hydrophilic material. In some embodiments, the hydrophilic material is made of: (a) glass fiber; (b) cellulose; (c) silica beads or particles; or (d) silica beads or particles covalently bonded with a plurality of aminopropyl, glycol, carbamoyl, zwitterionic groups, or combinations of two or more of the above groups. In some embodiments, the first porous solid carrier is composed of glass fiber. In some embodiments, the hydrophilic material of the second porous solid carrier is made of: (a) glass fiber; (b) cellulose; (c) silica; (d) a surface covalently bonded with a plurality of aminopropyl, glycol, carbamoyl, zwitterionic groups, or combinations of two or more of the above groups; or (e) silica covalently bonded with a plurality of aminopropyl, glycol, carbamoyl, or combinations of two or more of the above groups.In some embodiments, the hydrophilic material of the second porous solid support is composed of glass fiber. In some embodiments, the silica is in the form of beads or particles. In some embodiments, the beads or particles are covalently linked with multiple carbamoyl groups. In some embodiments, the silica beads or particles having multiple carbamoyl groups are Amide-80. In some embodiments, the first porous solid support is polyethylene with pores or openings having a width of 10 micrometers or less, and the second porous solid support consists of silica beads or particles covalently linked with multiple carbamoyl groups. In some embodiments, the silica beads or particles having multiple carbamoyl groups are Amide-80. In some embodiments, the second opening has an openable cap to retain the solution in the reaction vessel until the solution needs to be removed from the reaction vessel.

[0011] In another set of embodiments, the present invention provides a kit for reducing carbohydrates or labeling carbohydrates by reductive amination. The kit includes: (a) a reaction vessel having a body having (a) a first opening; (b) a first segment having a first chamber having a first cross-section over a defined area, the first chamber being fluidly connected to the first opening; (c) a second segment having a second diameter and a second cross-section over a defined area, and a second chamber having a cross-section over a defined area, the second chamber being fluidly connected to the first chamber of the first segment; (d) a second opening fluidly connected to the second chamber; and (e) a first porous solid support disposed between the first opening and the second opening and filling the first chamber. The first cross-sectional area of ​​the chamber, and (f) a second porous solid support composed of or having a surface of a hydrophilic material, wherein the second porous solid support is disposed between the first porous solid support and the second opening and fills the area of ​​the first or second cross-section; (b) a reducing agent or a label suitable for labeling carbohydrates by reductive amination, or the aforementioned reducing agent and label, and (c) instructions for reducing carbohydrates or instructions for labeling carbohydrates by reductive amination, or instructions for reducing carbohydrates and instructions for labeling carbohydrates by reductive amination. In some embodiments, the reaction vessel is a hole in a tube, cylinder, or porous plate. In some embodiments, the reaction vessel is a hole in a porous plate. In some embodiments, a second section of the hole is a pointed tip protruding from the bottom of the hole. In some embodiments, the first porous solid support is polyethylene having pores or openings with a width of 10 micrometers or less, and the second porous solid support consists of beads or particles of silica covalently linked with a plurality of carbamoyl groups. In some embodiments, silica beads or particles covalently linked with multiple carbamoyl groups are disposed in a pointed tip protruding from the bottom of a pore. In some embodiments, the marker is 2-aminobenzamide (2-AB), anthranilic acid (2-AA), 8-aminopyrene-1,3,6-trisulfonic acid (APTS), or procainamide hydrochloride. In some embodiments, the reducing agent is sodium cyanoborohydride or methylpyridineborane, or sodium cyanoborohydride and methylpyridineborane. In some embodiments, the kit further comprises one or more reagents for deglycosylation of the glycoconjugate. In some embodiments, one or more reagents are deglycosylation enzymes. In some embodiments, the deglycosylation enzyme is PNGase F. Attached Figure Description

[0012] Figure 1 : Figure 1This diagram illustrates an exemplary embodiment of the method of the present invention, in which glycans are used as exemplary carbohydrates to be reduced or labeled. Step 1 on the left represents a container holding an aqueous solution of a glycoprotein. Legend: Folded solid lines: protein components of glycoprotein molecules; light and dark geometry: glycans attached to glycoproteins; thin straight lines between the shapes of glycans and the thicker lines representing protein components: covalent linkages between glycans and proteins; the arrow between steps 1 and 2 indicates the addition of a deglycosylation enzyme to the aqueous solution, followed by enzymatic digestion of the glycoprotein, resulting in a starting aqueous solution sample. To the right of step 2 are arrows pointing to workflows 1A, 1B, and 1C. These three workflows exemplify how practitioners can choose and adjust workflows based on the solubility of the first porous solid support for hydrophilic (“H”) or non-hydrophilic (“non-H”) materials and the reducing agent or dye used to reduce or label glycans in organic solvents. Workflow 1A shows an embodiment where the dye or reducing agent is well soluble in an organic solvent and can be used regardless of whether the first porous solid support is hydrophilic or non-hydrophilic. Workflow 1B illustrates an embodiment where the dye or reducing agent is poorly soluble in an organic solvent, and the first porous solid support is non-hydrophilic. Workflow 1C illustrates an embodiment where the dye or reducing agent is either well soluble or poorly soluble in an organic solvent, and the first porous solid support is hydrophilic. The arrows between steps 2 and 3 indicate the addition of sufficient organic solvent to the initial aqueous solution containing the glycans released from the glycoprotein to form a mixture having the organic solvent concentrations shown in step 3 of the respective workflow. Step 3 of each workflow shows the concentration of the organic solvent in the solution used to immobilize the glycans on the first porous solid support, and steps 4 and 6 show the concentration of the organic solvent in the washing solution used to remove unwanted components from the first porous solid support washing solution. In step 7 of each workflow, the glycans are eluted with an aqueous solution for separation, analysis, or separation and analysis. Not shown: an opening at the top of the container for introducing reagents, and an opening at the bottom of the container for discharging reagents when necessary. Detailed Implementation

[0013] introduce

[0014] As described in the background section, the analysis of carbohydrates, and especially glycans linked to glycoproteins, is important for various regulatory and quality control purposes. In particular, analyzing the type and amount of glycans linked to therapeutic glycoproteins, such as monoclonal antibodies, has become an important quality control measure for the production of such glycoproteins and for confirming that they will possess the desired pharmacological activities.

[0015] Carbohydrates are typically analyzed by labeling them and then analyzing the labeled carbohydrates using appropriate instruments. For example, as described in common U.S. Patent No. 5,747,347 (hereinafter referred to as “347 Patent”), glycans released from glycoproteins via enzymatic digestion can be labeled by subjecting them to reductive amination with a suitable dye, and the glycans can then be analyzed by detecting the fluorescence of the dye bound to the labeled glycans (these contents are incorporated herein by reference). However, the release of glycans via enzymatic digestion typically occurs in an aqueous solution, and glycans released from glycoproteins are typically in an aqueous solvent. Furthermore, carbohydrates from other biomolecules and other biological sources are typically present in an aqueous environment. Labeling carbohydrates via reductive amination usually requires anhydrous conditions. Therefore, carbohydrates, such as glycans released from glycoproteins, must be dried to provide anhydrous conditions that allow reductive amination to occur. This drying step typically introduces considerable delays to carbohydrate labeling schemes and often also introduces problems related to handling and variability.

[0016] Furthermore, protocols for reductive amination typically require the separation of carbohydrates from other types of molecules that may be present in the sample mixture. For example, free carbohydrates in nature are often found in complex aqueous biological matrices that may contain salts, proteins, lipids, and other small metabolites, while protocols for releasing glycans from glycoproteins or other glycoconjugates often result in the released glycans being in solution along with other reagents such as buffer salts, denaturants, and reducing agents. Therefore, the glycans or other carbohydrates of interest in the mixture are typically purified by, for example, solid-phase extraction, filtration, or gel separation, followed by elution of the glycans and other carbohydrates of interest in a suitable solvent, and then removal of the solvent by evaporation. However, these purification steps often increase the amount of solvent that must subsequently be dried, and increase the overall time required for the protocol. Once dried, the purified glycans or other carbohydrates of interest are typically reconstituted or redissolved in a suitable non-aqueous solvent and labeled by reductive amination. After the reaction is complete, further purification is usually performed to remove any excess reactants, and in particular any excess free dye. This second purification is typically carried out by solid-phase extraction using a stationary phase, which may be the same as or different from the stationary phase used to purify unlabeled glycans or carbohydrates.

[0017] Surprisingly, the method of the present invention reduces the time and steps required to label carbohydrates by reductive amination and prepare them for analysis, while also reducing potential sample loss. More specifically, the method of the present invention surprisingly eliminates the need for a separation step and eliminates the need to first transfer the carbohydrate of interest to and then from a solid-phase extraction (“SPE”) cartridge, and eliminates the need for a separate drying step to remove the carbohydrate from the aqueous solution in which it is typically initially present, thus preparing it for reductive amination. Therefore, in the analysis of carbohydrates of interest, the combination of features provided by the method of the present invention offers significant advancements in speed, efficiency, and simplicity. This combination of features is particularly useful for labeling glycans released from glycoproteins by enzymatic digestion. In another aspect, the present invention provides an apparatus for labeling carbohydrates with the label APTS (8-aminopyrene-1,3,6-trisulfonic acid, CAS No. 196504-57-1) or other reductive amination dyes that are poorly soluble in organic solvents. Similarly, the apparatus of the present invention utilizes the method of the present invention to perform a workflow that both increases the labeling speed and reduces the number of transfers between containers that may result in sample loss.

[0018] Surprisingly, in some embodiments, when the glycan or other carbohydrate is immobilized on a solid support, the method of the present invention can be further used to reduce the glycan or other carbohydrate without labeling it. As known to those skilled in the art, glycans and other carbohydrates exist in two epimers, designated α and β, respectively. When unreduced carbohydrates are present in the sample being analyzed, each epimer of the carbohydrate produces a separate signal (e.g., two peaks are observed for each unreduced carbohydrate by mass spectrometry), while reduced carbohydrates have only one epimer form, and therefore only one signal or peak for each carbohydrate. Therefore, in embodiments where the carbohydrate is immobilized on a first porous solid support and reduced but not labeled, the sensitivity for detecting the presence of carbohydrates in the sample is improved, and the analysis of the present carbohydrates is simplified.

[0019] This section begins by outlining aspects of the method of the invention, and then describes more specifically the specific aspects and variations that enable processing, for example, different starting solutions, different reducing agents or dyes for labeling carbohydrates, and different types of containers. Furthermore, this section describes an inventive apparatus for carrying out a workflow using reducing agents or labeling dyes (such as APTS) that are not well soluble in organic solvents.

[0020] The starting point of this invention is that a sample contains one or more carbohydrates, and practitioners wish to label these carbohydrates by reductive amination for subsequent analysis, such as by measuring the fluorescence of the labeled carbohydrates, or practitioners wish to reduce but not label these carbohydrates and then analyze them by mass spectrometry. Glycans or other carbohydrates are typically present in aqueous solutions, although in some cases they may be present in dry samples, which are subsequently redissolved in an aqueous solution. For example, the aqueous solution may be a solution obtained by enzymatic digestion of glycoproteins with a deglycosylation enzyme and contains (i) an aqueous buffer in which the enzymatic digestion took place, (ii) fully or partially deglycosylated proteins remaining after the enzyme has released the glycans, (iii) any glycans released by the enzymatic digestion, (iv) a deglycosylation enzyme, (v) a buffer salt, (vi) a reducing agent, and (vii) other denaturing agents. In other embodiments, the carbohydrates may be in biological samples or other samples that also contain lipids, proteins, salts, and other metabolites. For ease of reference, an aqueous solution containing the carbohydrates to be labeled, along with any other reagents present in the solution, is sometimes referred to as the “starting aqueous solution sample.” Depending on the equipment used, the initial aqueous solution sample is typically contained in a container, such as a well, Eppendorf tube, or chamber of a microfluidic device. The container providing the initial aqueous solution sample is sometimes referred to below as the “initial container.”

[0021] In typical current protocols, a purification step is used to separate carbohydrates from any proteins, lipids, or other non-carbohydrate molecules present in the sample, resulting in isolated carbohydrates, which can then be subjected to reductive amination. This purification step is typically accomplished by transferring the sample into a solid-phase extraction (“SPE”) cartridge, eluting the non-carbohydrate components from the column, and then eluting the now-separated carbohydrates into individual wells or vials for reductive amination, or by using a material that retains the non-carbohydrate components but not the carbohydrates, allowing the carbohydrates to flow through and be collected.

[0022] Surprisingly, the method of the present invention eliminates the need for separate purification steps and multiple transfers of samples and carbohydrates in the workflow, thereby reducing process time and the chance of sample loss during multiple transfers from one container to another. Furthermore, the materials used in many embodiments are inexpensive and easily automated. Therefore, the method of the present invention offers a combination of advantages previously unavailable in the art.

[0023] The method of this invention utilizes the properties of glycans or other carbohydrates in organic solvents, such as reduced solubility in organic solvents compared to their solubility in aqueous solutions. This allows glycans or other carbohydrates in a sample to be immobilized on a first porous solid support while washing away molecules dissolved in the organic solvent and providing anhydrous conditions. The glycans or other carbohydrates in the sample can then be reduced or labeled under anhydrous conditions by reductive amination, while remaining immobilized on the first porous solid support. They can then be eluted from the first porous solid support by redissolving them in an aqueous solution (typically after washing away any reducing agent, or, in the case of reductive amination, after washing away any labels not bound to the glycans or other carbohydrates). The reduced or labeled glycans or other carbohydrates can then be separated and analyzed by providing them to a separation device, an analytical device, or preferably first to a separation device and then to an analytical device.

[0024] In some examples of these methods of the invention, the first porous solid support is a hydrophilic material, and an organic solvent is added to an initial aqueous solution containing polysaccharides or other carbohydrates of interest to produce, for example... Figure 1 The organic solvent concentration shown in workflow 1A is 75% to 95%, or a mixture with an organic solvent concentration higher than 95% is produced as shown in workflow 1C. Without being bound by theory, it is believed that when the organic solvent concentration is 75% to 95%, the glycans or other carbohydrates present in the sample are retained on the hydrophilic first porous solid support through hydrophilic interactions, while when the organic solvent concentration exceeds 95%, it is believed that the glycans or other carbohydrates in the sample will precipitate or aggregate and be captured and retained in the first porous solid support by simple filtration through the pores or openings in the first porous solid support, regardless of whether the first porous solid support is hydrophilic or non-hydrophilic. Therefore, the range of materials permissible for the first porous solid support is wider when adding an organic solvent to the initial aqueous solution sample to produce a mixture with an organic solvent concentration higher than 95% compared to using an organic solvent to produce a mixture with an organic solvent concentration equal to or lower than 95%.

[0025] In the method of this invention, a starting aqueous solution sample containing polysaccharides or other carbohydrates of interest (and which may also contain proteins, denaturants, salts, enzymes, or other compounds) is placed in an initial container. The starting aqueous solution sample is then mixed with an organic solvent such that when the mixture of the organic solvent and the starting aqueous solution sample comes into contact with a first porous solid support, it is in an organic solvent of a selected concentration (i.e., 75% to 95% when used with a hydrophilic first porous solid support for capture via hydrophilic interaction, or greater than 95% when used with a hydrophilic or non-hydrophilic first porous solid support for capture via simple filtration). Those skilled in the art will understand that many methods can achieve this. For example, the organic solvent is added to the container initially containing the starting aqueous solution sample at a concentration capable of producing the desired concentration. Alternatively, the starting aqueous solution sample can be transferred to a larger container and mixed with an organic solvent to produce a mixture with the desired organic solvent concentration, which is then added to the container containing the first porous solid support. Alternatively, a certain amount of organic solvent may be present in the container holding the first porous solid support, such that when the initial aqueous solution sample is added to the container, the mixture of the organic solvent and the initial aqueous solution sample produces a mixture with the desired organic solvent concentration. Finally, the desired amount of organic solvent may be added to the container holding the first porous solid support, and sufficient initial aqueous solution sample may be slowly added to the container, such that the mixture of organic solvent and initial aqueous solution sample in the container at the first porous solid support is always equal to or higher than the desired organic solvent concentration of the initial aqueous solution sample.

[0026] In studies based on this invention, a certain amount of organic solvent is typically added to a second container to produce a mixture with an organic solvent concentration of 80% or higher, and then a sample is added to the organic solvent by adding an initial aqueous solution sample. For example, in these studies, 80 μL of organic solvent is added to a 20 μL initial aqueous solution sample. The resulting mixture is then transferred to a container holding a first porous solid support.

[0027] For ease of reference, the container holding the first porous solid support is sometimes referred to herein as a "reaction vessel". (In microfluidic devices, the initial aqueous solution sample is not transferred to a separate container holding the first porous solid support, but rather from a first section of a channel, conduit, etc., to a second section of that channel, conduit, etc., which contains the first porous solid support. The phrase "reaction chamber" is sometimes used herein to refer to a portion of a microfluidic device configured for carrying out the method of the present invention. For ease of reference, unless otherwise stated, the following will generally discuss embodiments in which the initial aqueous solution sample is transferred to a reaction vessel; however, it should be understood that in microfluidic applications, the initial aqueous solution sample is also transferred to a portion having a sufficiently large space to accommodate a mixture of the organic solvent and the initial aqueous solution sample, as well as the first porous solid support. The reaction vessel typically has: The container has a first opening at the top through which solutions and reagents can be introduced; a body, typically cylindrical, housing a first porous solid support; and a second opening, typically located at the bottom of the container opposite the first opening. The second opening may be closable to prevent solution from leaving the reaction vessel until necessary. The body of the container has a chamber with a cross-section (circular in the case of a cylindrical body) having an area defined by the dimensions of the body. The first porous solid support is placed in the reaction vessel, filling the cross-sectional area of ​​the reaction chamber such that the mixture of the organic solvent and the initial aqueous solution sample containing polysaccharides or other carbohydrates must pass through the pores or openings in the first porous solid support to reach the second opening.

[0028] Because polysaccharides and other carbohydrates are hydrophilic, they tend to remain on hydrophilic surfaces when in solutions containing 75% to 95% organic solvent. In some preferred embodiments, the concentration of the organic solvent is about 80%, where “about” means ±2% herein. It is not desirable to be bound by theory that polysaccharides or other carbohydrates are retained on hydrophilic surfaces under these conditions through non-covalent hydrophilic interactions rather than through filtration. Therefore, in these embodiments, the pore size of the first porous solid support is not critical, but should be small enough to prevent polysaccharides or other carbohydrates from contacting the hydrophilic surface of the first porous solid support. For example, the pores or openings in the first porous solid support should not be so large that droplets of the initial aqueous solution sample can pass through the pores or openings without contacting the hydrophilic surface. The suitability of any particular size of pore or opening can be readily tested by the assays described below. Preferred materials for these embodiments include glass fiber; cellulose; alumina; aminopropyl; asparagine; cyclodextrin; triazole; diethylaminoethyl; silica; or silica derivatized by chemical groups such as glycols, cyanopropyl, ethylenediamine-N-propyl, aminopropyl, amides (carbamoyl), zwitterionic groups, or combinations thereof; wherein glass fiber and derivatized silica are preferred, and glass fiber is particularly preferred. The first porous solid support is a support that is not derivatized with carboxyl groups or does not use carboxyl groups to capture carbohydrates present in the initial aqueous solution sample, and should not be made of a material or derivatized material capable of reacting with the reducing agent or labeling agent used in reductive amination. For example, the material should not be derivatized with multiple aldehydes. The material can be shaped as a membrane filling a region of the cross-section of the container or as a monolithic material, such that the solution must pass through the first porous solid support. However, in some embodiments, particularly in the case of silica, the material used for the solid support can be in the form of beads or particles, which can be held in place within the reaction vessel by, for example, a plastic frame with cross-hatching smaller than the diameter of the beads or particles, positioned below the beads or particles within the vessel's cavity. In some embodiments, the first porous solid support can be made of a material with a non-hydrophilic bottom layer but a hydrophilic surface layer to allow polysaccharides or other carbohydrates to remain on the hydrophilic surface. In a preferred embodiment, the material used to prepare the hydrophilic first porous solid support is hydrophilic.

[0029] If practitioners choose to increase the concentration of the organic solvent to 95% or higher, for example, to 99% or higher, thereby diluting the aqueous component to the point where the polysaccharide or other carbohydrate is almost in the pure organic solvent, then at lower concentrations, both hydrophilic and non-hydrophilic materials can be used as the first porous solid support. This allows practitioners to use less expensive materials that readily form pores or openings of the required size (e.g., 10 micrometers or less) for the first porous solid support, such as polyethylene, nylon, polyvinylidene fluoride, or polypropylene. As with the hydrophilic materials described above, the non-hydrophilic material chosen for use as the first porous solid support should not be a material containing chemical groups (such as aldehydes) that are expected to react with reducing agents or markers that are reductively amination-enhanced, or a material derivatized with chemical groups (such as aldehydes) that are expected to react with reducing agents or markers that are reductively amination-enhanced, or a material derivatized with chemical groups (such as aldehydes). Unwilling to be bound by theory, it is believed that at organic solvent concentrations above 95%, polysaccharides or other carbohydrates aggregate or precipitate from the initial aqueous solution sample and can be captured and retained on the first porous solid support by filtration through pores or openings of 10 micrometers or smaller.

[0030] Using hydrophilic materials with pore sizes of 10 micrometers or smaller and organic solvents with a concentration greater than 95% also allows for the retention of monosaccharides and other small polysaccharides or carbohydrates that do not precipitate well and have weak hydrophilicity. Therefore, depending on the specific polysaccharide or carbohydrate of interest and the material used for the first porous solid support, an organic solvent is added to the initial aqueous solution sample to produce a mixture of 80% to 99% or more organic solvent, for example, by adding the organic solvent to the initial aqueous solution sample in a ratio of 4:1 to 49:1. The appropriate ratio of organic solvent to the initial aqueous solution sample and the appropriate material used for the first porous solid support will be discussed in more detail below.

[0031] As noted, the material comprising the first solid support is porous, with pores or openings sized to allow the initial aqueous solution sample to permeate or filter through the first porous solid support and retain the glycans or other carbohydrates at the concentration of the organic solvent present, while allowing liquids such as the aqueous components of the initial aqueous solution sample, as well as solutes still dissolved in the mixture of the organic solvent and the initial aqueous solution, such as deglycosylation enzymes, proteins (glycosylated or deglycosylated), buffer salts, water, and reducing agents, to elute from the first porous solid support and exit the reaction vessel. In studies based on this invention, it was found that proteins precipitate from the solution along with the glycans or other carbohydrates and deposit on the first porous solid support. The presence of the precipitated proteins does not affect the reduction or labeling of the glycans or other carbohydrates.

[0032] The interaction between the first porous solid support and the carbohydrate is non-covalent, and therefore does not require the time or conditions necessary for covalent interactions to form between the carbohydrate and the first porous solid support. In studies based on this invention, a series of glycoproteins are deglycosylated and the resulting glycan is labeled by reductive amination according to the methods described herein, wherein proteins, such as any aglycosylated proteins remaining after the deglycosylation reaction, and any deglycosylating enzymes that may bind to the first porous solid support, do not interfere with the labeling or subsequent separation and analysis of the carbohydrate.

[0033] Preferably, the first porous solid support containing retained polysaccharides or other carbohydrates is washed with an organic solvent, either a pure organic solvent or a mixture in which the organic solvent concentration is 95% or higher. This washing is typically very fast, but requires the removal of any remaining water from the initial aqueous solution sample, or requires dilution to a level below the level that would interfere with carbohydrate reduction or reductive amination. Therefore, washing with an organic solvent or a mixture in which the organic solvent concentration is 95% or higher eliminates the need for a separate drying step. Since drying steps typically take a considerable amount of time to evaporate water from the initial aqueous solution sample, or require equipment to provide a vacuum for rapid evaporation of water, eliminating the drying step significantly reduces the time (in the case of simple evaporation) or cost and equipment (in the case of generating a vacuum to accelerate evaporation) required to prepare the carbohydrates needed for reductive amination. Furthermore, since the separation of carbohydrates from other components and the removal of the aqueous solution do not require (a) transfer to an SPE cartridge and (b) elution of the purified carbohydrates from the SPE cartridge into a container for drying, the time required for these steps and the potential loss of carbohydrates during the transfer process are eliminated.

[0034] Now, certain aspects of various embodiments of the method of the present invention will be discussed to provide further explanation and guidance.

[0035] Reduction or reductive amination of polysaccharides or other carbohydrates on a first porous solid support

[0036] In some embodiments, a reducing agent is used to reduce the glycan or other carbohydrate, or, under conditions (such as pH, temperature, and time) that allow reductive amination to occur to label the carbohydrate, the glycan or other carbohydrate is subjected to reductive amination with a suitable label, such as 2-AB (2-aminobenzamide), 2-AA (o-aminobenzoic acid), APTS (8-aminopyrene-1,3,6-trisulfonic acid, CAS No. 196504-57-1), or procainamide hydrochloride (CAS No. 614-39-1). Of course, the conditions used for reducing and reductively amination of glycans or other carbohydrates are well known in the art. For example, U.S. Patent No. 5,747,347, published in 1998, teaches the labeling of carbohydrates by reductive amination using 2-AB. Therefore, it is anticipated that those skilled in the art are well familiar with the reaction conditions suitable for reductive amination reactions using markers such as those listed above (for clarity, note that markers used in reductive amination reactions, such as 2-AB, 2-AA, or APTS, are sometimes referred to as dyes by practitioners. Following this usage in the art, the two terms may be used interchangeably herein). In other embodiments, the polysaccharides or other carbohydrates are reduced by using a reducing agent.

[0037] In a preferred embodiment, the polysaccharide or other carbohydrate is immobilized on a first porous solid support during reduction or reductive amination. As practitioners understand, the reagent volume used in the analytical method is very small. Once the polysaccharide or other carbohydrate is retained on the first porous solid support (e.g., a membrane, bulk material, or silica beads), a reducing agent or labeling material is typically added in amounts as small as 10 to 20 microliters. It is conceivable, without being bound by theory, that such a small amount of reagent will be retained on the first porous solid support by capillary action or surface tension. In a typical embodiment, the container can be opened at the end where it is designed to allow liquid to flow out (typically at the bottom if the container is designed to allow liquid to move vertically from top to bottom, or at the bottom if the container is designed to allow fluid to move horizontally, e.g., in some microfluidic applications, a microfluidic tube may be designed to introduce fluid from one side and exit from the other, where the inlet and outlet openings are different). However, if desired, the container can have a manual or automatic way to seal the bottom or outlet area, thereby retaining the reagent in the container during steps requiring the reagent's presence, and opening the bottom or outlet area when needed to allow it to drain or be eluted from the container. For example, the bottom can have a sheet, cap, or other covering that allows fluid to remain in the container when closed above the opening, and allows fluid to flow out of the container when opened. In microfluidic applications, reductive amination occurs in a reaction chamber that can be placed vertically or horizontally. For example, one or more valves may be present between the reaction chamber and a channel or other pathway, allowing the practitioner to continue the reaction by opening the valve separating the reaction chamber from a specific channel so that the solution and solvent can be eluted from the reaction chamber along the desired path. If the practitioner wishes to reduce or label glycans or other carbohydrates in larger volumes for use at a preparative scale rather than an analytical scale, a removable cap at the bottom of the container is preferred.

[0038] After reducing the glycan or other carbohydrate, they can be washed to remove excess reducing agent. Following reductive amination, the now-labeled glycan or other carbohydrate can be washed to remove any marker not bound to the carbohydrate (“excess” or “free” dye or marker). The solvent or solution used for this washing step depends on the solubility of the reducing agent or marker in the organic solvent used to wash the glycan or other carbohydrate, and whether the material of the first porous solid support is hydrophilic. For example, dyes 2-AA and 2-AB are well soluble in organic solvents, while APTS is not. For water-soluble carbohydrates reduced with a reducing agent or dye-labeled carbohydrates, practitioners can use workflow 1A, or workflow 1C if the practitioner has used a hydrophilic first porous solid support.

[0039] In workflow 1A, the hydrophilic or non-hydrophilic solid support is simply washed with a pure organic solvent or a solution of organic solvent with a concentration greater than 95% (correspondingly less than 5% water or aqueous solution), preferably using a higher ratio of organic solvent to water or aqueous solution. In these embodiments, the reduced or labeled polysaccharide or other carbohydrate is retained on the first porous solid support, while during washing with an organic solvent or a high concentration of organic solvent, the reducing agent or free dye is eluted from the container into the water or aqueous solution. The reduced or labeled polysaccharide or other carbohydrate is then eluted from the reaction vessel or reaction chamber into the receiving container by washing the membrane with an aqueous solution. The reduced or labeled carbohydrate can then be provided to the analytical element for analysis.

[0040] If the first porous solid carrier is hydrophilic, then practitioners can use workflow 1C instead. For example... Figure 1 As shown, in step 6 of workflow 1C, the first porous solid support is washed with an organic solvent mixture having an organic solvent concentration of 75% or higher. In a preferred embodiment, the concentration of the organic solvent is 78% or higher, and preferably about 80%, where "about" means ±2%. As previously stated, at these organic solvent concentrations, the polysaccharide will remain on the hydrophilic first porous solid support, thereby washing away the reducing agent or excess label.

[0041] Unlike 2-AA and 2-AB, APTS is poorly soluble in organic solvents. In embodiments where the reducing agent and dye chosen by the practitioner are not well soluble in organic solvents and the first porous solid support selected for reduction or labeling by reductive amination is non-hydrophilic, the workflow will be changed as follows: Figure 1 The workflow 1B is shown. This workflow differs from workflow 1A in the following ways. First, because the reducing agent or dye is poorly soluble in organic solvents, a solution with a higher concentration of organic solvent relative to water is used to wash the reducing agent or excess dye off the first porous solid support compared to the solution used in workflow 1A. Typically, the washing solution for APTS or other dyes or reducing agents poorly soluble in organic solvents is about 75% to 90% organic solvent, preferably about 80%, with the term "about" here meaning ±2% (for convenience, this washing solution will sometimes be referred to below as "organic solvent / aqueous washing solution"). For any specific reducing agent or dye, the appropriate concentration of organic solvent relative to the aqueous buffer for removing the reducing agent or APTS or other dyes poorly soluble in organic solvents can be determined by referring to the guide concentration of the solvent used in the HILIC for separating carbohydrates.

[0042] Secondly, the reduced or labeled glycans or other carbohydrates will not remain aggregated or precipitated at the concentrations of the organic solvent / aqueous cleaning solution used in these embodiments, and will therefore redissolve and detach from the first porous solid support. Furthermore, since the first porous solid support in these embodiments is made of a non-hydrophilic material, the reduced or labeled glycans or other carbohydrates will not be retained on the support through hydrophilic interactions. Therefore, in these embodiments, as the reduced or labeled glycans or other carbohydrates are released from the first porous solid support due to organic solvent / aqueous cleaning, a second porous solid support can be used to capture these reduced or labeled glycans or other carbohydrates, since this second porous solid support is made of a hydrophilic material capable of retaining the glycans (or other carbohydrates in other carbohydrate embodiments) released from the first porous solid support. Not wishing to be bound by theory, in the presence of the organic solvent / aqueous cleaning solution, the glycans released from the non-hydrophilic first porous solid support will be retained on the hydrophilic second porous solid support through hydrophilic interactions.

[0043] In a preferred embodiment, the second porous solid support is disposed downstream of the first solid support along the path of the solvent and solution moving through the container. In a preferred embodiment, the second porous solid support is in the same container (or a section of a microfluidic channel, tube, etc.) as the first porous solid support. Like the first solid support, the second solid support is porous, allowing the solution and dissolved reducing agent or excess dye to flow through and be eluted from the container; however, as described above, the second solid material is made of a hydrophilic material, which is capable of retaining the reduced or labeled polysaccharides in the presence of an organic solvent / aqueous cleaning solution used to wash the reducing agent or excess labeling off the first porous solid support. In these embodiments, the hydrophilic second porous solid carrier used to capture polysaccharides or other carbohydrates detached from the non-hydrophilic first porous solid carrier is made of any hydrophilic material suitable for manufacturing the hydrophilic first porous solid carrier as described above (e.g., glass; cellulose; alumina; aminopropyl; asparagine; cyclodextrin; triazole; diethylaminoethyl; silica; or silica functionalized or derivatized with chemical groups such as diol, cyanopropyl, ethylenediamine-N-propyl, amide (carbamoyl), one or more zwitterionic groups, or combinations of two or more of these). In some preferred embodiments, the silica or functionalized silica is in the form of beads or particles. In some preferred embodiments, the silica is carbamoyl-functionalized or derivatized. In some embodiments, the carbamoyl-derived silica is Amide-80, and in some embodiments it consists of beads with a size of 30 micrometers. Hydrophilic materials cannot be materials containing chemical groups (such as aldehydes) that are expected to undergo reductive amination with reducing agents or markers, or materials derived from chemical groups (such as aldehydes) that are expected to undergo reductive amination with reducing agents or markers.

[0044] Any particular material or its functional groups can be readily tested to determine whether it is hydrophilic enough to be used as a hydrophilic material for preparing a second porous solid support or a hydrophilic first porous solid support. For example, the material of interest or the functionalized material can be placed in a column, and a known amount of labeled carbohydrates can be introduced into the column in a selected organic solvent / aqueous cleaning solution. The fluid flowing out of the column enters an analytical instrument (such as a fluorescence detector) to measure how much of the known amount of labeled carbohydrates has left the column. The difference between the amount of labeled carbohydrates introduced into the column and the amount that leaves the column is the amount retained by the material being tested. Materials that retain a higher amount of labeled carbohydrates are preferred.

[0045] Returning to the overall description of the method of the present invention, once the reducing agent or excess label has been washed away, the reduced or labeled polysaccharides or other carbohydrates are eluted from the second porous solid support by washing it with an aqueous solution. Figure 1 In the depicted embodiments, this step is shown as step 7 of the various workflows. Typically, up to 20% of the solution used to elute carbohydrates may be an organic solvent, but at least 80% may be an aqueous solution, with a higher concentration of aqueous solution preferred. It should be noted that the “aqueous solution” can be water, but is preferably a buffer solution, such as HEPES (2-[4-(2-(hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) buffer, phosphate-buffered saline, or ammonium formate buffer. The presence of salt in the buffer solution increases the polarity of the aqueous solution, thereby enhancing the release of glycans or other carbohydrates from the solid carrier during elution. When the “glycan or other carbohydrate” is a labeled glycan, the buffer helps to maintain the glycan in a pH-controlled solution, thereby increasing its stability.

[0046] The eluted, reduced or labeled carbohydrates are then collected and fed to analytical instruments for analysis. For example, the reduced or labeled glycans or other carbohydrates can be separated using high-performance liquid chromatography (HPLC), capillary electrophoresis, microfluidic separation, or hydrophilic interaction liquid chromatography (“HILIC”). The labeled glycans or other carbohydrates can then be analyzed by detecting the fluorescence of the labeled glycans or other carbohydrates and measuring the intensity of that fluorescence, or by mass spectrometry, or by a combination of detecting fluorescence intensity and mass spectrometry. Reduced glycans or other carbohydrates are typically analyzed by mass spectrometry.

[0047] As those skilled in the art will understand, it is easy to determine whether any particular dye is considered to be well soluble or poorly soluble in any particular organic solvent. For example, in a transparent container, if a reducing agent or dye can be dissolved in an organic solvent at a concentration of 10 mg / ml, it is considered to have achieved high solubility. If the tested label cannot reach this concentration in the selected organic solvent without visible precipitation or remaining undissolved, it indicates that the label has low solubility in that solvent, and if the first porous solid support is non-hydrophilic or its surface is non-hydrophilic, then workflow 1B should be used.

[0048] Reference Figure 1 Some embodiments of the present invention method discussed above can be used to better illustrate the implementation of the present invention. Figure 1 The figures illustrate several exemplary embodiments of the method of the present invention. Figure 1Step 1 shows a vial containing an aqueous solution of the glycoprotein. The protein component of the glycoprotein molecule is represented by folded solid lines, while the glycan component is represented by light and dark geometric shapes. The covalent bonds between the glycan and the protein component are represented by thin straight lines between the shapes representing the glycan and the thick lines representing the protein component. Arrows indicate the addition of a deglycosylation enzyme to the aqueous solution, and Step 2 describes the result of the enzymatic digestion of the glycoprotein, thereby releasing the glycan. To the right of Step 2 are arrows pointing to workflows 1A through 1C.

[0049] In all workflows, the arrows between steps 2 and 3 indicate the addition of an organic solvent to the aqueous solution that has undergone enzymatic digestion, to produce a mixture with a certain organic solvent concentration, as shown in the corresponding workflow. This organic solvent concentration is approximately 80% or more when the first porous solid support is hydrophilic or has a hydrophilic material surface; and in workflows where the first porous solid support is non-hydrophilic, the organic solvent concentration is greater than 95%. In step 3 of each workflow, a mixture having the desired concentration of organic solvent and the initial aqueous solution sample is introduced into a reaction vessel containing the first porous solid support. In each exemplary workflow, the first porous solid support is shown as a membrane. In step 3, the first porous solid support (in this case, a membrane) captures and retains the glycan, which, as previously described, is represented by light and dark geometry. In this figure, the reaction vessel in the illustrated workflow has a cylindrical body and an inverted conical bottom, wherein the solution is introduced from the top of the vessel and passes through the membrane, which is positioned just below the middle of the cylindrical body. In workflow 1B, the reaction vessel further comprises a second porous solid support of hydrophilic material disposed within a conical portion (shown as a shaded area within the conical portion of the vessel). In all three workflows, the glycan remains on the membrane while the mixture of the initial aqueous solution and organic solvent passes through the membrane. In step 4 of each workflow, the membrane is washed with an organic solvent or a solution of organic solvent greater than 95% to remove salts, cleaning agents, and other unwanted components that may be present on the membrane, while the glycan remains on the first porous solid support. In step 5, an agent (including a marker of choice for the practitioner, typically a reducing agent) for reducing but not labeling the glycan, or for labeling the glycan by reductive amination, is introduced into the vessel, and reduction or reductive amination occurs as the glycan is immobilized on the membrane. Figure 1In the illustrated embodiment, the tip of the inverted conical portion has an opening (not shown) that allows the washing solution and eluted reagents to exit the container. In other embodiments, the bottom of the container may have an opening releasably covered by a sheet, cap, or other covering, which can be manually or automatically removed by an operator to allow the solution or reagent to drain from the container when needed. In step 6 of workflow 1A, the membrane is washed with an organic solvent or an aqueous solution containing more than 95% organic solvent and less than 5% aqueous solution to remove reducing agents or any markers not bound to carbohydrates. In step 6 of workflow 1B, the membrane is washed with a solution containing a lower percentage of organic solvent relative to aqueous solution than the solution used in workflow 1A, because this workflow is employed when the reducing agent or dye is poorly soluble in organic solvents and the first porous solid support is not hydrophilic. The washing solution in the example shown in workflow 1B is approximately 80% organic solvent and approximately 20% aqueous solution (e.g., water or an aqueous buffer). With such a high water content in the washing solution, the glycans will not remain on the non-hydrophilic solid support and will flow into the hydrophilic second porous solid support along with excess dye. The glycans are then captured and retained on the hydrophilic second porous solid support, while the reducing agent or excess dye will flow out through the opening at the tip of the inverted cone. In step 7 of all workflows, an aqueous solution is used to elute the glycans from the porous solid support on which they are held (the first porous solid support in workflows 1A and 1C; the second porous solid support in workflow 1B). Typically, the reduced or labeled glycans are eluted into wells, vials, or other collection containers.

[0050] It should be noted that the container with two porous solid carriers shown in workflow 1B can of course also be used in the workflow shown in workflow 1A. Figure 1 In step 3 of step A, a mixture of an organic solvent (over 95%) and an aqueous solution is used to retain the polysaccharide on the first porous solid support, and in step 6, a washing solution containing over 95% organic solvent is used to retain the polysaccharide on the first porous solid support. Regardless of the material used to prepare the first porous solid support, a second porous solid support is unnecessary. Therefore, a container with two supports can be flexibly used in either workflow.

[0051] In some preferred embodiments, the eluted reduced or labeled glycans or other carbohydrates are then separated by, for example, high-performance liquid chromatography, capillary electrophoresis, microfluidic separation, or hydrophilic interaction liquid chromatography, and then analyzed by feeding them to a selected analytical instrument. Typically, the above analysis is performed by feeding the glycans or other carbohydrates to one or both of a fluorescence detector or a mass spectrometer.

[0052] Reduced without labeling glycans or other carbohydrates

[0053] As mentioned above, carbohydrates exist in two epimeric forms, resulting in two signals for each carbohydrate during analysis. Reduction of the carbohydrate's reducing end ensures that each carbohydrate in the sample exists in only one epimeric form, making signal analysis easier and improving detection sensitivity. Therefore, in some embodiments, practitioners can choose to reduce the polysaccharide or other carbohydrate immobilized on the first porous solid support without labeling it.

[0054] In these cases, following the steps outlined above for preparing polysaccharides or other carbohydrates for reductive amination, the polysaccharides or other carbohydrates are immobilized on a first porous solid support. The immobilized polysaccharides or other carbohydrates are then typically contacted with one or more reducing agents in an organic solvent, without contacting the dyes (such as 2-AA or 2-AB) used in labeling the polysaccharides or other carbohydrates by reductive amination. Suitable reducing agents and processes will be discussed in separate sections below.

[0055] Once the glycans or other carbohydrates are reduced, they can be eluted from the first porous solid support, as described above regarding glycans or other carbohydrates labeled with dyes highly soluble in organic solvents. In some preferred embodiments, the labeled glycans or other carbohydrates are then separated by, for example, high-performance liquid chromatography, capillary electrophoresis, microfluidic separation, or hydrophilic interaction liquid chromatography, and then analyzed by feeding them to a selected analytical instrument. Typically, the analysis of reduced but unlabeled glycans or other carbohydrates is performed by feeding them to a mass spectrometer.

[0056] carbohydrate

[0057] The method of this invention can generally be used for any carbohydrate with a reducing end. In some preferred embodiments, the carbohydrate is a polysaccharide; in some embodiments, the carbohydrate is an oligosaccharide, and in others, they may be a disaccharide or a monosaccharide. Non-starch polysaccharides (such as cellulose and hemicellulose), as well as pectin, and cross-linked polysaccharides (such as...) This is a preferred embodiment. In some preferred embodiments, the carbohydrate to be reductively amination and labeled is a polysaccharide released from the glycoconjugate by enzymatic digestion. In some preferred embodiments, the polysaccharide is an O-polysaccharide. In some preferred embodiments, the polysaccharide is an N-polysaccharide. In some preferred embodiments, either the O-polysaccharide or the N-polysaccharide has been released from the glycoconjugate by enzymatic digestion, as described below.

[0058] Glycoproteins and enzymes that release glycans digestion

[0059] Glycoproteins are produced by eukaryotic cells after protein translation by covalently adding linear or straight-chain carbohydrates. These protein-carbohydrate conjugates are called glycoproteins; the junctions of the carbohydrates are called glycosylation sites. The polysaccharides or oligosaccharides attached to the protein are called glycans. A wide variety of glycans have been found at different glycosylation sites on specific glycoproteins. The specific pattern of glycans on a particular glycoprotein depends on the specific cell line that produces that protein and the conditions under which the cells grow.

[0060] Glycans are typically linked to glycoproteins in one of two ways. The first way, known as N-glycans, involves the glycan being linked at an asparagine residue via an N-glycosidic bond. The second way, known as O-glycans, involves the glycan being linked to the oxygen atom of an amino acid residue. For example, N-acetylgalactosamine can be enzymatically linked to the oxygen atom of a serine or threonine residue.

[0061] N-Glycans can be enzymatically released from glycoproteins via enzymatic hydrolysis by various enzymes, such as PNGase F (peptide-N4-(acetyl-β-glucosamine)-asparaginamide, EC 3.5.1.52). The removal of glycans from glycoconjugates by enzymatic activity is generally referred to as “enzymatic digestion.” Enzymatic digestion of N-glycans, such as that via PNGase F, is typically carried out in an aqueous solution and results in the initial release of N-glycans as β-glycosamines, where the free reducing end of the released glycan binds to ammonia (see, for example, Tarentino, et al. TIGG 1993, 23, 163-170; Rasmussen J.R. J Am. Chem. Soc. 1992, 114, 1124-1126; Risley, et al. J. Biol. Chem. 1985, 260, 15488-15494, 1985). As described in the Background section, N-glycans released by PNGase F are typically labeled via reductive amination, in which the free reduced end of the glycan is conjugated to the free amino group of a label (such as a fluorescent dye or a charged moiety). Depending on the label used, the labeled glycan can then be analyzed by any of a variety of analytical methods, such as high-performance liquid chromatography (“HPLC”), capillary electrophoresis (“CE”), carbohydrate gel electrophoresis, or microfluidic separation.

[0062] organic solvents

[0063] In embodiments of the method of the present invention, an organic solvent is used to reduce the concentration of the initial aqueous solution sample to or below the point at which carbohydrates in the sample would aggregate or precipitate, or aggregate and precipitate on the first porous solid support, so that other compounds in the sample can be removed by washing the first porous solid support, for example, with an organic solvent. In some studies based on the present invention, acetonitrile has been used as the organic solvent, and is particularly preferred. Acetonitrile is hydrophobic and aprotic. It is contemplated that other organic solvents may be used in embodiments of the method of the present invention, and hydrophobic and aprotic organic solvents are preferred. A variety of other organic solvents considered suitable for embodiments of the present invention include: anhydrous ethanol, anhydrous methanol, isopropanol, butanol, toluene, ethyl acetate, acetone, tetrahydrofuran, diethyl ether, dichloromethane, chloroform, tert-butyl methyl ether, benzene, carbon tetrachloride, isooctane, and hexane. Mixtures of two or more of the organic solvents such as those shown above may also be used.

[0064] Dimethyl sulfoxide (“DMSO”) and dimethylformamide (“DMF”) are less preferred as the main organic solvent or main component of a mixture of organic solvents used in embodiments of the method of the present invention. It is believed that DMSO or DMF can be mixed in a relatively suitable amount (such as 0.1% to 2%) with one of the organic solvents mentioned in the preceding paragraph or a mixture of the aforementioned organic solvents without affecting the ability of the solid support to retain carbohydrates and enable the reductive amination reaction to proceed in the presence of the organic solvent or mixture of organic solvents.

[0065] Those skilled in the art will understand that no single solvent is suitable for all cases, regardless of the solid carrier that may be used in the embodiments of the methods of the present invention. Furthermore, those skilled in the art who label glycans released from glycoproteins know that it is generally necessary to test combinations of reagents to determine whether such combinations are suitable for the release, labeling, and analysis of glycans present on a variety of glycoproteins, and such testing is considered routine in the art. Practitioners can readily test the suitability of any specific organic solvent for any specific sample and any specific solid support, wherein the sample contains a carbohydrate that the practitioner wishes to reduce or label with a specific marker by a specific reducing agent, and the solid support on which the practitioner wishes to retain the carbohydrate for reduction or labeling. The test involves: adding an excess of the organic solvent to be tested to an initial aqueous solution sample containing a known amount of the selected carbohydrate; contacting the solvent / solution mixture with the selected solid support (regardless of whether a protocol for labeling carbohydrates by reductive amination is known or a solid support being tested for this purpose); reducing or labeling the carbohydrate by reductive amination with 2-AB; washing the solid support with the organic solvent to remove any marker not bound to the carbohydrate; eluting the carbohydrate from the solid support with an aqueous buffer; and then analyzing the eluted solution to determine whether the carbohydrate has been reduced or labeled, and if so, whether it is present in the expected amount. If the eluent analyzed is found to contain reduced or labeled carbohydrates not in the expected amounts, it indicates that the specific combination of organic solvent, reducing agent or labeling agent, and solid carrier is unsuitable for reducing or labeling the selected carbohydrates using the chosen reducing agent or labeling agent.

[0066] Contact the initial aqueous solution sample with an organic solvent.

[0067] The glycan or other carbohydrate to be labeled, or the glycan or other carbohydrate to be reduced without labeling, can be obtained in various ways. In some embodiments, the carbohydrate of interest is a glycan linked to a glycoprotein, which can be released into an aqueous solution by incubating the glycoprotein with a deglycosylation enzyme, such as the exemplary deglycosylation enzyme PNGase F. In other embodiments, the glycan or other carbohydrate of interest is already present in the aqueous solution. In any embodiment, the aqueous solution containing the glycan or other carbohydrate of interest is contained in a first container (which, for convenience, is sometimes referred to herein as a “sample container”). The aqueous solution is transferred from the sample container to a second container, such as a tube, cylinder, the pores of a porous plate, or a discrete section of a microfluidic device. The first porous solid support may already be disposed in the second container, or the first porous solid support may be disposed in the second container after the aqueous solution has been transferred to the second container. For ease of reference, the second container is sometimes referred to herein as a “second container” or a “reaction container”. When the aqueous solution is introduced into the second container, the second container may already contain a sufficient amount of organic solvent to retain the polysaccharides or other carbohydrates in the aqueous sample on the first porous solid support, or after the aqueous solution containing polysaccharides or other carbohydrates is transferred to the second container, a sufficient amount of organic solvent may be introduced into the second container to retain the polysaccharides or other carbohydrates in the aqueous sample on the first porous solid support.

[0068] As noted, in some embodiments, the reduction or labeling of glycans or carbohydrates of interest can be performed in a microfluidic device. Some microfluidic devices have been used to release glycans from glycoproteins via enzymatic digestion. Typically, the region in such a device where enzymatic digestion takes place may be conveniently referred to as the "first chamber," where these regions are not designed to have sufficient space for a first solid support and a sufficient amount of organic solvent to be introduced into the aqueous solution to retain the glycans or other carbohydrates in the aqueous solution sample on the first porous solid support. Conveniently, the device can be designed to allow the aqueous solution containing the released glycans to flow from the first chamber into a larger second chamber within the microfluidic device, which contains the first solid support and also has sufficient space to introduce a sufficient amount of organic solvent to cause the glycans to aggregate or precipitate, or aggregate and precipitate on the first solid support. Once the glycans have bound to the first solid support, any residual aqueous solution is immediately washed away with an organic solvent to provide anhydrous conditions, enabling the reduction of the glycans in the second chamber or labeling of the glycans by reductive amination.

[0069] In some implementations, carbohydrates may be present in the mixture or sample. For example, practitioners may wish to reduce or label carbohydrates present in ammoniated cell lysates or sonicated tissue samples by reducing their presence. The lysates or sonicated tissue can be placed in a pore, tube, cylinder, or other container with a solid support; or, the solid support can be added to the container after the lysates or sonicated tissue has been placed in it. An organic solvent may be present in the container when the lysates or sonicated tissue are added; or, the organic solvent may be added subsequently to retain carbohydrates in the lysates or tissue, or to retain glycans or other carbohydrates in an aqueous sample, on a first porous solid support. The organic solvent and the lysates or sonicated tissue are placed in the container such that the lysates or sonicated tissue are completely submerged in the solvent. It should also be noted that, regardless of the ratio of organic solvent to aqueous solution used to retain carbohydrates or glycans or other carbohydrates in an aqueous sample on the first porous solid support, once the carbohydrates are retained on the solid support, any residual aqueous solution is washed away with the organic solvent to provide anhydrous conditions conducive to the reduction of carbohydrates or labeling of carbohydrates by reducing amination.

[0070] The volume of organic solvent that reduces the concentration of aqueous solutions

[0071] As described in the preceding sections, standard protocols for reducing glycans or other carbohydrates, or for labeling them via reductive amination, rely on drying the glycans or carbohydrates prior to reductive amination, as these processes typically require anhydrous conditions. Similarly, as noted above, glycans and carbohydrates in lysates or other biological samples released from glycoproteins via enzymatic digestion are typically present in aqueous solutions and often require a drying step to provide the anhydrous conditions necessary for reduction or reductive amination.

[0072] In embodiments of the method of the present invention, the drying step and thus the time required for drying are eliminated by introducing an excess of organic solvent into an aqueous solution containing polysaccharides or other carbohydrates. As used herein, the phrase “excess organic solvent” refers to an amount of organic solvent that reduces the ratio of the aqueous solution to the organic solvent to such a point that the carbohydrates in the sample remain on the hydrophilic surface or cause them to aggregate or precipitate, or aggregate and precipitate, thereby retaining them on the first porous solid support by the excess aggregated or precipitated polysaccharides or carbohydrates.

[0073] In the study based on this invention, a standard deglycosylation protocol is employed, wherein the glycoprotein is enzymatically digested in an aqueous solution, thereby releasing the glycans into the aqueous solution. Typically, this protocol produces 25 μl to 50 μl of aqueous solution containing fully or partially deglycosylated glycoproteins, deglycosylation enzymes, glycans released from the glycoproteins, buffer salts, reducing agents, and other denaturing agents (consistent with those used elsewhere in this invention; this portion is referred to in this section as the “starting aqueous solution sample”). A large excess of organic solvent, 675 μl, is then added to the starting aqueous solution sample, resulting in a solvent / solution mixture of approximately 19 parts organic solvent to 1 part starting aqueous solution sample (assuming an average starting aqueous solution sample size of 35 μl). A porous glass fiber solid support is then contacted with the solvent / solution mixture, allowing the glycans in the sample to be retained on the first porous solid support. Not wishing to be bound by theory, since the average concentration of the organic solvent exceeds 95%, it can be assumed that the glycans will precipitate or aggregate and be retained on the glass fiber by filtration (although hydrophilic interactions may also play a role). The polysaccharides immobilized on the solid support were then labeled by reductive amination while maintaining their immobilization on the first porous solid support. The labeled polysaccharides were then eluted from the first porous solid support for analysis.

[0074] In these initial studies, a ratio of 95% or more organic solvent to 5% or less aqueous solution was selected to test the retention rate of carbohydrates on a first porous solid support in the presence of a significant excess of organic solvent relative to the initial aqueous solution sample. In other studies, different ratios of organic solvent to aqueous solution and different materials (aminopropyl-derived silica particles) were used as the first porous solid support. These studies showed that at a 1:1 ratio of organic solvent to initial aqueous solution sample, at least some carbohydrates were retained on the solid support; and at higher ratios, the carbohydrates present in the sample were retained on the solid support at a higher proportion. The ratio of organic solvent to aqueous solution will result in more complete or complete capture of carbohydrates present in a particular sample, and it is expected that this ratio will vary depending on the specific organic solvent used and the material used as the first porous solid support. Those skilled in the art are familiar with solid-phase extraction materials and procedures for capturing carbohydrates, and it is expected that knowledge of useful solvents and materials in solid-phase extraction procedures for carbohydrates will provide practitioners with the necessary guidance in selecting suitable solvents and materials useful in embodiments of the methods of the present invention.

[0075] Based on these results, an organic solvent concentration of 70% to 100% is considered suitable for retaining carbohydrates on a solid support, with each percentage exceeding 75% being more preferred, and 80% or higher being more preferred than a concentration of 75% to 79%. As mentioned above, it is contemplated that the specific minimum ratio of organic solvent to aqueous solution that allows carbohydrates to be retained, or retained, on any specific solid support depends on the specific organic solvent and the specific solid support chosen. Any specific ratio or concentration of organic solvent to aqueous solution can be readily tested relative to a specific organic solvent and a solid support of any specific material, for example, by performing two parallel determinations according to the protocol described in the examples, wherein the ratio or concentration of organic solvent to aqueous solution used in the first determination is 19:1 (“known ratio” or “known concentration”), and the specific ratio or concentration to be tested is used in the second determination (“test ratio” or “test concentration”). If the amount and type of reduced or labeled glycans analyzed at the test ratio or concentration are within acceptable limits for measurements of the amount and type of reduced or labeled glycans at known ratios or concentrations, then that test ratio or concentration is acceptable for the reduction and labeling of glycans using the specific solid carrier being tested. Since different carbohydrates and different solid carriers may require higher ratios or concentrations of organic solvents with aqueous solutions, or may also function well at lower ratios or concentrations, similar parallel assays can be used to determine the ratio of organic solvent to aqueous solution suitable for any combination of the carbohydrate to be labeled and the solid carrier to be tested for capturing the carbohydrate in solution. As those skilled in the art will understand, the analysis of glycans linked to glycoproteins typically requires testing to determine, for example, how optimally to deglycosylate a specific glycoprotein of interest, which may be somewhat resistant to deglycosylation due to various factors, such as the protein's tertiary structure, the availability of deglycosylation enzymes to the glycosyl groups, and the glycoprotein's resistance to denaturation that can acquire glycans. Therefore, the aforementioned tests for quantity and type are considered routine in the art.

[0076] While the discussion above focused on determining a lower ratio of organic solvent to aqueous solution, it is also expected that ratios higher than 19:1, such as 20:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1 to 39:1, or 40:1 to 49:1, would also be useful. However, ratios higher than approximately 25:1 increase the amount and cost of reagents used, as well as the size of the containers required for mixing the reagents, without necessarily increasing the retention of carbohydrates on the solid support. Furthermore, higher ratios can dilute carbohydrates to the point where longer incubation times are required for them to contact and remain on the solid support. Therefore, ratios higher than 49:1 are less preferred. It is believed that the preferred ratio of organic solvent to aqueous solution is sufficient to retain the polysaccharides or other carbohydrates in the initial aqueous solution sample on the solid support, and then the solid support is washed with organic solvent to remove any residual aqueous solution, thereby enabling reduction or reductive amination.

[0077] Containers for holding solid carriers

[0078] As described above, in a typical embodiment, the glycan or other carbohydrate to be reduced or labeled by reductive amination is initially in an aqueous solution, which is then diluted by adding the aqueous solution to a larger volume of organic solvent (or conversely, by diluting the aqueous solution containing the glycan or other carbohydrate by adding a large volume of organic solvent). Conveniently, the resulting organic solvent / aqueous solution containing the glycan or other carbohydrate to be reduced or labeled is placed in a second container designed for this purpose. In a typical embodiment, the container has a body of a certain length and two ends. In some embodiments, the two ends are positioned opposite each other along the length of the container body. Each of the two ends is independently openable or capable of being independently opened to allow the introduction of solution and reagents at the first end and the discharge of solution and reagents at the second end. In some embodiments, the container is cylindrical. In some embodiments, the container is cylindrical but narrows at the second end to form an orifice to facilitate the capture of the eluted labeled carbohydrates during elution for analysis. In some embodiments, the container may be an Eppendorf tube, a microcentrifuge tube, or a centrifuge tube. Typically, such a tube has: a first section having an opening or a first open end; a cylindrical body connected to a second section opposite the first end, the second section having a tapered shape that narrows away from the cylindrical first portion until reaching the second end. The second end can be opened, but in some embodiments it is capable of being closed, so that when the bottom end is closed, the solution in the container can be incubated in the container, and when the second end is open, the solution can flow out of the container. For example, the bottom end can be fitted with a cap or removable cover so that a user or automatic device can open the bottom end and allow the solution in the container to flow out. In other embodiments, the container may typically be cylindrical, conical, tetrahedral, or cubic in shape. Containers can be shaped to be housed in devices designed to house them, in which case they may be referred to as "cartidges".

[0079] In some embodiments, the second container is a pore in a multi-well plate, and in a preferred embodiment, each pore of the multi-well plate is a container adapted to capture and retain carbohydrates (such as glycans) as described above. Such pores typically have a cylindrical body that narrows towards the bottom, the bottom section including an opening to allow solution to exit the pore. The outlet located at the bottom of the pore preferably has a diameter narrower than the pore, and especially in cases where a plate is required in a system that uses automated sample collection, it can be a pointed tip protruding from the bottom of the pore.

[0080] Regardless of whether the second container is a tube, cylinder, or orifice, it has a first porous solid carrier disposed between the top of the container and the outlet. In some embodiments, the second container further has a second porous solid carrier disposed between the first porous solid carrier and the outlet. In some preferred embodiments, the first porous solid carrier is in the form of a membrane or a monolithic material. In a preferred embodiment, the first porous solid carrier is disposed just above the bottom of the container. In orifices with nozzles, the first porous solid carrier is preferably disposed just above the nozzle. In another preferred embodiment, the second porous solid carrier is a hydrophilic material disposed between the first porous solid carrier and the outlet. In embodiments where the second porous solid carrier is, for example, composed of beads, such as bare silica beads or derived silica beads, wherein the diameter of the beads may be smaller than the diameter of the container outlet, and the beads or other second porous solid carrier can be held in place by conventional methods (such as by using one or more porous compressed glass blocks, or by having a plastic retainer below the beads or other second porous solid carrier (internal cross members)).

[0081] Solid-phase extraction (SPE) cartridges for retaining carbohydrates and other devices for retaining carbohydrates, including hydrophilic polymers, typically have components for retaining the polymer within the device. These components are usually chosen to be non-reactive with the carbohydrates or reagents to be exposed therein and to allow fluids such as cleaning solutions to exit the device through predetermined outlets (such as nozzles). Any of these conventional means can be used or adapted to retain a first or second porous solid support within the device of the present invention.

[0082] The reaction vessel itself is made of a material that is non-reactive to the reagents and solutions used therein. Typically, the reaction vessel is plastic. SPE cylinders and containers for carbohydrates are well known in the art and are available from many suppliers. Plastics or other materials used for SPE cylinders to separate carbohydrates from other types of compounds, such as proteins, are suitable for the methods and apparatus of this invention.

[0083] Regardless of shape, the container includes a chamber containing a first porous solid support, and in embodiments using APTS or other markers that are not readily soluble in organic solvents, a second porous hydrophilic solid support is positioned downstream of the first porous solid support along the path through which the manufacturer intends the reagent to enter the container and exit through its outlet. The first and second porous solid supports are further described below.

[0084] In some embodiments, the reaction vessel is designed such that the solution used in the method travels vertically through the vessel, allowing gravity to aid in the movement of the solution through a first porous solid carrier, and in embodiments having a second porous solid carrier, also through that carrier. In some embodiments, the vessel is designed such that the solution moves the reagent through the vessel under pressure. For example, the method can be used in a microfluidic tube or channel having chambers by placing a first solid carrier in a portion of the chamber of the tube or channel (thus serving as the reaction vessel), and placing a second porous solid carrier in the same portion of the chamber or in a portion of another section of the tube or channel positioned horizontally or vertically to the reaction chamber, wherein the solution flows from the reaction chamber to the second solid carrier by a pressure gradient in the tube or channel. In embodiments where the second porous solid carrier is in a second section of the tube or channel, the chamber containing the reaction chamber is fluidly connected to the chamber of the tube or channel in which the second porous solid carrier is disposed; in other cases, the two chambers may be separated by a valve or other component, wherein the valve or other component allows the two chambers to be fluidly connected when needed.

[0085] First porous solid carrier, second porous solid carrier, and pore size

[0086] The method of the present invention uses a first porous solid support to capture and retain carbohydrates present in solution, wherein the solution is a pure organic solvent or a mixture with a high ratio of organic solvent to aqueous solution. In some embodiments, the method of the present invention further uses a second porous solid support capable of capturing and retaining labeled carbohydrates released from the first solid support in the presence of a solution with a lower ratio of organic solvent to aqueous solution.

[0087] The term "solid carrier" refers to a carrier having a solid surface, but the solid carrier does not necessarily have to be made from a single piece of material. A "solid carrier" can, for example, consist of various derived silica beads that, when combined and arranged throughout a second container arrangement, provide a hydrophilic surface on which glycans or other carbohydrates can be retained. If the solid carrier is made of beads or similar material particles, the beads can be compressed or held, for example, in a plastic retainer to hold the beads in the desired position within the second container. In such an embodiment, the plastic retainer can consist of a plastic ring sized to fit precisely into the cavity of the second container and attached to plastic cross-sections extending from the ring, much like a tennis racket, wherein the cross-sections have holes smaller than the diameter of the beads, thereby holding the beads in place while allowing liquid to flow through. Similarly, the solid carrier can be made of glass fiber, which can be stacked on top of each other to provide a surface on which glycans or other carbohydrates can be retained. If desired, the glass fiber can be held in place by a plastic retainer as described above. In embodiments that do not use a second porous solid carrier, beads, glass fibers, or other instances where the solid carrier is not a single structure can be packaged into a bed at the bottom of a second container. Beads and other particles can be retained, for example, by narrowing the walls of the container so that their diameter is smaller than the diameter of the beads or particles, or by using a suitable retainer with protruding cross-sections whose pores or openings are too small for the beads or particles to pass through.

[0088] In some embodiments, the hydrophilic material is formed into a rigid and porous membrane or monolithic material. The membrane or monolithic material can then be shaped to precisely fit the chamber of the second container and fill the cross-sectional shape of the chamber at the desired location.

[0089] Regardless of whether the first porous solid support is a bead, particle, membrane, or monolith, it is positioned throughout the entire cross-section of the container chamber at the desired location within the container, such that the solution or solvent in the container must pass through the first porous solid support before exiting. If the second container narrows from a cylindrical section to a tapered section (e.g., in a typical Eppendorf tube), the size of the bead, particle, membrane, or monolith can be set to completely cover the bottom area of ​​the cylindrical section and held in place by narrowing the container wall below to form the tapered section. Similarly, if the second container is a cylindrical section of a hole in a porous plate, and the hole has a pointed tip with an opening at the bottom, allowing the solution to exit the hole when needed, then the size of the bead, particle, membrane, or monolith can be set to completely cover the bottom area of ​​the cylindrical section of the hole and held in place by narrowing the hole wall below the membrane or monolith to form the pointed tip.

[0090] In embodiments where the hydrophilic material is used for the first porous solid support and the glycan or carbohydrate is not a monosaccharide or is very small, the size of the pores or openings can be larger than in the embodiments discussed below, as long as the pores or openings force the glycan or other carbohydrate to contact the hydrophilic material as the organic solvent / initial aqueous solution sample flows through the first porous solid support. When the concentration of the organic solvent in the organic solvent is 80% or more and the aqueous solution is 20% or less, the glycan or other carbohydrate will tend to interact with and remain on the hydrophilic material of the solid support.

[0091] As used herein, the terms "porous" and "permeable" in describing solid supports are intended to indicate that the solid support causes solvents and solutions to be gradually filtered as they pass through it. Unless the context otherwise specifies or requires, all solid supports described herein are porous solid supports. It should be noted that in some embodiments, porous solid supports may be held or maintained in place by, for example, fittings, narrowing of container walls, or internal structures of the container in which they are housed. Such fittings, narrowings, or internal structures are solid and may hold or support first and second porous solid supports; however, they are generally made of the same material as the reaction vessel and should not be construed as first or second porous solid supports simply because of the use of these terms in the foregoing disclosure.

[0092] The material of the first porous solid carrier

[0093] As described in the introduction, when an organic solvent is added to the initial aqueous solution sample to a concentration of 80% to 95%, most polysaccharides and other carbohydrates, except for monosaccharides and other carbohydrates with only weak hydrophilicity, will remain on a solid hydrophilic surface. Therefore, in many embodiments, the first porous solid support can be any hydrophilic material that does not chemically react with the reagent to be used, presents a solid surface, and can be made such that a liquid can permeate through it. A variety of materials are currently used in cylinders and columns to retain carbohydrates during SPE processes and can be used in embodiments of the method of the present invention. For example, the solid support can be made of cellulose; glass (such as glass fiber); alumina; functionalized surfaces containing glycols, aminopropyl, amides, cyanopropyl, ethylenediamine-N-propyl; or porous hydrophilic materials known to retain carbohydrates capable of retaining organic solvents. In some embodiments, the solid support is made of silica beads, and in some embodiments, the silica beads are derivatized with aminopropyl, glycol, or carbamoyl groups. The first porous solid support preferably does not have usable carboxyl groups on its surface. As used herein, the terms “functionalized” and “derivatively derived” are equivalent and refer to the surface of the material forming the solid support host being covalently bonded to a molecule of one of the listed functional groups (e.g., aminopropyl, glycol, carbamoyl), or in some embodiments, to molecules of two or more of these functional groups (e.g., both glycol and carbamoyl molecules are covalently attached to the material surface of the first porous solid support). In some preferred embodiments, the material is silica. When contacted with carbohydrates under conditions that enable interaction (such as temperature and pH), the molecules of the functional groups can exhibit non-covalent interactions with the carbohydrates. Solid-phase extraction of carbohydrates with other polar and hydrophilic molecules has been performed in the art for many years, and the conditions for performing SPE are generally suitable for implementing embodiments of the method of the present invention. It is anticipated that those skilled in the art are familiar with the conditions for performing SPE.

[0094] In some embodiments, the organic solvent will be present at a concentration higher than 95%. These embodiments allow for the capture of monosaccharides or other polysaccharides or carbohydrates that are only weakly hydrophilic and cannot be well precipitated in the organic solvent when water is present at a concentration higher than 5%. Alternatively, practitioners may wish to capture and analyze larger polysaccharides or carbohydrates present in a sample that could be captured by the aforementioned hydrophilic materials, and they may also wish to capture monosaccharides that cannot be retained on the solid support when using organic solvents at concentrations of 95% or lower. For these embodiments, a wide range of materials can be used for the first porous solid support, including the materials named in the preceding paragraph and other non-hydrophilic materials, because it is anticipated that the polysaccharides or other carbohydrates will precipitate from the solution and aggregate or precipitate, or aggregate and precipitate, thereby enabling them to be captured on the first porous solid support by physical filtration rather than by relying on hydrophilic interactions between the polysaccharides or other carbohydrates and the solid support material. Therefore, in these embodiments, the first porous solid support can be any material having the following characteristics: (a) solid, (b) not reacting with the reagent to be used, (c) capable of being shaped to remain within the second container and fill the cross-section of the container chamber so that the solution or solvent must be filtered through the pores or openings in the material, and (d) can have pores or other openings from 10 micrometers to 0.05 micrometers, or (d') can be produced in the form of beads or other particles, which can be packaged into a bed or retainer and have pores or openings within that size range. Thus, the hydrophilic materials described above can be used, but ceramic filters with suitable pore sizes can also be used, for example. The suitability of any particular material for implementing these embodiments can be tested by the assays taught in the section on testing the combination of organic solvents and materials for the solid support.

[0095] In studies based on this invention using polyethylene (“PE”) as the first porous solid carrier, it was demonstrated that polysaccharides could be effectively captured if the pore size of the first porous solid carrier was 10 micrometers or smaller. Studies based on this invention using glass fiber with a pore size of approximately 0.7 micrometers were found to perform well. It is anticipated that smaller pore sizes can be used as long as the pores are not so small that the solvent and solution filter too slowly, excessively slowing down the operation or even preventing the solvent or solution from flowing out of the container. Any specific pore or opening size can be readily tested to determine whether the solvent and unwanted reagents filter through the first or second porous solid carrier, respectively, within a time deemed reasonable by a practitioner. Those skilled in the art will further appreciate that the solid carrier should have a large number of pores or openings to facilitate the flow of solvent and unwanted reagents through the solid carrier, as having only a few pores or openings will slow their passage through the solid carrier without providing any benefit in separating and immobilizing polysaccharides or other carbohydrates on the solid carrier. Since SPE cartridges for separating carbohydrates have been available for many years, it is expected that those skilled in the art can use the pore size and composition of such cartridges as a guide to determine suitable materials and pore sizes for use as porous solid carriers in the methods and apparatus of the present invention.

[0096] In some particularly preferred embodiments, the material used for the first porous solid carrier is PE (polyethylene) because it is inexpensive and can be produced in suitable thicknesses with appropriately sized pores, and can be easily shaped to remain within a container, cylinder, hole, or column, filling the cross-section of the container chamber. In preferred embodiments, the pore size of PE is from 10 micrometers to 0.25 micrometers. In preferred embodiments, PE can have pores or openings of 10, 9, 8, 7, 6, 5, 4, 3, or 2 micrometers, 1 micrometer, or 0.5 micrometers, or any size within this range. In some preferred embodiments, PE is a monolithic material. In some embodiments, PE is a membrane.

[0097] For analytical applications, the total volume of the solution used is typically 1 mL. For preparative-scale applications, the volume of the solution used may be 1 liter or more, and the thickness of the material may be considerable. The first porous solid support preferably has multiple pores that are small enough to prevent the passage of aggregated or precipitated polysaccharides or other carbohydrates, but also large enough to allow the passage of liquids and dissolved reagents (suitable pore sizes were discussed in the preceding sections). Those skilled in the art should be able to select a material suitable for the first porous solid support.

[0098] Initially, it was thought that polysaccharides or other carbohydrates bound to a first solid support in the presence of an organic solvent or a mixture of an organic solvent and an aqueous solution in a high proportion, while in an aqueous solution, the carbohydrates were provided to the first solid support. Not wishing to be bound by theory, it is now considered that in embodiments using a hydrophilic material as the first porous solid support and with an organic solvent concentration of 80% to 95%, the polysaccharides or other carbohydrates are retained on the support by hydrophilic interactions; while at higher organic solvent concentrations, the polysaccharides or other carbohydrates precipitate from the solution and aggregate or precipitate. It is further considered that one or both of these aggregates or precipitates are collected on the first porous solid support primarily due to filtration. However, depending on the chemical properties of the porous solid support, other interactions between the carbohydrates and the first porous solid support may also occur in the presence of the solvent / solution mixture. For example, the solvent / solution mixture may be provided to the first solid support under conditions where ionic interactions or hydrogen bonding may exist at certain pH, temperature, and salt concentrations, and the solid support and carbohydrates will further undergo this interaction during subsequent steps of the method of the present invention. Regardless of the mechanism of action, the result is that, in the presence of an organic solvent or a mixture of a high proportion of organic solvent and an aqueous solution, most or all of the polysaccharides or other carbohydrates are non-covalently retained on the first porous solid support, rather than when passing through the first porous solid support in the presence of that solvent or a mixture of a high proportion of organic solvent and an aqueous solution. Not wishing to be bound by theory, it is assumed that, according to the embodiment of workflow 1A, carbohydrates are retained on the first porous solid support by filtration in step 3 and retained by filtration during the washing process in step 6; according to the embodiment of workflow 1B, carbohydrates are retained on the first porous solid support by filtration in step 3 and captured and retained on the second porous solid support by hydrophilic interaction during the washing process in step 6; and according to the embodiment of workflow 1C, carbohydrates are retained on the first porous solid support by hydrophilic interaction in step 3 and retained by hydrophilic interaction during the washing process in step 6.

[0099] Initially, graphitized carbon was considered suitable as a first solid carrier in the methods and kits of the present invention. However, graphitized carbon is unsuitable as a first porous solid carrier for several reasons. First, as mentioned above, the first porous solid carrier should not be a carrier that retains glycans or other carbohydrates in organic solvents or solvent mixtures consisting mainly of organic solvents. When carbohydrates are in a solution containing a high proportion of organic solvents and aqueous solutions, graphitized carbon will not retain glycans or other carbohydrates. Second, some embodiments of the method of the present invention use glycans or other carbohydrates in a solution that also contains glycosylated proteins, including glycans or other carbohydrates released by enzymatic digestion and may also contain deglycosylation enzymes (which are also proteins). It is believed that graphitized carbon will retain the glycosylated proteins and enzymes present in the solution, and the retained proteins will block the pores of the solid carrier. Therefore, graphitized carbon cannot be used as a first or second porous solid carrier.

[0100] In some embodiments, the first porous solid carrier is made of glass. Preferably, the glass is in a form with a high surface area to promote retention of carbohydrates in the solvent / solution mixture, and preferably the glass is in a form that allows the mixture to flow through to promote this capture. For example, the glass may have multiple small pores to allow fluid to flow through for filtration, or it may be in the form of beads or particles. In some preferred embodiments, the glass is in the form of glass fibers. In some embodiments, the glass fibers may be loose. In some embodiments, the glass fibers may be woven. In embodiments where the glass fibers are loose, they are typically used in conjunction with a lower structural carrier that holds the fibers in the container while allowing the solvent, solution, and unwanted reagents to flow through. In these embodiments, the structural carrier is positioned between the glass fibers and an opening through which the solvent, solution, and unwanted components exit the container.

[0101] In some embodiments, the first porous solid support is made in the form of aminopropyl. Various forms of aminopropyl for separating carbohydrates are known in the art, and several are commercially available. For example, aminopropyl AP(NH2) HPLC columns for separating carbohydrates are available from Separation Methods Technologies, Inc. (Newark, DE). TMThe NH2 HPLC column was sold by Sigma-Aldrich Co. (St. Louis, Missouri). Aminopropylsilanes are used in the art for the HILIC separation of sugars. It is anticipated that those skilled in the art are familiar with various methods of using aminopropyls to separate carbohydrates in processes such as HPLC and HILIC, and that suitable forms of aminopropyls can be selected in embodiments of the methods using aminopropyls of the present invention to bind carbohydrates, such as N-glycans.

[0102] In some embodiments, the first porous solid support is made of cellulose. Cellulose is used in sheet form, but is typically used in solid-phase extraction in the form of microcrystalline powder, which is preferred due to the larger surface area provided for binding carbohydrates. As practitioners will understand, solid supports used in powder, nanoparticle, or other small particle form are typically used in conjunction with filters or other structures that allow solvents, solutions, and unwanted reagents to flow through during elution while the powder or nanoparticles or other small particles remain in the container. In these embodiments, the filter or other structure is positioned between the powder, nanoparticles, or other small particles and an opening through which the solvent, solution, and unwanted components exit the container.

[0103] In some embodiments, the first solid carrier may be a porous hydrophilic material that preferentially retains carbohydrates over proteins, buffer salts, reducing agents or other reagents known to be present in the specific mixture of carbohydrates to be separated.

[0104] Materials for the second solid carrier

[0105] As described above, when carbohydrates on the first porous solid support are reduced by a reducing agent poorly soluble in organic solvents, or labeled by reductive amination with APTS or other labels poorly soluble in organic solvents, a solution of organic solvent mixed with some water is needed to remove the reducing agent or excess label, wherein the water concentration is such that the reduced or labeled carbohydrates detach from the first porous solid support. This washing solution is typically 60% to 90% organic solvent and 40% to 10% aqueous solution, preferably about 75% to 85% organic solvent and 25% to 15% aqueous solution, and 80% to 85% organic solvent and 20% to 15% aqueous solution is preferred (of course, any specific percentage of organic solvent chosen by the practitioner will match the percentage of aqueous solution required to bring the mixture to a total of 100%). In these cases, the second porous solid support used allows the labeled carbohydrates to be captured and retained in the presence of this solution. Therefore, in some embodiments, the present invention provides a container having a first porous solid carrier and a second porous solid carrier as described above, wherein the second porous solid is arranged along the solution flow path between the first porous solid carrier and an opening through which it exits the container as a reducing agent or marker.

[0106] As the name suggests, the second porous solid support is porous and preferably a hydrophilic material, such as resins commonly used for HILIC separation or solid-phase extraction (SPE) of carbohydrates. It is anticipated that materials used in HILIC separation and SPE extraction of carbohydrates are generally suitable for the second porous solid support. SPE is widely used in the art, and there are numerous related teachings, such as Thurman and Mills, *SOLID-PHASE EXTRACTION: PRINCIPLES AND PRACTICE*, John Wiley & Sons Inc. (New York, NY, 1998); N. Simpson, *Solid-Phase Extraction: Principles, Techniques, and Applications*, Marcel Dekker Inc. (New York, NY, 2000); and Waters Corp., *BEGINNER'SGUIDE TO SPE: SOLID-PHASE EXTRACTION*, John Wiley & Sons Inc. (New York, NY, 2014). Therefore, it is expected that technicians will be able to easily select materials suitable as a second porous solid carrier.

[0107] Examples of porous hydrophilic materials suitable for a second porous solid carrier include: cellulose; glass; alumina; aminopropyl; or silica modified with diol, cyanopropyl, ethylenediamine-N-propyl, amide (carbamoyl), asparagine, cyclodextrin, triazole, diethylaminoethyl; or combinations of two or more of these materials.

[0108] In some preferred embodiments, the second porous solid support is silica beads or particles covalently bonded with carbamoyl groups. In some particularly preferred embodiments, the covalently bonded spherical silica beads or particles are Amide-80. In preferred embodiments, the size of the Amide-80 beads or particles is from 3 micrometers to 60 micrometers, more preferably from 5 micrometers to 50 micrometers, and even more preferably about 30 micrometers, where "about" means ±2 micrometers. The second porous solid support, in the presence of the organic solvent / water washing solution described above, captures any reduced or labeled polysaccharides or other carbohydrates that have detached from the first porous solid support.

[0109] It is further anticipated that the material used for the second porous solid support, when washed with an aqueous solution such as phosphate-buffered saline, will release the retained reduced or labeled carbohydrates. (For clarity, it should be noted that the aqueous solution used in this step to elute the reduced or labeled carbohydrates from the second porous solid support is different from the organic solvent / aqueous solution mixture used to wash away excess reducing agent or excess labeling from the first porous solid support, as it does not contain a washing solution with approximately 80% organic solvent concentration.) Typically, the washing solution used in this step does not contain organic solvents.

[0110] Material configuration of the first solid carrier and the second solid carrier

[0111] As described above, the first and second solid carriers are porous to allow for filtration of the solution, thereby enabling the capture and retention of carbohydrates in the solution. The materials chosen for the first and second porous solid carriers are either configured with a large surface area, such as being woven to allow solvent / solution contact, or have multiple pores or openings to achieve the same function. Preferably, the solid carriers are not in the form of magnetic beads. Since the method of the present invention takes into account the fixed position of the first and second solid carriers within the second container and their inactivity or lack thereof, the use of magnetic beads would increase costs without providing any advantage. Therefore, their use is not preferred in the method or apparatus of the present invention.

[0112] In some embodiments, one or both of the first or second porous solid support is a membrane. In some embodiments, one or both of the first or second porous solid support is a monolithic material. In some embodiments, one or both of the first or second porous solid support may be in the form of resin, granules, or powder. In these embodiments, the resin, granules, or powder will typically be held in a second container by a structural carrier to prevent the resin, granules, or powder from leaving the second container with the solution and solvent flowing through them. In some embodiments, one or both of the first or second porous solid support is in the form of a packed bed, which is held in the container by the design of the container or other structural carriers. In a preferred embodiment, the first or second porous solid support is both porous and rigid. In some embodiments, one or both of the first or second porous solid support may be disposed in a microfluidic channel. In embodiments where one or both of the first or second porous solid support is not properly positioned in the container, the container may further have a structure or insert that holds one or both of the first or second porous solid support in the desired position within the container. For example, the container may be formed with an internal component extending across the entire width of the container and having pores or other openings sized to prevent material from moving out of the intended position from the first or second porous solid carrier, but large enough to allow the flow of solvents, solutions, and reagents. Alternatively, the container may include one or more structures (such as frits) disposed between the first or second porous solid carrier to hold the first or second porous solid carrier in the intended position. The structure or frit itself is shaped and sized to match annular rings or other structural arrangements on the sides of the container, and the annular rings or other structures may be, for example, sized and shaped to mate with flanges inside the container. In some embodiments, the rings or other structures may be secured to flanges or structures inside the container to hold the annular rings or other structures (and filters attached to the rings or other structures) in the desired position. The dimensions of the rings or other structures may be configured to match, for example, matching grooves or notches surrounding the interior of the container, or they may have spikes or other protrusions extending from the rings or other structures to match matching grooves disposed inside. In some implementations, the ring or other structure can be secured to the container by snap-fit. For example, the ring or other structure may have multiple protrusions that engage with matching recesses on the container wall. Alternatively, the ring or other structure can be placed inside the container and then secured to the desired location on the wall by adhesive or ultrasonic welding.

[0113] The solution flows through a first porous solid support and (if applicable) also through a second porous solid support.

[0114] The intended outcome is that, as the solvent / solution mixture (which further includes unwanted reagents such as buffer salts, deglycosylation enzymes, deglycosylated glycoproteins, etc.) flows through the solid support, carbohydrates are retained on the first porous solid support. The carbohydrates on the first solid support are then washed with an organic solvent (the same or different from the organic solvent used in the aqueous solution mixture) to remove any residual aqueous solution and to perform reductive amination. Those skilled in the art will understand that carbohydrates can be retained on the first solid support within seconds, but it is undesirable for carbohydrates to flow through the first solid support too quickly (e.g., less than 1 second) so that retention does not occur, nor too slowly (e.g., more than 15 minutes) so that unnecessary time is added to the process. Therefore, the flow rate through the container should be slow enough to allow carbohydrates a chance to be retained on the solid support, but fast enough to avoid delays that would add unnecessary time to the workflow. Those skilled in the art will be familiar with the materials used in the cartridge of the solid-phase extraction process, the pore size, and other characteristics that would result in the desired flow rate, as well as the selection of various conditions, such as positive pressure, centrifugation, or the use of a vacuum manifold, to increase the flow rate of liquid through or across the solid support. It is anticipated that practitioners will be familiar with the extraction of carbohydrates from samples using solid-phase extraction procedures by selecting the materials, pore size, and flow rate used in the extraction, and this familiarity can provide sufficient guidance in various embodiments of the method of the present invention for selecting the material, pore size, and flow rate of the first solid support as the solvent / solution mixture flows through it. In embodiments where a second porous solid support is present, similar considerations can be used to determine the appropriate size of the pores or openings in the second porous solid support.

[0115] markers

[0116] As described in the background section, reductive amination and labeling with 2-AB is disclosed in common U.S. Patent No. 5,747,347, published in 1998. Since the publication of Patent 347, reductive amination has been widely used for labeling glycans and other carbohydrates over the past 20 years, and it is assumed that practitioners are familiar with the process and conditions for labeling carbohydrates by reductive amination.

[0117] In general, in the method of the present invention, any marker or dye suitable for labeling carbohydrates by reductive amination can be used to label glycans or other carbohydrates. Conveniently, the marker is a fluorophore, such as 2-AA (o-aminobenzoic acid) or 2-AB (2-aminobenzamide). In some preferred embodiments, the marker is 2-AB. In some preferred embodiments, the marker is 2-AA. In some preferred embodiments, the marker is APTS (8-aminopyrene-1,3,6-trisulfonic acid, CAS No. 196504-57-1). In some preferred embodiments, the marker is procainamide hydrochloride (CAS No. 614-39-1). In some embodiments, the marker comprises a primary amine group that reacts with the reducing end of the carbohydrate by reductive amination and has fluorescent properties, a chromogenic moiety, or a detectable charge. In some embodiments, the marker is a chemical moiety having a detectable charge.

[0118] reducing agent

[0119] A reducing agent is used in embodiments where it is desired to reduce glycans or other carbohydrates without labeling, and in embodiments where glycans or other carbohydrates are labeled via reductive amination. The use of reducing agents in the reduction of glycans or other carbohydrates and in reductive amination is well known in the art and is therefore only briefly described herein. Reducing agents suitable for the reductive amination of glycans include sodium cyanoborohydride and methylpyridineborane. Reducing agents are typically in organic solvents, such as DMSO. Sometimes tetrahydrofuran (THF), the organic solvent used as a reducing agent, is incompatible with plastics; therefore, embodiments where tetrahydrofuran is used in the reaction vessel or any component of the reaction vessel (such as the internal support for holding a second porous solid support in place) is made of plastic cannot be used as an organic solvent for the reducing agent. Typically, a solution containing both the solvent and the reducing agent is added to a solution containing the label. The concentration of the reducing agent in the final mixture is typically from 0.5 M to 2 M.

[0120] Reduce but do not label glycans or other carbohydrates

[0121] In the absence of a label, the reducing agents and concentrations described above can be used to reduce glycans or other carbohydrates. The reduction of glycans or other carbohydrates is carried out conventionally according to the invention, and it is anticipated that practitioners are familiar with suitable solvents and concentrations. Suitable reducing agents and concentrations were discussed in the previous section. Once the glycans or other carbohydrates have been reduced, they can be eluted from the first porous solid support (or, in embodiments using a second porous solid support) by washing the support as described in the next section.

[0122] Retained carbohydrates were eluted from the first porous solid support.

[0123] Once any excess reducing agent or excess labeling has been removed from the reduced or labeled glycans or other carbohydrates retained on the first porous solid support, the retained reduced or labeled glycans or other carbohydrates can be eluted from the first porous solid support by washing the support with an aqueous solution, wherein the aqueous solution redissolves the aggregated or precipitated glycans or other carbohydrates. Preferably, an aqueous solution with added salt is used. Combinations of solutions can also be used. The aqueous solution may contain up to 20% organic solvent or may be water or a buffer solution only. As described above, the suitability of any particular solution or combination for eluting carbohydrates from a solid support made of any particular material can be readily tested by performing parallel assays.

[0124] Eluting carbohydrates from a second porous solid support

[0125] As described in the previous chapter, if the selected first porous solid support is non-hydrophilic, and the glycan or other carbohydrate has been reduced by a reducing agent poorly soluble in organic solvents, or has been labeled with APTS or another dye poorly soluble in organic solvents, the glycan or other carbohydrate will detach from the first porous solid support and enter the organic solvent / aqueous solution washing solution used to remove excess reducing agent or labeling. The glycan or other carbohydrate detached from the first porous solid support will be captured by the hydrophilic second porous solid support. Once excess reducing agent or excess labeling has been washed away from the reaction vessel, the reduced or labeled glycan or other carbohydrate can be eluted from the second porous solid support using one or more of the aqueous solutions described in the previous chapter.

[0126] Reagent test kit

[0127] In some embodiments, the present invention provides a kit for immobilizing glycans or other carbohydrates on a first porous solid support, and for reducing or labeling the glycans or other carbohydrates while immobilizing them on the first porous solid support, or by reductive amination. In some embodiments, the kit provides a container that sequentially comprises: a first opening for introducing a solution; a first section for receiving a mixture containing glycans or carbohydrates of interest; a first porous solid support; a second section in which a hydrophilic second porous solid support is disposed; and a second opening through which the solution can exit the container.

[0128] Conveniently, the size of the first section between the first opening and the first porous solid support is set to receive the volume of the mixture of the organic solvent and the starting aqueous solution sample used in the intended analytical or preparative-scale reduction or labeling procedure. In a preferred embodiment, the first porous solid support is non-hydrophilic. In some of these embodiments, the non-hydrophilic first porous solid support is made of polyethylene, nylon, polyvinylidene fluoride, or polypropylene. In a preferred embodiment, the first porous solid support has pores or openings with a width of 10 micrometers or less, thereby allowing the mixture of the organic solvent and the starting aqueous solution sample to be filtered through the first porous solid support. In some preferred embodiments, the first porous solid support is a monolithic material or membrane that extends and fills the cross-sectional arrangement of the container, such that the mixture flowing into the first section of the container must be filtered through the first porous solid support. In other embodiments, it may not consist of a single solid block, but rather an aggregate of blocky materials such as fibers, beads, or granules. A first porous solid carrier consisting of a blocky assembly of materials such as glass fibers or silica beads can be held in a desired position within a container by standard means, such as arranging these blocks on a retainer, such as a plastic insert, or on an internal container cross member in which the cross member is smaller than the size of the block.

[0129] In some embodiments, the container is a tube or cylinder. In some embodiments, particularly for automated analysis, the container is a well in a multi-well plate. The outlet at the bottom of the well may be a pointed nozzle, or the bottom of the well may be narrowed to facilitate elution of the solution from the well into the desired container. For clarity, it should be noted that the container provided in the kit is used as the "reaction vessel" as described in the method of the present invention, because samples containing the polysaccharides or other carbohydrates to be reduced or labeled are typically mixed with organic solvents in a separate container before use of the kit.

[0130] As noted, the container in the kit of the present invention further comprises a hydrophilic second porous solid carrier disposed between the first porous solid carrier and the second opening. Like the first porous solid carrier, the second porous solid carrier is disposed throughout and fills the cross-sectional area of ​​a second section of the container such that fluid containing polysaccharides or other carbohydrates detached from the first porous solid carrier must contact and be filtered through the second porous solid carrier. In some embodiments, the second porous solid carrier is a monolithic material or a membrane. In other embodiments, the second porous solid carrier is not composed of a single solid block, but rather of an aggregate of blocks such as fibers, beads, or particles. The second porous solid carrier prevents its movement toward the second opening by being arranged on a retainer, membrane, or filter. In some embodiments, both the first and second porous solid carriers have retainers, membranes, or filters to hold them in their respective positions within the container. In some embodiments where the second porous solid carrier is composed of beads or particles, the walls of the container narrow as it approaches the second opening (e.g., forming a pointed tip at the bottom of the opening) to a size smaller than the diameter of the beads or particles, preventing the beads or particles from leaving the container through the second opening. Other means known in the art for retaining beads or other materials within an SPE cylinder or other separation device may also be used. The choice of the specific method for retaining one or both of the first or second porous solid support is not critical, as long as it allows the solution to exit the container when needed and does not react with the intended reagent during use. One or both of the first or second opening may have a removable cap.

[0131] The kit may contain one or more deglycosylation enzymes, one or more denaturing agents (such as sodium dodecyl sulfate), and preferably one or more labels for reductive amination. The labels may be, for example, 2-AA, 2-AB, APTS, procainamide hydrochloride, or two or more of these. The kit may contain a reducing agent, which may be in powder form or soluble in an organic solvent. The reducing agent may be, for example, sodium cyanoborohydride or methylpyridineborane. The kit may further contain printed instructions on how to immobilize glycans or other carbohydrates on a first porous solid support; and how to reduce them or label them by reductive amination while still immobilizing them on the first porous solid support in the container; and how to elute the reduced or labeled glycans or other carbohydrates from the container according to the reducing agent or labeling agent used in the procedure.

[0132] Example

[0133] Example 1

[0134] This embodiment lists abbreviations for some of the reagents used in the exemplary workflow of the deglycosylation and labeling process described in the following embodiments.

[0135] "PNGase F mixture": a 1:1 mixture of PNGase F (~1 mg / ml) and 750 mM ammonium bicarbonate pH 8.0 buffer.

[0136] “2-AB”: 2-aminobenzamide.

[0137] "2-AB labeled mixture": 2 mg / ml 2-aminobenzamide, 40 mM cyanoborohydride, and 200 mM acetic acid in 90:10 acetonitrile:DMSO.

[0138] “DTT”: Dithiothreitol.

[0139] “APTS”: 8-Aminopyrene-1,3,6-trisulfonic acid.

[0140] “APTS-labeled mixture”: 2 mg / ml of 8-aminopyrene-1,3,6-trisulfonic acid, 40 mM of cyanoborohydride, and 200 mM of acetic acid in 90:10 acetonitrile:DMSO.

[0141] Example 2

[0142] This embodiment illustrates an exemplary workflow for releasing N-glycans via enzymatic digestion and labeling them with 2-AB using an exemplary solid carrier.

[0143] N-Glycan Release and Preparation Steps

[0144] 20 μl of a 2 mg / ml glycoprotein was denatured at 90 °C for half an hour in the presence of DTT (1 μL, 550 mM). The solution was cooled, 2 μl of PNGase F mixture was added, and the mixture was incubated at 37 °C for 16 hours to release N-glycans from the glycoprotein.

[0145] N-Glycan Precipitation and Purification Steps

[0146] The mixture (approximately 23 μl) was mixed with 600 μl of acetonitrile and added to a glass fiber membrane mounted in a 1 mL column. Under these conditions, N-glycans precipitated onto the glass fiber membrane, while contaminants, such as denaturants, were washed away by the solvent. Additional washing with acetonitrile can be performed to reduce undesirable contaminants remaining on the glass fiber membrane.

[0147] N-glycan labeling

[0148] 25 μL of the 2-AB labeling mixture was added to the glass fiber membrane. The labeling reaction was carried out at 70 °C for one hour.

[0149] Washing and elution steps for excess reactants:

[0150] If necessary, the solid support can be washed with acetonitrile to remove any excess dye. The purified labeled polysaccharide can then be eluted from the solid support with water.

[0151] Analysis steps

[0152] The labeled N-glycans eluted from the membrane are then analyzed. For example, 1 μl of eluted N-glycans is fed to a high-performance liquid chromatography (HPLC) instrument to separate the glycans, and then fed to a device for detecting and measuring the fluorescence of the separated glycans for analysis.

[0153] Example 3

[0154] This embodiment illustrates an exemplary workflow for labeling free oligosaccharides with APTS using an exemplary cellulose solid carrier.

[0155] N-Glycan Release and Preparation Steps

[0156] A 20 μl aqueous solution of 0.5 mg / mL maltodextrin was mixed with 600 μl of acetonitrile (an organic solvent) and added to a cellulose membrane mounted in a 1 mL column. Under these conditions, the free polysaccharides aggregated or precipitated on the solid support, while other components and water were washed away by the organic solvent. Washing with acetonitrile was then performed to further reduce the presence of water remaining on the support after the initial acetonitrile / aqueous solution was drained from the container.

[0157] N-glycan labeling

[0158] Add 15 μL of the APTS labeling mixture to the cellulose membrane. Allow the labeling reaction to proceed at 70 °C for one hour.

[0159] Washing and elution steps for excess reactants:

[0160] If necessary, the solid support can be washed with 85% acetonitrile / 15% water to remove any excess dye. The purified labeled oligosaccharides can then be eluted from the porous solid support with water.

[0161] Analysis steps

[0162] After elution, the labeled oligosaccharides are analyzed. For example, the labeled oligosaccharides can be separated by feeding 1 μl of the eluted sample to a high-performance liquid chromatography (HPLC) instrument, and then analyzed using a device that detects and measures fluorescence to measure the fluorescence of the labeled oligosaccharides.

[0163] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or alterations may be made by those skilled in the art based on their purpose, and all such modifications or alterations are included within the spirit and scope of this application and the scope of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. An apparatus for reducing or labeling carbohydrates provided in an aqueous solution by reductive amination, the apparatus comprising: A reaction vessel having a body having (a) a first opening, (b) a first section having a first chamber having a first cross-section having a defined area and a first diameter, wherein the first chamber is fluidly connected to the first opening, (c) a second section having a second chamber having a second cross-section having a defined area and a second diameter, wherein the second chamber is fluidly connected to the first chamber of the first section, (d) a second opening fluidly connected to the second chamber, and (e) a first porous solid carrier for capturing the carbohydrates originally provided in the aqueous solution, the first porous solid carrier being disposed within the... Between the first opening and the second opening, the region of the first cross-section of the first chamber is filled, and the entire region of the first cross-section is fluidly connected to the first chamber and the second chamber; the first porous solid carrier (1) is composed of a non-hydrophilic material or has a non-hydrophilic material surface, or (2) is composed of a hydrophilic material; and (f) a second porous solid carrier for capturing the carbohydrate labeled or reduced by reductive amination, the second porous solid carrier being composed of a hydrophilic material or having a hydrophilic material surface, wherein the second porous solid carrier is disposed between the first porous solid carrier and the second opening and fills the region of the first cross-section or the second cross-section.

2. The apparatus according to claim 1, wherein, The diameter of the first section of the body of the reaction vessel is wider than the second diameter of the second section.

3. The apparatus according to claim 1, wherein, The first porous solid support is disposed in the first section of the reaction vessel, and the second porous solid support is disposed in the second section of the reaction vessel.

4. The apparatus according to claim 1, wherein, The reaction vessel is a tube or cylinder.

5. The apparatus according to claim 1, wherein, The reaction vessel is a hole in a porous plate.

6. The apparatus according to claim 5, wherein, The second section of the hole is a pointed tip that protrudes from the bottom of the hole.

7. The apparatus according to claim 6, wherein, The second porous solid carrier is disposed in the nozzle.

8. The apparatus according to claim 1, wherein, The reaction vessel is located within a microfluidic device.

9. The apparatus according to claim 1, wherein, The first porous solid carrier is made of a non-hydrophilic material or has a surface of a non-hydrophilic material.

10. The apparatus according to claim 9, wherein, The non-hydrophilic material of the first porous solid carrier has pores or openings with a width of 10 micrometers or less.

11. The apparatus according to claim 9, wherein, The non-hydrophilic material is polyethylene and has pores or openings with a width of 10 micrometers or less.

12. The apparatus according to claim 1, wherein, The first porous solid carrier is composed of a hydrophilic material.

13. The apparatus according to claim 12, wherein, The hydrophilic material is made of: (a) glass; (b) cellulose; (c) silica; (d) a surface covalently bonded with a plurality of aminopropyl, glycol, carbamoyl, zwitterionic groups, or combinations of two or more of the groups; and (e) silica covalently bonded with a plurality of aminopropyl, glycol, or carbamoyl groups, or combinations of two or more of the groups.

14. The apparatus according to claim 13, wherein, The first porous solid carrier is composed of glass fiber.

15. The apparatus according to claim 1, wherein, The hydrophilic material of the second porous solid carrier is made of the following materials: (a) glass; (b) cellulose; (c) silica; (d) a surface covalently bonded with a plurality of aminopropyl, glycol, carbamoyl, zwitterionic groups, or combinations of two or more of the groups; and (e) silica covalently bonded with a plurality of aminopropyl, glycol, or carbamoyl groups, or combinations of two or more of the groups.

16. The apparatus according to claim 15, wherein, The hydrophilic material of the second porous solid carrier is composed of glass fiber.

17. The apparatus according to claim 15, wherein, The silica is in the form of beads or particles.

18. The apparatus according to claim 17, wherein, The beads or particles are covalently linked with multiple carbamoyl groups.

19. The apparatus according to claim 18, wherein, The silica beads or particles having multiple carbamoyl groups are Amide-80.

20. The apparatus according to claim 1, wherein, The first porous solid carrier is polyethylene with pores or openings having a width of 10 micrometers or less, and the second porous solid carrier is composed of silica beads or particles covalently linked with multiple carbamoyl groups.

21. The apparatus according to claim 20, wherein, The silica beads or particles having multiple carbamoyl groups are Amide-80.

22. The apparatus according to claim 1, wherein, The second opening has an openable cap to retain the solution in the reaction vessel until it is necessary to remove the solution from the reaction vessel.

23. A kit for reducing carbohydrates or labeling carbohydrates by reductive amination, comprising: (a) A reaction vessel having a body comprising: (a) a first opening; (b) a first segment having a first chamber having a first cross-section having a defined area, wherein the first chamber is fluidly connected to the first opening; (c) a second segment having a second diameter and a second cross-section having a defined area and a second chamber having a defined area of ​​cross-section, wherein the second chamber is fluidly connected to the first chamber of the first segment; (d) a second opening having a second opening fluidly connected to the second chamber; and (e) a first porous solid support for capturing carbohydrates provided in a solution containing ≥95% organic solvent. The first porous solid carrier is disposed between the first opening and the second opening and fills the region of the first cross-section of the first chamber. The first porous solid carrier (1) is composed of a non-hydrophilic material or has a non-hydrophilic material surface, or (2) is composed of a hydrophilic material or has a hydrophilic material surface; and (f) a second porous solid carrier for capturing the reduced carbohydrate or the carbohydrate labeled by reductive amination, the second porous solid carrier being composed of a hydrophilic material, wherein the second porous solid carrier is disposed between the first porous solid carrier and the second opening and fills the region of the first cross-section or the second cross-section. (b) A reducing agent or a marker suitable for labeling carbohydrates by reductive amination, or a reducing agent and a marker suitable for labeling carbohydrates by reductive amination, and, (c) Description of carbohydrate reduction or description of carbohydrate labeling by reductive amination, or description of carbohydrate reduction and description of carbohydrate labeling by reductive amination.

24. The kit according to claim 23, further wherein, The reaction vessel is: a hole in a porous plate; a tube; or a cylinder.

25. The kit according to claim 24, wherein, The reaction vessel is a hole in a porous plate.

26. The kit according to claim 25, wherein, The second section of the hole is a pointed tip that protrudes from the bottom of the hole.

27. The kit according to claim 23, wherein, The first porous solid carrier is polyethylene with pores or openings having a width of 10 micrometers or less.

28. The kit according to claim 27, wherein, The second porous solid carrier is composed of silica beads or particles covalently linked with multiple carbamoyl groups.

29. The kit according to claim 28, wherein, The reaction vessel is a hole in a porous plate, and the silica beads or particles covalently bonded with multiple carbamoyl groups are disposed in a pointed tip protruding from the bottom of the hole.

30. The kit according to claim 23, wherein, The marker is 2-aminobenzoamide (2-AB), anthranilic acid (2-AA), 8-aminopyrene-1,3,6-trisulfonic acid (APTS), or procainamide hydrochloride.

31. The kit according to claim 23, wherein, The reducing agent is sodium cyanoborohydride or methylpyridineborane, or sodium cyanoborohydride and methylpyridineborane.

32. The kit according to claim 23, further comprising one or more reagents for deglycosylation of the glycoconjugate.

33. The kit according to claim 32, wherein, One or more of the reagents are deglycosylation enzymes.

34. The kit according to claim 33, wherein, The deglycosylation enzyme is PNGase F.

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