Rapid glycan sample preparation method, system and kit

Abstract

Disclosed are methods, systems, and kits for isolating and recovering glycans from glycosylated substrates. The magnetic particles are incorporated into a sample containing a glycosylated substrate. Magnetic particles are used to efficiently mix samples through multiple steps of a separation and collection workflow. The magnetic particles are surface functionalized to attach the released glycans to the surface of the particles. The disclosed system includes a device capable of generating an electromagnetic field in which a sample containing magnetic particles is placed for treatment.

Description

[0001] Priority

[0002] This application is being filed on April 24, 2024 as a PCT International Patent Application and claims priority to U.S. Provisional Patent Application No. 63 / 497,916, filed April 24, 2023 and titled Rapid Glycan Samples Preparation Methods, System and Kits. The disclosure of this provisional application is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to methods, systems and kits for biological sample preparation. More specifically, the present disclosure relates to rapid and automatable methods, systems and kits for glycan sample preparation, i.e., isolation and harvesting of glycans from glycosylated substrates. BACKGROUND

[0004] Glycans are chain-like structures composed of individual sugar molecules (monosaccharides) connected together by chemical bonds. Glycans are commonly found attached to proteins and lipids in living organisms. Glycosylation of proteins is the process in which complex glycan chemistry is attached to proteins to form glycoproteins. It is estimated that more than half of all human proteins are glycosylated. The attached glycans provide important structural and functional properties to proteins. The constituent monosaccharides, their combinations and bonding positions, and the types of branching can all affect the properties and roles of these glycosylated proteins.

[0005] Glycans are important players in cell-cell interactions and communication, and they also control the behavior of the immune system in many different ways. For example, glycans are an indicator of whether a cell or protein belongs to the body. Foreign glycan structures and patterns are recognized by the immune system as “non-self” and activate an immune response. In contrast, the presence of “self” glycans can act as a signal to prevent the immune system from overreacting. Recently, there has been growing interest in the key role of glycans as components of antibodies, affecting their binding to pathogens and infected cells, and neutralizing or marking them for removal by white blood cells. Differences in glycan structures can change the target, binding ability, and activity of antibodies.

[0006] Given their various importance in biological pathways, the study of glycans has become indispensable in the biopharmaceutical industry, for example, with respect to biomarker discovery, cancer drug development, and with respect to efficacy and safety assessment of protein therapeutics.

[0007] Current analytical methods for glycan-related research include the use of capillary electrophoresis and hydrophilic interaction liquid chromatography, and while these can be effective, they require extensive sample preparation, including glycoprotein capture, glycan release, labeling, purification, and pre-concentration steps. It is known in the industry that current standard methods for glycan sample preparation include many steps that require repeated and laborious human "hands-on" time, including multiple pipetting steps and sample transfer steps. Additionally, current protocols are time-consuming, and they require many laboratory pieces of equipment, including centrifuges, vacuum manifolds, heaters, well plates. During each step, the user needs to move the sample from one particular piece of laboratory equipment or device to another. Furthermore, current protocols often require the user to manually mix the sample in each step by pipetting up and down. This is inefficient and introduces multiple opportunities for human error or sample mishandling. These steps result in time-consuming protocols and inherently have multiple potential points of human error, affecting the reliability, robustness, and reproducibility of the results, which again increases laboratory costs if the protocol needs to be repeated due to error.

[0008] Accordingly, there remains a need for efficient and less error-prone methods of glycan sample preparation. More specifically, there is a need for methods that reduce human interaction and reduce "hands-on" time with the sample. There is also a need for methods that introduce efficient but gentle mixing of the sample and do not rely on crude methods such as pipetting, which does not result in efficient mixing and can damage the protein sample.

[0009] Additionally, there remains a need for systems that reduce the number of sample transfers from one piece of equipment to another, thereby reducing the number of devices needed to complete the protocol and reducing the time and cost associated with each sample preparation. SUMMARY

[0010] Disclosed herein are methods, systems, and kits for improved glycan sample preparation. More specifically, the present disclosure relates to the isolation and recovery of glycans from glycosylated substrates by the use of mixed-in magnetic particles into a sample containing glycosylated substrates. The magnetic particles are used to efficiently mix the sample through multiple steps during the disclosed workflow. The high and gentle mixing provided by the magnetic particles results in increased reaction kinetics and increased reaction yield. The mixing of the particles is actuated by the incorporation of the sample container within a device capable of generating a magnetic and more specifically an electromagnetic field.

[0011] The disclosed methods, systems and kits provide a glycan sample preparation workflow that is less time consuming and reduces potential human error and "hands-on" time when compared to current benchmark methods in the industry. The current methods provide improved reliability, robustness and reproducibility of results by reducing human interaction with the sample and by improving the reaction kinetics of protein digestion, labeling of glycans and attachment and release of glycans from magnetic particle surfaces.

[0012] In one aspect, a method for isolating and harvesting glycans from glycosylated substrates is presented. The method comprises the following steps:

[0013] a) adding an enzymatic reagent to a sample containing glycosylated substrates and releasing glycans from the glycosylated substrates;

[0014] b) labeling the released glycans;

[0015] c) attaching the labeled glycans to magnetic particles; and

[0016] d) dissociating the labeled glycans from the magnetic particles;

[0017] wherein a magnetic field is applied during each of steps a) to d).

[0018] In one embodiment, a further step of collecting the dissociated labeled glycans is performed. In one embodiment, the glycosylated substrates are prepared in the sample prior to the addition of the enzymatic reagent, wherein the preparation optionally includes a denaturing step prior to the introduction of the enzymatic reagent. The denaturing step can include various known techniques and workflows for denaturing the glycosylated substrates, e.g., proteins. For example, in various embodiments, the glycosylated substrates can be denatured by adding heat to the sample, by mechanical agitation, or by adding a denaturing reagent, which can be specific to the substrate / protein of interest, or by a combination of any of these known techniques.

[0019] According to some aspects, the glycosylated substrates refer to glycoconjugates, glycoproteins, glycolipids, glycopeptides, peptidoglycans, glycosides, antibodies, antibody-drug conjugates and / or lipopolysaccharides, or any molecule or compound to which a glycan structure is attached.

[0020] In other aspects, a system for isolating and harvesting glycans from glycosylated substrates is disclosed. The system comprises a device capable of generating a magnetic field, more specifically an electromagnetic field. By the inclusion of magnetic particles contained in the sample, the device can generate an electromagnetic field for mixing the sample contained therein. Accordingly, the device can also be referred to as an electromagnetic mixer.

[0021] The disclosed system's apparatus can generate a varying magnetic field and utilize an oscillating electromagnetic field to completely deagglomerate magnetic particles, thereby allowing for optimal exposure and enhanced mixing with the surrounding solution. Therefore, reaction kinetics are significantly improved compared to more conventional mixing methods that utilize mechanical vortex oscillators or pipettes to mix samples.

[0022] In other aspects of this disclosure, kits for preparing glycan samples are provided. Kits for separating and collecting glycans from glycosylated substrates include: magnetic particles, at least one enzymatic reagent, at least one labeling reagent; and optionally at least one reagent for attaching and / or dissociating the glycans from the plurality of magnetic particles.

[0023] The kit contains multiple magnetic particles that conform to the description provided herein and can be used with the devices disclosed herein. The magnetic particles are provided in a container, which is also included in the kit. Additional containers suitable for use with the devices disclosed in the system may also be provided. The kit may further include various buffers and reagents used to process the workflow described herein regarding the separation and harvesting of glycans from glycosylated substrates. Attached Figure Description

[0024] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the applicant's teachings in any way.

[0025] Figure 1 This is the result of using the workflow, magnetic particles, and system described in the embodiments of this disclosure to capture glycans.

[0026] Figure 2 The sample purification efficiency of the disclosed workflow is shown. Figure A shows the chromatogram of the polysaccharide-containing sample before cleaning, and Figure B shows the chromatogram of the polysaccharide-containing sample after cleaning according to the workflow disclosed herein.

[0027] Figure 3 The chromatogram of a sample processed according to the workflow disclosed herein is shown, wherein the magnetic particles have carboxyl surface functional groups. Detailed Implementation

[0028] This document discloses methods for more efficient and automated glycan sample preparation. More specifically, the present disclosure provides methods, systems, and kits for isolating and harvesting glycans from glycosylated substrates. The methods and workflows outlined herein demonstrate improved efficiency, can be performed with significantly reduced hands-on time, and can be implemented using a single laboratory apparatus, eliminating the need for sample transfers from multiple devices that could lead to errors and sample mishandling. Therefore, the methods disclosed herein provide robustness, reliability, and reproducibility of results—all key characteristics of glycan analysis in the biopharmaceutical industry and in any laboratory setting where glycan sample preparation is performed.

[0029] Selected definition

[0030] The terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the scope of this disclosure.

[0031] Unless the context clearly indicates otherwise, the singular forms “a (kind)” and “the” are intended to include the plural forms as well.

[0032] The term “and / or” or “and or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0033] The term “about”, when referring to measurable values ​​such as length, width, diameter, radius, or the amount, dosage, time, temperature, etc. of a compound, means covering a variation of 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0034] When used in this specification, the term "comprises" (comprising) designates the presence of the stated feature, integer, step, operation, element, and / or component (part), but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof. Unless otherwise defined, all terms used in this specification (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the event of a conflict of terminology, this specification shall prevail.

[0035] All patents, patent applications and publications mentioned in this article are incorporated herein by reference in their entirety.

[0036] The embodiments described in one aspect of this disclosure are not limited to those described. Embodiments may also be applied to different aspects of this disclosure, provided that they do not prevent those aspects of this disclosure from being used for their intended purpose.

[0037] The term "ferrimagnetic particles" refers to particles comprising ferrimagnetic materials. Ferromagnetic particles are responsive to external magnetic fields (e.g., changing magnetic fields) but demagnetize when the external magnetic field is removed. Therefore, ferrimagnetic particles can be efficiently mixed with a sample by an external magnetic field and efficiently separated from the sample using a magnet or electromagnet, while remaining suspended without magnetically induced aggregation.

[0038] The term "remanence" refers to the residual magnetism retained by a material after the magnetic field is removed. Materials with high remanence retain a large magnetic field strength after the magnetic field is removed, while materials with low remanence retain a small magnetic field strength or zero magnetic field strength. The remanence of magnetic materials can range from about 0 emu / g to about 30 emu / g, from about 0 emu / g to about 20 emu / g, from about 1 emu / g to about 10 emu / g, from about 3 emu / g to about 5 emu / g, or less than, equal to, or greater than about 0 emu / g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 emu / g.

[0039] As used herein, the term "functionalized surface" or "surface functionality" refers to a surface coated with moieties, each moiety having free functional groups bound to magnetic particles; consequently, the surface of the magnetic particles is coated with moieties containing functional groups. Functional groups can be used to covalently attach bioaffinity absorbers for biomolecules in solution. In one example, the functional group is a carboxylic acid. A suitable moiety with free carboxylic acid functional groups is a succinic acid moiety, wherein one of the carboxylic acid groups is bonded to an amine of an aminosilane via an amide bond, and the second carboxylic acid is a free carboxylic acid group attached or tethered to the surface of the magnetic particles. The functionalized surface can be selected from carboxyl, amide, amino, hydroxyl, thiol, toluenesulfonyl, epoxy, alkyl, vinyl, aryl, or silica groups.

[0040] As used herein, the term "glycosylated substrate" refers to a protein structure or molecule containing a protein that is covalently bonded to a carbohydrate molecule at several specific amino acid residues via a glycosylation mechanism. Glycosylated substrates may include glycoconjugates, glycoproteins, glycolipids, glycopeptides, peptidoglycans, glycosides, antibodies, antibody-drug conjugates, proteoglycans, glycosphingolipids, chondroitin sulfate, heparin sulfate, hyaluronic acid, glycosaminoglycans, fusion glycoproteins, lipopolysaccharides, or any molecule or compound thereof with a glycan structure attached thereto.

[0041] As used in this article, the term "polysaccharide" refers to a chain-like structure composed of individual sugar molecules (monosaccharides) linked together by chemical bonds.

[0042] method

[0043] In some embodiments, a method for separating and collecting glycans from glycosylated substrates is presented. The method includes the following steps:

[0044] a) Add an enzymatic reagent to a sample containing a glycosylated substrate and release the polysaccharide from the glycosylated substrate;

[0045] b) Labeled released glycans;

[0046] c) Attaching the labeled polysaccharide to the magnetic particles;

[0047] d) Dissociate the labeled polysaccharide from the magnetic particles; and

[0048] A magnetic field is applied during each of steps a) to d).

[0049] In one embodiment, a further step includes collecting the dissociated labeled glycans. In another embodiment, a glycosylated substrate is prepared in the sample prior to the addition of an enzymatic reagent, wherein the preparation optionally includes a denaturation step prior to the introduction of the enzymatic reagent. The denaturation step may include various known techniques and workflows for denaturing glycosylated substrates, such as proteins. For example, in many embodiments, the glycosylated substrate may be denatured by: applying heat to the sample, by mechanical stirring, or by adding a denaturing reagent (which may be specific to the substrate / protein of interest), or by any combination of these known techniques.

[0050] According to some sources, the substrate for glycosylation refers to glycoconjugates, glycoproteins, glycolipids, glycopeptides, peptidoglycans, glycosides, antibodies, antibody-drug conjugates and / or lipopolysaccharides, or any molecule or compound to which a glycan structure is attached.

[0051] In a preferred embodiment, the glycosylated substrate undergoes a denaturation step by heating and mixing or stirring the sample. During this step, the sample temperature can range from room temperature to 100°C, or preferably from about 55°C to 75°C, or even more preferably 65°C. The duration of this step will depend on the chosen temperature, with higher temperatures requiring less time and lower temperatures requiring longer reaction times, as those skilled in the art will understand. At 65°C, the sample duration for the denaturation step can be about 10 minutes. This step is optional, although recommended, because it unfolds the substrate (e.g., protein), making sites for glycan cleavage more readily available and exposed to the enzymatic reagent that will be introduced into the sample in subsequent steps. Theoretically, once the protocol is complete, performing the denaturation step should yield a higher amount of glycan because more sites on the protein are exposed for cleavage, and therefore more glycan can be separated from the substrate and ultimately collected during further steps of the workflow / method.

[0052] The disclosed method or workflow relies on the inclusion of multiple magnetic particles placed in a sample container (e.g., a tube, well, or plate). The magnetic particles are used to efficiently mix the sample throughout the workflow. To achieve this, the sample containing the magnetic particles is placed in a device capable of generating a magnetic field, more specifically an electromagnetic field. The electromagnetic field generated by this device can have various properties; for example, it can be rotating or static. This means that it can cause the magnetic particles to move and remain in motion (rotation) in the x, y, or z directions to achieve efficient mixing of the sample and reagents therein, or it can cause the particles to aggregate on the sides of the tube without moving (stationary), which allows for the separation of sample components and particles where it is necessary to remove sample components from the sample container. Further details and aspects regarding the device and magnetic particles will be disclosed in detail in subsequent sections of this disclosure.

[0053] In one implementation, steps a) through e) outlined above can all occur within the same sample container without requiring sample transfer. This means that at any point during the various steps of the workflow, the sample does not need to be removed from the original sample container and can be kept there by adding buffer and reagents until the collection step where the polysaccharide-containing solution is removed from the tube.

[0054] Once the glycosylated substrate in the sample has denatured, an enzymatic reagent is added to the sample. This reagent reacts with the substrate at specific attachment sites and is able to cleave glycosidic bonds, releasing or deactivating the glycan from the glycosylated substrate. After protein denaturation is complete, a static magnetic force is applied to the device containing the sample. This causes the magnetic particles to be attracted to the walls of the sample container and cease to disperse and move within the sample. This efficiently stops the mixing of the sample and particles and allows the enzymatic reagent to be added to the tube. Subsequently, the rotating magnetic field is reactivated to force the magnetic particles back into mixing mode. This step can be performed from approximately 1 minute to 24 hours and occurs at temperatures ranging from room temperature to 100°C. More preferably, the mixing of the enzymatic reagent with the sample is performed at 50°C for approximately 15 minutes.

[0055] In current methods known in the art, the reactions and molecular interactions occurring in the sample during denaturation and enzymatic cleavage steps typically take place on a vortex oscillator, which mechanically oscillates the entire container (typically a 96-well plate). This type of container mixing is less efficient than that of our method, which utilizes magnetic particles within the sample itself to actuate highly efficient mixing of the sample. The more efficient the mixing of substances within the sample, the more kinetic energy and forces are generated, leading to increased substance interactions and increased reaction yields. This, in turn, increases the amount of glycan cleavage and the release of glycans, which can be separated and retrieved from the substrate.

[0056] In some aspects, the enzymatic reagent is selected from peptidyl-N-glycosidase F (PNGase F), endoglycosidase H (EndoH), endo-α-N-acetylgalactosaminease, α-mannosidase, or O-glycosidase, or combinations thereof. The type of reagent selected will cleave a specific type of glycosidic bond, such as N-glycans or O-glycans. "O"-linked glycans are attached to the oxygen atom of an amino acid residue in a protein, while "N"-linked glycans are attached to the amide nitrogen of an asparagine residue in a protein. Those skilled in the art will readily understand that different enzymes can be used to cleave various types of glycosidic bonds specific to the target protein, whether N-glycans, O-glycans, C-glycans, or any other known type of bond. In a preferred embodiment, the enzymatic reagent is peptidyl-N-glycosidase F (PNGase F), which cleaves N-glycans.

[0057] Once the glycosylated substrate has undergone enzymatic digestion, the static magnetic field is reactivated to cause the magnetic particles to aggregate away from the mixing mode and onto the container wall where they are no longer moving. The labeling step can then be initiated by adding a dye reagent, which results in the release of labeled glycans for further analysis in subsequent processes such as capillary electrophoresis, mass spectrometry (MS), etc. Labeling reagents or dyes are added while the magnetic beads are in static mode, and once the reagent is added to the container, the rotating field in the apparatus is turned on again, allowing for efficient mixing of the release glycans and the labeling reagent, thus producing labeled glycans.

[0058] The duration of the labeling step will depend on the chemistry and reaction kinetics of the type of labeling reagent selected. In some aspects, the labeling dye to be used may be selected from 2-o-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 1-aminopyrene-3,6,8-trisulfonic acid (APTS), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), or dyes containing a rapid labeling functional group such as N-hydroxysuccinimide carbamate, or combinations thereof. In a preferred embodiment, the labeling reagent is 2-aminobenzamide (2-AB). The labeling step can be carried out at a temperature range of room temperature to 100°C for a duration of 1 minute to 24 hours. More preferably, the labeling of the released glycan is carried out at a temperature of 65°C for about 60 minutes.

[0059] Although the labeling of the released glycan can be performed at different times or steps throughout the workflow, such as once the released glycan has attached to the magnetic particles, it is preferable that the labeling occurs before the glycan attaches to the particles. In this preferred embodiment, higher labeling reaction or efficiency is believed to be achieved because more reaction sites are available if the glycan is free in solution rather than attached to the particles.

[0060] Next, the labeled glycan is attached to the magnetic particles. This is achieved by adding a diluent to the sample container containing the labeled glycan. Again, a static magnetic force is applied within the device to pull the magnetic particles from the sample solution, and a reagent is added, which attaches the labeled glycan to the surface of the magnetic particles. Once the reagent has been added, a suitable magnetic field is turned on to efficiently mix the labeled glycan and attach it to the particles. The reaction can proceed for a duration of approximately 1 to 100 minutes, or 2 to 30 minutes, or 3 to 15 minutes, or 4 to 8 minutes, or more preferably approximately 5 minutes. In one embodiment, the reagent used is acetonitrile (ACN).

[0061] Magnetic particles may include surface functional groups, meaning they possess specific reactive groups on their surface that can be used to bind glycans in solution to the particle surface. In one embodiment, the particles have already been carboxyl-functionalized on their surface. It is believed that the attachment mechanism between the glycans and the surface groups of the particles occurs through hydrophilic interactions and is not driven by charge attraction.

[0062] Once the glycans have been attached to the particle surface, a static magnetic field is applied to pull down (i.e., stop the mixing process and gather the particles together at one location within the container so they no longer disperse) the magnetic particles and remove the supernatant solution from the sample container. The supernatant at this stage will contain various unwanted substances that must be removed before processing the sample in subsequent analytical steps. The supernatant may include protein fragments, unreacted dyes, reagent residues from previous steps, buffer solutions, etc., which, if not removed, will affect sample integrity. After this cleaning step, the labeled glycans attached to the surface of the magnetic particles can then dissociate from the particles—in other words, be released.

[0063] To dissociate the attached labeled glycans from the surface of the magnetic particles, an eluent (e.g., water) is added to the sample container, and a rotating magnetic field is actuated to initiate efficient mixing of the particles with the eluent. This step may be repeated several times; for example, it may be necessary to repeat the step at least twice in a mixing mode to ensure that all attached labeled glycans are dissociated from the particle surface and are now in solution. This ensures the efficient release of any remaining glycans still attached and can increase the final yield of glycan collection. A washing step may also be performed before eluting the glycans. Acetonitrile buffer may be added at least once and the mixing mode actuated to wash away any remaining unwanted substances from the sample. This step is preferably repeated multiple times, for example, three times.

[0064] The supernatant obtained after the dissociation step (i.e., the elution step) is then collected, concentrated, or otherwise prepared for further analysis. To achieve this, a static magnetic field is turned on to pull down the particles, and the supernatant can be removed from the sample container.

[0065] The sample containing the dissociated labeled glycan can then be prepared for the desired analysis using an appropriate protocol for that particular type of analytical tool. For example, in one embodiment, the glycan sample is further analyzed by capillary electrophoresis, liquid chromatography such as reversed-phase liquid chromatography or hydrophilic interaction chromatography (HILIC), mass spectrometry (MS), or nuclear magnetic resonance (NMR), or a combination of these techniques.

[0066] Figure 1The image shows chromatographic readings of the glycan profile of the Aflibercept (Zaltrap) model protein obtained from the method disclosed above. Hydrophilic interaction liquid chromatography (HILIC) is used to separate 2-AB-labeled free N-glycans based on their hydrophobicity, where less polar glycans elute earlier and more polar glycans elute later. For example, it can be... Figure 1 The chromatogram shows well-resolved peaks for the main glycans. The first large peak on the left is 2-AB dye and buffer reagent. The peaks below at 10 to 30 minutes include unbound glycans, neutral and acidic glycans with varying levels of sialic acid and / or negative charge, highly sialylated glycans, and a mixture of fucoidylated glycans.

[0067] system

[0068] A system for preparing glycan samples is also disclosed. In one embodiment, a system for separating and collecting glycans from glycosylated substrates is disclosed, the system comprising:

[0069] - A device capable of generating a magnetic field;

[0070] - Magnetic particles;

[0071] - At least one enzyme-catalyzing reagent; and

[0072] - At least one labeling reagent.

[0073] In some embodiments, the system further includes reagents for attaching or dissociating the glycan with or from a plurality of magnetic particles. In other embodiments, the system further includes at least one denaturing reagent. In further embodiments, the system includes a buffer solution, a washing solution, a reducing agent, and other reactants for performing the glycan sample preparation workflow disclosed herein.

[0074] The device is capable of generating a magnetic field, and more specifically, an electromagnetic field. By incorporating magnetic particles contained in a sample, the device can generate an electromagnetic field for the purpose of mixing the sample contained therein. Therefore, the device can also be referred to as an electromagnetic mixer. For the purposes of the following disclosure, the terms "device," "electromagnetic mixer," or "magnetic assembly" are used interchangeably and refer to the same components of the disclosed system.

[0075] The disclosed system's apparatus generates a varying magnetic field and utilizes an oscillating electromagnetic field to completely deagglomerate magnetic particles, thereby allowing for optimal exposure and enhanced mixing with the surrounding solution. Therefore, reaction kinetics are significantly improved compared to more conventional mixing methods that utilize mechanical vortex oscillators or pipettes. This reduces sample transfer and also decreases hands-on time, human interference, or potential sample contamination and loss. The use of such an apparatus not only provides a significantly more efficient mixing device, which enhances reaction kinetics, but also offers a means for automating sample workflows.

[0076] Suitable such devices (magnetic components or electromagnetic mixers) for processing fluid samples are described in the following: WO2017093896, US 2018-0369831, US 2020-0011773, WO 2020016854, Arnold et al., US20200011773, Arnold et al., US 10656147, Campbell et al., and WO 2021203005, Corpstein et al., each of which is incorporated herein by reference in its entirety.

[0077] In several aspects, the disclosed system may include a magnetic component comprising a plurality of magnetic structures configured to generate a magnetic field gradient within one or more chambers in which a fluid / sample containment container is placed. The magnetic structures may be formed as a plurality of electromagnets configured to be individually actuated by a controller. Each electromagnet can generate a magnetic field within the fluid container. The electromagnets may be differentially actuated to generate magnetic field gradients within the fluid container to agitate, mix, or otherwise influence magnetic particles disposed within the fluid container. Activation of the electromagnets of the electromagnetic structures can generate magnetic field gradients that influence the magnetic particles in the xy directions. Furthermore, activation of the electromagnets of the plurality of electromagnetic structures can generate magnetic field gradients that influence the magnetic particles in both the xy and z directions. This can be characterized as generating a rotating magnetic field utilized during the mixing step of the disclosed glycan sample preparation method and workflow.

[0078] Magnetic particles can be placed inside a sample container and dispersed within a fluid, and can be configured to be agitated by a magnetic field (or gradient) generated by a magnetic assembly arranged in adjacent fluid chambers (e.g., surrounding a peripheral chamber in which the sample container is placed) to facilitate movement of the magnetic particles within the fluid. The magnetic assembly may include one or more magnetic structures arranged in a horizontal or substantially horizontal layer. Each magnetic structure may be formed by one or more magnets, such as electromagnets. The vertical position of one or more magnetic structures relative to the fluid may be movable or adjustable, for example, before, during, or after facilitating the movement of the magnetic particles within the fluid. Adjusting the vertical position of one or more magnetic structures before facilitating the movement of the magnetic particles can be used, for example, to handle different sample volumes and / or to influence the characteristics of the magnetic field generated by the magnetic assembly. The vertical movement of the magnetic structures, while facilitating the movement of the magnetic particles, may add, for example, a vertical movement component within the particles to provide more efficient or effective mixing of the particles in the fluid.

[0079] Alternatively or alternatively, electrodes of various magnetic structures can be selectively excited (e.g., different vertically spaced layers) to handle different sample volumes and / or affect the characteristics of the magnetic field generated by the magnetic components.

[0080] The magnetic component structure can be formed by multiple electromagnets arranged around a fluid chamber at one or more different vertical heights, wherein each electromagnet is individually controlled to generate a desired magnetic field within the fluid chamber that effectively influences the magnetic particles disposed therein. Based on the selective application of electrical signals to the multiple electromagnets surrounding the fluid chamber, the rotation, spin, horizontal left-right movement, and / or vertical up-down movement of the magnetic particles within the fluid sample, or any combination of these movements, can be influenced by the combined effect of the magnetic field gradients generated by the various electromagnets.

[0081] For example, the signal applied to an electromagnet for each magnetic structure (e.g., in a single horizontal layer) can be configured to generate a magnetic field gradient essentially in the xy plane, while the signal applied to an electromagnet for different magnetic structures (if present) (e.g., electromagnets in different horizontal layers) can result in a magnetic field gradient exhibiting a z-direction or vertical component. In this way, the combined effect of multiple electromagnets can generate magnetic fields with different characteristics, such as different intensities and / or directions, within the sample container, so as to rapidly and efficiently mix fluids and / or trap target analytes within the fluid, as a non-limiting example.

[0082] According to one aspect of this disclosure, by applying alternating current (AC) with different phase delays to multiple electromagnets in the array, all pores can be activated simultaneously, thereby minimizing diffusion distance and improving reaction kinetics in a homogeneous suspension containing magnetic particles generated in each sample container within the chamber. During any buffer exchange process, a steady-state magnetic field (DC) can be used to trap the beads at the corner walls of the sample containers (this is also characterized as a magnetostatic force). The system may also include a temperature control module, means for heating the samples, and includes multiple temperature sensors and a cooling fan.

[0083] Efficient mixing of large-volume samples (>1 mL) via mechanical agitation is challenging because classic two-dimensional shaking cannot bring particles to the top of the solution, and extensive vertical shaking causes solution splashing and droplet adhesion to vial caps / caps. A two-layer electromagnet structure enables efficient and uniform three-dimensional particle mixing for large sample volumes.

[0084] For a broad measurement coverage, the system is designed to be compatible with both high-response particles (e.g., ferrimagnetic beads) and low-response particles (e.g., superparamagnetic beads) by tuning the AC waveform applied to the electromagnet assembly. Even for samples containing low-response superparamagnetic beads, power consumption is less than 5 W.

[0085] The applied magnetic field strength, type (rotating or static), temperature, time sequence, and other parameters are selected and adjusted via the user interface on the device. Optimal levitation of the magnetic particles can be achieved by controlling different signal phases for each electromagnet. The system can be extended from a single sample container controlled by four electromagnets to 96 wells (sample containers) controlled by 117 electromagnets, with each electromagnet shared by four wells.

[0086] Magnetic particles

[0087] Magnetic beads have been widely used in sample preparation. However, the magnetism of the particles is typically only utilized during the buffer exchange (supernatant removal) process, rather than during the mixing step as is the case in this disclosure. In previously known workflows, mixing of the sample with surface-functionalized beads is usually still achieved by mechanical agitation (e.g., shaking, pipetting). Using these types of conventional methods, the magnetic particles may aggregate and cluster in discrete regions near the container walls, significantly reducing mixing efficiency.

[0088] Suitable magnetic particles for use in the systems and methods described herein include, but are not limited to, paramagnetic particles, such as AMPure XP beads available from Beckman Coulter, Inc., Brea, CA. Suitable magnetic particles also include those described in: US Patent Nos. 5,705,628; 5,898,071; and 6,534,262, and in published PCT applications WO 2020 / 018919, published January 23, 2020, and US 2021 / 0139953, published May 313, 2021, all of which are incorporated herein by reference as if fully set forth herein.

[0089] In some respects, magnetic particles can be ferrimagnetic, meaning they are composed of ferrimagnetic materials. Ferromagnetic particles can respond to an external magnetic field (e.g., a changing magnetic field) but can be demagnetized when the external magnetic field is removed. Thus, ferrimagnetic particles can be efficiently mixed with a sample by an external magnetic field and efficiently separated from the sample using a magnet or electromagnet, while remaining suspended without magnetically induced aggregation.

[0090] In some respects, the magnetic particles described herein are sufficiently responsive to magnetic fields, enabling them to move efficiently through a sample. Typically, the range of field strength can be the same as that of any electromagnet, as long as it is capable of moving the particles. For example, magnetic fields may have strengths between about 10 mT and about 250 mT, between about 20 mT and about 80 mT, and between about 30 mT and about 50 mT.

[0091] In some instances, more powerful electromagnets can be used to mix microparticles with lower responsiveness. In others, the magnetic field can be focused as much as possible onto the sample. Furthermore, the electromagnet can be placed as close to the sample as possible, since magnetic field strength decreases with the square of the distance.

[0092] Magnetic particles can be of various shapes, both regular and irregular. In some instances, the shape maximizes the surface area of ​​the particle. For example, magnetic particles can be spherical, strip-shaped, elliptical, or any other suitable shape. Magnetic particles can have various densities, which can be determined by the composition of the core. In some instances, the density of magnetic particles can be adjusted using a coating.

[0093] Ferromagnetic particles may include ferrites. Ferrites include ceramic materials, which comprise combinations of iron oxides with inorganic compounds of metallic, nonmetallic, or quasi-metallic atoms. For example, ferrites may comprise iron(III) oxides (Fe₂O₃) blended with one or more other metallic elements such as barium, manganese, nickel, zinc, titanium, or any other suitable metallic element. Other examples of ferrites include Fe₂TiO₂, FeTiO₂, MnFeO₄, NiFe₂O₄, and MgFe₂O₄. Further examples of ferrites include an iron core comprising sulfides or hydroxyl oxides, such as Fe₃S₈, Fe₃S₄, FeS, or FeOOH.

[0094] Magnetite (Fe3O4) is an example of a magnetic material that can be used in the examples described herein; it is an example of a ferrite. Magnetite contains Fe. 2+ and Fe 3+ Both ions. In some cases, Fe... 2+ and Fe 3+ The electron spins of ions can couple in a crystal structure, making magnetite ferrimagnetic, as described herein. However, in some instances, ferrimagnetic particles include any ferrimagnetic material (e.g., ferrite). According to some instances, the ferrimagnetic material (e.g., ferrite) may not be magnetite (Fe3O4), however, in some instances, magnetite is a suitable ferrimagnetic material.

[0095] Ferrites can be divided into two main families (hard ferrites and soft ferrites) based on their magnetic coercivity (e.g., the ability of a material to withstand an external magnetic field without becoming demagnetized).

[0096] Hard ferrites possess high magnetic coercivity and high remanence after magnetization. They can be used to manufacture permanent magnets because they are not easily demagnetized in the absence of an external magnetic field due to their high remanence. Examples of hard ferrites include strontium ferrite and barium ferrite.

[0097] Soft ferrites exhibit low magnetic coercivity. They also possess low remanence after magnetization. The magnetization of soft ferrites is more easily altered than that of hard ferrites. Furthermore, the magnetization of soft ferrites can be easily reversed without significant energy dissipation (e.g., via hysteresis losses). Soft ferrites can also possess high resistivity, thus preventing the formation of eddy currents in the material, another source of energy loss.

[0098] Soft ferrites may include manganese-zinc (MnZn) ferrites and nickel-zinc (NiZn) ferrites. Therefore, in some instances, the ferrimagnetic particles comprise MnZn ferrites. In other instances, the ferrimagnetic particles comprise NiZn ferrites. Ferromagnetic particles comprising MnZn ferrites and / or NiZn ferrites can be magnetized in the presence of an external magnetic field and are thus capable of moving in the presence of an external magnetic field, but will not significantly aggregate due to magnetic induction after the external magnetic field is removed because they have low remanence.

[0099] Some ferrites can be considered semi-hard ferrites. Semi-hard ferrites possess properties between those of soft and hard ferrites. For example, cobalt ferrite (CoFe2O4) is a semi-hard ferrite that can be magnetized in the presence of an external magnetic field (e.g., a changing magnetic field generated by a magnetic component), but does not have high remanence after the external magnetic field is removed, so that the subferromagnetic particles, including the cobalt ferrite core, do not significantly aggregate due to magnetically induced aggregation.

[0100] Magnetic particles can be of various shapes, both regular and irregular. In some instances, the shape maximizes the surface area of ​​the particle. For example, magnetic particles can be spherical, strip-shaped, elliptical, or any other suitable shape. Magnetic particles can have various densities, which can be determined by the composition of the core. In some instances, as described herein, the density of magnetic particles can be adjusted using coatings.

[0101] The strength of a magnetic field can be determined using a gaussmeter. A gauss is the unit of magnetic flux density B in a system of gauss units, and is equal to Mx / cm² or g / Bi / s. 2 A gauss is a unit used to measure the strength of a magnetic field, and a gaussmeter can be used to perform this measurement. The higher the gauss number, the stronger the magnetic field will be, and therefore the greater the distance that the field will need to travel to from surrounding magnets.

[0102] Magnetic particles may include magnetic materials having a maximum magnetic field strength (Bmax) in the range of about 20 emu / g to about 250 emu / g, 40 emu / g to 200 emu / g, 50 emu / g to 150 emu / g, or about 80 emu / g to 100 emu / g. Bmax may be >40, >50, >60, or >70 emu / g. Bmax can be measured using a SQUID (superconducting quantum interference device). Magnetic particles or magnetic beads may have ultra-high magnetic responses, for example, having a Bmax in the range of about 80-100 emu / g or about 89 emu / g. This is compared to many commercially available magnetic beads with <40 emu / g.

[0103] In some embodiments, the magnetic particles may include magnetic materials having a maximum magnetic field strength (Bmax) in the range of about 20 emu / g to about 250 emu / g and a remanence in the range of about 0 emu / g to about 30 emu / g.

[0104] Magnetic particles can range in average diameter from about 1 nm to about 1 mm. In some instances, magnetic particles can have an average diameter in the range of 50 nm to 500 nm. In some instances, magnetic particles can have an average diameter in the range of 100 to 200 nm. The diameter of magnetic beads can range from about 80 to about 120 nm or about 100 nm.

[0105] Magnetic particles can be substantially solid or may have a degree of porosity. Where the magnetic particles do indeed include a degree of porosity, the diameter of individual pores can range from about 5 Å to about 1000 Å, or from about 50 Å to about 500 Å. At least multiple pores can be through-holes (e.g., extending completely between opposing surfaces). The pore size or total porosity of the magnetic particles can be determined by a number of suitable methods. For example, the total volume of an ideal (e.g., non-porous) magnetic particle can be determined, and then the volume of the actual porous framework material can be determined. The porosity is then calculated by subtracting the volume of the actual porous framework material from the ideal magnetic particle. The porosity or individual pore size of the magnetic particles can also be determined by measuring individual pores using optical measurements with a microscope and processing the images.

[0106] Magnetic particles possess sufficient surface area to allow for efficient molecular bonding. In some instances, the surface area of ​​magnetic particles can be as small as approximately 0.1 m². 2 / g to approximately 500 m 2 / g, approximately 1 m 2 / g to approximately 200 m 2 / g, or approximately 10 mg 2 / g to approximately 100 m 2 Within the range of / g. In some embodiments, the magnetic particles or magnetic beads have a >5 μm 2 / g、>7 m 2 / g, or >10 m 2 / g surface area. Surface area can be measured by Brunauer-Emmett-Teller (BET) surface analysis. Brunauer-Emmett-Teller (BET) surface area analysis provides an assessment of the specific surface area of ​​a material using a fully automated analyzer, measured as a function of relative pressure, of nitrogen multilayer adsorption. This technique encompasses assessment of external area and pore area to determine the specific surface area in m². 2 Total specific surface area calculated per g.

[0107] The magnetic particles described herein may include a variety of different materials. In the presence of mixtures of materials, the total magnetic content of the magnetic particles may constitute at least 50% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, or even 100% by weight of the magnetic particles.

[0108] Magnetic particles may include any of those described herein. Non-magnetic materials constituting the remainder of the magnetic particles may include, for example, any coating materials described herein. Non-magnetic materials may be used as coatings to encapsulate the magnetic portion of the magnetic particles, or they may be used as functional components to interact with and bind to the analyte of interest. Non-magnetic materials may also serve as filler components.

[0109] Magnetic particles or beads can be surface-functionalized with carboxyl, amino, hydroxyl, silica, streptavidin, or endopeptidase groups. Magnetite particles or beads may have a magnetite core. Magnetite particles or beads may have a magnetite core coated with a silica shielding layer. The silica shielding layer may be attached to a silane binder. The silane binder may be attached to a polymer. The polymer may be surface-functionalized with carboxyl, hydroxyl, silica, amine, amide, and combinations of different functional groups, or other known surface functional groups. In a specific embodiment, the magnetic beads or particles have carboxyl surface functional groups.

[0110] Magnetic particles can be coated and / or functionalized by any method known in the art. The coating can be, for example, a polymer layer or a silica layer. Examples of polymer layers may include polyethylene, polystyrene, polymethyl methacrylate, polyvinyl alcohol, or any other suitable polymer.

[0111] For example, the synthesis of core-shell Fe3O4 nanoparticles (NPs) can be carried out via the hydrolysis of tetraethyl orthosilicate (TEOS) in the presence of Fe3O4 nanoparticles to provide silica-coated magnetite core-shell particles. For instance, Fe3O4 NPs can be dispersed in water using an ultrasonic water bath, followed by mixing with an ammonia solution (25 wt% in water) and ethanol. TEOS can be added dropwise to the Fe3O4 suspension with stirring overnight at room temperature. The product can be separated using an external magnet, washed with water, and dried at 50 °C. The particles can be characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and UV-Vis absorption spectroscopy. See WO2020018919, which is incorporated herein by reference in its entirety.

[0112] Polymer-coated magnetic cores can be prepared, for example, by dispersing ~4 g of 100 nm magnetite cores in 100 mL of water under stirring. 10 mL of acrylic acid is added to a flask under stirring with K₂S₂O₈ to obtain a homogeneous suspension. The suspension is heated to 80 °C and stirred for 15 hours. The suspension is cooled to room temperature, and the solid is collected from the suspension using a permanent magnet. The collected solid is washed with water and dried at 60 °C to provide poly(acrylic acid) coated magnetic beads.

[0113] An example of the type of magnetic beads that can be used with the methods, systems, and kits of this disclosure includes a magnetite core surrounded from the inside out by a silica shield, a silane linker, and a polymer. The polymer can be covalently attached to surface functional groups such as carboxyl, amino, amide, or silica groups bonded to another functional group. According to the workflow disclosed herein, the surface functional groups can be used to bind and capture glycans released from glycosylated substrates. The magnetic particles can exhibit high magnetic response, for example, a Bmax of approximately 89 emu / g compared to <40 emu / g for most commercial magnetic beads. The magnetic particles can exhibit a magnetic response >10 μm. 2 / g high surface area.

[0114] Kit for preparing polysaccharide samples

[0115] In some aspects of this disclosure, kits for preparing glycan samples are disclosed. Kits for separating and collecting glycans from glycosylated substrates include:

[0116] - Multiple magnetic particles;

[0117] - At least one enzyme-catalyzing reagent;

[0118] - At least one labeling reagent; and

[0119] - Optionally, at least one agent for attaching and / or dissociating the polysaccharide to a plurality of magnetic particles.

[0120] The multiple magnetic particles provided with the kit conform to the description provided herein and are incorporated into this section of the disclosure and related embodiments. The magnetic particles are provided in a container, which is also included in the kit. Further containers suitable for use with the devices disclosed in the system may also be provided. The kit may further include various buffers and reagents used to process the workflow described herein regarding the separation and harvesting of glycans from glycosylated substrates.

[0121] The at least one enzymatic reagent provided in the kit will depend on the type of substrate for glycosylation of interest and the type of glycosidic bond prevalent in the glycoprotein of interest. The kit may include at least one enzyme or a combination of enzymes. In some aspects, the enzymatic reagent includes peptidyl-N-glycosidase F (PNGase F), endoglucosidase H (EndoH), endoglucosidase-α-N-acetylgalactosaminease, α-mannosidase, or O-glycosidase, or combinations thereof. In one embodiment, the enzymatic reagent provided in the kit is peptidyl-N-glycosidase F (PNGase F).

[0122] In some aspects, the labeling reagent / dye included in the kit is selected from 2-o-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 1-aminopyrene-3,6,8-trisulfonic acid (APTS), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), or combinations thereof. In a preferred embodiment, the labeling reagent is 2-aminobenzamide (2-AB).

[0123] The kit may also include a variety of other buffers and reagents used in the polysaccharide separation and collection workflow disclosed herein. Some of these buffers, solvents, and reagents may include denaturing buffers and / or reagents, acetic acid, DMSO, glycerol, sodium cyanoborohydride, tetrahydrofuran (THF), acetonitrile (ACN), and other dilution and elution buffers known to those skilled in the art.

[0124] Example

[0125] The following examples illustrate a method or workflow for preparing polysaccharide samples according to the embodiments described herein.

[0126] Example A

[0127] In this embodiment, the silica-coated magnetic particles are achieved through the following process steps.

[0128] Silica-encapsulated magnetite cores were prepared by dispersing 20 g of 100 nm magnetite cores in 800 mL of methanol in a 1 L crusher. The mixture was sonicated for 30 min to achieve a homogeneous suspension. 370 mL of 28% ammonium hydroxide was added to the suspension, and it was stirred for 30 min. Following sonication, a liquid mixture comprising 0.5 mL of tetraethyl orthosilicate and 4.5 mL of methanol was added dropwise to the suspension over a 0.5 h time span for further sonication. After sonication, the beaker was capped, and the suspension was continuously stirred for 15 h. After stirring, the silica-encapsulated particles were captured with a permanent magnet. The silica-encapsulated particles were then washed five times with water. The particles were then dried in an oven at 80 °C for 24 h.

[0129] Example B

[0130] The carboxylated silica-coated magnetic core was prepared as follows: 4 g of silica-coated magnetic core particles prepared according to Example A, dispersed in 150 ml toluene, were dispersed in a 500 ml flask with stirring. 20 g of (3-triethoxysilyl)propylsuccinic anhydride was added to the flask with stirring. Subsequently, 0.2 g of imidazole was added with stirring to produce a homogeneous suspension. The suspension was refluxed at approximately 114°C with stirring for 15 hours. After reflux, the suspension was cooled to room temperature, and the solid was collected from the suspension using a permanent magnet. The solid was first washed once with methanol, then washed five times with water, and transferred to a 500 ml flask. 150 ml of 0.1 M acetic acid in water was added to the flask with stirring to obtain a homogeneous suspension. The suspension was heated to 90°C and held for 15 hours. The suspension was cooled to room temperature. The solid was collected from the suspension using a permanent magnet. The solid was washed five times with water and dried in an oven at 60°C for 15 hours.

[0131] Example 1

[0132] Denaturation and deglycosylation of glycosylated substrates

[0133] Place PCR tubes (0.2 mL) in the holder (chamber) of the electromagnetic mixer and add 83.3 μL of 3% carboxylated magnetic particles (prepared as in Example B) (50% glycerol / H2O) to each PCR tube. Turn on the electromagnetic mixer and select the desired mixing mode. Mix the contents of the tubes at room temperature for 2 minutes. Stop the mixing function on the device (i.e., rotating magnetic field) and apply a static magnetic field to remove the particles from the mixing mode (i.e., pull-down) so that 50% glycerol / water can be removed from each tube. Subsequently, add 100 μL of fresh LC-MS grade water to each vial and mix for 1 minute. Repeat this washing step 3 times. Apply the static magnetic field again to remove the magnetic particles from the mixing mode and remove water from each vial.

[0134] Next, prepare a 6 mg / mL solution of the glycosylated substrate, namely the glycoprotein (abflibercept, brand name "Zaltrap"). Water can be used for dilution to achieve the desired concentration. For example, to prepare a 72.9 μL solution of 6 mg / mL, add 55.4 μL of water to 17.5 μL of 25 mg / mL stock solution. Add 16 μL of the glycoprotein solution (6 mg / mL) to the tube. Then add 4 μL of Rapid PNGase F buffer (5X) to each tube. Turn the heater on the device to 65°C and mix for 10 minutes. After 10 minutes, turn off the mixing mode and the heater, and allow the sample to cool for 5 minutes. Once the sample has cooled, add 2 μL of Rapid PNGase F enzyme to each vial and turn the mixing back on at 50°C for 15 minutes.

[0135] Labeling of released glycans

[0136] During the deglycosylation step, a fresh 2-AB labeling reagent solution is prepared by measuring 5 mg of 2-AB and transferring it to a 0.5 mL tube. Then, 130 μL of DMSO and 70 μL of acetic acid are added to the tube. The amount of labeling reagent prepared during this step will depend on the number of samples processed at one time. If less labeling reagent is required, smaller amounts of 2-AB, acetic acid, and DMSO are used while maintaining a constant ratio (i.e., 2.5 mg 2-AB with 65 μL DMSO and 35 μL acetic acid).

[0137] Next, prepare 5 μL of a 1M sodium cyanoborohydride THF solution. Add 20 μL of the labeling reagent and 5 μL of the 1M sodium cyanoborohydride THF solution to each sample tube and mix at 65°C for 60 minutes. Protect the samples from light during this step, as the 2-AB labeling dye is photosensitive (the electromagnetic mixer can be covered with aluminum foil during this step). After the labeling reaction is complete, add 150 μL of acetonitrile to each tube and mix. Adding acetonitrile to the tubes causes the labeled glycans in the solution to attach to the surface of the magnetic particles through hydrophilic interactions.

[0138] Cleaning and elution

[0139] A static magnetic field is applied to pull down the magnetic particles in the solution and remove ACN and dye solutions. Add 100 μL of fresh acetonitrile and mix for 1 minute. Then remove the supernatant. Repeat this step three times. To elute the attached glycans, add 50 μL of H2O to the tube to elute and dissociate the labeled glycans from the surface of the magnetic particles. Mix for 3 minutes, then remove the supernatant and transfer it to a separate tube (sample vial). Repeat this elution step twice. The final sample volume should be 2 × 50 μL = 100 μL. Then prepare the sample for HPLC analysis. Inject the glycan sample into LC for UHPLC characterization using the following conditions:

[0140] Sample: N-glycan analysis of aflibercept

[0141] Column: ACQUITY UPLC Glycan BEHamide column, 130 Å, 1.7 μm, 2.1 mm x 150 mm

[0142] Mobile phase: Buffer A, 50 mM ammonium formate aqueous solution, pH 4.4; Buffer B, 100% acetonitrile.

[0143] Flow rate: 0.4 mL / min

[0144] Temperature: 60℃

[0145] FLD detector: excitation 360 nm, emission 428 nm

[0146] Injection: 20 μL

[0147] Gradient of the mobile phase in the column:

[0148]

[0149] Figure 3 The chromatogram shown illustrates the results of the above-described N-glycan sample preparation workflow of aflibercept according to Example 1, which uses magnetic particles that have been surface-functionalized with carboxyl groups.

[0150] The following numbered entries define further instances and features of this disclosure:

[0151] 1. A method for collecting glycans from a glycosylated substrate, the method comprising:

[0152] a) Add an enzymatic reagent to a sample containing a glycosylated substrate and release the polysaccharide from the glycosylated substrate;

[0153] b) Labeled released glycans;

[0154] c) Attaching the labeled polysaccharide to the magnetic particles;

[0155] d) Dissociate the labeled polysaccharide from the magnetic particles; and

[0156] A magnetic field is applied during each of steps a) to d).

[0157] 2. The method according to item 1 further includes the step of denaturing the glycosylated substrate before adding the enzymatic reagent.

[0158] 3. The method according to item 1 further includes the step of collecting the labeled glycan after step d).

[0159] 4. The method according to item 1, wherein steps a) through d) occur in the same sample container.

[0160] 5. The method according to item 1, wherein the magnetic particles are present in the sample of steps a) to d).

[0161] 6. The method according to item 1, wherein the magnetic field is an electromagnetic field.

[0162] 7. The method according to item 1, wherein steps a) to d) are performed in a device capable of generating an electromagnetic field.

[0163] 8. The method according to item 7, wherein the device comprises a plurality of electromagnetic structures.

[0164] 9. The method according to any one of items 1-8, wherein the magnetic field includes a rotating magnetic field or a static magnetic field.

[0165] 10. The method according to item 9, wherein the rotating magnetic field is applied at intervals during steps a) to d) to mix the sample.

[0166] 11. The method according to entry 9, wherein a static magnetic field is applied at least during steps a), b) and the step of collecting the labeled glycan.

[0167] 12. The method according to any one of items 1-11, wherein the glycosylated substrate comprises a glycoconjugate, a glycoprotein, a glycolipid, a glycopeptide, a peptidoglycan, a glycoside, an antibody, an antibody-drug conjugate, and / or a lipopolysaccharide.

[0168] 13. The method according to any one of items 1-12, wherein releasing the glycan from the glycosylated substrate in step a) comprises releasing O-glycan or N-glycan.

[0169] 14. The method according to any one of items 1-13, wherein the enzymatic reagent comprises peptidyl-N-glycosidase F (PNGase F), endoglycosidase H (EndoH), endo-α-N-acetylgalactosaminease, α-mannosidase, or O-glycosidase, or a combination thereof.

[0170] 15. The method according to any one of items 1-14, wherein step b) comprises adding a labeling reagent selected from 2-o-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 1-aminopyrene-3,6,8-trisulfonic acid (APTS), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), or combinations thereof.

[0171] 16. The method according to item 15, wherein the labeling reagent is 2-aminobenzamide (2-AB).

[0172] 17. The method according to any one of entries 1-16, wherein step c) comprises adding acetonitrile (ACN) to link the labeled polysaccharide to the magnetic particles.

[0173] 18. The method according to item 2, wherein the step of denaturing the glycosylated substrate is carried out at a temperature ranging from room temperature to 100°C for a duration ranging from 1 minute to 24 hours.

[0174] 19. The method according to item 18, wherein the temperature is about 55°C to 75°C and the duration is about 5 to 15 minutes.

[0175] 20. The method according to any one of items 1-19, wherein steps a) and b) are each performed in a temperature range of room temperature to 100°C for a duration ranging from 1 minute to 24 hours.

[0176] 21. The method according to item 20, wherein the temperature during step a) is about 40°C-60°C and the duration is about 5-25 minutes.

[0177] 22. The method according to item 20, wherein the temperature during step b) is about 55°C-75°C and the duration is about 50-70 minutes.

[0178] 23. The method according to any one of items 1-22, wherein step d) and the step of collecting the labeled glycan are performed at room temperature for a combined duration of about 5-25 minutes.

[0179] 24. The method according to any one of items 1-23, wherein the magnetic particles are surface functionalized with carboxyl groups.

[0180] 25. The method according to any one of items 1-24, wherein the magnetic particles comprise a polymer surface coating.

[0181] 26. The method according to any one of items 1-25, wherein the magnetic particles are particles having a magnetic strength of about 20 emu / g to 250 emu / g.

[0182] 27. The method according to any one of items 1-26, wherein the magnetic particles are ferrimagnetic particles comprising ferrite compounds.

[0183] 28. The method according to any one of items 1-27, wherein step c) comprises adding acetonitrile buffer to facilitate the attachment of released glycans to functional groups on the surface of magnetic particles.

[0184] 29. The method according to any one of entries 1 to 28, wherein step d) comprises adding acetonitrile buffer to promote the dissociation of the labeled glycan from the surface of the magnetic particles.

[0185] 30. The method according to item 15, wherein the labeling reagent is 2-o-aminobenzoic acid (2-AA).

[0186] 31. The method according to any one of items 1 to 30, further comprising analyzing the collected labeled glycans by capillary electrophoresis, reversed-phase liquid chromatography, hydrophilic interaction chromatography (HILIC), mass spectrometry (MS), or nuclear magnetic resonance (NMR).

[0187] 32. A system for collecting glycans from a glycosylated substrate, the system comprising:

[0188] - A device capable of generating a magnetic field;

[0189] - Multiple magnetic particles;

[0190] - At least one enzyme-catalyzing reagent; and

[0191] - At least one labeling reagent.

[0192] 33. The system described in item 32, wherein the magnetic field is an electromagnetic field.

[0193] 34. The system according to item 32, wherein the apparatus comprises a plurality of electromagnetic structures.

[0194] 35. The system according to any one of items 33-34, wherein the electromagnetic field comprises a rotating field and / or a static field.

[0195] 36. The system according to item 35, wherein a rotating magnetic field generated by the device actuates the movement of magnetic particles to mix a sample placed within the device.

[0196] 37. The system according to item 35, wherein the static magnetic field generated by actuation is used to pull magnetic particles down from the sample.

[0197] 38. The system according to item 32 further includes at least one agent for attaching and / or dissociating the polysaccharide with a plurality of magnetic particles.

[0198] 39. The system according to any one of items 32-38 further includes a denaturing agent.

[0199] 40. The system according to any one of items 32-39, wherein at least one enzymatic reagent comprises peptidyl-N-glycosidase F (PNGase F), endoglycosidase H (EndoH), endo-α-N-acetylgalactosaminease, α-mannosidase, or O-glycosidase, or a combination thereof.

[0200] 41. The system according to any one of items 32-40, wherein at least one labeling reagent comprises 2-o-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 1-aminopyrene-3,6,8-trisulfonic acid (APTS), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), or a combination thereof.

[0201] 42. The system according to any one of items 32-41, wherein a plurality of magnetic particles are surface functionalized with carboxyl groups.

[0202] 43. A kit for recovering glycans from glycosylated substrates, the kit comprising:

[0203] - Magnetic particles;

[0204] - At least one enzyme-catalyzing reagent;

[0205] - At least one labeling reagent; and

[0206] - Optionally, at least one agent for attaching and / or dissociating the polysaccharide from the magnetic particles.

[0207] 44. The kit according to item 43, wherein the magnetic particles are particles having a magnetic strength of about 20 emu / g to 250 emu / g.

[0208] 45. The kit according to any one of items 42-43, wherein the magnetic particles are ferrimagnetic particles comprising ferrite.

[0209] 46. ​​The kit according to any one of items 43-45, wherein the magnetic particles have an average diameter in the range of 50 nm to 500 nm.

[0210] 47. The kit according to any one of items 43-46, wherein the magnetic particles are surface functionalized with carboxyl groups.

[0211] 48. The kit according to any one of items 43-47, wherein at least one enzymatic reagent comprises peptidyl-N-glycosidase F (PNGase F), endoglycosidase H (EndoH), endo-α-N-acetylgalactosaminease, α-mannosidase, or O-glycosidase, or a combination thereof.

[0212] 49. The kit according to any one of items 43-48, wherein at least one labeling reagent comprises 2-o-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 1-aminopyrene-3,6,8-trisulfonic acid (APTS), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), or a combination thereof.

[0213] 50. The kit according to any one of entries 43 to 49, wherein at least one reagent for attaching and / or dissociating the polysaccharide to the magnetic particles comprises acetonitrile.

[0214] It will be understood that the various features and functions disclosed above, as well as other features and functions or alternatives thereof, can be expected to be combined into many other different systems or applications. It will also be understood that various substitutions, modifications, variations, or improvements that are not currently foreseen or anticipated can subsequently be made by those skilled in the art, and these substitutions, variations, and improvements are also intended to be covered by the appended claims.

Claims

1. A method for harvesting glycans from a glycosylated substrate, the method comprising: a) adding an enzymatic reagent to a sample containing a glycosylated substrate and releasing glycans from the glycosylated substrate; b) labeling the released glycans; c) attaching the labeled glycans to magnetic particles; d) dissociating the labeled glycans from the magnetic particles; and wherein a magnetic field is applied during each of steps a) to d).

2. The method of claim 1, further comprising a step of denaturing the glycosylated substrate prior to adding the enzymatic reagent.

3. The method of claim 1, further comprising a step of collecting the labeled glycans after step d).

4. The method of claim 1, wherein steps a) to d) occur in the same sample container.

5. The method of claim 1, wherein the magnetic particles are present in the sample during steps a) to d).

6. The method of claim 1, wherein the magnetic field is an electromagnetic field.

7. The method of claim 1, wherein steps a) to d) are performed in a device capable of generating an electromagnetic field.

8. The method of claim 7, wherein the device comprises a plurality of electromagnetic structures.

9. The method of any one of claims 1-8, wherein the magnetic field comprises a rotating magnetic field or a static magnetic field.

10. The method of claim 9, wherein a rotating magnetic field is applied at intervals during steps a) to d) to mix the sample.

11. The method of claim 9, wherein a static magnetic field is applied at least during steps a), b), and the step of collecting the labeled glycans.

12. The method of claim 1, wherein releasing glycans from the glycosylated substrate in step a) comprises releasing O-glycans or N-glycans.

13. The method of claim 1, wherein step b) comprises adding a labeling reagent selected from 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 1-aminopyrene-3,6,8- trisulfonic acid (APTS), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), or a combination thereof.

14. A system for harvesting glycans from a glycosylated substrate, the system comprising: - a device capable of generating a magnetic field; - a plurality of magnetic particles; - at least one enzymatic reagent; and - at least one labeling reagent.

15. The system of claim 14, wherein the magnetic field is an electromagnetic field.

16. The system of any one of claim 15, wherein the electromagnetic field comprises a rotating field and / or a static field.

17. The system of claim 14, wherein the device comprises a plurality of electromagnetic structures.

18. The system of claim 14, further comprising at least one reagent for attaching and / or dissociating glycans from the plurality of magnetic particles.

19. The system of claim 14, further comprising a denaturation reagent.

20. The system of claim 14, wherein the plurality of magnetic particles are surface functionalized with carboxyl groups. ​

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