Liquid chromatography calibration method for fast labeling of n-glycans

Abstract

The present invention provides methods for preparing a fast-labeled dextran ladder and other calibrants for liquid chromatography. The methods include a two-step process comprising a reductive amination step that provides a reducing glycan and reacts it with a compound having a primary amine to produce an intermediate compound. The intermediate compound is then fast-labeled with a fast-labeling reagent to produce the fast-labeled dextran ladder.

Description

[0001] This application is a divisional application of application No. 202010662332.1, filed on November 12, 2015; application No. 202010662332.1 is a divisional application of the following application: filed on November 12, 2015, application No. 201580072776.7, entitled "Liquid Chromatography Calibration Method for Rapidly Labeled N-Glycans".

[0002] Cross-references to related applications

[0003] none. Background Technology

[0004] Hydrophilic interaction chromatography (“HILIC”) coupled with fluorescence detection on a UPLC platform can be used to separate complex N-linked glycan groups because it can separate neutral and charged glycans in a single chromatographic run. Elucidating the structures of complex N-linked glycans can be a significant analytical challenge due to the heterogeneity, isomerization, and end-group isomerism of glycans. A dextran ladder can be used to calibrate hydrophilic interaction chromatography (“HILIC”) separation of glycans and convert peak retention times into glucose unit (“GU”) values. Each individual glycan structure has a GU value directly related to its bonds and its constituent monosaccharides. Because each component of a glycan structure contributes to the GU value in a specific way for a given glycan, GU values ​​can be used to predict the structure.

[0005] Therefore, by referencing separation calibrators, such as those based on a dextran ladder, the elution time of a glycan can be expressed in glucose units. The dextran ladder can be used to calibrate liquid chromatography (“LC”) operations, preventing variations due to different time periods or systems. GU values ​​can be calculated by fitting a fifth-order polynomial distribution curve or a cubic spline-fitted curve to the retention time of the dextran ladder. Using this curve, GU values ​​can then be assigned based on the retention times observed in the test sample. GU values ​​for N-glycans are reproducible as long as the inter-column standard deviation is less than 0.3. This allows for direct comparison with database values ​​collected from a range of instruments over a period of time. With GU values, it is possible to query a database of glycans stored in GU values, thereby helping to elucidate the potential glycan structures present within a glycan group.

[0006] Furthermore, even when comparing different labeling methods, HILIC separation reveals that it breaks down labeled N-glycans into very similar characteristic spectra. Generating a dextran ladder suitable for rapidly labeled glycans using rapid labeling reagents is not a straightforward task. Unlike N-glycosylamines released via enzymatic deglycosylation of N-glycosylated proteins, dextran is a sugar with a reducing aldehyde terminus. Therefore, unlike N-glycosylamines, dextran cannot be modified with rapid labeling reagents targeting amine nucleophiles. Because dextran does not contain suitable nucleophiles, it cannot be rapidly labeled. Therefore, it is necessary to calibrate the liquid chromatography process using standards with the same optical and / or other physicochemical properties as the rapidly labeled N-glycan. Summary of the Invention

[0007] This document provides a method for preparing rapidly labeled dextran ladders and other calibrators that can be used in liquid chromatography. The method includes the following steps: providing a reducing glycan having an aldehyde group, and reacting the reducing glycan with a compound having a primary amine to generate an intermediate compound. The intermediate compound is rapidly labeled with a rapid-labeling reagent to generate a rapidly labeled dextran ladder, which has substantially the same optical properties as a rapidly labeled N-glycan generated by rapidly labeling an N-glycan with the rapid-labeling reagent. Calibrators having the following structural formulas are also provided:

[0008]

[0009] Where R represents a glucose unit or a monosaccharide unit. The method presented in this article is applicable to the rapid labeling or double labeling of compounds containing aldehyde groups. Attached Figure Description

[0010] Figure 1 An implementation scheme for a workflow for preparing rapidly labeled N-glycans is shown.

[0011] Figure 2 The fluorescence emission spectrum of rapidly labeled N-glycans labeled with a rapid labeling reagent is shown, with an optimized excitation wavelength of 265 nm and an optimized emission wavelength of 425 nm.

[0012] Figure 3 The fluorescence emission spectra of analogs of the rapidly labeled reagents via reduction of the amination-labeled dextran ladder are shown, with a shift in fluorescence characteristics, having an optimized excitation wavelength of 370 nm and an optimized emission wavelength of 480 nm.

[0013] Figure 4A and Figure 4B The fluorescence properties of rapidly labeled dextran ladders produced using the methods provided herein are shown.

[0014] Figure 5A , Figure 5B and Figure 5C A representative HILIC fluorescence chromatogram of the rapidly labeled ethanolamine dextran ladder produced by the method provided herein is shown.

[0015] Figure 6A , Figure 6B and Figure 6C The corresponding base peak ion (“BPI”) chromatograms of each batch of rapidly labeled dextran ladder in Example 3 are shown.

[0016] Figure 7A and Figure 7B The following is a fluorescence chromatogram obtained by comparing rapidly labeled propylaminoglucan with rapidly labeled ethanolamine glucan using a glycan BEH amide 2.1×50 mm column. Detailed Implementation

[0017] In biopharmaceutical preparation, the glycosylation profile of biological samples is typically assessed by analyzing the released glycans. However, these sample preparation techniques are often time-consuming or can result in samples that are insensitive to mass spectrometry (“MS”) analysis. Enzymatic release and rapid labeling of N-glycans address many of these limitations, providing higher throughput for N-glycan sample preparation and improving the sensitivity of glycan detection. For example, as described herein, glycoproteins can now be deglycosylated to N-glycans in as little as 10 minutes via enzymatic release and rapid labeling of N-glycans, which are then rapidly reacted with a rapid labeling reagent. The resulting labeled glycans are then extracted from the labeled reaction byproducts using a SPE method for convenient sample analysis.

[0018] The N-glycan characterization profile of biopharmaceuticals is a crucial quality attribute because it serves as a measure of efficacy, safety, and preparation conditions. Therefore, glycan analysis methods for clinical and commercial biotherapeutic formulations require high sensitivity. Furthermore, rapid turnaround times and high throughput facilitate product development during analysis.

[0019] Most analytical strategies for evaluating N-glycans derived from glycoproteins involve deglycosylation via PNGase F and labeling the resulting N-glycan with a chemical moiety that imparts its detectable properties. In one method described herein, the labeled glycan is separated by hydrophilic interaction chromatography (“HILIC”) and detected by fluorescence (“FLR”) and mass spectrometry (“MS”).

[0020] In addition, we have previously developed a sample preparation solution that enables FLR and MS to be sensitive to glycan detection while increasing the throughput of N-glycan sample preparation. We have developed a rapid labeling reagent that can be synthesized and reacts rapidly with N-glycans when they are released from glycoproteins. (Brousmiche et al., U.S. Patent Application 2014 / 0350263, filed August 13, 2014,

[0008] -

[0022] ,

[0047] -

[0050] ,

[0053] -

[0182] ,

[0191] ,

[0228] , and

[0230] -

[0316] , incorporated herein by reference). By using this rapid labeling reagent, N-glycans can be labeled within a 5-minute reaction time. The rapid labeling reagents used in this paper contain a rapid labeling group for N-hydroxysuccinimide (NHS) carbamate, a highly efficient quinoline fluorophore, and a strong basic tertiary amine for enhancing ionization, as illustrated in Table 1 below.

[0021] Table 1

[0022] Exemplary rapid labeling reagent

[0023]

[0024] In the method of this invention, reducing sugars are labeled (tagged) via reductive amination, and the resulting secondary amine-containing sugars are rapidly labeled using a rapid labeling reagent. The method provided herein can be used to label or double-label any aldehyde-containing compound.

[0025] To further facilitate the preparation of N-glycans, the use of rapid labeling reagents can be directly combined with the deglycosylation process of PNGase F, which also involves surfactants and HILICμ elution solid-phase extraction (“SPE”) cleanup to provide quantitative recovery of the released and labeled glycans. This eliminates the need for solvent drying before liquid chromatography (“LC”) analysis of the sample, thus providing additional benefits.

[0026] Example 1

[0027] The released N-glycans were rapidly prepared using a rapid labeling reagent for HILIC analysis.

[0028] In this embodiment, we describe a method for preparing tagged (also referred to herein as “labeled”) N-glycans, from glycoprotein to analytically ready sample, with complete deglycosylation within 30 minutes. Our method is an improved scheme that can be implemented using a protocol called GLYCOWORKS. TM RAPIFLUOR-MS TMThe kit for N-glyans facilitates the implementation of this assay. Sensitivity to labeled N-glyans is increased by at least 2 to 100 times compared to previous fluorescence and MS detection, and accurate characteristic spectral analysis is provided based on robust solid-phase extraction (“SPE”) for neutralizing tetrasialylated N-glyans. (Lauber, M. et al., Rapid Preparation of Released N-Glyans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection, Anal. Chem. 2015, 97, 5401-5409), is incorporated herein by reference.

[0029] Based on rational design considerations to provide rapid labeling kinetics, high fluorescence quantum yield, and significantly improved MS detectability, a rapid tagging reagent was synthesized to facilitate our N-glycan analysis. N-glycan sample preparation depends on the reductive amination of aldehyde-terminated sugars. In this process, the glycan is reductively amination under anhydrous conditions to minimize desialylation. Sample preparation is thus transformed from aqueous to anhydrous conditions. Furthermore, by utilizing the rapid tagging reagent, reductive amination can be eliminated via an aqueous rapid tagging reaction.

[0030] If a glycan is not labeled by a rapid labeling reaction, reductive amination can be used to label the glycan at its reduced end. In this reaction, a labeling agent containing a primary amine undergoes a condensation reaction with the aldehyde group of the glycan to give an imine or Schiff base, which is reduced by a reducing agent to produce a secondary amine. This reaction is typically carried out in dimethyl sulfoxide containing acetic acid, but alternative methods using tetrahydrofuran and methanol have been described. Examples of amines (also referred to herein as primary amines or compounds having a primary amine) include ethanolamine, propylamine, aminobenzamide, peptides with a free amino terminus (as shown in Example 5 herein), N,N-dimethylethylenediamine, aminoanthracene, and aminobiotin. The advantage of this labeling method is that each glycan is stoichiometrically linked to a label, allowing for direct quantification based on fluorescence or UV absorbance intensity.

[0031]

[0032] As an alternative, use the GLYCOWORKS described in this article. TM RAPIFLUOR-MS TMThe N-glycan kit enables rapid labeling of glycans in the laboratory, and is designed to eliminate bottlenecks in all aspects of N-glycan sample preparation. See below. Figure 1 As shown, the optimized N-glycan sample preparation workflow requires three steps: (1) deglycosylation, releasing the glycan from the glycoprotein; (2) labeling, endowing the glycan with a detectable chemical entity; and (3) a cleanup step, eliminating potential interferences in the sample. These glycans are then rapidly reacted with one or more rapid labeling reagents, labeled with a tag consisting of a highly efficient fluorophore and a strongly basic tertiary amine, thereby enhancing the sensitivity for fluorescence and MS detection. A structural description of the rapidly labeled glycan is shown below. Notably, the rapidly labeled glycan has a very unique bond moiety, distinct from the secondary amine bond from the reductive amination reaction. The rapidly labeled N-glycan will contain a neutral (non-acidic) urea bond. This structure can influence the physicochemical properties and / or characteristics of rapidly labeled glycans, including their chromatographic retention and fluorescence properties, as well as other physicochemical properties such as isoelectric point (“pI”), acidity, basicity, hydrophobicity, hydrophilicity, ability to chelate metals, UV absorbance, fluorescence, absorbance in the visible spectrum, colorimetric changes, molecular size, affinity for binding partners (i.e., between biotinylated residues and avidin or streptavidin, between epitopes and paratopes), reduction / oxidation potential, crosslinking tendency, cleavageability (chemical and thermal), and different lengths of polymeric substituents (i.e., 4-repeating polyethylene glycol (PEG), 40-repeating PEG).

[0033]

[0034] As used in this article, the trademark GLYCOWORKS TM and RAPIFLUOR-MS TM The trademark GLYCOWORKS belongs to its applicant, Waters Technologies Corporation. TM This is used in conjunction with a sample preparation kit for laboratory use, which includes biological standards, a sample preparation device, a disposable blade holder, and chemical reagents for preparing chromatographic and mass spectrometric samples. Similarly, the trademark RAPIFLUOR-MS... TM It can be used in conjunction with chemical reagents for preparing chromatographic and mass spectrometric samples, as well as with sample preparation kits for laboratory use, which include biological standards, sample preparation devices, and disposable blade holders.

[0035] Therefore, GLYCOWORKS TM RAPIFLUOR-MS TMThe N-Glycan Kit is designed for the rapid enzymatic release and labeling of N-glycans. This protocol has been validated with monoclonal antibodies and has been tested on a wide range of other N-linked glycoproteins. This sample preparation kit utilizes optimized deglycosylation conditions and reagents for rapid release. The kit may include the rapid labeling reagents described in U.S. Patent Application 2014 / 0350263, filed August 13, 2014,

[0008] -

[0022] ,

[0047] -

[0050] ,

[0053] -

[0182] ,

[0191] ,

[0228] , and

[0230] -

[0316] , which is incorporated herein by reference, and the kit is designed to provide the dual benefits of sensitive fluorescence detection and appropriate signal intensity for quality assays.

[0036] As described in this article, the characterization and monitoring of protein N-glycosylation are of great significance for disease state detection and biopharmaceutical preparation. Glycosylation characterization profiles are typically assessed primarily by analyzing released glycans, but the techniques commonly used for sample preparation are notoriously time-consuming or result in samples being insensitive to MS analysis. With the development of the GlycoWorks Rapid Fluor-MS N-Glycan Kit described in this article, unprecedented sensitivity for glycan detection has been achieved, while simultaneously increasing the throughput of N-glycan sample preparation, thus overcoming these shortcomings.

[0037] Equally important as the efficiency and sensitivity gains offered by this new sample preparation method and its associated methods is its robustness and its ability to produce results consistent with historical N-glycan characterization analyses. Furthermore, the generated glycan data can be used to query glycan databases, but it first needs to be converted into standardized values ​​known as glucose unit (“GU”) values. Thus, another benefit of implementing a calibration benchmark is the ability to convert existing glycan data into a format suitable for searching potential glycan databases. The converted data can be used in the discovery process for samples investigating unknown glycan compositions. Once the glycans are converted into GU values, users can query online databases to gain deeper insights into potential glycan structures that may be present in the sample, potentially reducing the time required for typical characterization. In liquid chromatography, calibration is performed very frequently, sometimes so frequently that it is performed after each separation of a glycan mixture.

[0038] To detect fluorescently labeled (“FLR”) glycans, hydrophilic-interacting liquid chromatography (“HILIC”) can be coupled with fluorescence detection. For separation processes and compared to certain conventional high-performance liquid chromatography (“HPLC”) methods, ethylene-bridged glycan columns (hereinafter referred to as “BEH glycan columns” or “BEH columns”) operating in HILIC mode offer improved peak resolution due to their ability to separate both neutral and acidic glycans. BEH glycan columns enable and produce reproducible glycan separation data and optimize methods in less time.

[0039] BEH columns utilize mixed silica BEH and bridging technology particles functionalized with stable amide-containing substances. BEH technology produces stationary phases with a variety of particle sizes ranging from 1.7 to 5 μm in diameter, bridging HPLC and ultra-high performance liquid chromatography (“UPLC”) platforms. BEH particles provide peak shape and efficiency for basic analytes, a reasonable range of chromatographic selectivity, and improved chemical stability at mobile phase limits, particularly at elevated pH. The resolving power of these columns is partly due to the porous particles with optimal amide ligand concentrations for relevant applications. The column can be optimized for use in UPLC or HPLC systems with fluorescence (“FLR”) detection for the separation of released and labeled N-linked glycans from various biotherapeutic agents, achieving HILIC-based separation of both neutral and charged labeled glycans.

[0040] To fully utilize BEH glycan columns, or simply for any HILIC-based characterization of glycans, a dextran calibration ladder (sometimes referred to below as a "dextran ladder" or "dextran ladder standard") can be used. The glycan characterization profile obtained from the HILIC / FLR system can be calibrated against the dextran ladder, assigning glucose unit (GU) values. For example, one known such ladder, the 2AB-labeled dextran calibration ladder, differs from other commercially available products. This glucose homopolymer has a higher average molecular weight (approximately 4,500 Daltons), thus offering twice the range of "usable" GU values ​​compared to other dextran ladder standards, with observed GU values ​​ranging from 2 to 30. Consequently, the retention time allocation for high molecular weight glycans is improved. The purity and structural integrity of the dextran ladder can be assessed using HILIC and mass spectrometry ("MS").

[0041] The elution time of glycans can be referenced using a dextran ladder, denoted by glucose units (“GU”). Each individual glycan structure has a GU value directly related to its bonds and constituent monosaccharides. Since each monosaccharide in a particular bond contributes to the GU value in a specific way for a given glycan, the GU value can be used to predict the structure. Therefore, the dextran ladder provided in this paper can be used to calibrate LC operations, preventing variations due to different time periods or systems. GU values ​​can be calculated by fitting a fifth-order polynomial distribution curve or a cubic spline-fitted curve to the dextran ladder, and then using this curve to allocate GU values ​​according to retention time. The GU values ​​of N-glycans exhibit high reproducibility as long as the inter-column standard deviation is less than 0.3. This allows for direct comparison with database values ​​collected from a range of instruments over a period of time.

[0042] With GU values, it is possible to query a database of glycans stored in GU values, thereby helping to elucidate the potential glycan structures present in glycan groups. The dextran ladder also provides quality-controlled standards that can be used to calibrate chromatograms obtained in different laboratories using different instruments. Glycan retention times acquired using a HILIC-FLR instrument will vary depending on the instrument and laboratory. By converting retention times to GU values, the resulting data can be used to compare information between different locations, both in-situ and off-site. While the use of GU values ​​is only one example here, other chromatographic methods can benefit from using dextran calibrators prepared by the methods of this invention, including but not limited to reversed-phase chromatography and combined iterative reversed-phase and HILIC methods.

[0043] Rapid labeling of N-glycans simplifies the preparation of glycan samples for analysis. However, generating a dextran ladder suitable for rapid labeling of glycans with rapid labeling reagents is not a straightforward task. This article presents a two-step method for labeling reducing glycans that allows for tuning chromatographic responses and chemical properties such as fluorescence and MS activity, as well as tuning multiple tags with different detection properties. Each step differs in the properties linked to the reducing glycan. The term “reducing glycan” refers to reducing sugars, reducing polysaccharides (different sugar units of heteropolysaccharides, or different monosaccharides, or homopolysaccharides), and includes any aldehyde-terminated sugar such as chitosan, chitobiose, galactose-β-(1-3)-GalNAc-glycan, mannose-di-(N-acetyl-D-glucosamine), and maltose. Reducing glycans or reducing sugars are any sugars capable of acting as reducing agents because they have a free aldehyde group. Therefore, the method presented herein can be used for labeling or double-labeling any aldehyde-containing (sometimes also called aldehyde group) compound, including O-glycans.

[0044] As described below, the first step utilizes a reductive amination method, which involves reacting a reducing polysaccharide (aldehyde-terminated sugar) with a compound having a primary amine to produce an intermediate compound such as ethanolamine, propylamine, or other compounds having an aldehyde that has been converted to a secondary amine, or being converted into a reducing polysaccharide (reducing sugar) with a secondary amine terminator. Primary amines (also referred to herein as "compounds having a primary amine" or amines) that exhibit the desired properties to produce an intermediate compound or a compound whose aldehyde has been converted to a secondary amine include ethanolamine, propylamine, aminobenzamide, peptides with a free amino terminus (as shown in Example 5 herein), N,N-dimethylethylenediamine / aminoanthracene, and aminobiotin. The second step involves reaction with a rapid labeling reagent that can impart different properties. In other words, the properties of the intermediate compound differ from those of the rapid labeling compound (i.e., the rapid labeling polysaccharide).

[0045]

[0046] R-NH2 is an ethanolamine or a compound of a similar type that is a primary amine.

[0047] This method was initially developed to meet the need for rapid labeling reagents. Figure 2 The optical properties of the tagged N-linked glycans are compared with those of the precursors shown below, which are obtained by reductive amination of the tagged dextran standard (also referred to herein as reducing sugar). Figure 3 The properties match.

[0048]

[0049] It has been found that the fluorescence properties (i.e., excitation and emission peaks) of these two types of substances are unfavorably different. Therefore, this dextran ladder is unsuitable as a calibrator for the analysis of rapidly labeled glycans in liquid chromatography.

[0050] The method presented herein utilizes a rapid tagging / amine-reactive labeling pathway for tagging reduced glycans (or any aldehyde-terminated glycans), providing detection of N-glycans by their fluorescence and / or MS activity. Conventional tagging of reduced glycans involves reductive amination using specific tagging reagents (a lengthy process, i.e., reaction time of 1 to 4 hours, and possibly up to 8 hours, to achieve high yields). However, the method of this invention utilizes reductive amination, immediately followed by a second step (rapid, approximately 5 minutes) to introduce a tag (or label) with fluorescence / MS activity, yielding a sugar molecule having the following structural formula I. As provided in this embodiment, the terminal monosaccharide residue is shown as an N-acetylated glucosamine (GlcNAc) linked to the remaining sugar structure via its 4-OH position. However, the terminal monosaccharide residue does not necessarily have to be GlcNAc; the glucan ladder can be capped with a glucose monosaccharide linked to the structure via its 6-OH position. Alternatively, the terminal monosaccharide residue can be linked via its 4-OH position. The dextran ladder can be capped with any glucose monosaccharide exemplified by Formula II below, which is attached to the remaining sugar structure via its 6-OH position.

[0051]

[0052] Where R can be a monosaccharide unit or a glucose residue, R1 is -CH2CH2OH, and R2 is the portion of the fast-tagged reagent that incorporates the FLR-MS functional group into the structure, illustrated by the following structure:

[0053]

[0054] The method presented herein allows for the tagging of reduced glycans with fast-tag reagents that possess the same optical and MS properties as other molecules already labeled with the fast-tag reagent. In other words, reductive amination of a primary amine with a fast-tag reagent produces molecules with different absorption and emission properties than a contrasting molecule labeled with the fast-tag reagent (N-linked molecule). The linker can be different (amine vs. urea), resulting in altered chromophore properties. Optical properties are maintained by reductive amination with a compound containing a primary amine followed by tagging the amine with the fast-tag reagent.

[0055] Figure 4A The optical properties of the rapidly labeled dextran ladder produced by the method described herein are shown, wherein the rapidly labeling reagent is:

[0056]

[0057] Figure 4B The results show that the dextran ladder has substantially the same fluorescent properties as rapidly labeled glycans produced using the same rapid labeling reagent, exemplified below:

[0058]

[0059] The method of this invention allows for adjustment of the relative retention of glucose homopolymers (referred to herein as "dextran ladders") by changing their overall polarity. For example, different retention characteristics are produced by reductive amination with a) ethanolamine or b) propylamine followed by reaction with a rapid labeling reagent. Therefore, the chromatographic retention characteristics of the dextran ladder, among other things, can be adjusted as a calibration benchmark.

[0060] Therefore, the method of the present invention can produce dextran ladders with chemical properties highly similar to those of N-glycans labeled with a rapid labeling reagent.

[0061] As provided in the recommended steps listed in Example 3, one embodiment of such glucose homopolymer includes reductive amination with ethanolamine followed by labeling with a rapid labeling reagent to produce a rapidly labeled ethanolamine dextran ladder as shown below (embodiment of rapidly labeled dextran ladder):

[0062]

[0063] In another embodiment, ethanolamine in the steps listed in Example 3 can be replaced with propylamine. Figure 7B Compared to what is shown, when using Figure 7A When separated by the BEH Amide stationary phase, the resulting rapidly labeled propyl aminoglucan (shown below) exhibits altered chromatographic retention.

[0064]

[0065] Rapidly labeled propylaminoglucan, a rapidly labeled glucan ladder

[0066] Example 2

[0067] Preparation of rapidly labeled dextran ladder

[0068] Reductive amination and coupling process of dextran

[0069] Accurately weigh 100 mg of Dextran 5000 and place it in an 8 mL vial. Then, using a pipette, add 2800 μL of DMSO to the 8 mL vial and stir until the dextran dissolves. Record the actual weight.

[0070] Add 1200 μL of glacial acetic acid, 90.0 mg (91.0 mL) of redistilled ethanolamine (MW = 61.08, d = 1.012), and 128 mg of sodium cyanoborohydride sequentially to the 8 mL vial. Gently mix the slurry and incubate at 70 °C for 3 hours with magnetic stirring on a heating block. After 3 hours of incubation, remove the resulting reaction solution from the heating block and cool to below 40 °C. Transfer the reaction contents from the 8 mL vial to a balanced 50 mL centrifuge and add 40 mL of ACN solution. Measure and record the tare weight, and place the vial in a refrigerator for 30 minutes. Then, centrifuge the mixture at 4000 PRM for 5 minutes and decant the supernatant. Resuspend the resulting particles in 40 mL of ACN solution and vortex vigorously. Repeat the centrifugation, decanting, and resuspension steps for a total of three washes. A 30-minute settling time is not required. The particles were dried under a nitrogen stream for 20 minutes, and then incubated overnight under vacuum at room temperature. The particles were weighed to determine the final recovery. The final weight, tare weight, and recovered weight were recorded.

[0071] Rapid labeling process for producing ethanolamine-labeled dextran ladder

[0072] This procedure aims to perform a rapid labeling reaction at a molar volume exceeding that of the rapid labeling reagent by 100-200 times. Dissolve 4.0 ± 0.3 mg of ethanolamine dextran in 500 μL of 50 mM HEPES solution, pH 7.9 (obtained by titration of free acid with sodium hydroxide). Add 300 μL of anhydrous DMF. Dissolve 100 ± 0.5 mg of the rapid labeling reagent in this dextran solution. Allow the reaction to proceed at room temperature for 10 minutes. Periodically stir / shake the reaction mixture (every 20 to 30 seconds). After incubation at room temperature, dilute the reaction mixture with 7.6 mL of ACN. Ideally, the reaction mixture should be diluted just before loading into the SPE column.

[0073] HILIC SPE Cleanup

[0074] The mixture was washed with 6 mL of water and equilibrated with 6 mL of 85% ACN solution. The ACN-diluted reaction mixture was divided into two (2) 4.5 mL aliquots (approximate volumes) and loaded onto the column. It was washed three times with 6 mL of 1:9:90 formic acid / water / ACN. It was eluted with three (3) 4 mL aliquots of unadjusted 200 mM ammonium acetate and 5% ACN. The eluent was distributed in 600 μL volumes and dried by centrifugal vacuum evaporation.

[0075] The above process can produce up to approximately 80 standard samples per batch of labeled dextran prepared from ethanolamine dextran intermediates.

[0076] Example 3

[0077] Experimental conditions and representative data for rapidly labeled ethanolamine dextran (rapidly labeled dextran).

[0078] Experimental conditions for 1 to 3 batches of rapidly labeled ethanolamine dextran were described, and the fluorescence and MS properties of the labeled dextran ladder prepared as described in Example 2 were analyzed using rapid-labeled dextran liquid chromatography. The column was washed with 70% HPLC-grade acetonitrile (ACN) / 30% HPLC-grade water (v / v). The column was then equilibrated with the mobile phase conditions before the first injection. The HILIC UPLC / FLR / MS conditions used in the analysis are provided in Tables 2 and 3 below.

[0079] Table 2

[0080]

[0081] Table 3

[0082] Mass spectrometry analysis

[0083]

[0084] Figure 5A , Figure 5B and Figure 5C Fluorescence chromatograms of each of the three batches of the prepared (ethanolamine) dextran ladder are shown. Figure 6A , Figure 6B and Figure 6C Chromatograms of the corresponding base ion (“BPI”) for each batch of rapidly labeled dextran ladder are shown. The mass of the labeled glucose unit (GU) is shown in Table 4.

[0085] Table 4

[0086]

[0087] Example 4

[0088] Chromatographic retention was adjusted by reducing and amination with compounds containing primary amines.

[0089] A second rapidly labeled dextran was prepared by replacing the ethanolamine listed in Example 2 with propylamine. The chromatographic retention of the resulting rapidly labeled propylamino dextran was compared with that of the aforementioned rapidly labeled ethanolamine dextran under the experimental conditions outlined below:

[0090] The column was washed with 70% HPLC-grade acetonitrile (ACN) / 30% HPLC-grade water (v / v). The column was then equilibrated with the mobile phase conditions before the first injection. Table 5 provides the HILIC UPLC / FLR conditions used in the analysis.

[0091] Table 5

[0092]

[0093] Figure 7A and Figure 7B The following is a fluorescence chromatogram obtained by comparing rapidly labeled propylaminoglycan with rapidly labeled ethanolamine glycan when using a BEH Amide 2.1×50 mm column.

[0094] Predictive Example 5

[0095] Introducing multiple functions into reducing sugars

[0096] More broadly, the proposed method can impart certain chemical properties to reducing glycans and reducing sugars by introducing separate labeling components (or referred to herein as “tags” or “labels”), one through reductive amination and the other through rapid labeling.

[0097] In one embodiment of the method of the present invention, the double-labeled N-glycan can be purified from the sample matrix by streptavidin pull-down, followed by detection via a rapid tagging reagent to impart fluorescence and / or enhanced ionization efficiency. Such "double-labeled" sugars, as shown below, can simultaneously possess biotin labeling and highly fluorescent, MS-active labeling. In one embodiment shown below, reductive amination can be performed using an aminobiotin molecule, and the reductively amination sugar can be labeled via a rapid tagging reaction.

[0098]

[0099] In one implementation, if this method is applied to the reduction of aminobenzamide-modified polysaccharides and subsequent labeling with a rapid labeling reagent, it may be possible to achieve multi-wavelength detection of a single sugar substance using more than one fluorophore / chromophore.

[0100]

[0101] The method of this invention also allows for the use of epitope tags to label sugars / glycans, thereby enabling immunoaffinity enrichment or immuno-based detection. In one specific embodiment, the amination sugar can be reduced using a hemagglutinin (HA) epitope tag (a peptide with the sequence YPYDVPDYA) and then labeled with a rapid tagging reagent.

[0102]

Claims

1. A compound having structural formula I or II: Equation I Formula II, Where R is a glucose residue, and R1 is selected from –CH2CH2OH, –CH2CH2CH3. and And R2 is selected from 、 、 、 , ,and .

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