Rapid preparation of labeled glycosylamines and methods for analyzing glycosylated biomolecules that produce them

Abstract

To provide methods for producing derivatized glycosylamines in high yield without causing degradation of a biological sample in analyzing glycosylated biomolecules.SOLUTION: A deglycosylation mixture of biomolecules deglycosylated enzymatically or chemically is mixed with a reagent solution, which has a labeling reagent contained in a polar aprotic, non-nucleophilic organic solvent, so as to produce derivatized glycosylamine products. The derivatization reaction is carried out by selecting conditions of not depleting protein. The derivatized glycosylamines are separated from the reaction mixture and detected by chromatographic detection, fluorescence detection, mass spectrometry or ultraviolet detection and / or a combination thereof.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 072,747, filed Oct. 30, 2014, and U.S. Provisional Application No. 62 / 107,994, filed Jan. 26, 2015, which are hereby incorporated by reference in their entirety.

[0002] Statement Regarding Federally Sponsored Research or Development None

[0003] Names of Parties to a Joint Research Agreement None

Background Art

[0004] Methods for analyzing compounds derived from biological sources often include a derivatization step that introduces a phosphor to facilitate detection after separation by chromatography. Although there are various derivatization methods, the long processing time is a serious factor in effectively using the once-obtained analysis data. Reagents and related methods have been developed in recent years to reduce the lead time, especially for the analysis of compounds having an amine or amino group. However, the desired reaction selectivity between a primary amine and a hydroxyl group is often not obtained. The yield of the labeled compound is not optimized. In addition, "over-labeled" compounds frequently occur in prior art methods. Furthermore, the solubility of the labeled or tagged compound in a high organic solvent is low, which may interfere with downstream solid phase extraction methods ("SPE"), especially SPE based on hydrophilic interaction chromatography. Also, prior art analysis generally requires that the compound be separated from the biological source and / or be subjected to a washing step prior to derivatization, resulting in additional steps and slowing down the analysis procedure.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Accordingly, there is a need for a method of analyzing a compound, in which the derivatization step produces a tagged compound that is selectively labeled in high yield without causing degradation of the biological sample or over-labeling such that high resolution is possible during subsequent analysis of the labeled compound. **Means for Solving the Problems**

[0006] (Summary of the Invention) A method for analyzing a glycosylated biomolecule, comprising: (a) generating a deglycosylated mixture, wherein the glycosylated biomolecule is deglycosylated by a technique using a natural or synthetic enzyme or a chemical technique; (b) preparing a reagent solution containing a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; (c) mixing the deglycosylated mixture with the reagent solution in a reaction mixture at a volume ratio of about 2.5:about 1; and (d) detecting the derivatized glycosylamine. The reaction mixture contains a molar excess of the labeling reagent over the amount of modifiable amine in the range of about 10 to about 2000, and can include free glycosylamine, proteinaceous amine, and derivatized glycosylamine. These steps can be carried out without intentionally depleting the proteome. The derivatized glycosylamine may then be separated from the reaction mixture using on-line or off-line SPE and / or other separation methods, or may not be separated from the reaction mixture. Optionally, a quenching solution can be added to the reaction mixture such that the pH of the reaction mixture is shifted above 10. The yield of the derivatized glycosylamine can be in the amount of about 80 to about 100 mole percent of the reaction mixture. The derivatized glycosylamine is then separated from the reaction mixture and detected by chromatography detection, fluorescence detection, mass spectrometry ("MS") or ultraviolet ("UV") detection and / or combinations thereof. In some embodiments, the method can further include contacting the glycosylated biomolecule with an enzyme to generate a deglycosylated mixture, or can be deglycosylated by other enzymatic techniques or chemical techniques.

[0007] In another aspect, methods for rapid derivatization of glycosylamines are also described herein. In some embodiments, the methods for rapid derivatization of glycosylamines include: (1) providing a biological sample; (2) combining the biological sample with peptide N-glycosidase F to produce a deglycosylation mixture; (3) providing a reagent solution comprising a labeling reagent combined with anhydrous dimethylformamide (DMF); and (4) mixing the deglycosylation mixture with the reagent solution to produce a reaction mixture comprising the labeling reagent, liberated glycosylamines, proteinaceous amines, and derivatized glycosylamines. The reaction mixture can have a molar excess of labeling reagent over the modifiable amine in an amount of about 10 to about 1000. For some embodiments, other ranges of molar excess are also described herein.

[0008] In the described methods, the concentration of organic solvent in the reaction mixture can be about 0 to about 50 percent. In some embodiments, the range of organic solvent in the reaction mixture can be about 10 to about 40 percent. In some exemplary embodiments, the range of organic solvent in the reaction mixture can be about 20 to about 30 percent by volume. The deglycosylation mixture is mixed with the reagent solution in a volume ratio of about 9:1 to about 1:9. In exemplary embodiments, the deglycosylation mixture is mixed with the reagent solution in a volume ratio of about 2.5 to about 1, resulting in a reaction mixture having about 25 to about 30 percent reagent solution. In some embodiments, the amount of reagent solution in the reaction mixture is 28.6 percent. The reagent solution can be maintained at about ambient temperature or below ambient temperature to about 4°C. A buffer solution can be added to the deglycosylation mixture. The buffer solution can be sodium phosphate or HEPES. Alternatively, the biological sample can be deglycosylated in a buffer solution such as HEPES to facilitate subsequent derivatization reactions with fewer steps.

[0009] Optionally, a quenching solution can be added to the reaction mixture. The quenching solution can contain ethylenediamine. The ratio of ethylenediamine to water to acetonitrile can be about 5 to about 5 to about 90 by volume. Then, after the deglycosylation mixture is mixed with the reagent solution for about 2 minutes to about 10 minutes, the quenching solution can be added to the reaction mixture. The pH of the reaction mixture can shift above about 10.

[0010] Generally, to prepare a glycosylated biomolecule for analysis, this can be deglycosylated either synthetically or naturally. More specifically, to prepare a labeled N-glycan, a biological sample can have a glycosylated biomolecule such as a glycoprotein that is deglycosylated by peptide N-glycosidase F (PNGase F) to produce a deglycosylation mixture. The labeling reagent can be combined in anhydrous dimethylformamide (DMF) and / or other polar aprotic, non-nucleophilic organic solvents to yield a reagent solution. The deglycosylation mixture and the reagent solution are subsequently mixed at a volume ratio of about 2.5 to about 1 (2.5:1, v / v / v) to produce a reaction mixture, and the glycoamine can be rapidly labeled by the labeling reagent. The derivatized glycoamine can be subjected to chromatographic analysis, mass spectrometry, ultraviolet detection, and / or fluorescence detection.

[0011] The rapid labeling method of glycoamine described herein can include alternative forms with respect to separation methods and detection methods. Separation methods include, but are not limited to, hydrophilic interaction chromatography ("HILIC"), solid phase extraction ("SPE"), capillary electrophoresis, high pH anion exchange chromatography, or reverse phase liquid chromatography. Detection methods can include chromatographic detection such as high performance liquid chromatography ("HPLC"), ultra-high performance liquid chromatography (UHPLC), supercritical fluid chromatography, ultraviolet ("UV") detection, fluorescence detection, matrix-assisted laser desorption ionization mass spectrometry, electrospray ionization mass spectrometry, and / or pulsed amperometric detection.

Brief Description of the Drawings

[0012] [Figure 1] 1 is a flow diagram illustrating steps of the method described herein. [Figure 2] 1 is a chart showing the effect of temperature on the yield of amino-labeled groups and on the reaction selectivity between the amine and hydroxyl groups of bradykinin. [Figure 3A] 1 is a graph showing the effect of the organic solvent DMSO and the effect of the organic solvent concentration on the yield of amino-labeled groups and on the reaction selectivity between the amine and hydroxyl groups of bradykinin. [Figure 3B] 1 is a graph showing the effect of the organic solvent DMF and the effect of the organic solvent concentration on the yield of amino-labeled groups and on the reaction selectivity between the amine and hydroxyl groups of bradykinin. [Figure 4A] 1 is a graph showing the effect of buffer 200 mM sodium borate on the yield of amino-labeled groups. [Figure 4B] 1 is a graph showing the effect of buffer 200 mM sodium phosphate on the yield of amino-labeled groups. [Figure 4C] FIG. 1 shows the % of glycosylamines modified at the hydroxyl group when 200 mM sodium borate buffer and 200 mM sodium phosphate buffer are used in the reaction mixture. [Figure 5A] 1A and 1B are graphs showing the effect of buffer concentration and ionic strength on the yield of amino-labeled groups and hydroxyl groups and on the reaction selectivity between the amine and hydroxyl groups of bradykinin. 1C and 1D are graphs showing the effect of buffer concentration and ionic strength on the yield of amino-labeled groups of bradykinin. [Figure 5B] 1A and 1B are graphs showing the effect of buffer concentration and ionic strength on the yield of amino-labeled groups and hydroxyl groups and on the reaction selectivity between the amine and hydroxyl groups of bradykinin. 1C and 1D are graphs showing the effect of buffer concentration and ionic strength on the yield of hydroxyl groups of bradykinin. [Figure 6]It is a graph showing the influence of reaction time on the yield of the amino-labeled group and on the reaction selectivity between the amine group and the hydroxyl group of bradykinin. [Figure 7A] It is a chromatogram showing the influence of the molar excess of the tagging reagent on the yield of the amino-labeled group and on the level of the over-labeled glycan. [Figure 7B] It is a chromatogram showing the influence of the molar excess of the tagging reagent on the yield of the amino-labeled group and on the level of the over-labeled glycan. [Figure 7C] It is a chromatogram showing the influence of the molar excess of the tagging reagent on the yield of the amino-labeled group and on the level of the over-labeled glycan. [Figure 7D] It is a chromatogram showing the influence of the molar excess of the tagging reagent on the yield of the amino-labeled group and on the level of the over-labeled glycan. [Figure 7E] It is a chromatogram showing the influence of the molar excess of the tagging reagent on the yield of the amino-labeled group and on the level of the over-labeled glycan. These figures demonstrate how the molar excess of the reagent to the glycosylamine must be optimized in order to obtain high yields (above 80%) without the introduction of high (above 0.2 mole percent) levels of the "over-labeled" species. [Figure 8A] It is a chromatogram showing the results of the fluorescently labeled N-glycosylamine released from pooled human IgG analyzed by LC with fluorescence detection and a chromatogram showing the influence of the quenching solution composition on the fluorescence background level. The figure shows the results after quenching by the addition of ethylenediamine after the reaction and after washing by 1:14:85 formic acid / water / ACN washing. [Figure 8B]1 shows chromatograms showing the results of fluorescently labeled N-glycosylamines released from pooled human IgG analyzed by LC with fluorescence detection and the effect of quenching solution composition on fluorescence background levels, after post-reaction quenching by addition of isopropylamine and after washing with a 1:14:85 formic acid / water / ACN wash. [Figure 8C] 1 shows chromatograms depicting the results of fluorescently labeled N-glycosylamines released from pooled human IgG analyzed by LC with fluorescence detection and the effect of quenching solution composition on fluorescence background levels, after post-reaction quenching with the addition of hexylamine and after washing with a 1:14:85 formic acid / water / ACN wash. [Figure 9A] Fluorescence chromatograms obtained for labeled glycosylamines showing the different effects of various washes used during SPE, after washing with DMF / ACN in a ratio of about 15 to about 85 by volume (15:85 (v / v)). [Figure 9B] Fluorescence chromatograms obtained for labeled glycosylamines showing that various washes used during SPE have different effects. The chromatogram is after washing with approximately 85 percent ACN. [Figure 9C] Fluorescence chromatograms obtained for labeled glycosylamines showing the different effects of various washes used during SPE after washing with formic acid / water / ACN in a ratio of about 1:14:85 by volume (v / v / v). [Figure 10] 1 is a fluorescence chromatogram obtained for glycosylamines of pooled human IgG labeled with Labeling Reagent-1 as described in Example 2 using the methods described herein. [Figure 11A]1 is a fluorescence chromatogram obtained for cetuximab glycosylamine labeled with Labeling Reagent-1 as described in Example 3 using the methods described herein. [Figure 11B] 11B is a reference peak intensity chromatogram corresponding to FIG. 11A, as described in Example 3 using the methods described herein. [Figure 12A] HILIC fluorescence chromatogram obtained for anti-citrinin mouse IgG1 glycosylamine labeled with labeling reagent-1 by SPE of the reaction in HEPES buffer. [Figure 12B] HILIC fluorescence chromatogram obtained for anti-citrinin mouse IgG1 glycosylamine labeled with labeling reagent-1 by SPE of the reaction in phosphate buffer. [Figure 13A] HILIC fluorescence chromatogram obtained for anti-citrinin mouse IgG1 glycosylamine labeled with labeling reagent-1 by direct HILIC analysis of the mixture after the labeling reaction. [Figure 13B] 1 shows a HILIC fluorescence chromatogram obtained for anti-citrinin mouse IgG1 glycosylamine labeled with labeling reagent-1 by online SPE-HILIC analysis of the post-labeling reaction mixture. [Figure 13C] HILIC fluorescence chromatogram obtained for anti-citrinin mouse IgG1 glycosylamine labeled with labeling reagent-1 by high pH / low pH online SPE-HILIC analysis of the post-labeling reaction mixture. [Figure 14A] HILIC-FLR-MS of RFMS-labeled N-glycans derived from anti-citrinin mouse IgG1. [Figure 14B] Figure 1 shows HILIC-FLR-MS of IAB-labeled N-glycans derived from anti-citrinin mouse IgG1. [Figure 14C] FIG. 1 shows response factors for RFMS-labeled and IAB-labeled glycans. [Figure 15A] HILIC-FLR-MS of RFMS-labeled N-glycans from pooled human IgG. [Figure 15B] It is a diagram of HILIC-FLR-MS of 2AB-labeled N-glycans derived from pooled human IgG. [Figure 15C] It is a diagram showing the response coefficients of RFMS-labeled glycans and 2AB-labeled glycans. [Figure 16] It is a diagram showing the relative performance of glycan labels.

Mode for Carrying Out the Invention

[0013] A novel method for analyzing compounds in a biological sample is provided herein. To analyze a glycosylated biomolecule, the molecule is deglycosylated either naturally or by synthetic treatment and then contacted with a labeling reagent under selected conditions to yield a derivatized glycosylamine with optimal yield and minimal excess labeling. Derivatization is carried out without first separating the biomolecules of the sample. Chromatographic analysis can also be carried out directly on the mixture of derivatives obtained, and such analysis includes, but is not limited to, high performance liquid chromatography ("HPLC"), ultra-high performance liquid chromatography (UHPLC), mass spectrometry, supercritical fluid chromatography, ultraviolet ("UV") detection and / or fluorescence detection ("FLR"). However, as separation, there are optionally SPE offline techniques and SPE online techniques as described herein.

[0014] The methods described herein are suitable for use in automated analysis and processing systems, such as those described by Hewitson et al. in U.S. Patent Application No. 62 / 100,252, which is incorporated herein by reference. Provided herein are these automated sampling and reaction systems for high performance liquid chromatography or detectors for a wide range of applications, including but not limited to the identification and quantification of proteins and peptides, and the monitoring and analysis of nutritional components in cell culture media and food and feed. The automated sampling and reaction systems can provide turnkey analysis that can be optimized for high performance liquid chromatography processes and detection. The disclosed methods and systems can be used with various types of detectors, TUV, PDA, or FLR detectors. The automated sampling and reaction systems are also effective for application-specific performance accuracy and provide the same results daily, instrument-to-instrument, and laboratory-to-laboratory worldwide.

[0015] In addition, the methods described herein are suitable for use in various automated workflow systems and solutions for production and distribution. The methods described herein are used in automated workflow systems that use automated workflow systems for liquid handling and robotic systems to increase throughput and provide increased efficiency and safety in the laboratory. Automated workflow systems often function as platforms for biopharma, research, and clinical diagnostics and include, but are not limited to, digital dispensers, DNA extraction, PCR setup, ELISA, multi-channel pipetting, pipetting platforms, and related equipment and software incorporated to create high performance workflows in the laboratory.

[0016] In the method of the present invention, the glycosylamine is labeled in solution under conditions where the proteome has not been depleted from the mixture. The conditions of the reaction mixture, including temperature, organic solvent composition, organic solvent concentration, buffer composition, pH, ionic strength, molar excess of reagent, and time, are selected and controlled such that the desired reaction selectivity between the primary amine and the hydroxyl group of the glycosylamine can be achieved. In the methods described herein, the conditions for labeling or tagging the glycosylamine using various labeling reagents, particularly rapid tagging labeling reagents, are optimal.

[0017] As used herein, the phrase "glycosylated biomolecule" means and includes proteins, peptides, glycans, amino acids, lipids, DNA, RNA, and nucleic acids. The glycosylated biomolecule to be analyzed (in singular or plural, sometimes referred to as a biomolecule or compound) may naturally possess or be capable of generating a glycan having a primary amine, secondary amine, or tertiary amine. The primary amine or secondary amine may be present alone or in plurality. The primary amine and secondary amine are considered "modifiable" amines with respect to the labeling reagents described herein. More specifically, an amine is an organic compound having a functional group containing a nitrogen atom with a lone pair of electrons. Generally, an amine is a derivative of ammonia, where one or more hydrogen atoms are replaced by substituents such as an alkyl group or an aryl group. Exemplary amines include amino acids, biogenic amines, trimethylamine, and aniline. Inorganic derivatives of ammonia are also referred to as amines herein. Further, the glycosylated biomolecule may be a single species or a mixture of multiple types in the sample. The glycosylated biomolecule may include, but is not limited to, amines (primary, secondary, etc.), amino acids, peptides, proteins, polyamines, glycosylated compounds, compounds having glycan groups, or glycoproteins.

[0018] More generally, a glycosylated biomolecule can mean any molecule to which a sugar has been added either naturally or by synthetic treatment. Thus, a glycosylated biomolecule, glycosylated compound or glycoprotein is a compound or molecule that undergoes post-translational modification or post-translational modification and contains one or more carbohydrates and glycan groups attached thereto. Such glycosylated compounds can occur naturally or be produced synthetically, and these include proteins, lipids, amino acids (even when present as residues in peptides or proteins), antibodies, peptides or other organic molecules. An N-glycan (or N-linked glycan) can be attached to the nitrogen of the amide group or amino group of an asparagine side chain or an arginine side chain. On the other hand, an O-linked glycan can be attached to the hydroxyl oxygen of a serine side chain, threonine side chain, tyrosine side chain, hydroxylysine side chain or hydroxyproline side chain or to the oxygen of a lipid. In the context of the methods presented herein, the glycosylated compound may be present singly or in plurality in the sample.

[0019] Glycosylamines, or N-glycosides, are a class of compounds consisting of amines with a β-N-glycosidic bond to a carbohydrate, forming a cyclic hemiaminal ether bond (α-amino ether). In other words, a glycosylamine is a compound having a glycosyl group bonded to an amino group. Examples of glycosylamines include, but are not limited to, nucleosides such as adenosine and glycosides having an amine group such as N,N-dimethyl-β-D-glucopyranosylamine, glucosylamine, glucosyl-n-butylamine, glucosyl-n-hexylamine, glucosyl-n-octylamine, glucosyl-n-decylamine, glucosyl-n-dodecylamine, maltosyl-n-dodecylamine. Furthermore, D-glucose, D-galactose, lactose, cellobiose and maltose can be converted to the corresponding glycosylamines, 1-amino-1-deoxy-D-glucose, 1-amino-1-deoxy-D-galactose, 1-amino-1-deoxylactose, 1-amino-1-deoxycellobiose and 1-amino-1-deoxymaltose by treatment with an aqueous solution of ammonia in the presence of one equivalent of ammonium hydrogen carbonate.

[0020] Certain glycosylation patterns have been associated with health and disease states, and N-glycan analysis is increasingly being applied by multiple industries, including pharmaceutical biomanufacturing. Many protein-based biopharmaceuticals are glycosylated proteins, and uncontrolled variations in protein glycosylation are a major regulatory concern. For example, the relative amounts of individual glycan structures need to be monitored during the process to establish the stabilization of the production and purification steps. Fluorescent labeling of N-glycans (in glycosylamine form) is beneficial for detecting the amino acid distribution profile of N-glycans in biological samples, as it improves both the sensitivity and selectivity of detection as well as the chromatographic behavior of glycans.

[0021] Thus, a wide range of enzymatic and chemical techniques are effective for protein deglycosylation and glycan release. Enzymatic deglycosylation techniques utilize glycosidases, examples of which include, but are not limited to, N-glycosidase A (PNGase A), N-glycosidase F (PNGase F), O-glycosidase, neuraminidase, β1-4 galactosidase, and β-N-acetylglucosaminidase. For example, as described in detail herein, glycoprotein samples can be deglycosylated with an enzyme, namely peptide N-glycosidase F (PNGase F), which removes N-linked oligosaccharides from glycoproteins, except for compounds containing α(1-3)-linked fucose at the reducing end. However, N-glycosidase A (PNGase A) can remove all N-glycans. Other useful enzymes include endoglycosidases such as endoglycosidases or glycoamidases like N-glycanase. In the case of enzymatic deglycosylation, N-glycans are released from asparagine residues as glycosylamines.

[0022] In the case of chemical release of N-glycans from glycoproteins, the glycoprotein can be treated with hydrazine anhydrous at 90 °C for several hours. To release O-glycans from glycoproteins, the glycoprotein is treated with O-glycanase or chemically with hydrazine anhydrous, specifically at 60 °C for up to 6 hours. Alternatively, O-glycans can be released by reductive alkaline-catalyzed β-elimination, using sodium borohydride for non-reductive β-elimination, or by using other releasing reagents such as trifluoromethanesulfonic acid or various amines. Other chemical techniques include, but are not limited to, hydrazinolysis and trifluoromethanesulfonic (TFMS) acid treatment.

[0023] Furthermore, glycan analysis has been increasingly applied in biological research and clinical analysis. Specific glycosylation patterns have been associated with health and disease states. Additionally, changes in glycosylation can modulate the biological activity of proteins, as shown, for example, for the glycosylation of the Fc portion of recombinant immunoglobulins. Therefore, approaches to the analysis of oligosaccharides derived from glycoproteins often focus on the analysis of related and subsequently derivatized glycans. However, while these approaches enable the detailed analysis of oligosaccharide structures independent of the carrier glycoprotein, they provide no information regarding the glycan attachment sites.

[0024] Therefore, the characterization of protein glycosylation profiles is of great importance as it is required for various regulatory purposes and the manufacture of biopharmaceutical drugs. The pool of free glycans is extremely complex and structurally heterogeneous, which necessitates efficient methods for separation and sensitive detection methods. The relative amounts of individual glycan structures need to be monitored during the process to establish the stabilization of the manufacturing and purification steps.

[0025] Accordingly, methods for preparing labeled glycosylamines are presented herein. Techniques not previously described are used in conjunction with labeling reagents to rapidly produce glycans that are immediately analyzable. These techniques include the step of labeling glycosylamines with labeling reagents composed of a fluorescent moiety, a proton affinity / charge tag group, and an N-hydroxysuccinimide ester or carbamate reactive group, as specified below as Labeling Reagent-1, Labeling Reagent-2, Labeling Reagent-3, and Labeling Reagent-4 in Table 1.

[0026]

Table 1

[0027] Other labeling reagents that may be used in connection with the methods described herein include those identified in unpublished U.S. patent application Ser. No. 14 / 458,760, entitled "Rapid Fluorescence Tagging of Glycans and Other Biomolecules with Enchanced MS Signals." See page 2, line 4 to page 4, line 9; page 11, line 4 to page 25, line 18, and page 29, line 1 to page 30, line 10, which are incorporated herein by reference. Additional labeling reagents can also be found in U.S. Pat. No. 7,148,069, from Col. 8, 1.56 to Col. 9, 1.54 and Col. 15, 1.22 to 29, which are incorporated herein by reference; in U.S. Pat. No. 7,494,815, from Col. 7, 1.19 to Col. 11, 1.24, which are incorporated herein by reference; in U.S. Pat. No. 8,124,792, from Col. 2, 1.13 to Col. 4, 1.5 and Col. 7, 1.11 to Col. 17, 1.20, which are incorporated herein by reference; and in U.S. Pat. No. 5,296,599, from Col. 4, 1.66 to Col. 5, 1.32 and Col. 5, 1.66 to Col. 7, 1.28, which are incorporated herein by reference.

[0028] Additionally, the described methods can serve as a basis for the preparation of other labeled glycosylamines, including those derived from alternative labeling reagents. As described herein, such labeling reagents can have alternative reactive groups, such as isocyanates, isothiocyanates, or imidates / thioimidates, and / or alternative functionalities, such as charge tags, that enhance negative ion mode mass spectrometry.

[0029] Labeling reaction conditions, including temperature, organic solvent composition, organic solvent concentration, buffer composition, pH, ionic strength, molar excess of reagents, and time, are selected and controlled to achieve the desired reaction selectivity between primary amines and hydroxyl groups. The yield of labeled glycans is optimized, and the generation of so-called "overlabeled" glycans (glycans / glycosylamines modified with >1 label) is minimized. A quenching solution composed of a hydrophilic amine-containing compound, i.e., ethylenediamine, can also be used. This quenching solution controls the time glycosylamines are allowed to react with the labeling reagent and also shifts the pH of the reaction to a higher pH (>10), which increases the solubility of labeled glycans in highly organic solvents (i.e., >50% acetonitrile), thereby facilitating downstream SPE procedures based on hydrophilic interaction chromatography ("HILIC").

[0030] The glycans released from the glycoproteins are labeled in solution under conditions where the protein bodies are not specifically depleted from the mixture. An exemplary labeling reaction is shown immediately below.

[0031] [ka]

[0032] To develop and optimize a method for labeling glycosylamines with NHS carbamate reagents and / or NHS ester reagents, N-succinimidyl N-methylcarbamate, a low molecular weight, NHS carbamate reagent (Labeling Reagent-4), was reacted with the simple peptide, bradykinin. This peptide was used as a surrogate for glycosylamines, but such could not be readily tested in separate experiments considering that they degrade to reducing sugars with aldehyde termini. Tarentino, A.L. et al., 2-Iminothiolane: A Reagent for the Introduction of Sulphydryl Groups into Oligosaccharides Derived from Asparagine-linked Glycans, Glycobiology 1993, 3(3), 279-85. Bradykinin contains only one primary amine (its N-terminus) as well as one hydroxyl group and is thus a useful tool in optimizing labeling yields and selectivity for glycosylamine derivatization. Bradykinin also contains two arginine residues which are highly basic and enable the assay of reaction products by LC-MS without concern for significant deviations in ionization efficiency when the N-terminus is labeled.

[0033] By measuring the % of the bradykinin population modified (with single amine modification) together with bradykinin modified at its hydroxyl group (single hydroxyl modification or modification at two sites), the inventors discovered that the labeling yield and reaction selectivity were optimized for amines compared to hydroxyl groups. As shown in FIGS. 2 to 5, a number of plots of % modification versus molar excess of reagent are provided. Further, the discovery of optimal conditions for this type of reaction is illustrated including temperature as shown in FIG. 2, organic solvent composition as shown in FIG. 3, organic solvent concentration as shown in FIG. 3, buffer composition as shown in FIG. 4, pH as shown in FIG. 4 and ionic strength as shown in FIG. 5.

[0034] Put simply, a high labeling rate of the derivatized compound was achieved with a minimal level of over-labeling when (1) the temperature was from ambient temperature to sub-ambient temperature; (2) dimethylformamide (DMF) was used as the organic solvent; (3) DMF accounted for 20% - 30% or less of the reaction mixture; (4) a sodium phosphate solution buffer between pH 7.9 and pH 8.2 was used; and (5) the phosphate concentration was maintained at ≤50 mM. The over-labeling is less than about 1 mole percent, more preferably from about 0.0 to about 0.5 mole percent, and in some embodiments from about 0.0 to about 0.2 percent. Also, the buffer concentration can be between about 5 mM and about 1000 mM, or in some embodiments between about 5 mM and about 200 mM or between about 5 mM and about 100 mM or between about 5 mM and about 50 mM. A high yield of the labeled glycosylamine can be achieved with a molar excess of the labeling reagent over the modifiable amine in an amount ranging from about 10 to about 2000, from about 30 to about 1000, from about 40 to about 500, or from about 50 to about 300. Further, although not necessarily advantageous in terms of yield, it has been shown that an organic solvent is useful for solubilizing some labeling reagents such as Labeling Reagent - 1. The inventors further discovered that 20 - 30% DMF is sufficient to increase solubility without significantly affecting the yield and the yield and selectivity of the labeling reaction (Figure 3). For this reason, a reaction mixture composed of 20 - 30% DMF is preferred.

[0035] As provided in Figure 3, the composition and concentration of organic solvents were tested as co-solvents for the labeling reaction for dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). Both DMSO and DMF are polar aprotic solvents and have both a large relative permittivity (>20) and a large dipole moment. Other polar aprotic solvents include tetrahydrofuran (THF) and acetonitrile. With high polarity, these solvents dissolve charged species, including various anions and nucleophilic groups such as CN(-) and HO(-). Without hydrogen bonding, the nucleophilic groups are relatively "free" in solution and more reactive. Thus, solvents particularly useful in the method of the present invention include non-nucleophilic, polar aprotic solvents.

[0036] Generally, common characteristics for aprotic solvents include: (1) solvents capable of accepting hydrogen bonds, (2) solvents having no acidic hydrogen centers (acetone and esters lack this criterion); and (3) solvents that dissolve organic salts. Polar aprotic solvents are solvents that dissolve many salts but lack acidic hydrogens. This type of solvent often has intermediate dielectric constants and polarities. However, as described above, certain polar aprotic solvents have both a high relative permittivity and a high dipole moment, another example being acetonitrile ("MeCN") and HMPA (hexamethylphosphoramide).

[0037] To determine the optimal reaction time, and more specifically, to determine the time for which the reaction must proceed before it is terminated, a time-course study based on the bradykinin assay was performed. Figure 6 shows the results from a time-course experiment in which the labeled reagent-4 was reacted with bradykinin for various lengths of time before the reaction was terminated by the addition of diethylamine. As shown in Figure 6, the labeling reaction was carried out using labeled reagent-1 at concentrations of 500-fold, 400-fold, 300-fold, 200-fold, and 100-fold molar excess to obtain the optimal molar ratio of the tagged compound to the total primary amine concentration. This time-course reveals that the reaction of the NHS carbamate under the conditions outlined is efficiently complete after 120 seconds. To achieve glycosylamine labeling, the NHS carbamate reaction is preferably allowed to proceed for between 2 and 10 minutes before further sample processing is carried out. Thus, the reaction time can range between about 10 seconds and about 30 minutes or in some embodiments the reaction time is between about 30 seconds and about 10 minutes, and preferably the reaction time is between about 2 minutes and about 5 minutes.

[0038] The above method was used to label N-glycans released from several monoclonal antibodies, including mouse IgG and chimeric IgG (cetuximab) expressed from the mouse Sp20 cell line. To prepare the labeled N-glycans, glycoprotein samples were deglycosylated with peptide N-glycosidase F (PNGase F) and subsequently reacted with a labeling reagent at room temperature. The labeling reagent was dissolved at a concentration of 127 mM in anhydrous dimethylformamide (DMF). The deglycosylation mixture and the reagent solution were subsequently mixed at a volume ratio of 2.5:1, resulting in a reaction mixture composed of approximately 36 mM of the labeling reagent and 4.8 μM of free N-glycan (in the form of glycosylamine). Under these conditions, the glycosylamine is present in the reaction mixture along with other amine species derived from the sample and, most importantly, the proteinaceous amines of the precursor glycoprotein. Glycoproteins tend to contain a larger number of proteinaceous amines (i.e., lysine residues) than N-glycan sites. Thus, the most abundant amine in the deglycosylation mixture is the proteinaceous amine.

[0039] The inventors further discovered that it is advantageous from the points of speed and reproducibility to maintain the proteinaceous amines in the sample with a special purpose. Thus, a higher concentration of the labeling reagent (i.e., Labeling Reagent-1) can be added to the reaction mixture, and any other amines and nucleophilic groups without any other explanation have a negligible effect on the labeling yield. The labeling of glycosylamine is, as a result, more reproducible between samples. In addition, the amount of the labeling reagent can be more easily adjusted towards the desired properties of the amine modification. For example, a high yield of labeled glycosylamine can be more easily obtained without much concern that over-labeled glycan species will occur.

[0040] As an alternative, the labeling reaction (also referred to herein as "derivatization" or "derivatization step") can be performed even after depleting the proteome from the deglycosylation mixture. An IgG sample contains approximately 75 proteinaceous amines. Thus, an IgG-based free glycan mixture (two glycans; one on each heavy chain) containing approximately 4.8 μM of glycosylamine contains at least 180 μm of total primary amine composition. The inventors have found that under these conditions, the desired modification of a biological sample can be obtained by achieving a labeling reagent concentration of about 18 to about 180 mM, which is in the range of about 100 to about 1000-fold molar excess of the reagent over the total primary amine concentration. Conditions using at least about 200-fold molar excess are preferred to yield optimal labeling rates and relatively low levels of over-labeled glycans (≤0.24%) (Figure 7).

[0041] Generally, the labeling reaction is carried out for a minimum of about 5 minutes. Nevertheless, the tagging reaction can proceed in an even shorter time (i.e., seconds). By adding a quenching solution, the labeling reaction can be terminated. The quenching solution contains ethylene diamine / pure water / acetonitrile in a mixture of approximately 5:5:90 (v / v / v). By adding this quenching solution, the labeling reaction is terminated by removing the unreacted labeling reagent remaining after adding a significant concentration of amine (>100 mM). The quenching solution also shifts the pH of the reaction mixture from about 6.5 to a pH greater than 10, from a pH up to about 9. The shift to a basic pH ensures that the labeled glycosylamine as well as reaction by-products, which may include urea-linked molecules as these by-products, remain soluble. The high pH obtained by adding the quenching solution deprotonates the proton affinity / charge tag groups of the label and thus enhances solubility in a highly organic / low polarity solution. This facilitates sample preparation procedures that rely on the polarity of the glycan, as in the case where HILIC-based SPE is used to concentrate the labeled glycan from the labeling reaction mixture. Yu, Y. et al., A Rapid Sample Preparation Method for Mass Spectrometric Characterization of N-Linked Glycans, Rapid Commun Mass Spectrom 2005, 19(16), 2331-6. If the pH of the solution does not change, especially when a phosphate buffer is used during the derivatization step, both the reaction by-products and the labeled glycosylamine can co-precipitate.

[0042] As described in Example II below, the quenching solution is added to the labeling reaction mixture at a volume ratio of about 9 to about 1 (9:1 (v / v)). At this step, the labeling reaction is terminated and the reaction product is dissolved in a solution compatible with HILIC SPE. The properties of the amine in the quenching solution are important for this procedure. The amine must be significantly hydrophilic. Ethylenediamine is therefore a preferred amine for the quenching solution. On the other hand, as shown in the data of Figure 8, when other hydrophobic amines such as isopropylamine and hexylamine are tested and the samples are analyzed by LC with a fluorescence detector, it has been found that undesirable background levels due to reaction by-products occur.

[0043] To separate the derivatized glycosylamines, hydrophilic SPE sorbents such as Waters Sep-Pak Aminopropyl, Waters Oasis HLB or Waters Oasis WAX can be conditioned for SPE and then used to process the quenched, ACN-diluted samples. The Waters Sep-Pak Amino Propyl cartridge has a moderately polar, silica-based bonded phase with a weakly basic surface. This sorbent can be used as a polar adsorbent with various selectivities for acidic / basic analytes or as a weak anion exchanger in aqueous media with a pH below 8. Applications for this type of cartridge include the extraction of phenols and phenolic dyes, petroleum fractions, sugars and drugs and metabolites. The cartridge is designed to be connected to a pump and syringe for positive pressure flow to speed up sample processing time. The cartridge also has a removable reservoir and is used on vacuum-assisted flow equipment and certain automated systems. SPE can likewise be performed using miniaturized, 96-well format equipment.

[0044] After loading, the SPE adsorbent can be washed to further facilitate the removal of reaction by-products. Conditions have been discovered that facilitate the selective concentration of labeled glycosylamines from the reaction by-products. The inventors have found that an SPE washing step containing formic acid / water / acetonitrile in a ratio of 1:14:85 (v / v / v) is effective in reducing reaction by-products that appear in the LC chromatogram of the resulting sample as void peaks and sloping baselines that saturate the detector. Another HILIC SPE washing solvent useful in this regard is phosphoric acid / water / acetonitrile in a ratio of 0.3:14.7:85 (v / v / v). Useful HILIC SPE washing solvents can vary in acid composition, for example, from about 0.01 to about 10% when the organic solvent composition is from about 50 to about 100 volume %, from about 0.05 to about 5% when the organic solvent composition is from about 60 to about 98 volume %, or from about 0.3 to about 3% when the organic solvent composition is from about 70 to about 96 volume %, and from about 0.1 to about 3% when the organic solvent composition is from about 80 to about 95 volume %.

[0045] Several washing conditions were used in SPE. As shown in FIGS. 9A, 9B, and 9C, the washing conditions affected the fluorescence chromatogram obtained for the labeled glycosylamine. 1:14:85 (v / v / v) formic acid / water / acetonitrile was effective in reducing the fluorescence background and in shifting the pH of the loaded sample from high pH to low pH, and the shift is an important consideration given that high pH has been reported to induce epimerization of N-acetylglucosamine residues to N-acetylmannosamine. Liu, Y. et al., Investigation of Sample Preparation Artifacts Formed During the Enzymatic Release of N-linked Glycans Prior to Analysis by Capillary Electrophoresis, Anal Chem. 2009, 81(16), 6823-9. It is also ensured that the degradation of the silica-based SPE adsorbent is minimized by rapidly shifting the pH of the loaded sample by washing with an acidic 1:14:85 (v / v / v) formic acid / water / acetonitrile wash solution. Pettersson, S.W. et al., Chemical Stability of Reversed Phase High Performance Liquid Chromatography Silica under Sodium Hydroxide Regeneration Conditions, J Chromatogr A 2007, 1142(1), 93-7.

[0046] As described herein, the high-pH / low-pH HILIC SPE process reduces the chromatographic background encountered during the analysis of labeled glycosylamines. Nevertheless, the chromatographic background and peaks eluting near the void time of the HILIC column were found to be further reduced in intensity by using HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) rather than phosphate to buffer the labeling reagent. This resulted in SPE elution with a less intense chromatographic background. See Figures 12A and 12B.

[0047] With HEPES, the distribution, solubility, or SPE selectivity of reagent by-products changed conveniently, but it was possible to change in this way without the need for a quenching solution. Thus, for deglycosylation and subsequent labeling of the released N-glycosylamines, a solution buffered with HEPES can be used. Also, other buffering zwitterionic compounds having a pKa between about 7 and about 9 and being non-nucleophilic, such as, but not limited to, ADA (N-(2-acetamido)-2-imino diacetic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), BICINE (N,N-bis(2-hydroxyethyl)glycine), DIPSO (3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPBS (N-(2-hydroxyethyl)piperazine-N‘-(4-butanesulfonic acid)), MOBS (4-(N-morpholino)-butanesulfonic acid), MOPS (3-(N-morpholino)-propanesulfonic acid), MOPSO (3-(N-morpholinyl)-2-hydroxypropanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid), POPSO (piperazine-N,N‘-bis(2-hydroxypropanesulfonic acid)) can be used. The ionization state of the buffer compound being neutral or positive rather than negative can further reduce the chromatography background. Thus, cationic, non-nucleophilic buffer compounds such as tertiary amines: TEA (triethylamine), BIS-TRIS (2,2-bis(hydroxymethyl)-2,2‘,2“-nitrilotriethanol), BIS-TRIS propane (1,3-bis[tris(hydroxymethyl)methylamino]propane) can be used.

[0048] Alternatively, online SPE can be used alternatively in place of offline SPE. Online SPE is generally performed in the form of one-dimensional trap elution chromatography, where the so-called "trapping" column is paired with the analytical column by a fluidics switching mechanism. The trapping column allows for loading at high linear velocities and is generally composed of a large particle adsorbent such that the eluate of the online SPE is diverted to waste. The sample is injected onto the trap / online SPE column and washed free of matrix components and impurities. Subsequently, the eluate from the trap column is directed to the analytical column and a gradient is developed to elute and separate the components of the sample. To assist in performing optimal chromatography, the stationary phase of the trapping column needs to exhibit a lower retention force than that of the analytical column. This matching between the trapping column and the analytical column ensures that the analyte compounds are re-concentrated onto the analytical column. As a result, gradient elution provides a separation with a resolution comparable to that obtained by directly injecting the analyte compounds onto the analytical column instead. Trap-elution chromatography has generally been realized for reversed-phase separations but not for HILIC separations. In this way, the inventors have discovered the retention force of HILIC stationary phases for a new type of analyte compounds (i.e., glycans, glycosylamines or labeled glycosylamines including labeled glycans).

[0049] HILIC adsorbents and stationary phases for optimal online SPE / trap-elution chromatography have also been discovered. In particular, the inventors have found that a polymeric hydrophilic-lipophilic balance (HLB) adsorbent (Waters Oasis HLB) exhibits ideal glycan retention for a trapping stationary phase. Specifically, the inventors have found that Oasis HLB retains glycans under conditions of approximately 10:90 water / acetonitrile. On the other hand, glycans begin to elute from Oasis HLB at an aqueous strength of 11 to 15% water. This HILIC retention is unique in that it is approximately 10% weaker in acetonitrile compared to the retention of stationary phases commonly used in glycan HILIC chromatography, such as amide-bonded stationary phases or polyol (polyhydroxyl)-bonded stationary phases. Oasis HLB is a polymeric adsorbent composed of repeating hydrophilic, amide moieties along with nonpolar moieties. This structure imparts to Oasis HLB a particularly noteworthy HILIC retention that has been found to be beneficial for trapping / online SPE columns.

[0050] More specifically, for online SPE of glycosylamines (glycans), an Oasis HLB trapping column (i.e., 2.1×30 mm Waters Oasis HLB, 20 μm particle diameter) can be paired with an analytical column filled with amide-bonded organosilica particles (i.e., 2.1×50 mm, Waters Glycan BEH Amide 130 Å 1.7 μm). The glycan is loaded onto the online SPE / trapping column while the eluate is diverted to waste by some fluidic mechanism. The labeled glycosylamine adsorbs to the adsorbent of the SPE column and is washed at a relatively fast flow rate using a highly organic mobile phase such as 88% ACN. The mobile phase composition can also be modified with additives such as 1% strength ammonia solution in 12:88 (v / v) H2O:ACN. The eluate from the Oasis HLB SPE column can be re-directed to the analytical HILIC column after the wash step. During the wash step, derivatization by-products and other components of the sample matrix are flushed to waste. Thus, the wash is designed to prevent interference. Also, by using online SPE, the wash solution generally contacts the adsorbent for a very short time period further avoiding matrix interference. Online SPE also reduces the amount of time the analyst has to consume to perform manual sample preparation techniques.

[0051] Next, chromatographic conditions such as those described in Example 2 and suitable for gradient elution of glycans are used such that separation and chromatograms are obtained.

Example

[0052] [Example 1] Discovery of Conditions for Maximizing Labeling Rate and Minimizing Over-Labeled Species The labeling reagents (i.e., labeling reagents 1-4) were reacted with 10 μM bradykinin. The reactions were carried out under different conditions; all reactions were quenched by adding 5 M diethylamine HCl (pH 9.8) solution so that the final diethylamine concentration was approximately 800 mM. The resulting reaction products were assayed by LC-UV-MS using a Waters ACQUITY UPLC H-Class Bio coupled with a Waters Synapt G2-S. Samples were injected in a volume of 10 μL onto an ACQUITY UPLC CSH C18 C18, 130 Å, 1.7 μm, 2.1 × 50 mm column. Separation was performed using a quaternary gradient between mobile phases consisting of 0.1% TFA (v / v) in water, 0.1% TFA (v / v) in ACN, 0.1% FA (v / v) in ACN, and 0.1% FA (v / v) in ACN at a temperature of 60°C and a flow rate of 0.3 mL / min. Eluting species were detected by UV absorption (10 Hz, 214 nm) and electrospray ionization mass spectrometry (100-2000 m / z, 2 Hz). Data obtained by the above method are shown in Figures 2 through 6.

[0053] The Waters ACQUITY UPLC H-Class Bio is a chromatography system designed with a bio-inert flow path made of non-stainless steel material to allow large biomolecules to pass through the chromatography column intact. It utilizes sub-2-μm hybrid particle chemistry, which is particularly intended for the analysis of proteins, peptides, nucleic acids, and glycans. This provides a flow-through needle injector and a quaternary solvent delivery system that enables running multiple chromatography modes, including but not limited to reversed-phase (RP), ion-exchange (EX), size-exclusion (SEC), or hydrophilic interaction (HILIC) chromatography. This chromatography system uses multi-solvent mixing with an optional 6-port solvent selection valve for additional solvents intended for use, enabling binary, ternary, or quaternary gradient operation. Also, this system allows the user to vary the concentration of the mobile phase modifier and the organic solvent. Instead of manually preparing the solvent mixture, this system creates blends from pure solvents and creates concentrated stocks on demand. It also enables automatically adjusting and calculating the proportion of buffer stock required for the desired conditions when the pH and ionic strength are specified by the user. Furthermore, this system reduces volume and minimizes band broadening to maintain high separation efficiency. This system uses an ultra-low dispersion detector that maintains peak integrity, allowing for multiple detections in biological applications. Also, this system uses various heaters and a multi-column manager (along with column switching) that enable low-diffusion and accurate temperature control for chromatography selectivity and control of the highest retention time accuracy.

[0054] The Waters ACQUITY UPLC H-Class Bio can be combined with the Waters Synapt G2-Si high-resolution mass spectrometer. The Waters Synapt G2-Si is a high-resolution mass spectrometry system that utilizes the ion collision cross section (CCS) characteristics using T-Wave ion mobility to enhance the peak capacity, specificity, and sensitivity of biomolecule analysis. The Waters Synapt G2-Si is equipped with an enlarged ion sampling orifice, an enhanced vacuum pump configuration, and ion conduction optics. This dual T-Wave, off-axis design ensures that ions are efficiently introduced from the ion source to the quadrupole MS analyzer while simultaneously filtering out unwanted neutral impurities. This results in minimized background noise and increased MS ion intensity.

[0055] The Waters ACQUITY UPLC CSH C18 (phenyl-hexyl and fluoro-phenyl) columns provide mutual selectivity compared to other reversed-phase UPLC columns. These columns have a spherical particle shape with a hybrid particle substrate such as hybrid organic / inorganic particles, use 15% carbon loading, C18 chemistry, have an inner diameter of 2.1 mm, a length of 30 mm, and are end-capped. These columns include a 130 Å pore size and low silanol activity and are capable of operating the pH in the range of approximately pH 1 to approximately pH 11.

[0056] [Example 2] Rapid Preparation of Labeled Glycosylamines Release from Pooled Human IgG and Mouse IgG1 Pooled human IgG (serum-derived) samples and mouse IgG1 samples were prepared similarly. Lyophilized IgG samples were reconstituted in 50 mM sodium phosphate at pH 7.9 and mixed with peptide N-glycosidase F (PNGase F, New England BioLabs, P0705). This mixture was prepared such that the IgG concentration was approximately 1 mg / mL and the active concentration of PNGase F was approximately 25 units / μL. Subsequently, this mixture was incubated at 37 °C for 1 hour under conditions that result in complete deglycosylation of the Fc region of mouse IgG. At the completion of the 1-hour incubation, the deglycosylated mixture was cooled to room temperature and then diluted with an equal volume of 50 mM sodium phosphate at pH 7.9. Meanwhile, labeling reagent-1 was dissolved in anhydrous dimethylformamide (DMF) to a concentration of 127 mM (assuming that labeling reagent-1 was purified as a 1:1 complex with NHS). The deglycosylated mixture and the reagent solution were subsequently mixed in a volume ratio of 2.5:1 to produce a reaction mixture composed of approximately 36 mM labeling reagent-1 and 4.8 μM free N-glycan (and 180 μM total primary amine concentration) and reaction by-products. Reaction by-products include, but are not limited to, compounds corresponding to amine hydrolysis products of the labeling reagent and reaction products generated by this amine and the labeling reagent (i.e., urea-linked molecules).

[0057] The reaction was allowed to proceed for 5 minutes and then terminated by the addition of a quenching solution (5:5:95 (v / v / v) ethylenediamine / water / ACN). The quenching solution was added to the reaction mixture in a 9:1 volume ratio. Subsequently, the resulting quenched, ACN-diluted sample was subjected to solid-phase extraction (SPE) using a Waters Sep-Pak Aminopropyl μElution plate and vacuum-driven SPE. The wells were first washed with water and then conditioned with 15:85 (v / v) water / ACN. Subsequently, the quenched, ACN-diluted sample was loaded onto the wells. The adsorbed sample was then washed with a solution composed of 1:14:85 (v / v / v) formic acid / water / ACN. Finally, the concentrated, labeled glycosylamine was eluted from the SPE sorbent using an eluent composed of 100 mM ammonium acetate (pH 7), 5% ACN. The resulting labeled glycosylamine was analyzed either directly in the form of the SPE eluate or alternatively as a dried (by centrifugal evaporation) and reconstituted sample.

[0058] Analysis of the labeled glycosylamine was performed by a combination of hydrophilic interaction liquid chromatography (HILIC) separation and fluorescence detection and mass spectrometry detection (Waters ACQUITY UPLC H-Class Bio System paired with a Waters Synapt G2-S Mass Spectrometer). A 2.1×50 mm column packed with 1.7 μm amide-bonded organosilica particles was used with an aqueous mobile phase (mobile phase A) composed of 50 mM ammonium formate (pH 4.5) and a mobile phase (mobile phase B) composed of ACN. The aqueous sample was injected in a 1 μL volume and separated at a temperature of 60 °C according to the gradient in Table 2. The labeled glycosylamine was detected using a fluorescence detector (5 Hz, excitation λ = 265 nm, emission λ = 425 nm) and electrospray ionization mass spectrometry (600 - 2500 m / z, 1 Hz). Figure 10 shows representative chromatographic data of the sample obtained from this procedure.

[0059]

Table 2

[0060] [Example 3] Rapid preparation of labeled glycosylamines released from cetuximab Labeled glycosylamines were prepared from cetuximab (chimeric IgG1 expressed from murine Sp20 cells) using a procedure similar to that described in Example 2, except that a unique deglycosylation method was used. Figures 11A and 11B show representative chromatographic data for a cetuximab sample obtained from this procedure.

[0061] [Example 4] Figures 13A, 13B, and 13C provide exemplary HILIC fluorescence chromatograms obtained for N-glycosylamines labeled with Labeling Reagent-1, purified by online SPE with Waters Oasis HLB, and then subsequently separated using an analytical column packed with an amide-bonded stationary phase. Alternatives to Waters Oasis HLB can be used. Non-bonded or diol-bonded sorbents constructed with silica-based or organosilica-based particles have been found to exhibit retention capabilities that make them useful alternatives to trapping stationary phases.

[0062] More specifically, Figure 13B shows a chromatogram obtained for N-glycosylamines purified by online SPE with a mobile phase A of 50 mM ammonium formate at pH 4.5 and a mobile phase B of ACN. Further, Table 3 immediately below shows the corresponding SPE gradient table. Figure 13C shows a chromatogram obtained for N-glycosylamines purified by online SPE with a mobile phase A of 50 mM ammonium formate at pH 4.5, a mobile phase B of ACN, and a mobile phase C of 1% (v / v) ammonia hydroxide. Table 4 shows the corresponding SPE gradient table.

[0063] [Table 3]

[0064]

Table 4

[0065] As described herein, variants of hydrophilic-lipophilic balance polymer adsorbents include, but are not limited to, Phenomenex Strata X (a functionalized polymer adsorbent containing N-vinylpyrrolidone having multiple modes with respect to retention of analyte-adsorbent interactions), Thermo Hypersep PEP (a cartridge filled with a porous DVB material modified with a urea functional group), Agilent SampliQ OPT (filled with an amide-modified divinylbenzene polymer resin), Waters Oasis WAX, Waters Oasis WCX, Waters Oasis MAX, and Waters Oasis MCX. The Phenomenex Strata X adsorbent is a polymer-based reverse-phase functionalized adsorbent with a surface area of 800 sq.m / g that targets neutral and aromatic analytes and enables retention of neutral, acidic, or basic compounds under aggressive, highly organic washing conditions. This adsorbent relies on three mechanisms for retention: pi-pi bonding, hydrogen bonding (dipolar-dipolar interactions), and hydrophobic interactions. This type of adsorbent is pH-resistant and can handle a pH range of 0 to 14. The Thermo HyperSep Retain PEP cartridge is filled with a porous polystyrene DVB material modified with a urea functional group that enables recovery of polar or non-polar analytes. These columns provide an adsorbent with a particle size of 30-50 μm and a bed weight of 30 mg.

[0066] The Waters Oasis adsorbents are a family of SPE adsorbents currently sold under the OASIS trademark. These adsorbents are stable at both extremes of pH and in a wide range of solvents. These adsorbents provide good retention of polar compounds, and C 18It has a relative hydrophobicity retention capacity three times higher than that of conventional silica-based SPE adsorbents such as. Further extraction products include products such as Waters Oasis HLB, a general-purpose adsorbent for acidic, neutral, and basic compounds. Oasis HLB is a hydrophilic-lipophilic balanced, water-wettable, reversed-phase adsorbent made of a specific ratio of two monomers, hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene. The Waters Oasis WAX cartridge is a polymer reversed-phase, weak anion exchange, water-wettable mixed-mode polymer adsorbent optimized for the high selectivity of strongly acidic compounds. Waters Oasis WCX (weak cation exchanger) is a mixed-mode, water-wettable SPE adsorbent made of a copolymer matrix that enables the retention of all species of test compounds, especially strong bases (pKa > 10) and quaternary amines. This retention mechanism is both mixed-mode, ion exchange, and reversed-phase. Waters Oasis MAX (mixed-mode anion exchanger) is a mixed-mode polymer adsorbent optimized to achieve high selectivity and high sensitivity for extracting acidic compounds (pKa < 1) with an anion exchange group. This adsorbent is water-wettable. Waters Oasis MCX (mixed-mode cation exchanger) is a mixed-mode polymer adsorbent optimized for the retention of basic compounds with a pKa of 2 - 10.

[0067] [Example 5] HILIC-Fluorescence-ESI-MS (MS / MS) Analysis of Labeled N-Glycans To evaluate the sensitivity coefficient, the labeled N-glycan was analyzed by HILIC separation combined with fluorescence detection and mass spectrometry detection using UHPLC chromatography (ACQUITY UPLC H-Class Bio, Waters, Milford, MA). Either a 2.1×50 mm column or a 2.1×150 mm column packed with 1.7 μm amide-bonded organosilica particles (ACQUITY UPLC Glycan BEH Amide 130Å, Waters, Milford, MA) was used with an aqueous mobile phase composed of 50 mM ammonium formate (pH 4.4) and another mobile phase composed of ACN. The sample was injected in 1 μL aqueous volume or 10 μL of ACN / DMF volume and separated at 60 °C according to the gradient. The labeled N-glycan was detected using a fluorescence detector (5 Hz scan rate, gain = 1, ACQUITY UPLC FLR, Waters, Milford, MA) using the above excitation and emission wavelengths. The eluted glycan was also detected by positive ion mode electrospray ionization mass spectrometry using an ion mobility-capable QTof mass spectrometer (Synapt G2-S, Waters, Milford, MA) operating at a capillary voltage of 3.0 kV, a source temperature of 120 °C, a desolvation temperature of 350 °C, and a sample cone voltage of 80 V. The mass spectra were acquired at a rate of 1 Hz with a resolution of approximately 20,000 over the range of 500 - 2500 m / z.

[0068] Table 5 below shows the structures and abbreviations associated with the glycan labels described in this example along with the reagents used.

[0069]

Table 5

[0070] Results and Discussion High-Sensitivity Fluorescence Detection and MS Detection The sensitivity of RFMS labeling for N-glycan analysis was evaluated. In particular, the sensitivity coefficients of RFMS-labeled glycans were benchmarked against those of glycans labeled with alternative reagents. The commercially available alternatives most closely related to RFMS are the aminobenzamide or NHS carbamate analog of IAB. Cook, K.S. et al., Biologicals, 40(2), 109-17, (2012).

[0071] Figures 14A and 14B are HILIC fluorescence chromatograms and base peak intensity (BPI) MS chromatograms for equal amounts of N-glycans released from mouse IgG1 monoclonal antibody and labeled with RFMS and IAB, respectively. Based on the observed chromatographic peak areas, sensitivity coefficients for fluorescence detection and MS detection were determined for the most abundant glycan in the IgG profile, the fucosylated, biantennary FA2 glycan (Oxford notation) (Figure 14C). Harvey, D. et al., Proteomics, 9(15), 3796-801 (2009); Glycobase3.2 http: / / glycobase.nibrt.ie (accessed January 6, 2015).

[0072] Figure 14A shows the results of HILIC-FLR-MS of RFMS-labeled N-glycans derived from anti-citrulline mouse IgG1, and Figure 14B shows the results of IAB-labeled N-glycans derived from anti-citrulline mouse IgG1. Fluorescence (FLR) chromatograms and base peak intensity (BPI) MS chromatograms are shown. Labeled glycans (from 0.4 μg of glycoprotein, 1 μL aqueous injection) were separated using a 2.1 × 50 mm column packed with a 1.7 μm amide-bonded organosilica (130 Å) stationary phase. Sensitivity coefficients for RFMA-labeled glycans and IAB-labeled N-glycans, as shown in Figure 14C (evaluated by FA2 peak area per sample of N-glycans from 1 μg of anti-citrulline mouse IgG1), are shown. Fluorescence (FLR) sensitivity coefficients and MS (base peak intensity) sensitivity coefficients are shown, respectively. The analysis was performed in duplicate.

[0073] The results of the inventors regarding FA2 glycans indicate that the RFMS-labeled glycans generate a fluorescence signal twice as high as that of the N-glycans labeled with IAB, and more surprisingly, an MS signal approximately 800 times larger. Similarly, the RFMS labeling was also compared with the conventional 2-AB labeling. For such a comparison, N-glycans prepared from pooled human IgG by either RFMA or 2-AB were analyzed by HILIC-FLR-MS by loading equal masses (Figure 15A and Figure 15B, respectively).

[0074] Figure 15A represents the HILIC-FLR-MS of RFMS-labeled N-glycans derived from pooled human IgG, and Figure 15B represents the HILIC-FLR-MS of 2-AB-labeled N-glycans derived from pooled human IgG. Fluorescence (FLR) chromatograms and base peak intensity (BPI) MS chromatograms are shown. The labeled glycans (total glycans approximately 14 pmol, 1 μL aqueous injection) were separated using a 2.1×50 mm column packed with a 1.7 μm amide-bonded organosilica (130 Å) stationary phase. The amount of FA2 glycans was calibrated by two-point external calibration using quantitative standards (RFMS derivatized propylamine and 2-AB-labeled triacetylchitotriose). Figure 15C shows the response coefficients for RFMS-labeled glycans and 2-AB-labeled glycans (evaluated by the FA2 peak area per 1 picomole of FA2 determined by external calibration). Fluorescence (FLR) response coefficients and base peak intensity (BPI) MS response coefficients are shown respectively. The analysis was performed in duplicate.

[0075] Considering that rapid tagging and reductive amination are performed in completely different procedures, external calibration was established using quantitative standards to determine the amount of FA2 glycans loaded onto and eluted from the HILIC column. The response coefficients calculated using these calibrated amounts of FA2 glycans are provided in Figure 15C. It was confirmed that the RFMS-labeled glycans were detected with excellent sensitivity, specifically, a fluorescence signal 14 times higher and an MS signal 160 times larger than that of the 2-AB-labeled glycans.

[0076] To summarize the above observations, the inventors plotted the response coefficients of IAB and 2-AB as a percentage of the response coefficient of RFMS (Figure 16). Figure 16 shows the relative percent (%) performance of the glycan labeling. The response coefficients are shown as a percentage of the fluorescence response coefficient and MS response coefficient of the RFMS-labeled N-glycan. The comparison results were estimated by extrapolation from the published comparison controls of N-glycan, where it was found that procainamide provides equivalent fluorescence sensitivity and up to 50-fold greater ESI-MS sensitivity compared to 2-AB.

[0077] This plot depicts the response coefficients of IAB and 2-AB normalized against the response coefficient of RFMS, and thus the improvements in fluorescence sensitivity and MS sensitivity are evident in this plot. In Figure 16, the relative performance of reductive amination with another alternative labeling reagent, procaine amide, is also provided. Procaine amide is a chemical analog of aminobenzamide that has been shown in recent years to enhance the ionization of these glycans when the reductively aminated glycans are analyzed by HILIC-ESI(+)-MS. Previous studies have shown that procaine amide-labeled glycans produce equivalent fluorescence signals and MS signals that are 10 to 50 times larger compared to 2-AB-labeled glycans (an observation confirmed by our own analysis of 2-AB-labeled N-glycans and procaine amide-labeled N-glycans). Klapoetke, S. et al., J. Pharm Biomed Anal 53(3), 315-24 (2010). Compared to procaine amide, RFMS is thus predicted to provide at least a 3-fold improvement in MS sensitivity. Since both of these labels contain a tertiary amine moiety, it is reasonable to suggest that the excellent ionization of RFMS-labeled glycans is due to the fact that the RFMS label is more hydrophobic than the procaine amide label. Indeed, previous studies have shown that the addition of a hydrophobic surface region to a glycan label enhances electrospray ionization. Walker, S.H. et al., J Am Soc Mass Spectrom 22(8), 1309-17 (2011); Bereman, M.S. et al., Chem Commun (Camb) 26(2), 237-9 (2010). Therefore, it is notable that the RFMS label has a strongly basic side chain in addition to a relatively hydrophobic core structure. More clearly, the above response coefficient data suggest that the RFMS label provides unprecedented fluorescence sensitivity and MS sensitivity for HILIC chromatography profiling of N-glycans.

[0078] [Example 6] A Predictive Method for the Rapid Tagging of Amino Acids The methods and reagents described herein can be used to effect rapid tagging of amino acids. With appropriate buffers and diluents, pre-column derivatization and analysis of amino acids can be carried out. To reconstitute the derivatization reagent in powder form, the vial of reagent and diluent is heated on a heating block or other device set at 55 °C. The reconstituted reagent is generally in the range of 10 mM in acetonitrile. The reconstituted reagent can generally be stored for up to one week at room temperature. However, alternative solvents and temperatures can be utilized. To derivatize a sample of amino acid, 60 μl of borate buffer is added to the reconstituted sample in a 6 × 50 mm test tube and vortexed. Then 20 μl of the reconstituted reagent is added and immediately vortexed for several seconds. The mixture is generally incubated for 1 to 5 minutes at room temperature. The contents of the test tube are then transferred to a vial and sealed with a cap containing a siliconized septum. The vial is heated at 55 °C for 10 minutes. The amino acid derivative can be stored for up to one week at room temperature if tightly sealed and protected from evaporation.

[0079] Thus, although designed for the preparation of glycosylamines, the methods described herein can be applied to amino acid labeling (regardless of whether the amino acid residue is free or is a constituent in proteins and peptides).

Claims

1. A method for the rapid derivatization of a glycosylamine, comprising: contacting a biological sample containing a glycoprotein with an enzyme to produce a deglycosylated mixture; combining (a) a reagent solution containing a molar excess of a labeling reagent combined with a polar, aprotic, and non-nucleophilic organic solvent, wherein the labeling reagent is an N-hydroxysuccinimide ester or an N-hydroxysuccinimide carbamate, (b) the deglycosylated mixture, and (c) a buffer solution at a temperature from ambient temperature to 4 °C to produce a reaction mixture, wherein the ambient temperature is higher than 4 °C and the reaction mixture comprises the labeling reagent, free glycosylamine, proteinaceous amine, and derivatized glycosylamine, and adding a quenching solution to the reaction mixture 2 to 10 minutes after the deglycosylated mixture is combined with the reagent solution, wherein the quenching solution contains ethylenediamine and the pH of the reaction mixture shifts above 10; the deglycosylated mixture is combined with the reagent solution at a volume ratio of 2.5:1; the buffer solution contains HEPES or sodium phosphate and has a pH of 7.9 to 8.2 and a concentration of HEPES or sodium phosphate between 5 mM and 50 mM; and the derivatized glycosylamine is obtained between 80 and 100 mole percent of the reaction mixture and the over-labeling of the glycosylamine is in an amount less than 0.2 mole percent comprising the method.

2. The method according to claim 1, further comprising separating the derivatized glycosylamine by on-line solid phase extraction.

3. The method according to claim 1, wherein the polar, aprotic, and non-nucleophilic organic solvent is selected from the group consisting of DMF, DMSO, and acetonitrile.

4. The method according to claim 3, wherein the concentration of DMF in the reaction mixture is from 20 volume percent to 30 volume percent or the concentration of DMSO in the reaction mixture is from 30 volume percent to 50 volume percent.

5. The method according to claim 1, wherein the enzyme is peptide N-glycosidase F.

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