Rapid Preparation of Labeled Glycosylamines and Method for Analyzing Glycosylated Biomolecules that Generate Them

Abstract

To provide methods for selectively labeling glycosylated biomolecules and generating tagged compounds in a high yield.SOLUTION: Methods include the steps of: generating biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; preparing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing a deglycosylation mixture with the reagent solution to generate derivatized glycosylamines. The steps can be carried out without depletion of protein matter. A quenching solution is added to a reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture. The derivatized glycosylamines are detected by chromatographic detection, fluorescence detection, mass spectrometry ("MS"), or ultraviolet ("UV") detection and / or a combination thereof.SELECTED DRAWING: None

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 of biological origin often include a derivatization step of introducing a phosphor that facilitates 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 the prior art methods. Further, 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 origin 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 a 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 contain 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 comprise 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 the rapid derivatization of glycosylamines are also described herein. In some embodiments, the method for the rapid derivatization of glycosylamines comprises: (1) preparing a biological sample; (2) combining the biological sample with peptide N-glycosidase F to produce a deglycosylated mixture; (3) preparing a reagent solution comprising a labeling reagent combined with anhydrous dimethylformamide (DMF); and (4) mixing the deglycosylated mixture with the reagent solution to produce a reaction mixture comprising the labeling reagent, free glycosylamine, proteinaceous amine, and derivatized glycosylamine. The reaction mixture can have a molar excess of the labeling reagent over the modifiable amine in an amount from about 10 to about 1000. For some embodiments, other ranges of molar excess are also described herein.

[0008] In the described method, the concentration of the organic solvent in the reaction mixture can be from about 0 to about 50 percent. In some embodiments, the range of the organic solvent in the reaction mixture can be from about 10 to about 40 percent. In some exemplary embodiments, the range of the organic solvent in the reaction mixture can be from about 20 volume percent to about 30 volume percent. The deglycosylated mixture is mixed with the reagent solution at a volume ratio from about 9:1 to about 1:9. In an exemplary embodiment, the deglycosylated mixture is mixed with the reagent solution at a volume ratio of about 2.5 to about 1 to yield a reaction mixture having about 25 to about 30 percent of the reagent solution. In some embodiments, the amount of the reagent solution in the reaction mixture is 28.6 percent. The reagent solution can be maintained at a temperature of about ambient temperature or at a temperature maintained from below ambient temperature to about 4 °C. A buffer solution can be added to the deglycosylated 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 the subsequent derivatization reaction in 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

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 4C

Figure 5A

Figure 5B

Figure 6

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 7E

Figure 8A

Figure 8B

Figure 8C

Figure 9A

Figure 9B

Figure 9C

Figure 10

Figure 11A

Figure 11B

Figure 12A

Figure 12B

Figure 13A

Figure 13B

Figure 13C

Figure 14A

Figure 14B

Figure 14C

Figure 15A

Figure 15B

Figure 15C

Figure 16

DETAILED DESCRIPTION OF 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 in optimal yield and with 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 resulting mixture of derivatives, 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, SPE offline techniques and SPE online techniques are optionally available 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 the nutritional components of 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. The 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 associated 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 biologic molecule can mean any molecule to which a sugar has been added either naturally or through synthetic processing. Thus, a glycosylated biologic molecule, 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 an 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 compounds may be present singly or in plurality in a 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 with 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 their 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 bicarbonate.

[0020] Certain glycosylation patterns have been associated with health and disease states, and N-glycan analysis is increasingly being applied by multiple industries, including biopharmaceutical manufacturing. 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 stability in the manufacturing scale-up and purification steps. Fluorescent labeling of N-glycans (in glycosylamine form) is useful 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, a glycoprotein sample 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 on 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 release 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. Thus, 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 so that the stabilization of the manufacturing and purification steps can be established.

[0025] Accordingly, methods for the preparation of labeled glycosylamines are presented herein. Previously undescribed techniques are used in conjunction with labeling reagents to rapidly produce glycans that are immediately analyzable. These techniques involve labeling the glycosylamine with a labeling reagent 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 can be used in connection with the methods described herein include those identified in U.S. Patent Application No. 14 / 458,760, entitled "Rapid Fluorescence Tagging of Glycans and Other Biomolecules with Enchanced MS Signals," which is unpublished. See incorporated herein by reference, pages 2, line 4 to page 4, line 9; pages 11, line 4 to page 25, line 18 and pages 29, line 1 to page 30, line 10. Further labeling reagents can also be found in U.S. Patent No. 7,148,069, Col. 8, l. 56 to Col. 9, l. 54 and Col. 15, l. 22 to 29, incorporated herein by reference; U.S. Patent No. 7,494,815, Col. 7, l. 19 to Col. 11, l. 24, incorporated herein by reference; U.S. Patent No. 8,124,792, Col. 2, l. 13 to Col. 4, l. 5 and Col. 7, l. 11 to Col. 17, l. 20, incorporated herein by reference; and U.S. Patent No. 5,296,599, Col. 4, l. 66 to Col. 5, l. 32 and Col. 5, l. 66 to Col. 7, l. 28, incorporated herein by reference.

[0028] Furthermore, 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 can have alternative functionalities such as charge tags that enhance negative ion mode mass spectrometry.

[0029] The labeling reaction conditions, 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 primary amines and hydroxyl groups is achieved. The yield of labeled glycan is optimized, and the generation of so-called "over-labeled" glycan (glycan / glycosylamine 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 during which glycosylamine is reacted with the labeling reagent and also shifts the pH of the reaction to a higher pH (>10), which increases the solubility of the labeled glycan in a highly organic solvent (i.e., >50% acetonitrile), thereby facilitating the downstream SPE procedure based on hydrophilic interaction chromatography ("HILIC").

[0030] Glycans released from glycoproteins are labeled in solution under conditions where the proteome is not depleted from the mixture for a particular purpose. An exemplary labeling reaction is shown immediately below.

[0031]

Chemical formula

[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 a simple peptide, bradykinin. This peptide was used as a surrogate for glycosylamines, but such a thing could not be easily 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 therefore a useful tool in optimizing labeling yields and selectivity for glycosylamine derivatization. Bradykinin also contains two highly basic arginine residues, allowing the reaction products to be assayed by LC-MS without concern for significant deviations in ionization efficiency when the N-terminus is labeled.

[0033] By measuring the percentage of the population of bradykinin modified (with single amine modification) together with bradykinin modified at its hydroxyl group (either single hydroxyl modification or modification at two sites), the inventors discovered that the labeling yield and reaction selectivity are 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 the 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] Briefly, 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 was 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 the 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 dielectric constant (>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 are more reactive. Thus, solvents particularly useful in the method of the present invention include non-nucleophilic, polar aprotic solvents.

[0036] Generally, characteristics common to aprotic solvents include: (1) solvents capable of accepting hydrogen bonds, (2) solvents without 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 dielectric constant 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. From this time-course, it can be seen that the reaction of the NHS carbamate under the conditions outlined is efficiently complete after 120 seconds. To achieve the glycosylamine labeling, it is therefore preferred that the NHS carbamate reaction 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 by peptide N-glycosidase F (PNGase F) and subsequently reacted with the 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 then 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 specific 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 further 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 characteristics of the amine modification. For example, a high yield of labeled glycosylamine can be more readily obtained without much concern that over-labeled glycan species will occur.

[0040] As an alternative method, the labeling reaction (also referred to herein as "derivatization" or "derivatization step") can be carried out even after depleting the proteome from the deglycosylation mixture. An IgG sample contains approximately 75 proteinaceous amines. Thus, an IgG-based free glycan mixture containing approximately 4.8 μM of glycosylamine (two glycans; one on each heavy chain) contains a total primary amine composition of at least 180 μm. The inventors have found that under these conditions, the desired modification of the 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 an optimal labeling rate and a relatively low level of over-labeled glycan (≤0.24%) (Figure 7).

[0041] Generally, the labeling reaction is carried out for about 5 minutes at the shortest. Nevertheless, the tagging reaction can proceed in an even shorter time (i.e., seconds). By adding a quenching solution, the labeling reaction is terminated. The quenching solution contains ethylenediamine / 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 by 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 above 10, from a pH of 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 its solubility in a highly organic / low polarity solution. This smooths the sample preparation procedure that depends on the polarity of the glycan, as in the case when 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)). During 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 highly 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 were tested and the samples were analyzed by LC with a fluorescence detector, it was found that they produced an undesirable background level due to reaction by-products.

[0043] For separating 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 also 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 with formic acid / water / acetonitrile in a ratio of 1:14:85 (v / v / v) is effective in reducing the reaction by-products that appear in the LC chromatogram of the obtained 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 chromatograms obtained for the labeled glycosylamines. 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. Shifting 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 sorbent 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 chromatography background encountered during the analysis of labeled glycosylamines. Nevertheless, peaks eluting near the chromatography background and 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 led to SPE elution with a low-intensity chromatography background. See FIGS. 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-glycosylamine, 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 buffer compounds that are 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 fluidic 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 reverse-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 compound (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 the polymeric hydrophilic-lipophilic balance (HLB) adsorbent (Waters Oasis HLB) exhibits an ideal glycan retention capacity for trapping stationary phases. Specifically, the inventors have discovered 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 capacity is unique in that it is approximately 10% weaker with acetonitrile compared to the retention capacity 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 and amide moieties along with nonpolar moieties. This structure imparts to Oasis HLB a noteworthy HILIC retention capacity 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 packed 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 a certain hydrodynamic 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 12:88 (v / v) H 2O: It can be modified with additives such as a slightly more than 1% ammonia solution in ACN. The eluate from the Oasis HLB SPE column can be re-introduced to the analytical HILIC column after the washing step. During the washing step, derivatization by-products and other components of the sample matrix are flushed to waste. Thus, the washing is designed to prevent interference. Also, by using online SPE, the washing solution generally contacts the adsorbent for a very short time period, further avoiding matrix interference. Online SPE also reduces the amount of time an analyst has to consume to perform manual sample preparation techniques.

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

Examples

[0052] [Example 1] Discovery of conditions for maximizing the labeling rate and minimizing over-labeled species The labeling reagent (i.e., labeling reagents 1-4) was reacted with 10 μM bradykinin. The reactions were carried out under different conditions; all reactions were quenched by adding a 5 M diethylamine HCl (pH 9.8) solution such that the final concentration of diethylamine was approximately 800 mM. The resulting reaction products were assayed by LC-UV-MS using Waters ACQUITY UPLC H-Class Bio combined with Waters Synapt G2-S. Samples were injected at a volume of 10 μL onto an ACQUITY UPLC CSH C18 column, 130 Å, 1.7 μm, 2.1×50 mm. Separation was carried out at 60 °C and a flow rate of 0.3 mL / min using a quaternary gradient between mobile phases composed 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. Eluted species were detected by UV absorption (10 Hz, 214 nm) and electrospray ionization mass spectrometry (100-2000 m / z, 2 Hz). The data obtained by the above method are shown in Figures 2 to 6.

[0053] The Waters ACQUITY UPLC H-Class Bio is a chromatography system designed with a biocompatible 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, especially 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 planned 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, enabling 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 precise 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 introduced efficiently from the ion source to the quadrupole MS analyzer while simultaneously filtering out unwanted neutral impurities. As a result, the background noise is minimized and the MS ion intensity is increased.

[0055] The Waters ACQUITY UPLC CSH C18 (phenyl-hexyl and fluoro-phenyl) columns offer mutual selectivity compared to other reversed-phase UPLC columns. These columns provide a spherical particle shape with a 15% carbon load using a hybrid particle substrate such as hybrid organic / inorganic particles, C18 chemistry, 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. The 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 end of the 1-hour incubation, the deglycosylation 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 deglycosylation mixture and the reagent solution were subsequently mixed at 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 a total primary amine concentration of 180 μM) and reaction by-products. The reaction by-products include, but are not limited to, compounds corresponding to the amine hydrolysis products of the labeling reagent and the 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 at a volume ratio of 9:1. 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. An aqueous sample was injected at a volume of 1 μL 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 Glycosylamine Released from Cetuximab The labeled glycosylamine was prepared from cetuximab (chimeric IgG1 expressed from mouse Sp20 cells) using a procedure similar to that described in Example 2, except that a unique deglycosylation method was used. Figures 11A and 11B are chromatographic data representative of the cetuximab sample obtained from this procedure.

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

[0062] More specifically, Figure 13B is the chromatography obtained for N-glycosylamine purified by online SPE with mobile phase A of 50 mM ammonium formate at pH 4.5 and mobile phase B of ACN. Further, Table 3 immediately below is its SPE gradient table. Figure 13C is the chromatography obtained for N-glycosylamine purified by online SPE with mobile phase A of 50 mM ammonium formate at pH 4.5, mobile phase B of ACN, and mobile phase C of 1% (v / v) ammonia hydroxide. Table 4 is its 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 urea functional groups), 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 from 0 to 14. The Thermo HyperSep Retain PEP cartridge is filled with a porous polystyrene DVB material modified with urea functional groups 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 those. 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 strong 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 pKa 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 a 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 spectrum was 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 from anti-citrulline mouse IgG1, and Figure 14B shows the results of IAB-labeled N-glycans 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 shown in Figure 14C (evaluated as the FA2 peak area per sample of N-glycans from 1 μg of anti-citrulline mouse IgG1). 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 present 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, generate an MS signal almost 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 the rapid tagging and reductive amination are carried out in completely different procedures, the 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 percentages relative to the response coefficient of RFMS (Figure 16). Figure 16 shows the relative percent (%) performance of the glycan labels. The response coefficients are shown as percentages relative to 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] Since this plot depicts the response coefficients of IAB and 2-AB normalized against the response coefficient of RFMS, the improvement in fluorescence sensitivity and MS sensitivity is 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, which 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 noteworthy 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 equipment set at 55 °C. The reconstituted reagent is generally in the range of 10 mM in acetonitrile. The reconstituted reagent can generally be stored at room temperature for up to one week. 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 x 50 mm test tube and vortexed. Then 20 μl of the reconstituted reagent is added and immediately vortexed for a few seconds. The mixture is generally incubated at room temperature for 1 to 5 minutes. 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 at room temperature for up to one week if it is sealed airtight 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 a constituent in proteins and peptides).

Claims

1. A method for the rapid derivatization of glycosylamines, comprising: preparing a biological sample containing glycoprotein; contacting the glycoprotein with an enzyme to produce a deglycosylated mixture; and (a) a reagent solution containing a labeling reagent selected from an N-hydroxysuccinimide ester reagent or an N-hydroxysuccinimide carbamate reagent, and a polar, aprotic, and non-nucleophilic organic solvent, (b) the deglycosylated mixture, and (c) a buffer solution, to produce a reaction mixture, wherein the reaction mixture is formed under conditions where the proteome is not depleted from the deglycosylated mixture, the reaction mixture contains a molar excess of about 200 to about 2000 of the labeling reagent, the reaction mixture has the labeling reagent, free glycosylamine, proteinaceous amine containing lysine residues, and derivatized glycosylamine, wherein the derivatized glycosylamine is obtained between about 80 and about 100 mole percent of the reaction mixture, and the excess labeling of glycosylamine is less than 0.2 mole percent, and the temperature, organic solvent composition, organic solvent concentration, buffer composition, pH, ionic strength, molar excess of the reagent, and time of the reaction mixture are selected and controlled A method comprising the steps of:

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 volume ratio of the deglycosylated mixture to the reagent solution is 2.5:

1.

6. The method according to claim 1, wherein the reagent solution to be mixed is maintained at a temperature from ambient temperature to sub-ambient temperature.

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

8. The method according to claim 1, wherein the buffer solution contains HEPES or sodium phosphate.

9. The method according to claim 8, wherein the buffer solution has a pH of from 7.9 to 8.2 and a concentration of HEPES or sodium phosphate between 5 mM and 50 mM.

Images