Using trihydroxy and tetrahydroxy quaternary ammonium compounds as resolving agents for electrophoretic separations

Abstract

The present disclosure provides a method of improving the resolution of analytes in an electrophoretic separation using a gel by incorporating into the gel an effective amount of one or more tri- or tetra-hydroxy quaternary ammonium compounds or a mixture of such compounds.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 617,110, filed January 12, 2018, the contents of which are incorporated herein by reference for all purposes.

[0003] Federal Funding Statement

[0004] not applicable. Background of the Invention

[0006] The present invention relates to the field of analysis of analytes by electrophoretic separation of gels containing the analytes.

[0007] During electrophoresis, the analytes are subjected to an electrical force, causing the analytes to migrate through the gel. Typically, one or more analytes of interest are labeled to facilitate their detection and quantification after separation from other components present in the sample.

[0008] Electrophoresis is one of a series of techniques for analyzing various analytes present in a sample. Typically, a sample comprising one or more analytes of interest is loaded onto a gel, and the analytes in the sample are subjected to the effect of potential over the entire gel length, so that they move through the gel. The various forms of electrophoretic separation are known in the art. These forms include, but are not limited to, slab gel, capillary electrophoresis ("CE"), electrofiltration, differential gel electrophoresis ("DIGE"), zone electrophoresis, isoelectric focusing ("IEF"), native electrophoresis, two-dimensional electrophoresis, immunochemistry / immunofixation electrophoresis, solid phase pH gradient electrophoresis, microchip, mobile interface electrophoresis, isotachophoresis, pulsed field electrophoresis, and combinations thereof. In capillary electrophoresis, technology includes capillary gel electrophoresis or "CGE" that separates molecules based on molecular size and capillary zone electrophoresis or "CZE" that separates molecules based on charge: mass ratio.

[0009] CE, CGE, and CZEE are well known in the art and are widely taught in the literature, including Beckman Coulter's pamphlet "Introduction to Capillary Electrophoresis" (undated, but between 1991-1992); Whatley, H. "Basic Principles and Modes of Capillary Electrophoresis", Petersen and Mohammad, eds., "Clinical and Forensic Applications of Capillary Electrophoresis", Humana Press, Inc. Totowa, NJ Totowa (2001), and Laur and Rozing, eds. "High Performance Capillary Electrophoresis", 2nd edition, Agilent Technologies Inc., CA Santa Clara (2014). CGE has been used to separate proteins and nucleic acids for decades. See, e.g., Zhu et al., Anal Chim Acta. 2012 Jan 4; 709: 21-31.

[0010] Different electrophoresis techniques can be used to separate various types of compounds. Separating polysaccharides from each other, especially N-glycans, by electrophoresis is more challenging than separating proteins or nucleic acids from each other due to the characteristics of each of these types of compounds. See, for example, Schwarzer et al., "N-glycan analysis by CGE-LIF; Profiling influenza A virus hemagglutinin N-glycosylation during vaccine production", Electrophoresis 2008, 29, 4203-4214, p. 4204. Typically, the polysaccharides to be analyzed by CGE are labeled with charged fluorophores or UV-visible dyes to allow detection by, for example, laser induced fluorescence ("LIT") or by UV light. See, e.g., Schwarzer (supra), Reusch et al., “High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer,” 2014, doi: 10.4161 / mabs.26712.

[0011] Detection systems for electrophoretic separations may include absorbance, fluorescence, mass spectrometry, conductivity, potentiometric or amperometric methods. However, under conditions where electrophoretic separations are performed, the presence of other compounds in the sample that co-migrate with the analyte of interest can make it difficult to accurately detect and quantify one or more analytes of interest. Reducing this co-migration can improve the separation of the analytes of interest present in the analytical sample. To date, compositions and methods for reducing the co-migration of sample components in electrophoretic separations and thereby improving the separation of the analyte of interest from other components in the sample have not been reported.

[0012] There remains a need for compositions and methods that improve the resolution of co-migrating analytes and components, particularly glycans, during electrophoretic separations such as CE or CGE. Surprisingly, the present invention satisfies these needs and others.

[0013] Brief description of the invention

[0014] The present invention provides compositions, methods, systems and kits for improving the electrophoretic separation of labeled analytes. In a first group of embodiments, the present invention provides a gel for the electrophoretic separation of analytes. The gel comprises one or more compounds of structure 1, or one or more compounds of structure 2, or a mixture of one or more compounds of structure 1 and one or more compounds of structure 2, wherein the structure 1 is:

[0015]

[0016] The structure 2 is:

[0017]

[0018] In some embodiments, the gel comprises two or more compounds of structure 1. In some embodiments, the gel comprises two or more compounds of structure 2. In some embodiments, the gel comprises one or more compounds of structure 1 and one or more compounds of structure 2. In some embodiments, the compound having structure 1 is tris(2-hydroxyethyl)methylammonium hydroxide ("THMH"). In some embodiments, THMH is stabilized by a stabilizer. In some embodiments, the electrophoretic separation is capillary electrophoresis. In some embodiments, the capillary electrophoresis is capillary gel electrophoresis. In some embodiments, the capillary electrophoresis is capillary zone electrophoresis. In some embodiments, one or more compounds of structure 1, or one or more compounds of structure 2, or a mixture of one or more compounds of structure 1 and one or more compounds of structure 2 is present in a concentration of 0.001% wt / wt to 5.0% wt / wt of the gel. In some embodiments, one or more compounds of structure 1, or one or more compounds of structure 2, or a mixture of one or more compounds of structure 1 and one or more compounds of structure 2 is present in a concentration of 0.08% wt / wt to 0.15% wt / wt of the gel.

[0019] In another set of embodiments, the present invention provides a cartridge or capillary for capillary electrophoresis (CE) separation of an analyte of interest, wherein the cartridge or capillary is loaded with a gel comprising one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2, wherein Structure 1 is:

[0020]

[0021] The structure 2 is:

[0022]

[0023] In some embodiments, the gel comprises two or more compounds of structure 1. In some embodiments, the gel comprises two or more compounds of structure 2. In some embodiments, the gel comprises one or more compounds of structure 1 and one or more compounds of structure 2. In some embodiments, one or more compounds of structure 1 are tris(2-hydroxyethylmethylammonium hydroxide ("THMH")). In some embodiments, THMH is stabilized by a stabilizer. In some embodiments, the gel further comprises polyethylene oxide. In some embodiments, the analyte of interest is a labeled glycan. In some embodiments, the cartridge or capillary is a cartridge. In some embodiments, the cartridge or capillary is a capillary. In some embodiments, the capillary has an inner cavity having uncoated walls. In some embodiments, the capillary has an inner cavity having walls coated with a coating, wherein the coating (a) does not interfere with the CE of the analyte of interest, and (b) conditions the capillary without conditioning the capillary between runs to prevent a decrease in the resolution of the analyte over the course of at least 5 runs. In some embodiments, the coating does not interfere with CE of the analyte of interest, and the conditioning prevents a decrease in the resolution of the analyte over the course of at least 10 runs without conditioning the capillary between runs. In some embodiments, the coating of the wall is polyvinyl alcohol. In some embodiments, one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2 are present in the gel at a concentration of 0.001% wt / wt to 5.0% wt / wt of the gel. In some embodiments, one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2 are present in the gel at a concentration of 0.08% wt / wt to 0.15% wt / wt of the gel.

[0024] In another set of embodiments, the present invention provides a system for electrophoretic separation of an analyte, the system comprising (a) an apparatus for performing electrophoretic separation, and (b) a gel in which the electrophoretic separation is performed, the gel comprising one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2, wherein Structure 1 is:

[0025]

[0026] The structure 2 is:

[0027]

[0028] In some embodiments, the gel comprises two or more compounds of Structure 1. In some embodiments, the gel comprises two or more compounds of Structure 2. In some embodiments, the gel comprises one or more compounds of Structure 1 and one or more compounds of Structure 2. In some embodiments, one or more compounds of Structure 1 are tris(2-hydroxyethyl)methylammonium hydroxide ("THMH"). In some embodiments, THMH is stabilized with a stabilizer. In some embodiments, the electrophoretic separation is capillary electrophoresis. In some embodiments, the capillary electrophoresis is capillary gel electrophoresis. In some embodiments, the capillary electrophoresis is capillary zone electrophoresis. In some embodiments, one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2 are present in the gel at a concentration of 0.001% wt / wt to 5.0% wt / wt of the gel. In some embodiments, one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2 are present in the gel at a concentration of 0.075% wt / wt to 0.2% wt / wt of the gel.

[0029] In yet another set of embodiments, the present invention provides a method for performing electrophoretic separation of a labeled analyte, the method comprising (a) loading the analyte onto a gel, wherein the gel comprises an effective amount of one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2, wherein the Structure 1 is:

[0030]

[0031] The structure 2 is:

[0032]

[0033] And subject the labeled analyte in the gel to the action of the electric potential, thereby implementing the electrophoretic separation of the labeled analyte. In some embodiments, the gel comprises two or more compounds of structure 1. In some embodiments, the gel comprises two or more compounds of structure 2. In some embodiments, the gel comprises one or more compounds of structure 1 and one or more compounds of structure 2. In some embodiments, one or more compounds of structure 1 are tris (2-hydroxyethyl) methyl ammonium hydroxide ("THMH"). In some embodiments, THMH is stabilized by a stabilizer. In some embodiments, the electrophoretic separation is capillary electrophoresis. In some embodiments, the capillary electrophoresis is capillary gel electrophoresis. In some embodiments, the capillary electrophoresis is capillary zone electrophoresis. In some embodiments, the labeled analyte is a labeled polysaccharide. In some embodiments, a mixture of one or more compounds of structure 1, or one or more compounds of structure 2, or one or more compounds of structure 1 and one or more compounds of structure 2 is present in the gel at a concentration of 0.001% wt / wt to 5.0% wt / wt of the gel. In some embodiments, one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2 are present in the gel at a concentration of 0.075% wt / wt to 0.15% wt / wt of the gel. In some embodiments, the labeled glycans are labeled with a dye having multiple sulfonic acid moieties. In some embodiments, the labeled glycans are labeled with APTS, ANTS, or InstantQ TM Marked.

[0034] In yet another set of embodiments, the present invention provides a kit for performing electrophoretic separation, the kit comprising (a) a container containing a gel, wherein the gel comprises an effective amount of one or more compounds of Structure 1, or one or more compounds of Structure 2, or a mixture of one or more compounds of Structure 1 and one or more compounds of Structure 2, wherein Structure 1 is:

[0035]

[0036] Structure 2 is:

[0037]

[0038] and (b) buffer. In some embodiments, the gel comprises two or more compounds of structure 1. In some embodiments, the gel comprises two or more compounds of structure 2. In some embodiments, the gel comprises one or more compounds of structure 1 and one or more compounds of structure 2. In some embodiments, one or more compounds of structure 1 are tris(2-hydroxyethyl)methylammonium hydroxide ("THMH"). In some embodiments, THMH is stabilized by a stabilizer. In some embodiments, the container holding the gel is a cartridge or a capillary. In some embodiments, the cartridge or capillary is a cartridge. In some embodiments, the cartridge or capillary is a capillary. In some embodiments, the capillary has an inner cavity with uncoated walls. In some embodiments, the capillary has an inner cavity with coated walls. In some embodiments, the wall coating of the capillary is polyvinyl alcohol.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1a and Figure 1b . Figure 1a and Figure 1b is an electropherogram comparing the resolution of APTS-labeled glycans released from etanercept, a fusion protein biotherapeutic. Figure 1a . Figure 1a is an electropherogram showing the resolution of APTS-labeled glycans using a standard gel. The separation in the peak pointed by the arrow fails to resolve Man5 from other glycans that closely co-migrate with Man5 - APTS-labeled A1F, G0, and G0F-N. Figure 1b . Figure 1b The electropherograms of FIG. 1 show the resolution of APTS-labeled glycans using the same reagents, but where the gel also contains 0.1% of an exemplary quaternary ammonium compound, THMH. As indicated by the arrows, Figure 1b In the figure, the peak of APTS-labeled Man 5 is separated from the peaks of A1F, G0, and G0F-N. GU = glucose unit. RFU = relative fluorescence unit.

[0041] Figure 2a and Figure 2b . Figure 2a and Figure 2b is an electropherogram comparing the first injection of the sample ( Figure 2a ) and after subsequent injection of the same sample ( Figure 2b ), released from human IgG and then using InstantQ TMResolution of labeled glycans in capillaries coated on the inside with polyvinyl alcohol. Capillaries were loaded with fresh gel with 0.1% wt / wt of the exemplary quaternary ammonium compound THMH added before each run, but capillaries were not conditioned between any two runs. GU = glucose units. RFU = relative fluorescence units.

[0042] Figure 2a . Figure 2a This is an electrophoresis diagram showing InstantQ released from human IgG TM Separation of labeled glycans in the first run of these glycans (since injection 1 was a step standard, this injection was labeled "Injection 2").

[0043] Figure 2b . Figure 2b This is an electropherogram showing the release of InstantQ from human IgG. TM Separation of labeled glycans after 234 additional separations in the same PVA-coated capillary (as noted above, this is “injection 235” because the first injection was of the step standard). Fresh gel was loaded in the capillary between runs, but Figure 2a The operation shown and Figure 2b No conditioning was performed between runs shown. DETAILED DESCRIPTION OF THE INVENTION

[0045] In many cases, it is important to determine which analytes are present in a sample. For example, determining which sugars (also known as "glycans") are attached to glycoconjugates (such as glycoproteins) is important for understanding the pharmacokinetics, immunogenicity, and potential therapeutic effects of glycoproteins. Therefore, it is desirable to improve the detection of the presence of analytes of interest (such as glycans) in a sample.

[0046] Typically, the analyte present in the sample is detected by electrophoretic separation, and then the separated analyte is detected. As described in "Background Technology", many electrophoretic separation techniques are known in the art. These techniques include, but are not limited to, slab gel, capillary electrophoresis ("CE"), electrofiltration, differential gel electrophoresis ("DIGE"), zone electrophoresis, isoelectric focusing ("EEF"), native electrophoresis, two-dimensional electrophoresis, immunochemistry / immunofixation electrophoresis, solid phase pH gradient electrophoresis, microchip, mobile interface electrophoresis, isotachophoresis, pulsed field electrophoresis, and combinations thereof. In capillary electrophoresis, techniques such as capillary gel electrophoresis (also known as "CGE"), which separates molecules based on molecular size, and capillary zone electrophoresis (also known as "CZE"), which separates molecules based on charge: mass ratio, are included.

[0047] Although electrophoresis techniques generally separate many analytes well, some analytes, especially glycans, co-migrate with each other, making it difficult to determine which analytes are present and in what amounts. Reducing the co-migration of analytes (e.g., labeled glycans) present in a given sample will improve the separation of the analytes present in the sample. Unfortunately, to date, compositions and methods for reducing the co-migration of analytes, such as labeled glycans, thereby improving the separation of analytes are almost absent.

[0048] Surprisingly, we have discovered that the presence of low concentrations of tris(2-hydroxyethyl)methylammonium hydroxide (CAS No. 33667-48-0, a compound sometimes referred to herein as "THMH" or "TEMAH" and which has been used primarily for cleaning semiconductors) in a gel can significantly improve the resolution of analytes labeled with certain markers when separated electrophoretically in the gel. For example, analytes such as glycans labeled with the following markers: APTS (8-aminopyrene-1,3,6-trisulfonic acid trisodium salt, CAS name / number: 8-amino-1,3,6-pyrenetrisulfonic acid trisodium salt 196504-57-1); activated forms of APTS as taught in U.S. Patent Nos. 8,124,792 and 8,445,292; or other markers having multiple sulfonic acid moieties can be better separated by CGE when low levels of THMH are present in the gel during electrophoresis. In the studies that form the basis of the present invention, the presence of THMH in the gel expanded the separation range and improved the resolution of analytes in CGE separations. Some of the studies that form the basis of the present invention tested the effect of THMH on the separation of Man5 (also sometimes referred to herein as "Man-5" or "Man-5 glycan"), a glycan that is sometimes particularly difficult to separate in CGE separations, using APTS, as APTS itself is not able to adequately separate Man5 in gels. The studies showed that THMH helped maintain the peak shape of Man5 throughout the gel separation and continued to do so after multiple runs. As will be appreciated by those skilled in the art, maintaining peak shape in the separation of analytes is desirable because it helps prevent peaks of closely co-migrating analytes from overlapping each other, which can occur if peaks broaden during the process.

[0049] Considering that all electrophoretic techniques rely on the movement of analytes through a gel under the influence of an electric field, it is believed that the results obtained in studies using the CGE system will also hold true in other forms of electrophoresis using gels, especially in embodiments where electrophoretic separations are performed in a microfluidic environment. In addition, since the movement of compounds of interest through a gel under the influence of an electric field is a feature of electrophoretic techniques and is independent of the type of compound being analyzed, it is believed that while improved resolution of glycans is a preferred embodiment, the use of THMH and other compounds discussed herein will also improve the resolution of other labeled sugars and compounds containing them.

[0050] THMH is a preferred embodiment of a quaternary ammonium compound used in some embodiments. However, it is believed that similar results will be obtained in terms of improving the resolution of analytes in electrophoretic separations using other quaternary ammonium compounds containing a methyl group and three terminal hydroxyl groups, wherein the methyl groups are not directly attached to the nitrogen as in THMH, but are attached through an alkyl linker of 1 to 5 carbons. Therefore, in these embodiments, the trihydroxy quaternary ammonium compound has the structure shown in Structure 1. Structure 1:

[0051]

[0052] Compounds of Structure 1 are sometimes referred to herein as "trihydroxy quaternary ammonium compounds."

[0053] It is further believed that the use of a tetrahydroxy quaternary ammonium compound with a fourth hydroxyl group replacing the methyl group of THMH, wherein the fourth hydroxyl group can be directly or through an alkyl linker of 1 to 5 carbons and nitrogen, will achieve similar results in terms of improving the resolution of analytes in electrophoretic separation. Therefore, in these embodiments, the tetrahydroxy quaternary ammonium compound has the structure shown in Structure 2.

[0054] Structure 2:

[0055]

[0056] Compounds of Structure 2 are sometimes referred to herein as "tetrahydroxyquaternary ammonium compounds". In preferred embodiments, the hydroxyl group of the compound of Structure 2 is directly bonded to the nitrogen. In some embodiments, the compound of Structure 2 has 1 carbon between the hydroxyl group and the nitrogen. In some embodiments, the compound of Structure 2 has 2 carbons between the hydroxyl group and the nitrogen. In some preferred embodiments, the compound of Structure 2 has 3 carbons between the hydroxyl group and the nitrogen. In some preferred embodiments, the compound of Structure 2 has 4 carbons between the hydroxyl group and the nitrogen. In some preferred embodiments, the compound of Structure 2 has 5 carbons between the hydroxyl group and the nitrogen.

[0057] The compositions and methods of the present invention are particularly suitable for electrophoretic separation using gels for size separation of analytes, including hybridization techniques. In some embodiments, the electrophoretic separation using gels is capillary electrophoresis. In some embodiments, the capillary electrophoresis is capillary gel electrophoresis.

[0058] The above trihydroxy quaternary ammonium compounds or tetrahydroxy quaternary ammonium compounds can be used alone, or in the form of a mixture of the two trihydroxy quaternary ammonium compounds or a mixture of two trihydroxy quaternary ammonium compounds or a mixture of two tetrahydroxy quaternary ammonium compounds. For any particular trihydroxy quaternary ammonium compound, tetrahydroxy quaternary ammonium compound or mixture thereof, it can be easily tested to determine whether it can improve the resolution of one or more analytes of interest as follows: a sample of the analyte or analytes to be separated is introduced into a first gel (gel 1) containing the test compound or mixture of compounds, and a duplicate sample is introduced into a second gel (gel 2) without the test compound, and the test sample separated in gel 1 is compared with the test sample separated in gel 2 to determine whether the resolution of the selected analyte is (a) improved, (b) the same, or (c) worse. For example, if the analyte of interest is a quaternary ammonium compound, such as ANTS, APTS or InstantQ TM (ProZyme, Inc., Hayward, CA), they can be subjected to CGE separation in a first gel containing a test amount of the test compound or mixture to see whether the different labeled glycans of interest are better separated from each other than by CGE separation in a second gel (the only difference from the first gel being the absence of the test compound or mixture).

[0059] Typically, for convenience, the compound or mixture is introduced into the gel composition in the form of a solution, but it can be mixed into a buffer for the gel matrix as a powder, or introduced by other convenient means known in the art. The solution of the trihydroxy quaternary ammonium compound or tetrahydroxy quaternary ammonium compound may further contain a small amount of an antimicrobial agent. For example, THMH can be purchased from TCI America (Portland OR) in a 40-50% aqueous solution stabilized with the reagent MEHQ (4-methoxyphenol, CAS No. 150-76-5). It is believed that the good results obtained in the studies conducted with THMH on which the present disclosure is based were due to the presence of THMH, rather than due to the presence of a small amount of MEHQ in the THMH used in the studies.

[0060] The formulations for preparing gels for different electrophoretic applications are well known in the art. It is expected that those skilled in the art will be familiar with the formulations suitable for specific electrophoretic techniques for analyzing one or more analytes of interest. In some preferred embodiments, the gel formulation is a gel for capillary electrophoresis. In some preferred embodiments, the gel formulation is a gel for capillary gel electrophoresis. The trihydroxy quaternary ammonium compound or tetrahydroxy quaternary ammonium compound, or a mixture of the two trihydroxy quaternary ammonium compounds or the two or more tetrahydroxy quaternary ammonium compounds, or a mixture of two or more trihydroxy quaternary ammonium compounds or two or more tetrahydroxy quaternary ammonium compounds is preferably present in an amount of 0.005% to 5%, 0.0075% to 4%, 0.01% to 3%, 0.025% to 2%, 0.025% to 1%, 0.05% to 0.75%, 0.05% to 0. .60%, 0.075% to 0.50%, 0.075% to 0.4%, 0.075% to 0.3%, 0.075% to 0.2%, 0.075% to 0.15%, 0.08% to 0.125%, 0.85% to 0.125%, 0.09% to 0.120%, 0.09% to 0.115%, 0.09% to 0.11% and 0.1% are added to the selected gel formulation. 0.1% was found to be the best percentage for separating Man5 from other tightly co-migrating polysaccharides. It is expected that the percentage of one or more quaternary ammonium compounds that is most effective for separating different polysaccharides of interest may be different from the most effective percentage found for separating Man5 from other polysaccharides, but still within the aforementioned percentage range, and the most effective percentage or concentration for any particular polysaccharide or other labeled carbohydrate can be easily determined using the assay method taught by the present disclosure. Percentages can be calculated on a wt / wt basis or on a v / v basis. Both calculations are very similar, but the optimal concentration of a compound may differ slightly depending on which calculation is used. In the studies in this work, both calculations were used to prepare the mixtures and no functional differences were found.

[0061] Small amounts of THMH, other trihydroxy quaternary ammonium compounds, tetrahydroxy quaternary ammonium compounds, or mixtures thereof, added to a formulation for the preparation of an electrophoresis gel may be difficult to add to the gel formulation by pipetting or otherwise manipulating. Therefore, a solution containing THMH or other trihydroxy quaternary ammonium compounds or tetrahydroxy quaternary ammonium compounds is sometimes first diluted 2 to 10 times to increase the amount of solution containing the compound to be added. For example, a THMH stock solution (45-50% w / w) can be diluted 10 times with water containing MEHQ to a working concentration of about 5%. This working concentration of THMH is then added to the gel mixture to form a gel having a final concentration of 0.05-0.5% THMH. In some embodiments, it is desirable to improve the accuracy of the final solution by omitting an intermediate solution. In these embodiments, the THMH or other trihydroxy quaternary ammonium compound or tetrahydroxy quaternary ammonium compound or mixture of these compounds to be added to the formulation ("additive") can be weighed, the weight of the gel solution required to dilute the additive to the desired concentration calculated, the additive placed in a container suitable for holding the amount of gel to be used to dilute the additive, and the gel solution added directly to the additive in the container. Preferably, the gel solution and additive are then mixed by techniques used in the laboratory for mixing viscous solutions, for example, by placing the container on a rotating platform or by using a magnetic stirrer.

[0062] Any particular concentration of trihydroxyquaternary ammonium compound, tetrahydroxyquaternary ammonium compound or mixture thereof present in the gel thus obtained can be easily tested to check whether the concentration results in a current in the electrophoresis apparatus exceeding the amount recommended for use; if so, the particular concentration of the particular trihydroxyquaternary ammonium compound, tetrahydroxyquaternary ammonium compound or mixture thereof is not preferred. The trihydroxyquaternary ammonium compound, tetrahydroxyquaternary ammonium compound or mixture thereof is preferably added to the remaining components of the selected gel formulation and stirred to obtain a uniform distribution of the trihydroxyquaternary ammonium compound, tetrahydroxyquaternary ammonium compound or mixture thereof in the gel.

[0063] Similarly, the effect of any particular concentration of any particular trihydroxy quaternary ammonium compound, tetrahydroxy quaternary ammonium compound, or mixture thereof, on improving the resolution of one or more analytes of interest can be readily tested by separating test samples of the analytes in a first gel (Gel 1) containing the test concentration of the one or more compounds and in a second gel (Gel 2) without the test compounds, and comparing the test samples separated in Gel 1 to the test samples separated in Gel 2 to determine whether the resolution of the selected analytes is improved, the same, or worse. For example, if the analytes of interest are those known in the art as Man5, A1F, and GO, and are separated using, for example, ANTS, APTS, or InstantQ TM(ProZyme, Inc., Hayward, CA), they can be subjected to CGE with test amounts of test compounds or mixtures to see if the gel successfully separates the different labeled glycans of interest from each other. TM Analytes labeled with markers other than quaternary ammonium compounds can also be used to determine whether a particular amount of a particular trihydroxy quaternary ammonium compound, tetrahydroxy quaternary ammonium compound, or mixture thereof successfully improves the resolution of a particular analyte as compared to the same separation performed in the absence of the trihydroxy quaternary ammonium compound, tetrahydroxy quaternary ammonium compound, or mixture thereof. In a given gel formulation, the amount of a particular trihydroxy quaternary ammonium compound, tetrahydroxy quaternary ammonium compound, or mixture thereof that improves the resolution of selected analytes but does not cause overheating of the equipment used to perform the electrophoretic separation is considered to be an effective amount of the trihydroxy quaternary ammonium compound, tetrahydroxy quaternary ammonium compound, or mixture thereof.

[0064] definition

[0065] As used herein, the phrases "gel," "gel matrix," and "sieving matrix" are synonymous.

[0066] In the context of performing an electrophoretic separation, such as by CGE or a hybrid CE procedure using a gel, the term "resolution" refers to the ability to distinguish between two analytes that would otherwise co-migrate in the analytical system used to separate them. For ease of reference, the term "CGE" as used herein includes hybrid CE procedures using a gel, unless the context requires otherwise.

[0067] The term "label", in the context of "labeling an analyte of interest", refers to chemically attaching a fluorescent moiety or UV-visible dye to the analyte of interest.

[0068] The phrase "the analyte has been labeled by reaction with a named compound" means that the label attached to the labeled analyte is the label retained after the analyte is contacted with the named compound under conditions that allow the named compound to label the analyte with a fluorescent moiety or UV-visible dye.

[0069] Man5

[0070] Mammalian antibodies and fusion proteins are important and very expensive therapeutic groups, which are usually produced in commercial fermentation systems. During the production of antibodies or fusion proteins, monitoring the level of a glycan called "Man5" (Mannopentaose-di-(N-acetyl-D-glucosamine) or "oligomannosyl-5 glycan") (CAS No. 66091-47-2, also known as "Man-5" or "Man-5 glycan") can be used as a sign that the fermentation system is stressed and allows intervention, thereby hopefully saving production batches in time. The following structure of the Man-5 glycan is from the Sigma-Aldrich website (Sigma-Aldrich, St. Louis, MO):

[0071]

[0072] CE and CGE

[0073] Capillary electrophoresis and CGE have been used to separate various analytes for more than two decades. Therefore, it is expected that those skilled in the art are familiar with the various protocols and reagents used in the art for CGE and hybrid CE methods using gels, and these protocols and reagents need not be discussed in detail here. For example, general CE, especially CGE, is discussed in detail in the following documents: "Capillary Electrophoresis Methods for Pharmaceutical Analysis," Vol. 9, Ahuja and Jimidar, ed., Elsevier, London (2008), "Capillary Electrophoresis Guidebook: Principles, Operation, and Applications," (Methods in Molecular Biology), Altria, ed., Humana Press, Totowa, NJ (2010), and "Capillary Electrophoresis: Methods and Potentials," Engelhardt et al., ed., Friedr, Vieweg & Sohn, Braunschweig / Wiesbaden (1996), etc. In particular, CE of carbohydrates is also taught in many documents, including "Capillary' Electrophoresis of Carbohydrates" (Methods in Molecular Biology, Vol. 213), edited by Thibault and Florida, Humana Press Inc, Totowa, NJ (2003), "Capillary Electrophoresis of Carbohydrates: From Monosaccharides to Complex Polysaccharides," edited by Volpi, Humana Press, Totowa, NJ (2011), and "Carbohydrate Analysis: High Performance Liquid Chromatography and Capillary' Electrophoresis," edited by El Rassi, Elsevier Science BV, Amsterdam, the Netherland (1995), etc. Chapter 8 of the last reference discussed the use of various CE techniques (including CZE and CGE) for analyzing carbohydrates and glycoconjugates.

[0074] Gels, Cartridges, Capillaries, and Markers

[0075] Gels for electrophoretic separation of various analytes are well known in the art and have different compositions that can be tailored by one skilled in the art to the properties of the analyte of interest in the particular electrophoretic technique selected. It is expected that one skilled in the art will be familiar with gels in the art for electrophoretic separation of a particular analyte of interest by a selected electrophoretic separation technique, including gels and techniques for glycans and other carbohydrate-containing compounds.

[0076] In some embodiments for separating polysaccharides, the gel used in the methods and compositions of the present invention, particularly the gel used for CGE separation, is the same as that taught in U.S. Pat. No. 8,163,152 (hereinafter referred to as "Patent 152"), page 90, column 12, lines 15-23. In some preferred embodiments, the gel is prepared according to the formula listed in column 12, lines 25-35 of Patent 152, but with the following modifications: (1) the 7,000,000 mwt g / mol of polyethylene oxide ("PEO") listed in the formula is replaced by 8,000,000 mwt g / mol of PEO, because PEO with a molecular weight of 7,000,000 is not currently commercially available; (2) EtBr is omitted (this modified formula is sometimes referred to herein as "modified PEO gel"). In some embodiments, the mwt g / mol of the larger and smaller PEOs listed in the formula can each independently be ±20% of the mwt g / mol value. Moreover, regardless of the size of the PEO used, it is preferred to omit EtBr from the gel. The '152 patent is incorporated herein by reference in its entirety, and the portions cited above are specifically incorporated herein by reference. As is known to those of skill, "PEO" and "polyethylene glycol" or "PEG" are chemically synonymous, but "PEG" is generally used to refer to polymers below 20,000 g / mol, while "PEO" is generally used to refer to polymers above this point.

[0077] In some embodiments where the analyte of interest is a polysaccharide, the gel may be composed of a non-PEO compound for capillary gel electrophoresis of the polysaccharide, such as hydroxypropylmethylcellulose (HPMC). Although PEO is a particularly preferred gel, other neutral hydrophilic polymers may be used, such as alkylcelluloses, polyvinyl alcohol, dextran, or polyacrylamide.

[0078] The gel for electrophoretic separation can be produced by any means known in the art. For convenience, the compound of structure 1, structure 2 or a mixture thereof can be added to the reagent before the gel is formed so that the compound of structure 1, structure 2 or a mixture thereof is evenly distributed in the gel. In many instruments, the gel is placed in one or more containers, the end of the capillary is introduced into the gel, and the gel is driven into the capillary (usually under the action of atmospheric pressure).

[0079] In some embodiments, the gel can be arranged in the cartridge and driven into a capillary fluidically connected to the cartridge to facilitate replacement of the gel in the capillary between sample analyses. The cartridge is typically designed for use in an analytical instrument configured to receive them, such as a gel cartridge configured to be used with a GL1000 glycan analyzer (BiOptic Inc., New Taipei City, Taiwan, China), and preferably the gel can be replaced quickly to speed up a series of analyses. Typically, gels are prepared in large quantities, dispensed into cartridges, then purchased by practitioners who intend to use the cartridges, and CGE separations are performed in an instrument configured to install the cartridges.

[0080] For glycan analysis, suitable markers include APTS and InstantQ TM (ProZyme, Inc., Hayward, CA). ANTS (commonly available as 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt, hydrated sodium salt, CAS No. 5398-34-5) and other polysulfonate labels for glycans may also be used. It is believed that compounds of Structure 1, Structure 2, or mixtures thereof are particularly useful in electrophoretic separations where the label used to label the analyte of interest comprises multiple sulfonic acid moieties.

[0081] Separation of Man-5 glycans from other glycans

[0082] As mentioned in the previous section, changes in Man5 levels in a fermenter can indicate stress in the fermentation reaction, which can result in the loss of a single production run of a valuable therapeutic, such as an antibody that is FDA-approved as an anti-cancer therapeutic. Therefore, it has become important to determine the amount of Man5 present in the system. Unfortunately, monitoring the amount of Man5 by CGE has always been a challenge because there may be other glycans that co-migrate with Man5 when using conventional CGE gels. When glycans were labeled with APTS, the glycans that co-migrated with Man5 were A1F, G0, and G0F-N. When labeled with InstantQ TMWhen glycans are labeled, the glycans that co-migrated with the MAN-5 glycans were G0F-N and A1F. As reported in the previous section, the studies that form the basis of this disclosure show that when Man-5 glycans are labeled with APTS, the exemplary quaternary ammonium compound THMH improves the resolution of Man-5 glycans from other closely co-migrating glycans. Specifically, the peak shape of the APTS-labeled Man-5 glycans is maintained throughout the separation, making it easier to distinguish Man-5 glycans from G0F-N, G0, and A1F.

[0083] Uncoated and coated capillaries

[0084] As described above, the research that is the basis of this disclosure found that the presence of the exemplary quaternary ammonium compound THMH throughout the capillary electrophoresis caused a significant improvement in the resolution of some difficult-to-separate polysaccharides. It can be seen that the presence of quaternary ammonium compounds is advantageous in the CE separation of analytes, and can be used to improve the analytical capability of the presence of analytes of interest in samples when the analytes of interest and other analytes that co-migrate therewith are originally difficult to distinguish. In some preferred embodiments, the quaternary ammonium compound is THMH. In some preferred embodiments, the analyte is a polysaccharide, and the gel used is suitable for analyzing polysaccharides.

[0085] Most existing CE instruments are designed to wash the capillary between two sample runs and introduce fresh gel into the capillary. Such washing is called "regeneration" or "conditioning" of the capillary, which is used to improve reproducibility in many CE programs. For example, D. Heiger, HIGH PERFORMANCE CAPILLARY ELECTROPHORESIS, AN INTRODUCTION (Agilent Technologies, Publication 5968-9963E, Germany, 2000) ("Heiger") page 92 points out that the capillary can be conditioned by washing the capillary with a strong base, a strong acid, an organic compound (such as methanol or DMSO) or a detergent and then washing with a rinse solution.

[0086] While conditioning the capillaries by washing between runs does maintain their resolution, this practice also adds considerable time when multiple analyses must be performed. The steps of washing, rinsing, and refilling the capillaries with fresh gel are time consuming and can result in at least doubling the time required to complete an analysis. It may be desirable to eliminate or reduce the time required for multiple runs by reducing the need to condition the capillaries between runs.

[0087] ProZyme’s Gly-Q TMThe glycan analysis system can provide reproducible results without conditioning the capillary between each run. When the exemplary quaternary ammonium compound THMH is added to the gel used in the system, the ability to separate the Man5 labeled in the sample and the other labeled glycans that would have co-migrated with it is significantly improved. However, after multiple runs, this ability decreases. Typically, after 20-50 runs, the peaks representing the labeled glycans will become wider and may overlap, reducing the ability to separate the Man 5 labeled in the sample and other labeled glycans that closely co-migrated with it.

[0088] In the studies that form the basis of the present disclosure, washing the capillaries with an acid solution (acetic acid in the studies) or a low pH solution (0.1 M MES, 2-(N-morpholino)ethanesulfonic acid, pH 3.5 in the studies), followed by rinsing with water and adding fresh gel containing an exemplary quaternary ammonium compound, maintained the excellent ability of the capillaries to separate polysaccharides used as exemplary analytes. Without wishing to be bound by theory, we believe that the reduction in resolution after multiple separations performed in the same capillary without conditioning may be due to the interaction between the amine of the quaternary ammonium compound and the capillary wall, which is eliminated after conditioning the capillary. We expect that the same results will be obtained when other reagents that are conventionally used to condition capillaries are used. Therefore, the compositions and methods of the present invention are suitable for use in instruments where the capillaries are washed between each run according to a standard protocol for conditioning the capillaries.

[0089] Although washing the capillary provides the opportunity to use THMH to improve the resolution of Man5 from other closely migrating glycans, it also reduces the TM The ability of the glycan analysis system to reduce the overall time for glycan analysis by eliminating the conditioning time required for the capillary used for analysis. To reduce the need to condition the capillary when THMH is present in the gel, an alternative strategy was employed.

[0090] The studies reported in the previous paragraphs of this section were conducted using capillaries whose interiors were uncoated. (As will be appreciated by those skilled in the art, the exteriors of most, if not all, capillaries are coated to reduce their brittleness.) Capillaries coated on the interiors with any of a number of coatings are commercially available, typically to prevent the analyte from interacting with the walls. We are not aware of coatings that have been used to improve the performance of gel additives such as quaternary ammonium compounds.

[0091] In the research that forms the basis of this disclosure, commercially available capillaries coated with a permanent attachment coating were modified to be suitable for use in this system, and the presence of the coating was tested to see if it affected the separation of analytes when an exemplary quaternary ammonium compound, THMH, was present in the gel. The test used glycans as exemplary analytes, and the difficult-to-separate glycan Man5 as the glycan that was expected to be separated from other closely migrating glycans. All capillaries tested were obtained from Agilent Technologies, Inc. (Santa Clara, CA). The coating names discussed below are those mentioned by Agilent on its website.

[0092] Five different types of coated capillaries were tested: capillaries coated with polyvinyl alcohol or "PVA," μSIL-WAX, DB-1, μSIL-DNA, or CEP. The Agilent website describes the μSIL-WAX coating as "a modified polyethylene oxide hydrophilic coating." DB-1 is described as dimethyl polysiloxane. The Agilent website describes μSIL-DNA as a proprietary fluorocarbon polymer, and CEP as a permanently bonded polymer coating that masks the silanol functionality on the capillary surface and helps prevent sample adsorption.

[0093] As shown in Example 3 below, capillaries coated with polyvinyl alcohol or "PVA" showed no interference with glycan resolution in the presence of THMH, and there was no reduction in the ability to separate Man5 from other glycans over multiple runs of samples and standards without conditioning between runs. The other two types of coated capillaries tested, capillaries coated with μSIL-WAX or DB-1, were found to not reduce the ability of the THMH-gel to separate Man5 from other closely migrating glycans. Although these coatings have not been tested in multiple runs to date of this disclosure, any of these coatings can be used at least for single-run applications (e.g., using a CE instrument with the capillaries washed and the gel replaced between each run, as described above), and may also be suitable for multiple-use applications.

[0094] The last two coated capillaries tested, μSIL-DNA and "CEP", impaired the separation of glycans as exemplary analytes. Therefore, capillaries coated with μSIL-DNA or CEP are not preferred for use when the analytes of interest are glycans.

[0095] In PVA coated capillaries, or capillaries coated internally with other coatings that prevent resolution loss and do not themselves interfere with CE of the analyte, multiple runs of analytes can be performed in the same capillary without resolution loss, thereby avoiding conditioning steps between runs. This reduces the time to perform analyte analysis by at least half, providing a significant advantage over existing instruments and protocols that rely on reconditioning the capillary between runs to improve reproducibility.

[0096] Capillaries coated with any particular coating other than those tested in the studies underlying the present disclosure can be readily tested to determine whether the coating prevents degradation of the resolution of any particular quaternary ammonium compound or mixture of such compounds and does not itself interfere with the CE of the analyte of interest. For example, two capillaries (one coated with the coating of interest and the other uncoated) can be filled with the same gel containing the particular quaternary ammonium compound in question or mixture thereof, injected with the analyte of interest labeled with a detectable marker (e.g., Man5 labeled with APTS) and other analytes (also labeled with a detectable marker and expected to co-migrate closely with the analyte of interest, such as G0F-N and A1F), subjected to electrophoresis, and thereby run and determine (a) the effect of the presence of the coating on the resolution of the analyte in the uncoated capillary as compared to the resolution of the analyte in the uncoated capillary after a first run. Whether the ability of the analyte of interest to be separated from other analytes that are expected to co-migrate with it is adversely affected, and if not, second, (b) performing a plurality of such runs, such as 5 runs, 10 runs, 15 runs, 20 runs, 50 runs, 75 runs, 100 runs, 150 runs, or 200 runs, without conditioning the capillary between each run, and analyzing whether the coating prevents a decrease in resolution after the desired number of runs, a coating that prevents a decrease in resolution in a greater number of runs being more preferred than a coating that prevents a decrease in only a smaller number of runs. As described above, PVA coated capillaries that do not show a decrease in resolution in more than 200 runs when tested in capillaries without conditioning between runs are particularly preferred.

[0097] Reagent test kit

[0098] In some embodiments, the gel of the present invention can be provided in a kit for performing capillary electrophoresis. The kit may include: a container containing a gel suitable for performing CE, the gel comprising a compound of structure 1, a compound of structure 2, or a mixture of one or more compounds having structures 1 and 2; and a container containing a buffer suitable for use in performing CE. In a preferred embodiment, one or more compounds having structure 1, structure 2, or both are present at a concentration of 0.05% wt / wt to 0.5% wt / wt (or a total concentration if more than one such compound is present), more preferably 0.075% wt / wt to 0.150% wt / wt. Suitable buffers, such as MES, are well known in the art. In some embodiments, the container containing the gel can be a cartridge configured to introduce the gel into one or more capillaries, or can be a capillary or multiple capillaries. One or more capillaries may be coated on their interior with a coating that does not interfere with CE of the analytes of interest, and preferably a coating that prevents the improvement in analyte resolution from decreasing after multiple runs (e.g., 50, 100, 200 runs). In some preferred embodiments, the coating may be polyvinyl alcohol. Example

[0099] Example 1

[0100] This example presents the protocol used for the CE separation of glycan samples in the studies demonstrated in the accompanying figures. The trademarked reagents and instruments listed in this protocol are products of ProZyme, Inc. (Hayward, CA).

[0101] All samples were purified using commercially available kits using APTS or InstantQ TM Use the following equipment to mark the Gly-Q TM Experiments on the Glycan Analysis System:

[0102] a. Separation Capillaries - Use one of the following in each experiment:

[0103] i.Gly-Q TM G1 cartridge (standard gel, using uncoated capillaries);

[0104] ii. Gly-Q TM G1 cartridge (same gel as (i) but with THMH added at 0.1% wt / wt, using uncoated capillaries), or

[0105] iii. Gly-Q TM G1 cassette (same gel as (i) but with addition of THMH, using PVA coated capillaries).

[0106] b. Separation buffer: Gly-QTM Separation buffer

[0107] c. Calibration standard: Gly-Q TM Gu Stairs

[0108] d. Calibration standard injection: 2kV, 2 seconds

[0109] e. Migration standard: Gly-Q TM Migration Standards (Upper and Lower)

[0110] f. Migration standard injection: 2kV, lasting 2 seconds

[0111] g. Sample injection: 2kV, lasting 2 seconds

[0112] h. Separation: 10kV, lasting 120 seconds

[0113] Use of Gly-Q TM Data were analyzed using the GC-MS / MS software. Results were expressed in glucose units (GU).

[0114] For calibration purposes, all analyses began with an injection of the calibration standard ("Injection 1"). Thus, in all studies, "Injection 2" was the first injection and analysis of the sample of interest.

[0115] Example 2

[0116] This example provides the results of a study comparing the use of TM Resolution of Man5 from the closely co-migrating glycans A1F and GOF-N when using a gel dispensed with the G1 cartridge and when using such a gel with the addition of 0.1% of the exemplary quaternary ammonium compound THMH.

[0117] Also known as etanercept, it is a fusion protein biologic that inhibits tumor necrosis factor and has been approved in the United States for the treatment of several forms of arthritis. Glycans were released from etanercept by enzymatic digestion, labeled with APTS, and then subjected to CE. Figure 1a is to show the released labeled glycans in the presence of Gly-Q TM Electropherogram of separation on the gel distributed with the G1 cartridge. Arrows point to peaks containing Man5, A1F, and G0F-N, the latter two being two other glycans that co-migrated closely with Man5. As can be seen, CE in this gel did not result in separation of Man5 from other closely co-migrating glycans. Figure 1b is an electropherogram showing the resolution of the same APTS-labeled glycans on the same platform using the same reagents, but where the gel also contains 0.1% of an exemplary quaternary ammonium compound, THMH. Figure 1bAs shown by the middle arrow, the peak of APTS-labeled Man5 is separated from the peaks of AlF and G0F-N.

[0118] Example 3

[0119] This example provides two electropherograms comparing the release of human IgG by enzymatic digestion and dyed with InstantQ TM Resolution of labeled glycans after CE in a capillary coated internally with polyvinyl alcohol.

[0120] Figure 2a InstantQ was shown to be released from human IgG TM Electropherogram showing the separation of labeled glycans after the first injection of sample into a PVA-coated capillary loaded with a standard gel supplemented with 0.1% wt / wt of an exemplary quaternary ammonium compound THMH (since the first injection was that of the step standard, this injection is labeled “Injection 2”).

[0121] Figure 2b InstantQ is shown to be released from human IgG TM Separation of labeled glycans in the same sample in the same PVA-coated capillary after 234 additional separations over four days (as noted above, this injection is labeled “injection 235” because the first injection was an injection of the step standard), which included separations of glycans labeled with different markers and an additional step standard. Fresh gel was loaded in the capillary after each injection and electrophoresis, but Figure 2a The running and Figure 2b Of the 234 runs shown in , no conditioning was performed between any two runs. Figure 2a and Figure 2b Comparison of the results shows that although the heights of some peaks in the two electropherograms have changed, the peaks of each component are still clearly distinguishable. It can be seen that the use of PVA coating in the capillary can provide excellent reproducibility for the separation of glycans using gels containing exemplary quaternary ammonium compounds without the need to condition the capillary between each separation, and also saves the time required for conditioning the capillary more than 200 times between the two separations.

[0122] It is understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art are motivated to make various modifications or changes based on them, which will be included in the connotation and extension of this application and fall within the scope of the appended claims. All publications, patents and patent applications cited herein are fully incorporated herein by reference for all purposes.

Claims

1. Use of a gel for capillary electrophoretic separation of analytes, the gel comprising tris(2-hydroxyethyl)methylammonium hydroxide, i.e. THMH, and The analyte comprises: 1) APTS-labeled Man-5 glycan and 2) one or more glycans selected from APTS-labeled A1F, G0 and G0F-N.

2. The use according to claim 1, wherein the THMH is stabilized by a stabilizer.

3. The use according to claim 1, wherein the capillary electrophoresis is capillary gel electrophoresis.

4. The use according to claim 1, wherein the THMH is present in a concentration of 0.001% wt / wt to 5.0% wt / wt of the gel.

5. The use according to claim 4, wherein the THMH is present in a concentration of 0.08% wt / wt to 0.15% wt / wt of the gel.

6. Use of a cartridge or a capillary for capillary electrophoretic separation of an analyte of interest, wherein the cartridge or the capillary is loaded with a gel comprising tris(2-hydroxyethyl)methylammonium hydroxide, i.e. THMH; The analyte comprises: 1) APTS-labeled Man-5 glycan, and 2) one or more glycans selected from APTS-labeled A1F, G0 and G0F-N.

7. The use according to claim 6, wherein the THMH is stabilized by a stabilizer.

8. The use according to claim 6, wherein the gel further comprises polyethylene oxide.

9. The use according to claim 6, wherein the capillary has an inner lumen having uncoated walls.

10. The use according to claim 6, wherein the capillary has an inner lumen having a wall coated with a coating.

11. The use according to claim 10, wherein the coating is polyvinyl alcohol.

12. The use according to claim 6, wherein the THMH is present in the gel at a concentration of 0.001% wt / wt to 5.0% wt / wt of the gel.

13. The use according to claim 12, wherein the THMH is present in the gel at a concentration of 0.08% wt / wt to 0.15% wt / wt of the gel.

14. Use of a system for capillary electrophoresis separation of an analyte, the system comprising: (a) an apparatus for carrying out the electrophoretic separation, (b) a gel in which the electrophoretic separation is carried out, the gel comprising tris(2-hydroxyethyl)methylammonium hydroxide, i.e., THMH, and The analyte comprises: 1) APTS-labeled Man-5 glycan, and 2) one or more glycans selected from APTS-labeled A1F, G0 and G0F-N.

15. The use according to claim 14, wherein the THMH is stabilized by a stabilizer.

16. The use according to claim 14, wherein the capillary electrophoresis is capillary gel electrophoresis.

17. The use according to claim 14, wherein the THMH is present in the gel at a concentration of 0.001% wt / wt to 5.0% wt / wt of the gel.

18. The use according to claim 17, wherein the THMH is present in the gel at a concentration of 0.075% wt / wt to 0.2% wt / wt of the gel.

19. A method for performing capillary electrophoresis separation on a labeled analyte, the method comprising (a) loading the analyte onto a gel containing an effective amount of tris(2-hydroxyethyl)methylammonium hydroxide (THMH), and The analyte comprises: 1) APTS-labeled Man-5 glycan, and 2) one or more glycans selected from APTS-labeled A1F, G0 and G0F-N.

20. The method of claim 19, further wherein the THMH is stabilized by a stabilizer.

21. The method of claim 19, wherein the capillary electrophoresis is capillary gel electrophoresis.

22. The method of claim 19, wherein the THMH is present in the gel at a concentration of 0.001% wt / wt to 5.0% wt / wt of the gel.

23. The method of claim 22, wherein the THMH is present in the gel at a concentration of 0.075% wt / wt to 0.15% wt / wt of the gel.

24. Use of a kit for performing capillary electrophoresis separation of polysaccharides, the kit comprising (a) a container containing a gel, the gel comprising an effective amount of tris(2-hydroxyethyl)methylammonium hydroxide, i.e., THMH, and The polysaccharides include: 1) APTS-labeled Man-5 polysaccharides, and 2) one or more polysaccharides selected from APTS-labeled A1F, G0 and G0F-N.

25. The use according to claim 24, wherein the THMH is stabilized by a stabilizer.

26. The use according to claim 24, wherein the container containing the gel is a cartridge or a capillary tube.

27. The use according to claim 24, wherein the capillary has an inner lumen with uncoated walls.

28. The use according to claim 24, wherein the capillary has an inner lumen with a coated wall.

29. Use according to claim 28, wherein the coating coating the walls of the capillaries is polyvinyl alcohol.

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