Method for purification of labeled carbohydrates by free capillary electrophoresis

Abstract

The present invention relates to a method for purifying labeled glycans by electroosmotic flow assisted capillary zone electrophoresis, or a method for on-line electrokinetic purification of a labeled reaction mixture of labeled glycans and free labeling agent, wherein the method is performed in a separation mode, wherein the free labeling agent and the labeled glycans migrate in opposite directions, thereby separating the free labeling agent from the glycans. The method according to the present invention allows for a purification-free capillary zone electrophoresis analysis of labeled glycans. Furthermore, the method allows for separation in a shorter analysis time without the need for a sample purification step.

Description

Technical Field

[0001] This invention relates to a method for purifying labeled glycans by electroosmotic flow-assisted capillary zone electrophoresis, for online electrodynamic purification of a labeled reaction mixture of labeled glycans and free labeling reagent. In a specific embodiment, the method is carried out under the following conditions: i) alkaline background electrolyte conditions; ii) normal polarity separation mode. The invention also relates to the use of alkaline electrolyte compositions in the capillary zone electrophoretic separation of carbohydrates. In another embodiment, the method is carried out under the following conditions: i) acidic background electrolyte conditions; ii) reverse polarity separation mode. The invention also relates to the use of acidic electrolyte compositions in the capillary zone electrophoretic separation of carbohydrates. Background Technology

[0002] Protein glycosylation analysis is of great significance in the biomedical and biopharmaceutical fields, as well as the food and beverage industry [Saldova et al., 2007 #1; Shrivastava et al., 2022 #2; Sarkozy et al., 2023 #3]. Traditionally, after the sugar moiety is released from the polypeptide backbone of glycoproteins, they are mostly analyzed using chromatography, electrophoresis, mass spectrometry, or NMR [Mechref et al., 2002 #4; Lu et al., 2018 #5].

[0003] Capillary electrophoresis (CE) is a commonly used separation technique for analyzing complex carbohydrates. Since carbohydrates lack chromophores or fluorophores, their capillary electrophoretic analysis requires labeling the free reduced ends of the released sugars with fluorescent dyes containing primary amines, which are also charged to support proper electrophoretic migration [Mechref et al., 2002 #4]. Large quantities of labeling reagents are typically used to accelerate derivatization reactions. Therefore, a purification step using an industry-standard low-pH gel buffer system is necessary before performing CE analysis.

[0004] In addition to the extra / added sample preparation steps and associated manpower and costs, purification processes also carry the risk of losing some sample components. Since carbohydrates do not possess chromophore or fluorophore properties—as mentioned above—analysis using liquid chromatography requires a pre-separation labeling step, typically via reductive amination for fluorophore labeling [Guttman et al., 1996 #6]. To ensure rapid, high-yield derivatization, large quantities of labeling reagents are required, thus necessitating sample purification before analysis [Ruhaak et al., 2010 #7]. The most commonly used purification methods are gel filtration [Vanderschaeghe et al., 2009 #8] and solid-phase extraction (SPE), the latter including porous graphitized carbon [Broberg et al., 2007 #9] and HILIC columns [Anumula et al., 1998 #10]. Cotton-filled pipette tips have also been introduced to remove excess derivatization reagents [Selman et al., 2011 #11]. For high-throughput applications, HILIC plates [Trbojevi] are recommended. -Akma i [Szigeti et al., 2023, #12] and carboxyl-coated paramagnetic microbeads [Szigeti et al., 2016, #13]. While all these methods have been shown to be effective in removing most of the excess labeling reagent from the reaction mixture, they always require additional sample preparation steps and may result in the loss of some analyte components.

[0005] When analyzing carbohydrates using electrophoresis (CE), fluorescent labels should be charged to ensure proper electrophoretic migration of labeled glycans [Guttman et al., 1996 #14]. The most commonly used fluorescent labels in CE are 8-aminopyrene-1,3,6-trisulfonic acid (APTS) [Guttman et al., 1996 #14] and aminonaphthyltrisulfonate (ANTS) [Chiesa et al., 1993 #15], both carrying three strong negative charges over a wide pH range. In conventionally used low-pH gel buffer systems, the separation of labeled glycans in reverse polarity mode is based on their mass-to-charge ratio, unaffected by any significant influence of electroosmotic flow on the resulting differential electromigration [Liu et al., 1991 #16]. In these cases, the excess labeling reagent migrates before the labeled carbohydrates [Váradi et al., 2014 #17], so the tailing portion of the large fluorophore peak may cover some of the rapidly migrating sample components and N-glycans, making them almost undetectable.

[0006] It should also be noted that EOF-driven methods for separating carbohydrates have not been previously recommended because electroosmotic rheological conditions (i.e., EOF is weakened or eliminated) were considered necessary for effective separation, which could be achieved at low pH using negatively charged labeling agents in bare fused silica capillaries. High pH and significant EOF were considered disadvantageous due to the non-uniform spacing of homologues in highly polymerized oligosaccharides [Chiesa et al., 1993 #15]. This invention demonstrates the advantages of EOF-driven capillary zone electrophoresis separation in a radically different manner, where analyte molecules migrate in opposite directions (“reverse migration”) in an easy-to-use, purification-free workflow.

[0007] The inventors have introduced an online electro-electro-assisted purification process utilizing electroosmotic flow (EOF)-assisted separation, in which charged labeled reagents migrate against the EOF direction, opposite to that of analyte molecules migrating towards the detector. In this embodiment, the inventors used exposed fused silica capillaries, an alkaline pH background electrolyte, and a normal polarity mode. An alkaline running buffer is applied for N-glycomic analysis, eliminating the need for additional sample purification steps. The same principle can be applied to any capillary with negatively charged walls in an alkaline pH environment. Similarly, the present invention relates to capillaries with positively charged walls, and under reverse polarity, the system exhibits reverse electroosmotic flow towards the positive electrode (anode).

[0008] Therefore, the method of the present invention exhibits unexpectedly good separation performance.

[0009] The alkaline running buffer was then applied to the N-glycomic analysis of human serum samples, and the results showed excellent separation performance. More importantly, no additional sample purification steps were required.

[0010] The purpose of this invention is to provide an improved method for separating labeled carbohydrates, preferably polysaccharides, in electroosmotic-assisted capillary zone electrophoresis analysis.

[0011] According to the discovery of the present invention To achieve the above objectives, the inventors of this invention conducted systematic experimental work, resulting in this invention. This invention is based on the discovery that, in capillary electrophoresis (CE) analysis, in a separation mode where EOF drives the migration of background electrolytes and labeled carbohydrates towards the detector, while the free labeling agent migrates upstream (i.e., moves), labeled carbohydrates can be separated from the free labeling agent. Unexpectedly, the free labeling agent can be electrokinetically removed using this method.

[0012] In a particularly preferred embodiment, if CE is performed under alkaline background electrolyte conditions, capillary zone electrophoresis (CZE) analysis is preferred. In normal polarity separation mode (anode at the injection end), within the CE capillary with negatively charged inner walls, the negatively charged free labeled reagent migrates upstream (i.e., countercurrently) and can be electrokinetically removed from the mixture by applying an electric field, while the target analyte molecules migrate towards the detection zone driven by a carefully designed electroosmotic flow. Therefore, this method allows for capillary zone electrophoresis analysis of the labeled reaction mixture without purification, thus avoiding qualitative and quantitative losses of sample components during separation or purification steps.

[0013] Furthermore, the inventors have unexpectedly discovered that, compared to low-pH gel buffer separation methods used in the prior art, the method of the present invention can achieve unexpectedly shorter analysis time (preferably reduced by about 30-40%, for example, 35%).

[0014] Furthermore, this invention demonstrates the practicality of EOF-assisted capillary zone electrophoresis analysis of labeled carbohydrates, overcoming the technical deficiencies existing in the prior art. Summary of the Invention

[0015] This invention relates to the technical solutions and preferred embodiments defined in the following numbered paragraphs.

[0016] 1. A capillary electrophoresis (CE) method for separating labeled carbohydrates (preferably glycans) using electroosmotic flow (EOF) assisted separation. The carbohydrate is labeled with a labeling agent, and the method includes the following steps: - Provides a reaction mixture comprising the labeled carbohydrates and an excess of free labeling agent. - A sample of the reaction mixture is applied to the CE capillary under the following conditions. The labeled carbohydrate and the free labeling agent are both charged. Among them, EOF mobility (µ EOF ) toward the detector, Wherein, the effective migration rate (µeff) of the labeled carbohydrate and the free labeling agent is related to the migration rate (µeff) of the EOF. EOF )different, - Separation of the labeled glycan is performed via CE in a separation mode in which the EOF-driven background electrolyte (particularly used as a background electrolyte or in a separation buffer) and the labeled carbohydrate migrate toward the detector, while the free label migrates in the opposite direction.

[0017] In some respects, the µ-weight of free labeling agents eff The absolute value is higher, while the µ value of labeled carbohydrate agents is higher. eff Absolute value less than µ EOF However, free labeling agent µ eff and µ of labeled carbohydrate agents eff The direction (i.e., the algebraic symbol) and µ EOF The directions are opposite.

[0018] In a preferred embodiment, the carbohydrate is a polysaccharide with biological / biotechnological objectives.

[0019] In a preferred embodiment, the carbohydrates to be separated are oligosaccharides. In a specific method, labeled monosaccharides (if any) also migrate in the opposite direction, i.e., upstream.

[0020] A preferred embodiment of the present invention relates to the purification of labeled carbohydrates.

[0021] Preferred embodiments of the present invention relate to the isolation of labeled oligosaccharides, and more particularly to the purification of labeled oligosaccharides.

[0022] In the specification, CE is preferably capillary zone electrophoresis (CZE).

[0023] In a specific implementation, the labeling agent is a labeling dye, particularly a charged fluorescent dye; that is, a free (or unreacted) labeling agent is a free labeling dye.

[0024] 2. The CE method according to paragraph 1, wherein the carbohydrate, particularly the carbohydrate to be separated from the labeling agent, is an oligosaccharide. In a preferred embodiment, the oligosaccharide is a polysaccharide. The polysaccharide can be an O-polysaccharide or an N-polysaccharide, with an N-polysaccharide being a particularly preferred embodiment. In a preferred embodiment, the carbohydrate is a released polysaccharide, preferably an N-polysaccharide, wherein an N-polysaccharide cleaved from a glycoprotein is preferred.

[0025] In the specific implementation plan, oligosaccharides are separated from free labeling agents and labeling dyes.

[0026] In a very specific implementation, maltodextrin is separated from the labeling dye.

[0027] 3. The CE method according to paragraph 1 or 2, wherein the CE background electrolyte comprises a carrier buffer, the pH of which is set to provide electroosmotic flow toward the detector, and wherein the CE is capillary zone electrophoresis (CZE).

[0028] In specific methods, the labeling agent is a labeling dye, especially a charged fluorescent dye.

[0029] 4. A capillary zone electrophoresis (CZE) method according to paragraph 3 or any of the preceding paragraphs, for separating (e.g., purifying) labeled carbohydrates, preferably glycans. The carbohydrate is labeled with a labeling agent that carries a negative charge in its free state. The labeled carbohydrate, preferably the labeled polysaccharide, is negatively charged. The method includes the following steps: - A reaction mixture sample containing labeled polysaccharides and an excess of free labeling agent (preferably dyes, especially fluorescent dyes) is applied into a CZE capillary. - The labeled polysaccharide and the free labeling agent are separated by CZE in an alkaline pH separation buffer (under alkaline background electrolyte conditions and / or using an alkaline pH background electrolyte). In this process, an electroosmotic flow is driven from the anode (positive polarity) to the cathode (negative polarity), and the labeled carbohydrate migrates towards the cathode while the free labeling agent migrates towards the anode.

[0030] 5. The CZE method according to paragraph 4 or any of the preceding paragraphs, wherein The effective migration rate (µm) of the labeled glycan. eff Less than EOF mobility (µ) EOF Therefore, negatively charged labeled glycans exhibit positive apparent mobility (µm). app ) migrate towards the cathode, Because the µeff of the labeled dye is higher than that of the electroosmotic flow. EOF The free labeled dye will have a negative µ value app Migrate towards the anode.

[0031] Define effective mobility (µ) eff Electroosmotic migration rate, i.e., EOF migration rate (µF) EOF ) and apparent mobility (µ) app The formula for the relationship between ) is shown in Formula 1.

[0032] µ app = µ EOF + µ eff (Formula 1).

[0033] It can be seen that if µ eff If it is negative, then µ app It is also a negative value, and its absolute value is higher than µ. EOF This is true for charged, free labeling agents (dyes), but for analytes (labeled carbohydrates or glycans), although µ... effIt is a negative value (i.e., the same as µ). EOF Conversely), but its absolute value is lower than µ. EOF Therefore, they will migrate towards the detector.

[0034] The present invention also relates to an electroosmotic flow-assisted electrodynamic purification method for separating labeled carbohydrates (preferably polysaccharides) and negatively charged labeling agents (preferably dyes, especially fluorescent dyes).

[0035] in, - Provide a reaction mixture comprising labeled carbohydrates and an excess of free labeling agent. - Excess free labeling agent is separated from labeled carbohydrates by capillary zone electrophoresis (CZE) in alkaline separation buffer (alkaline background electrolyte conditions / using alkaline pH background electrolyte) and normal polarity separation mode. In this process, the electroosmotic flow is driven from the anode (positive polarity) to the cathode (negative polarity). The labeled carbohydrates migrate towards the cathode under the drive of the EOF, while the free labeling agent migrates towards the anode, in the opposite direction to the electroosmotic flow.

[0036] 6. The method according to any one of paragraphs 4 to 5 or any one of the preceding paragraphs, wherein the pH of the alkaline background electrolyte is >7, preferably 7 to 9, and more preferably 7.5 to 8.5.

[0037] 7. The method according to any one of paragraphs 4 to 6, wherein the alkaline background is a composition comprising a nitrogen-containing base and a carboxylic acid coion, wherein the nitrogen-containing base is preferably an amine buffer, and the carboxylic acid coion is preferably a C4-10 carboxylic acid, preferably a C4-10 straight-chain carboxylic acid.

[0038] The molecular weight or size of anion (e.g., carboxylic acid) can be set according to the size of the analyte, depending on the position where we want to obtain higher resolution.

[0039] As for alkalis, they can be of several types, as long as they have sufficient buffering capacity and are effective at a given pH.

[0040] 8. The method according to paragraph 7, wherein the background electrolyte comprises 5 to 9 parts of an amine buffer and 1 to 5 parts of a carboxylic acid, preferably Tris and hexanoic acid; highly preferably, the alkaline background electrolyte is a composition comprising Tris and hexanoic acid, preferably 5 to 9 parts of Tris and 1 to 5 parts of hexanoic acid.

[0041] 9. The method according to any one of paragraphs 4 to 8, wherein the CZE capillary is a fused silica capillary.

[0042] For those skilled in the art, other types of capillaries (e.g., glass, plastic) can also be used to form EOF.

[0043] 10. The method according to any one of paragraphs 4 to 9 or any one of the preceding paragraphs, wherein the labeling agent is a fluorescent dye.

[0044] 11. The method according to paragraph 10, wherein the fluorescent dye labeling agent is a negatively charged sulfonate of an aromatic amine, preferably. Sulfonates of aromatic hydrocarbons having fused rings, preferably with two, three, or four benzene rings, preferably with one to four sulfonates, preferably with two to three sulfonates, and especially preferably with three sulfonates. Preferably, the fluorescent dye labeling agent has a primary amine group capable of forming a Schiff base with carbohydrates.

[0045] Other labeling agents include, for example, hydrazine derivatives.

[0046] Other specific markers include clickable instant markers.

[0047] In addition, markers with negative and positive charges are described.

[0048] Such reagents are described, for example, in Guttman A and Hajba L. 2022 #28 and Palaniappan KK et al., 2016 / 28.

[0049] 12. The method according to any one of paragraphs 1 to 9, wherein, The carbohydrates are N-glycans cleaved from glycoproteins. The buffer solution is an alkaline buffer solution with a pH of 7.5 to 8.5. The labeling agent is an aromatic sulfonate having a primary amine group capable of forming a Schiff base with carbohydrates, preferably aminopyrene trisulfonate (APTS) or aminonaphthalene trisulfonate (ANTS).

[0050] 13. The capillary electrophoresis (CE) method according to any one of paragraphs 1 to 4, for separating (e.g., purifying) labeled carbohydrates, preferably glycans, The carbohydrate is labeled with a labeling agent that is positively charged in its free state, and the method includes the following steps: - A reaction mixture containing labeled glycans and an excess of free labeling agent (preferably a dye, especially a fluorescent dye) is applied into a CE capillary with positively charged walls. - The labeled glycan and the free labeling agent are separated by CE in an acidic pH separation buffer (under acidic background electrolyte conditions and / or using an acidic pH background electrolyte). In this process, within the positively charged capillary wall, electroosmosis flows from the cathode (negative electrode) to the anode (positive electrode). Positively charged labeled carbohydrates with greater EOF mobility move toward the anode (i.e., migrate), while free labeling agents move toward the cathode.

[0051] The present invention also relates to an electroosmotic flow-assisted electrodynamic purification method for separating labeled carbohydrates (preferably polysaccharides) and negatively charged labeling agents (preferably dyes, especially fluorescent dyes).

[0052] in - Provide a reaction mixture comprising labeled carbohydrates and an excess of free labeling agent. - Excess free labeling agent is separated from labeled carbohydrates by capillary zone electrophoresis (CZE) in alkaline separation buffer (alkaline background electrolyte conditions / using acidic pH background electrolyte) and reverse polarity separation mode. In this process, within the positively charged capillary wall, electroosmotic flow is driven from the cathode (negative polarity) to the anode (positive polarity). The labeled carbohydrates migrate towards the anode under the drive of the EOF, while the free labeling agent migrates towards the cathode, in the opposite direction to the EOF.

[0053] In a preferred embodiment, the pH is 2 to -7, preferably 3 to 6.

[0054] 14. The CZE method according to paragraph 13, wherein the... The effective migration rate (µeff) of labeled glycans is less than the EOF migration rate (µeff). EOF Therefore, positively charged labeled glycans migrate toward the anode with a positive apparent mobility µapp. Because the µeff of the labeled dye is higher than that of the electroosmotic flow. EOF The free labeled dye will migrate toward the cathode with a negative µapp (see Formula 1).

[0055] 15. The method according to any one of paragraphs 1 to 14, wherein no additional purification step is required before applying the mixture comprising the labeled polysaccharide and an excess of free labeling agent (preferably a dye, especially a fluorescent dye) to the CZE capillary.

[0056] 16. Applications of alkaline CE background electrolyte and carbohydrate labeling reagents in the separation of free labeling reagents and labeled carbohydrates by capillary electrophoresis (CE) under alkaline capillary electrophoresis (CE) conditions. In methods for separating (e.g., purifying) labeled carbohydrates, preferably glycans, wherein The carbohydrate is labeled with a labeling agent that carries a negative charge in its free state. The labeled carbohydrate, preferably the labeled polysaccharide, is negatively charged. Preferably, the method includes the following steps: - A reaction mixture sample containing labeled polysaccharides and an excess of free labeling agent (preferably dyes, especially fluorescent dyes) is applied into a CZE capillary. - The labeled polysaccharide and the free labeling agent are separated by CZE in an alkaline pH separation buffer (under alkaline background electrolyte conditions / using alkaline pH background electrolyte). In this process, an electroosmotic flow is driven from the anode (positive polarity) to the cathode (negative polarity), and the labeled carbohydrate migrates towards the cathode while the free labeling agent migrates towards the anode.

[0057] 17. The use according to paragraph 11, wherein the pH is 7 to 10, preferably 7 to 9, and more preferably 7.5 to 8.5.

[0058] 18. Applications of separating free labeled reagents and labeled carbohydrates by capillary electrophoresis (CE) under acidic CE background electrolytes and carbohydrate labeling conditions. In methods for separating (e.g., purifying) labeled carbohydrates, preferably glycans, wherein The carbohydrate is labeled with a marker that carries a positive charge in its free state. The labeled carbohydrate, preferably the labeled polysaccharide, is positively charged. Preferably, the method includes the following steps: - A reaction mixture sample containing labeled polysaccharides and an excess of free labeling agent (preferably dyes, especially fluorescent dyes) is applied into a CZE capillary. - The labeled polysaccharide and the free labeling agent are separated by CZE in an acidic pH separation buffer (under acidic background electrolyte conditions and / or using alkaline pH background electrolyte). In this process, electroosmotic flow occurs from the cathode (negative polarity) to the anode (positive polarity), with the labeled carbohydrate migrating towards the anode and the free labeling agent migrating towards the cathode.

[0059] 19. The use as described in paragraph 18, wherein the pH is 2 to 7, preferably 3 to 6.

[0060] The invention also relates to any one of paragraphs 16 to 19, wherein the conditions are as defined in any one of paragraphs 1 to 15.

[0061] The present invention also relates to the use of any one of paragraphs 16 to 19 for the preparative separation of carbohydrates.

[0062] The present invention also relates to any one of paragraphs 16 to 19 for the analysis and separation of carbohydrates.

[0063] According to any one of paragraphs 16 to 19, in a high-throughput method, multiple CZE capillaries are preferably used in parallel.

[0064] According to any one of paragraphs 16 to 19, in the application, the target sample component enters the separating capillary, while Excess labeling agent is excluded, therefore it does not interfere with labeled carbohydrates, or Excess labeling agent is applied to the capillary and migrates toward the anode.

[0065] According to the above implementation scheme, an excess of the labeling agent is applied to the capillary and migrates towards the cathode.

[0066] 20. The use according to any one of paragraphs 16 to 19, Used for analyzing carbohydrate purification processes, for example - For example, HILIC columns used for HPLC analysis, or - Sample loss in the CE-MS method.

[0067] 21. A kit for carbohydrate analysis, the kit comprising: - A negatively charged marker for free carbohydrates. - Alkaline electrolyte buffer, and optionally - A CZE capillary with negatively charged walls, preferably a bare fused silica capillary. -or - Positively charged free carbohydrate markers, - Acidic electrolyte buffer, and optionally - CZE capillary with positively charged walls.

[0068] In the implementation scheme, the free carbohydrate labeler is a reagent as defined in any of paragraphs 1 to 12 above, or any of paragraphs 13 to 14 above.

[0069] In the implementation scheme, the alkaline electrolyte buffer is defined as in any of paragraphs 1 to 15 above, or any of paragraphs 16 to 17 above. Attached Figure Description

[0070] Figure 1 A schematic diagram of the electric cleaning process, showing the apparent electrophoretic mobility (µm). app) and effective electrophoretic mobility (µ) eff ), while also considering the downstream electroosmotic flow migration rate (µ). EOF ).

[0071] Figure 2 EOF-assisted capillary zone electrophoresis analysis of APTS-labeled maltodextrin (top panel) and sugar-free control derivatized reaction mixtures (bottom panel). The inset shows the magnified portion of DP 5–25. Conditions are detailed in Example 1.

[0072] Figure 3 EOF-assisted capillary zone electrophoresis analysis of APTS-labeled malt oligosaccharides at pH 8.1. Scans: a – unpurified; b – purified; c – first wash buffer; d – second wash buffer; e – third wash buffer. The numbers above the peaks indicate the number of glucose units in the oligomer. See Example 1 for details of the conditions.

[0073] Figure 4 EOF-assisted capillary zone electrophoresis analysis of IgG N-glycans released by PNGase F and APTS-labeled IgG N-glycans. Scans: a – unpurified; b – purified; c – first wash buffer; d – second wash buffer; e – third wash buffer. See Example 1 for details of conditions.

[0074] Figure 5 EOF-assisted capillary zone electrophoresis analysis of PNGase F-released ribonuclease B N-glycan and APTS-labeled ribonuclease B N-glycan. Scans: a – unpurified; b – purified; c – first wash buffer; d – second wash buffer; e – third wash buffer. See Example 1 for details of conditions.

[0075] Figure 6 EOF-assisted capillary zone electrophoresis analysis of PNGase F-released fetoglobin N-glycan and APTS-labeled fetoglobin N-glycan. Scans: a – unpurified; b – purified; c – first wash buffer; d – second wash buffer; e – third wash buffer. See Example 1 for details of conditions.

[0076] Figure 7Capillary electrophoresis analysis of human serum N-glucose released by PNGase F and APTS-labeled human serum N-glucose. Figure A: Standard low-pH separation gel-buffer system after purification (reference method); Figure B: EOF-assisted CZE separation using online electrokinetic sample purification in an alkaline pH background electrolyte (method of this invention). Table 1 shows the corresponding structures of the numbered peaks. The insets in the two figures depict the complete separation trajectories with and without sample purification, respectively. Conditions: (A) 30 cm effective length (40 cm total length, 30 μm inner diameter, 365 μm OD) BFS capillary; HR-NCHO low-pH gel buffer system, separation voltage: 30 kV (reverse polarity); capillary temperature: 20°C; injection: 2 psi for 5 seconds with a water plug and 5 kV for 5 seconds with sample, polarity reversed; (B) Same as Example 1.

[0077] Abbreviations: BGE = Background Electrolyte CE = Capillary electrophoresis; CZE = Capillary Zone Electrophoresis; EOF = Electroosmotic flow; BFS = Bare Fused Quartz; DP = Degree of Aggregation; APTS = 8-aminopyrene-1,3,6-trisulfonic acid; ANTS = 8-aminonaphthalene-1,3,6-trisulfonic acid; HILIC = Hydrophilic Interaction Liquid Chromatography; HPLC: High Performance Liquid Chromatography; IgG = Immunoglobulin G.

[0078] OD = Optical Density definition In this document, the term "separation" of a target compound (including one or more compounds, such as analytes) from one or more other compounds refers to the removal of certain components, i.e., "other compounds," from a mixture, thereby separating the target compound from the removed portion. Preferably, in separating the target compound from other compounds, the target compound is present in the mixture before separation and is free of other compounds in the mixture after separation. The fewer other compounds contained in the desired compound, the more successful the separation.

[0079] In a preferred embodiment or preferred step, the desired compound is a labeled oligosaccharide, preferably a labeled polysaccharide; in this case, “separating” the labeled oligosaccharide (preferably a labeled polysaccharide) means separating the oligosaccharide (e.g., a polysaccharide) from other compounds, preferably from free, unreacted labeling agents in a labeled reaction mixture or sample, and possibly also from other non-target compounds that react with the labeling agent but are different from the target compound; in a particular embodiment, the labeled monosaccharide or a fragment thereof.

[0080] Separation techniques are methods for separating a desired compound from one or more other compounds, wherein the method utilizes differences in the physical and / or chemical properties of the desired compound and one or more other compounds, preferably utilizing differences in the electrophoretic mobility of the compounds.

[0081] In this article, "purifying the target compound from a mixture (e.g., a reaction mixture)" is understood to mean that, after a purification step, the target compound is provided in higher purity, i.e., the resulting mixture contains the target compound and contains fewer other compounds.

[0082] As used herein, “sample” refers to a substance comprising a mixture of compounds prepared for separation (e.g., for analysis by capillary electrophoresis). For example, a sample may be derived from a substance obtained, for example, from an environmental source, such as bodily fluids or tissues of a subject, and optionally processed for analysis. Samples used herein preferably contain carbohydrates, and more preferably polysaccharides of biological or biotechnological interest, as defined, described, or illustrated herein.

[0083] The sample is typically a reaction mixture or a portion thereof, containing the carbohydrate (preferably an oligosaccharide) and a labeling agent. The labeling agent used herein is a charged molecule, for example, positively or negatively charged, wherein preferably, the labeled carbohydrate becomes charged, for example, positively or negatively charged, upon reaction with the labeling agent.

[0084] In a broad sense, "carbohydrates" refers to carbohydrates with the chemical stoichiometry C. n (H2O) n Compounds or derivatives thereof, for example, having a major moiety (preferably comprising at least 30%, 50%, 70%, or 80% of the total molecular weight of the compound) and having a stoichiometric formula C n (H2O) n"Carbohydrate" is preferably an aldose or ketose. The term carbohydrate includes monosaccharides, oligosaccharides, and polysaccharides, as well as substances derived from monosaccharides by reducing a carbonyl group (aldolol), oxidizing one or more terminal groups to a carboxylic acid, or replacing one or more hydroxyl groups with hydrogen atoms or substituents (especially substituents with a molecular weight of at most monosaccharides, such as functional groups, particularly amino, thiol, or similar groups). It also includes derivatives of these compounds.

[0085] In a sense, the terms “polysaccharide,” “oligosaccharide,” and “polysaccharide” can be used interchangeably in this article, referring to carbohydrates that contain or have multiple monosaccharide units, preferably linked by glycosidic bonds.

[0086] The narrow definitions of "polysaccharide", "oligosaccharide" and "polysaccharide" can be found in the IUPAC Chemical Terminology Compendium (IUPAC, 2019, #30).

[0087] In a preferred embodiment, "polysaccharide" refers to a polysaccharide of biological or biotechnological interest.

[0088] Broadly speaking, glycans can comprise monosaccharides. In a preferred embodiment, the term "glycan" refers to the carbohydrate portion of a glycocomplex (e.g., glycoprotein, glycolipid, or proteoglycan). Glycans can be homopolymers or heteropolymers of monosaccharide residues (comprising either a single type or multiple types of monosaccharide residues). Glycans can be linear or branched. Glycans are typically N-linked, O-linked, or glycosaminoglycans.

[0089] The term “CATRIS” as used herein is understood to mean a composition of hexanoic acid and Tris in an alkaline pH range as defined herein (e.g., the content of this invention). In a particularly preferred embodiment, CATRIS comprises 150 mM hexanoic acid and 253 mM Tris (pH 8.1). The terms “Tris” and “Tris-base” as known to those skilled in the art refer to 2-amino-2-(hydroxymethyl)propane-1,3-diol.

[0090] "Capproic acid" and "hexanoic acid" are interchangeable terms, well known to those skilled in the art, referring to carboxylic acids derived from hexane with the chemical formula CH3(CH2)4COOH.

[0091] As used herein, "fluorescent" or "fluorescently labeled" compounds refer to compounds that can be detected by exposure to ultraviolet or visible electromagnetic radiation, which absorb the irradiation light and emit light with a wavelength longer than the irradiation light (emission light). According to the invention, preferably, both the target "fluorescently labeled" compound and the fluorescent labeling agent are charged, which can be positive or negative; in a particularly preferred embodiment, they are negatively charged.

[0092] In particular, the absolute value of the effective electrophoretic mobility of the fluorescent labeling agents used in this invention is higher than that of the effective electrophoretic mobility of "fluorescently labeled" compounds (especially glycans) because their algebraic signs are opposite.

[0093] In this article, "detection" is used in a broad sense to refer to the observation results obtained as a result of a separation method on a sample, which relates to a target substance or compound (preferably an analyte).

[0094] A detector is a device used for detection, typically located at a specific position in the CE capillary, to detect target compounds migrating within it.

[0095] In this document, the term “comprises” (or “comprising” or “including”) should be interpreted as having a non-exhaustive meaning and allowing for the addition or incorporation of further features or method steps or components to anything that includes the listed features or method steps or components. “Comprising” may be replaced with “including” if the practice of a given language variant requires it; and if other members or components are not essential to carrying out the invention, it may be limited to “consisting primarily of”.

[0096] The singular form “a (a, an, the)” or at least “a (a, an)” includes plural reference unless the context clearly indicates otherwise. Detailed Implementation

[0097] In this invention, during capillary electrophoresis, preferably capillary zone electrophoresis analysis of labeled carbohydrates, appropriate separation conditions are applied to prevent unreacted labeling agents from entering the separation capillary, while simultaneously supporting EOF-assisted target sample components (which have a slower reverse migration rate) to migrate downstream electrophoretically, thereby skipping the sample purification step.

[0098] Electroosmosis plays a crucial role in this invention. This phenomenon is caused by the surface charge on the capillary wall. For example, fused silica capillaries have silanol groups on their inner surface. The degree of ionization of the silanol groups depends on the pH and ionic strength of the background electrolyte, which can be controlled by running a buffer solution.

[0099] Electroosmotic flow (EOF) can be described by the following formula (Formula 2). (Formula 2) in, Let η be the dielectric constant, η be the viscosity of the buffer solution, and ζ be the zeta potential. The charged walls of the capillary attract ions with opposite charges from the background electrolyte, thus forming an electrical double layer. If a voltage is applied to the capillary, ions in the electrolyte portion (i.e., the diffuse portion of the double layer) will migrate towards the cathode, carrying water molecules. If the capillary wall is negatively charged, the buffer solution will flow net towards the negative electrode; if the capillary wall is positively charged, the buffer solution will flow net towards the positive electrode.

[0100] The zeta potential (ξ) is defined as the potential at the interface of the electrical double layer, or as the potential of the diffused layer at a finite distance from the capillary wall. The zeta potential depends on the thickness of the electrical double layer, where a lower ionic strength in the buffer solution leads to a thicker electrical double layer and / or a larger charge density on the capillary wall, both of which result in an increased zeta potential and enhanced electroosmotic flow.

[0101] Electroosmotic flow is typically pH-dependent. In bare fused silica (BFS) capillaries, the formation of silanol groups is pH-dependent. Therefore, at high pH, ​​the capillary surface carries a greater negative charge, resulting in higher electroosmotic flow; while at low pH, the electroosmotic flow is much lower. When the pH is less than 2, the groups are protonated, and the zeta potential and electroosmotic flow decrease. In the prior art, this range is considered ideal for carbohydrate analysis.

[0102] The opposite is true for positively charged capillary walls (reverse polarity EC or CZE). These capillaries are typically coated with adsorbed or covalently positively charged coatings and exhibit reverse electroosmotic flow toward the positive electrode (anode). Reverse polarity CZE can be used for the analysis of cation analytes. [Tim Wehr et al., 2004 #25] For example, positively charged polymers such as polyethyleneimine (PEI) can be covalently attached to the inner wall of a capillary, thereby reversing the charge of ions on the capillary wall. (Santos, Marcia R et al., (26))

[0103] Various capillary coating methods are described, for example in Hajba, L. et al., 2017 #27 or Guttman A and Hajba L. 2022 #28 (in Palaniappan KK et al., 2016 / 28).

[0104] In addition, markers with negative and positive charges are described.

[0105] In the implementation scheme, during capillary zone electrophoresis analysis of APTS-labeled carbohydrates, appropriate separation conditions are applied to prevent unreacted labeling agents from entering the separation capillary, while simultaneously supporting the downstream migration of EOF-assisted target sample components (which have a slower reverse electrophoretic migration rate), thus skipping the sample purification step. The applicability of this method was first evaluated using a maltodextrin ladder, then applied to the analysis of human serum N-glycans, and compared with conventionally used low-pH gel buffer separation methods.

[0106] Considering the downstream electroosmotic flow (µL) in the normal polarity separation mode of the BFS column EOF ) and the reverse effective migration (µ) of sample components including free fluorophores. eff This should allow us to deduce which molecules will move towards the downstream detector window and which will not even enter the separation capillary. Of particular note is the excess of high-mobility, triple-charged APTS dyes.

[0107] Therefore, in a particularly preferred embodiment, if CE analysis is performed, it is carried out under alkaline background electrolyte conditions, in a normal polarity separation mode, within a CE capillary with a negatively charged inner wall. In this case, the negatively charged free labeling agent that migrates upstream (i.e., migrates in the opposite direction to the electroosmotic flow) is removed electrodynamically, while the target analyte molecules migrate towards the detection zone driven by a carefully designed electroosmotic flow. By applying an electric field, the free labeling agent migrates out from the capillary inlet side (e.g., in the case of pressure injection) or does not enter the capillary (e.g., in the case of electrodynamic injection).

[0108] Therefore, this method can perform capillary zone electrophoresis analysis of labeled reaction mixtures without purification, thereby avoiding qualitative and quantitative loss of sample components during purification steps.

[0109] In addition to the extra / added sample preparation steps and the associated manpower and costs, the purification process also carries the risk of losing some sample components, as shown in Examples 2 to 6.

[0110] Apparent mobility (µm) of each sample component app ) is the EOF mobility (µ) EOF ) and the effective migration rate (µ) of analyte molecules in reverse migration eff The algebraic sum of , as shown in Formula 1.

[0111] µ app =µ EOF +µ eff (Formula 1) EOF mobility is a parameter that needs to be carefully controlled in CE. In this invention, by setting conditions, derivatized carbohydrates and derivatizing reagents are made to migrate (i.e. move), so that the derivatized carbohydrates migrate toward the detector in the capillary at an apparent mobility, while the free derivatizing reagents migrate in the opposite direction, "countercurrent" or upstream. Their effective mobility (in absolute terms) is higher than that of electroosmotic mobility and in the opposite direction (negative), thereby producing an apparent mobility pointing toward the inlet (opposite to the detector).

[0112] The term "migration" (or movement) includes the ability to "migrate" (or move). For example, in the case of electro-injection, this property of free labeled agents can result in upstream or reverse apparent migration rates, potentially preventing them from entering the capillary.

[0113] Therefore, the electroosmotic flow is driven toward the detector, and the charged labeled carbohydrates are carried toward the detector by a greater EOF migration rate (i.e., migration), while the free labeling agent (with an effective migration rate greater than the EOF migration rate and in the opposite direction) will migrate in the opposite direction (or at least be able to migrate).

[0114] It is recommended to select a suitable buffer solution based on the separation method. Typically, in normal polarity separation mode, a negatively charged capillary wall (e.g., a bare fused silica capillary) is applied, with a separation pH above 7, preferably above 7.5, or preferably 7 to 10, particularly 7.5 to 9, more preferably 7.6 to 8.6, especially preferably around 8, for example 8.1, depending on the pKa of the BGE buffer component. Once the buffer solution is selected, the ionic strength of the background electrolyte is set according to the labeling agent and the labeled carbohydrate to obtain a suitable EOF mobility to ensure the µ-value of the free labeling agent. eff The absolute value of is higher than that of electroosmotic flow and vice versa, i.e., it is the same as µ. EOF The algebraic signs are opposite, which makes the apparent mobility (µm) of the free labeling agent and each sample component increase when an electric field is applied during the analysis. app The sample components are driven toward the detector by electroosmotic flow, while the free labeling agent migrates in the opposite direction or does not enter the capillary.

[0115] Therefore, as a first approximation, the inventors envision that all target sample components with an effective mobility less than EOF will be brought toward the detector window, such as... Figure 1 As shown. Therefore, due to the µ-value of the charged labeling dye... eff Above µ EOF , its µ app This will be a negative value, indicating migration from the capillary inlet outwards. Note that at higher buffer concentrations, µ... EOF Decrease, µ eff It may decrease accordingly, but the change is not µ EOFThe concentration and composition of the buffer system are designed accordingly because the variations are large.

[0116] Figure 1 The theoretical basis of an embodiment of the invention is illustrated, wherein a negatively charged fused silica capillary is used in normal polarity mode. The charged labeling dyes used herein must be negatively charged, such as APTS.

[0117] In the case of a positively charged capillary wall, a positively charged labeling agent is applied.

[0118] In addition, the mobility of the common ion buffer component in the background electrolyte should be matched as closely as possible to the mobility of the sample component to mitigate potential peak efficiency loss mediated by electromigration dispersion (i.e., asymmetry-based loss) [Mikkers et al., 1979 #20].

[0119] In a specific embodiment, when using negatively charged walls (e.g., BFS capillaries), the inventors unexpectedly discovered, in normal polarity mode, that Tris exhibits excellent buffering capacity over an alkaline pH range (particularly at pH 8.1), while hexanoic acid, as a matching mobility co-ion, clearly meets all the aforementioned criteria. In a specific embodiment, Tris and hexanoic acid are used in a composition of 150 mM hexanoic acid and 253 mM Tris (CATRIS).

[0120] Those skilled in the art will understand that, in addition to Tris, other buffers that function in the alkaline pH range and other alkyl acids, particularly C4-C10 alkyl acids, are also useful in this invention as a means of providing a co-ionic alkaline environment.

[0121] Based on the above considerations, the inventors have discovered that the presence of electroosmotic flow at alkaline pH in normal polarity mode (anode on the injection side) can reverse the migration sequence of negatively charged sample components. The use of alkaline pH in the art is considered disadvantageous because it produces higher EOF, while lower pH reduces or eliminates EOF, which, if necessary, is essential in the art for high-quality separation of fluorescently labeled carbohydrates, along with masking (e.g., by TEA). However, the present invention enables the selection of separation conditions such that charged (preferably multi-charged, especially tri-charged) fluorophore-labeled dyes pass through the capillary at the slowest possible rate, or even not enter the capillary at all. To utilize this concept, the inventors disclose herein an electroosmotic flow-assisted online electrodynamic purification method for removing negatively charged fluorophore-labeled carbohydrates and negatively charged labeled dyes, such as APTS-labeled carbohydrates, using an alkaline pH background electrolyte. The mobility difference between the electroosmotic flow and the countercurrent differential electromigration of the sample components allows target analyte molecules to pass through the separation capillary and reach the detector. Therefore, excess labeling agents with high electrophoretic mobility do not even enter the capillary and thus do not interfere with the analysis.

[0122] As a first example, the applicability of this method was evaluated using a maltodextrin ladder, and then applied to the analysis of human serum N-glycans, compared with conventional low-pH gel buffer separation methods. Analysis of the labeled maltodextrin (see Example 2) showed that the 150 mM hexanoic acid-253 mM Tris (CATRIS, pH = 8.1) background electrolyte composition worked as expected; that is, the target countercurrent-efficient electrophoretic mobility sample fraction (DP>2) was carried downstream of the separation capillary with EOF assistance, while unreacted labeling did not enter the column. Therefore, it did not interfere with the analysis. This simple online procedure allows for the skipping of time-consuming and expensive sample purification steps.

[0123] Furthermore, the inventors investigated (see Examples 3 to 6) the purification process steps following APTS labeling of carbohydrates to elucidate potential qualitative or quantitative separation chromatographic biases due to the washing away of sample components during the cleanup process. Capillary zone electrophoresis using an alkaline pH Tris-hexanoic acid (CATRIS) background electrolyte allows for sample analysis without interference from any residual fluorescent labeling agent in the reaction mixture. Sample purification leverages the high EOF and the contrasting high and low effective mobilities of free APTS and labeled sample components.

[0124] Alternative labeling agents are entirely within the skill of those skilled in the art; other embodiments include 2-aminonaphthalenetrisulfonic acid (ANTS) and 2-aminoacridone. Other variants are described in Ruhaak LR et al., (2010) #31 and Guttman A and Hajba L. (2022) #31 and the references cited therein.

[0125] In some embodiments, such as for relatively large glycans, the inventors have found that the method according to the invention can shorten the analysis time. For example, in analyzing human serum N-glycans released by PNGase F and APTS-labeled human serum N-glycan samples, a reduction of approximately 30-40%, preferably approximately 35%, was achieved compared to the low-pH gel buffer separation method used in the prior art.

[0126] This conceptually different separation modality allows for the revelation of the quantity and type of sample components significantly lost during sample purification, resulting in higher-quality separation. Analysis of the maltodextrin ladder diagram clearly shows a bias towards smaller oligosaccharides in the quantitative analysis. Analysis of APTS-labeled N-glycan libraries of several well-characterized glycoproteins (IgG, ribonuclease B, and fetoglobulin) also unexpectedly revealed qualitative and quantitative losses of sample components during the washing step, as confirmed by analysis of the wash buffer. Furthermore, while EOF-driven separation has previously been discouraged due to uneven peak spacing at higher DP levels, the data presented here conversely demonstrate the advantages of EOF-driven capillary band electrophoresis separation over separately reverse-migrating analytes and residual labeled molecules in an easy-to-use purification-free workflow.

[0127] Using alkaline pH capillary zone electrophoresis mode allows for the analysis of labeled reaction mixtures without purification, which was previously impossible with acidic gel buffer systems. Therefore, the entire purification process, including wash waste, can be investigated. This method utilizes the opposite migration of free labeled dye and target labeled analyte molecules mediated by carefully designed electroosmotic conditions within a normal polar electrophoretic separation apparatus. In other words, the countercurrent electrophoretic migration rate of negatively charged free APTS dye and the countercurrent electrophoretic migration rate of APTS-labeled target sample components are greater than and less than the electroosmotic migration rate, respectively. When this operation is performed in normal polar separation mode, free APTS migrates out from the capillary inlet side under an applied electric field, while the labeled target analyte molecules, despite undergoing opposite electromigration due to their negative charge, are still carried towards the detection zone by the high electroosmotic flow. After APTS labeling of maltodextrin ladder and N-glycans released from several standard glycoproteins (including IgG, ribonuclease B and fetoglobulin, representing complex, high-mannose and highly sialylated carbohydrate types, respectively), each step of the purification process (including the washing step) was carefully examined by CZE analysis under alkaline pH conditions (see Examples 4 to 6).

[0128] After demonstrating the applicability of the method using a maltodextrin ladder, CATRIS buffer was applied to the analysis of APTS-labeled human serum N-glycans. The results were compared with a conventional low-pH gel buffer separation method (as a reference method). The two methods were comparable in the number of separated peaks, but the use of a high-pH CATRIS buffer shortened the analysis time (see Example 7).

[0129] It should be noted that the method according to the invention is also advantageous for identifying sample losses that may occur during other commonly used carbohydrate purification processes, such as HILIC columns used for HPLC analysis, where the reverse migration of labeled dyes and labeled sample components cannot achieve online sample purification.

[0130] Based on the above, a first aspect of the present invention is a method for purification-free electroosmotic flow-assisted capillary zone electrophoresis analysis of labeled glycans, or for online electrodynamic purification of a labeled reaction mixture of labeled glycans and a free labeling agent, wherein the method is carried out in the following mode: the labeled glycans are carried to the detector by electroosmotic flow, while the free labeling agent migrates countercurrently (i.e., upstream).

[0131] In a specific implementation, the method is carried out under the following conditions: alkaline background electrolyte conditions; normal polarity separation mode.

[0132] In an embodiment of this method, the pH of the alkaline background electrolyte is 7-9, preferably 8.1.

[0133] In an embodiment of this method, the alkaline background electrolyte is a composition of Tris and hexanoic acid.

[0134] In a preferred embodiment of the method, the alkaline background electrolyte is a composition consisting of 253 mM Tris and 150 mM hexanoic acid.

[0135] In a preferred embodiment of the method, the polysaccharide is an N-polysaccharide.

[0136] A second aspect of the invention is a background electrolyte composition for capillary zone electrophoresis analysis comprising Tris and hexanoic acid.

[0137] In embodiments of the background electrolyte composition, the pH value of the background electrolyte composition is 7-9, preferably 8.1.

[0138] In a preferred embodiment of the background electrolyte composition, the alkaline background electrolyte is a composition consisting of 253 mM Tris and 150 mM hexanoic acid.

[0139] The present invention is further illustrated by the following non-limiting embodiments. Those skilled in the art will understand that other embodiments or variations thereof may also achieve the objectives of the present invention based on these embodiments.

[0140] Example Materials used in the examples: Human IgG, ribonuclease B, bovine placental protein, sodium cyanoborohydride (1 M THF solution), 8-aminopyrene-1,3,6-trisulfonic acid, and dithiothreitol were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Hexanoic acid, Tris base, glycerol, glacial acetic acid, acetonitrile, sodium dodecyl sulfate (SDS), and tetrahydrofuran (THF) were purchased from VWR Chemicals BDH (Radino, Pennsylvania, USA). PNGase F enzyme was developed in-house by the University of Pannonia (Westsplem, Hungary), as described in [Kovács et al., 2022 #18]. Agencourt CleanSEQ paramagnetic cleaning beads were from Bio-ScienceKft (Budapest, Hungary). M040 maltodextrin ladders were generously provided by Grain Processing Co. (Muscatine, Iowa), and 30 μm ID (365 μm OD) fused silica capillaries were from Polymicro Technologies (Phoenix, Arizona, USA). The carbohydrate separation gel buffer (HR-NCHO) commonly used in the reference methods known in the prior art is from Bio-Science Kft (Budapest, Hungary).

[0141] Example 1: General Method Preparation of caprylic acid-Tris (CATRIS) running buffer Add 15 mL of 1 M Tris base stock solution and 1.875 mL of hexanoic acid to 60 mL of HPLC-grade water in a 200 mL beaker and mix thoroughly. Then, adjust the pH to 8.10 with 1 M Tris base stock solution while continuously stirring. Finally, bring the volume of the mixture to 100 mL with HPLC-grade water in a volumetric flask to obtain a final concentration of 150 mM hexanoic acid and 253 mM Tris (CATRIS). Then, filter the prepared buffer solution through a 0.2 μm membrane and sonicate to remove any residual air bubbles that may interfere with the electrophoresis process.

[0142] PNGase F-mediated carbohydrate release and fluorescent labeling First, 10 μL of IgG, ribonuclease B, and fetoglobulin (each at 1 mg / mL stock solution) were denatured by adding 4 μL of denaturing solution (400 mM DTT and 5% SDS) and incubated at 70°C for 15 min. This step was then followed by adding 2 μL of PNGase F (1.35 mg / mL in 50% glycerol) and incubating at 37°C for 2.0 h. The released oligosaccharides and 10 µL of 1 mg / mL maltodextrin ladder were fluorescently labeled by adding 20 µL of a reducing amination reaction mixture containing 1 µL of 20 mM APTS, 10% 1 M sodium cyanoborohydride (1 M in THF), 15% HPLC-grade water, 35% glacial acetic acid, and 40% tetrahydrofuran, and then incubated at 60°C for 1 h using the evaporative derivatization protocol described in [Reider et al., 2018 #19]. Then, 100–100 µL of HPLC-grade water was added to the dried labeled reaction mixture, and the sample was analyzed by electroosmotic CZE as described in [Váradi et al, 2014 #17] before and after purification with Agencourt CleanSEQ paramagnetic beads.

[0143] Conditions for EOF-assisted capillary zone electrophoresis Capillary electrophoresis analysis was performed on a PA800 Plus instrument (Beckman Coulter, Brea, California) using laser-induced fluorescence detection (excitation wavelength 488 nm, emission wavelength 520 nm) and bare fused silica (BFS) capillaries with an effective length of 30 cm (total length 40 cm) and an ID of 30 μm (OD of 365 μm). Samples were analyzed in CZE mode using a 253 mM Tris-150 mM hexanoic acid (pH 8.1) background electrolyte. A potential of 30 kV and a temperature of 20°C were applied, with the anode on the injection side and the cathode on the detection side (positive polarity of the separation voltage). Between runs, the separation capillary was rinsed for 3 min with 0.5 N NaOH, followed by a 3 min rinse with the separation background electrolyte, and the sample was injected at a pressure of 1 psi / 5 sec. System control and data acquisition were performed using Karat 32 software (version 10.1).

[0144] Electroosmotic flow-assisted CZE separation of APTS-labeled samples in CATRIS background electrolyte was performed in a BFS capillary with an effective length of 30 cm (total length of 40 cm) and an inner diameter of 30 µm (365 μm OD). A voltage of 30 kV was applied, with the anode on the injection side and the cathode on the detection side (positive polarity separation voltage). The sample was injected at a pressure of 1 psi for 5 seconds. The BFS separation capillary was washed with 0.5 N NaOH for 3 minutes, and then rinsed with CATRIS buffer for 3 minutes between runs.

[0145] Example 2 – Analysis of Maltodextrin Gradient In this embodiment, APTS-labeled maltodextrins (a mixture of pattern carbohydrates representing a wide range of polymerization degrees (DP 1-25+)) were analyzed by EOF-assisted capillary zone electrophoresis under alkaline conditions to evaluate online electric purification options. Figure 2 The upper scan of the image shows the separation of the maltodextrin ladder, with the numbers above the peaks indicating the number of glucose units in the oligosaccharide. The last peak in the electrophoresis image (DP 2) corresponds to the maltose component in the sample mixture; the highest still detectable maltodextrin is the 25-mer, such as... Figure 2 The illustration is shown. Note that, generally, APTS-labeled glycans do not migrate at DP < 2 levels in biopharmaceutical or biomedical samples, equivalent to < 2 GU (glucose units). Although negative charge-mediated countercurrent electromigration occurs in sample components with DP > 2, they are convectively induced downstream by EOF (Equation 1, µ). eff <µ EOF That is, to make them detectable.

[0146] Consider Formula 1 for maltose (DP2), where µ EOF = 2.15x10 -8 m 2 V -1 s -1 µ eff DP2 = -1.66x10 -8 m 2 V - 1 s -1 This leads to µ app DP2 = 4.95x10 -9 m 2 V -1 s -1 Therefore, it migrates downstream, making it detectable. On the other hand, excess APTS-labeled dye µ eff APTS = -2.92x10 -8 m 2 V -1 s -1 This leads to µ app APTS = -7.66x10 -9 m 2 V -1 s -1 In other words, it is not carried to the detector by the electroosmotic flow, but migrates out from the capillary at the inlet end and does not interfere with the detection.

[0147] On the other hand, under alkaline background electrolyte conditions, excess APTS-labeled dye and APTS-glucose (both µ-) eff >µ EOF (Formula 1) will not be carried to the detector by the electroosmotic flow, but will migrate out from the capillary at the inlet end, thus not interfering with the analysis. Figure 2 The lower scan of the image shows a control sample without sugar in the reaction mixture, indicating that no APTS-related peaks appeared in the electrophoresis image within 30 minutes.

[0148] Example 3 - Monitoring of Malt Oligosaccharide Gradient Purification Steps To reveal potential sample component losses during purification, the inventors first analyzed the APTS-labeled reaction mixture of unpurified (Scan a) and purified (Scan b) maltodextrins, as well as the three wash solutions discarded during the purification step (Scan c e). Figure 3The alkaline Tris-hexanoate buffer system (pH = 8.1) used in the analysis supported sufficient EOF levels to drive the oppositely migrating, negatively charged APTS-glycans toward the detector, but not the high electrophoretic mobility of the free dye. Because EOF drives the analysis, the oligosaccharides with the highest and therefore slowest effective migration reach the detection zone first, followed by those with lower and therefore higher effective migration. Under these conditions, the remaining unreacted, negatively charged APTS do not even enter the separation capillary, allowing the purification process to be studied undisturbed.

[0149] Figure 3 Scans a and b clearly show a significant difference in the low-polymerization-degree glucose oligomers (GU 4–6) between the purified and unpurified samples. Interestingly, analysis of the first wash buffer showed no sample loss, likely due to the high concentration of APTS dye inhibiting the binding of maltodextrins. However, in the second and third wash steps, when APTS levels were low after the first wash, the entire maltodextrin spectrum was detected, indicating preferential loss of sample components to low-GU glucose oligomers. Note the higher maltotriose peak in scan d compared to scan e, highlighting a process deviation in the wash step that resulted in a total loss of 66% of this sample component after the entire purification process. Similarly, maltotetrasaccharides and maltopentoses were lost by 41% and 11%, respectively, during sample purification. On the other hand, due to the loss of these low-GU oligomers, the peak area percentage of higher GU fragments increased significantly by 15–78%, conforming to the 100% rule. Table 1 shows the quantitative peak area differences between the purified and unpurified samples, exhibiting significant differences in the low-GU range.

[0150] Table 1. Peak area differences between purified and unpurified APTS-labeled maltodextrins (peak distribution as follows) Figure 2 (As shown)

[0151] Example 4 - CZE analysis during the purification step of APTS-labeled N-glycans released from IgG glycoproteins PNGase F-mediated carbohydrate release, fluorophore labeling, and EOF-assisted CZE were performed as detailed in Example 1.

[0152] Figure 4The electrophoresis patterns of unpurified (scan a) and purified (scan b) IgG N-glycan pools were compared, along with those of three consecutive washes (scan ce). By comparing the scan patterns of the unpurified (a) and purified (b) samples, the peaks with the longest migration times (#24-25) (representing the smallest and / or highly sialylated glycans in this example) almost disappeared during purification. The proportions of other smaller or highly sialylated carbohydrates in the pools also decreased; for example, peaks #23 and #24 decreased by 10% and 6%, respectively. The electrophoresis pattern of the first wash was similar to that of the original IgG N-glycans, while the electrophoresis patterns of the second and third washes showed the same pattern at lower and different levels, i.e., the peak in scan ce was larger than the peak in scan d, indicating that the loss of sample components was unpredictable and independent of the actual number of wash steps. Table 2 shows the peak area differences between the major peaks in the purified and unpurified samples.

[0153] Table 2. Peak area differences between purified and unpurified APTS-labeled IgG N-glycans. (Peak allocation based on {Mészáros et al., 2020 #24}).

[0154]

[0155] Example 5 - CZE analysis in the purification step of APTS-labeled N-glycan released from ribonuclease B N-glycan.

[0156] PNGase F-mediated carbohydrate release, fluorophore labeling, and EOF-assisted CZE were performed as detailed in Example 1.

[0157] In this embodiment, we investigated the high-mannose N-glycosylation of ribonuclease B, and the results are as follows: Figure 5 As shown, the scan pattern distribution is the same as in Example 4. At first glance, the unpurified and purified scan patterns appear very similar (scans a and b). While the first wash buffer (scan c) contains a large amount of all sample components, and its distribution is similar to the unpurified sample, the second wash buffer shows a clear bias towards the distribution of larger mannose 8 and 9 components. Although the third wash buffer clearly contains excessive amounts of sample components, the difference in content between the first and second wash buffers indicates that the purification process caused some spectral bias, as shown by the peaks in Table 3. For example, the loss of the Man 9 structure is 16.4%, and the degree of loss of other components constituting the purified spectrum also varies; that is, sample components with high mannose content do not represent the original distribution.

[0158] Table 3. Peak area differences between purified and unpurified APTS-labeled ribonuclease B N-glycans (peak allocation based on [Guttman et al., 1995 #22]).

[0159]

[0160] Example 6 - CZE analysis during the purification step of APTS-labeled N-glycans released from fetal globulins PNGase F-mediated carbohydrate release, fluorophore labeling, and EOF-assisted CZE were performed as detailed in Example 1.

[0161] like Figure 6 As shown, by comparing scans a and b, subtle spectral differences were found in highly sialylated fetoglobulin N-glycans due to the purification process. Most notably, the peak area ratios of the two major components, A3G3S(3)2S(6)1 and A3G3S(3)1S(6)2, were interchanged during purification. Electrophoresis patterns of the wash buffer (scans ce) showed significant amounts of all sample components, which may have contributed to the spectral discrepancies between purified and unpurified samples, as shown in Table 4.

[0162] Table 4. Peak area differences between purified and unpurified APTS-labeled fetoglobulin N-glycans (peak allocation based on [Guttman et al., 1996 #23]).

[0163] Gal(β 1,3)[REF 14]. Antennae differences.

[0164] Example 7 - A comparison was made between the conventional low-pH gel buffer system known in the prior art as a reference method and the EOF-assisted alkaline CZE analysis in the method of the present invention, using PNGase F-released and APTS-labeled human serum N-glycan samples.

[0165] For the N-glycemic release assay, 40 μL of 10-fold diluted human serum was denatured by adding 4.0 μL of denaturing solution (an aqueous solution containing 0.375% NP-40, 12.75% glycerol, and 0.625% SDS) and then incubated at 70°C for 15.0 min. Afterward, 2 μL of 1.35 mg / mL PNGase F (50% glycerol) was added, and the sample was incubated at 37°C for 2.0 h.

[0166] For fluorescent labeling, 20 µL of a reducing amination reaction mixture (containing 15% HPLC-grade water, 35% glacial acetic acid, 40% tetrahydrofuran, and 10% 1 M sodium cyanoborohydride (1 M in THF)) was added to the deglycosylated human serum sample. The labeling reaction was initiated by adding 1 µL of 20 mM APTS, followed by incubation at 60°C for 1 hour, with the vial cap opened, and proceeded according to our previously published evaporative derivatization protocol [Reider et al., 2018 #19]. After labeling, the sample was dissolved in 100 µL of HPLC-grade water. Note that all volatile salt components were removed during the evaporative labeling process.

[0167] EOF-assisted CZE was performed under the conditions detailed in Example 1.

[0168] In the reference method, for low-pH gel buffer-mediated CGE analysis, the separation medium (HR-NCHO low-pH gel buffer system, e.g., pH below 5) was filled into a BFS capillary with an effective length of 30 cm (total length 40 cm) and an inner diameter of 30 μm (outer diameter 365 μm). A potential of 30 kV was applied, with the cathode on the injection side and the anode on the detection side (polarity reversed). The sample was injected at 5 kV for 5 seconds (polarity reversed), preceded by a pre-injection of 2 psi / 5 seconds with a water plug. Between runs, the capillary was rinsed with separation gel buffer for 3 minutes.

[0169] For comparison, we used traditional low-pH gel buffer systems as reference methods. Figure 7 A) and the EOF-assisted alkaline CZE method using online electric purification according to the present invention ( Figure 7 B) Human serum N-glycan samples were analyzed. The inset shows electrophoresis images before and after purification using two different separation systems, revealing... Figure 7 The large peak associated with APTS in the upper scan of the illustration A, due to the online electric purification process, Figure 2 The peak was not observed in illustration B. Due to the high concentration of APTS in the reaction mixture, the analysis of the purified sample was as follows... Figure 7 As shown in Figure A. In EOF-assisted separation, the order of the separated peaks was reversed, and the structures of the identified sample components are shown accordingly in Table 5. The circles in the inset depict some obvious changes in peak shape, which may be caused by the purification process. For example, the peaks corresponding to the FA2BG2S2 structure (peak #6 in Figure A and peak #31 in Figure B) are significantly lower after purification than before purification, as shown in Figure A. Figure 7 As shown in the illustration for B. Similarly, due to Figure 7The presence of numerous APTS peaks in A indicates that, when using a conventional low-pH gel buffer system, the APTS peaks are almost completely suppressed in the scan of the unpurified reaction mixture. Note that the alkaline buffer system exhibits the same separation performance as the conventional low-pH gel buffer system (i.e., the same number of features identified (37 peaks)), but the analysis time is reduced by approximately 35%. The migration time and peak area reproducibility of the alkaline buffer system are 1.09% RSD and 1.66% RSD, respectively.

[0170] Table 5 Figure 7 The structures of human serum N-glycans isolated from A and 7B. (Nomenclature based on [Harvey et al., 1996 #21])

[0171] Industrial applicability Based on the above findings, a capillary electrophoresis method with EOF-assisted separation of labeled carbohydrates can be used, in which excess labeling agent separates from the analyte carbohydrates, thus not interfering with the analysis. This invention utilizes the following principle: during the separation process, the effective migration rate (µm) of labeled carbohydrates and free labeling agent... eff Mobility relative to EOF (µ) EOF The EOF is sufficiently different from the background electrolyte and labeled carbohydrates, and while the EOF drives the background electrolyte and labeled carbohydrates toward the detector, the free labeling agent with a high negative effective mobility migrates upstream (in the opposite direction) and is thus excluded from the analysis.

[0172] Therefore, the method according to the invention allows for unpurified capillary zone electrophoretic analysis of labeled glycans.

[0173] In the embodiments, the use of an appropriate running buffer according to the present invention helps to introduce the labeled target sample components into the separation capillary, while excess labeling agent is excluded and therefore does not interfere with the analysis.

[0174] Furthermore, the results of N-glycan component analysis of human serum samples confirmed that the method according to the invention is comparable to the reference method in terms of the number of separated peaks, but unexpectedly, it allows the analysis to be completed in a shorter time without the need for time-consuming and expensive sample purification steps.

[0175] Furthermore, the method according to the invention is also advantageous for identifying sample losses that may occur during other commonly used carbohydrate purification processes, such as HILIC columns used for HPLC analysis, where the reverse migration of labeling dyes and sample components is not possible during online sample purification.

[0176] References (1)Saldova, R.; Royle, L.; Radcliffe, C.M.; Abd Hamid, UM; Evans, R.; Arnold, J.N.; Banks, R.E.; Hutson, R.; Harvey, D.J.; Antrobus, R.;Petrescu, SM; Dwek, R.A.; Rudd, PM Glycobiology 2007, 17, 1344-1356. (2)Shrivastava, A.; Joshi, S.; Guttman, A.; Rathore, AS Analyticachimica acta 2022, 1209, 339828. (3)Sarkozy, D.; Farsang, R.; Szigeti, M.; Austin, S.; Lock, S.;Guttman, A. Journal of pharmaceutical and biomedical analysis 2023, 233,115434. (4)Mechref, Y.; Novotny, MV Chemical reviews 2002, 102, 321-369. (5)Lu, G.; Crihfield, C.L.; Gattu, S.; Veltri, L.M.; Holland, LAChemical reviews 2018, 118, 7867–7885. (6)Guttman, A.; Chen, F.T.; Evangelist, R.A.; Cooke, N. Analyticalbiochemistry 1996, 233, 234-242. (7)Ruhaak, L.R.; Zauner, G.; Huhn, C.; Bruggink, C.; Deelder, AM;Wuhrer, M. Analytical and bioanalytical chemistry 2010, 397, 3457-3481. (8)Vanderschaeghe, D.; Debruyne, E.; Van Vlierberghe, H.; Callewaert,N.; Delanghe, J. Electrophoresis 2009, 30, 2617-2623. (9)Broberg, A. Carbohydrate research 2007, 342, 1462-1469. (10)Anumula, KR; Dhume, ST Glycobiology 1998, 8, 685-694. (11)Only, MH; Hemayatkar, M.; Deelder, AM; Wuhrer, M.Analytical chemistry 2011, 83,2492-2499. (12)Trbojevi -Akma i , I.; Vu covi , F.; Private , T.; Village, M.; ernigoj, U.; See , J.; Šimunov , J.; K pka, A.; Col i , I.; Clary , L.;Novokmet, M.; Pu i -Bakovi , M.; Rapp, E.; Štrancar, A.; Polašek, O.; Wilson,JF; Lauc , G. Communications biology 2023 , 6 , 312 . (13)Szygeti, M.; Lew, C.; Roby, K.; Guttman, A. Journal of laboratoryautomation 2016, 21, 281-286. (14)Guttman, A. Nature 1996, 380, 461-462. (15)Church, C.; Horváth, C. Journal of chromatography 1993, 645, 337-352. (16)Liu, JP; Shirota, O.; Novotny, M. Journal of chromatography1991, 559, 223-235. (17)Váradi, J.; Lew, C.; Guttman, A. Analytical chemistry 2014, 86,5682-5687. (18)Kovács, N.; Farsang, R.; Szigeti, M.; Vonderviszt, F.; Jankovics,H. Molecular biotechnology2022, 64, 914-918. (19)Reider, B.; Szigeti, M.; Guttman, A. Talent 2018, 185, 365-369. (20)Mikkers, FEP; Everaerts, FM; Verheggen, TPEMJournal of Chromatography A 1979, 169, 1-10. (21)Harvey, DJ; Merry, AH; Royle, L.; Campbell, MP; Rudd, PM Proteomics 2011, 11, 4291-4295. (22)A. Guttman, T. Pritchett, Electrophoresis, 16(1995)1906-1911. (23)A. Guttman, FTChen, RAEvangelista, Electrophoresis, 17(1996)412-417. (24)B. Mészáros, G. Járvás, A. Farkas, M. Szigeti, Z. Kovács, R. Kun,M. Szabó, E. Csánky, A. Guttman, J Chromatogr B Analyt Technol Biomed LifeSci.2020 Jan. 15;1137:121913. (25)Tim Wehr, Capillary Zone Electrophoresis, Editor(s): Robert A.Meyers, Encyclopedia of Physical Science and Technology(Third Edition),Academic Press, 2003, Pages 355-368 (26)Santos, Marcia R; Ratnayake, Chitra K.; Fonslow, Bryan; Guttman,Andras “A covalent, cationic polymer coating method for the CESI-MS analysisof intact proteins and polypeptides” Biomarkers and Omics2015 AB Sciex. SCIEXSeparations, Brea, CA Document number: RUO-MKT-18-2325-A (27)L. Hajba, A. Guttman, Recent advances in column coatings forcapillary electrophoresis of proteins, TrAC Trends in Analytical Chemistry 90(2017)38_44 (28)Guttman A and Hajba L. Capillary Electrophoresis, Elsevier OxfordOX5 1GB, United Kingdom, 2022 (29)Palaniappan KK, Bertozzi CR.Chemical Glycoproteomics.Chem Rev.2016 Dec 14;116(23):14277-14306. doi: 10.1021 / acs.chemrev.6b00023.Epub 2016Nov 18.PMID: 27960262; PMCID: PMC5327817. (30)IUPAC.Compendium of Chemical Terminology, 2nd ed.(the "GoldBook").Compiled by A. D. McNaught and A. Wilkinson.Blackwell ScientificPublications, Oxford(1997).Online version(2019-)created by S. J. Chalk.ISBN0-9678550-9-8.https: / / doi.org / 10.1351 / goldbook. (31)Ruhaak LR, Zauner G, Huhn C, Bruggink C, Deelder AM, Wuhrer M.Glycan labeling strategies and their use in identification andquantification.Anal Bioanal Chem.2010 Aug;397(8):3457-81.

Claims

1. A capillary electrophoresis (CE) method for electroosmotic flow (EOF)-assisted separation of labeled carbohydrates, wherein the carbohydrates are labeled with a labeling agent, preferably oligosaccharides. The method includes the following steps: - Provides a reaction mixture containing labeled carbohydrates and an excess of free labeling agent. - A sample of the reaction mixture is applied to the CE capillary under the following conditions. The labeled carbohydrate and the free labeling agent are both charged. Among them, EOF mobility (µ EOF ) toward the detector, The effective migration (µm) of the labeled carbohydrates and the free labeling agent. eff ) and the EOF mobility (µ) EOF )different, - Separation of the labeled carbohydrates is performed by EC in a separation mode in which the EOF drives the background electrolyte and the labeled carbohydrates toward the detector, and the free labeling agent migrates in the opposite direction.

2. The CE method according to claim 1, wherein, The carbohydrates are polysaccharides, particularly N-polysaccharides.

3. The CE method according to claim 1 or 2, wherein, The CE background electrolyte includes a carrier buffer with a pH setting to provide electroosmotic flow toward the detector, and wherein the CE is capillary zone electrophoresis (CZE).

4. The capillary zone electrophoresis (CZE) method according to claim 3, wherein the method is used to separate labeled carbohydrates, preferably polysaccharides. in, The carbohydrate is labeled with a marker that carries a negative charge in its free form, and The labeled carbohydrates, preferably labeled polysaccharides, are negatively charged. The method includes the following steps: - A sample containing a reaction mixture of labeled polysaccharides and an excess of free labeling agent (preferably a dye, especially a fluorescent dye) is applied into the CZE capillary. - The labeled glycan and the free labeling agent are separated by CZE in an alkaline pH separation buffer with an alkaline pH background electrolyte. In this process, an electroosmotic flow is driven from the anode (positive polarity) to the cathode (negative polarity), and the labeled carbohydrate migrates towards the cathode, while the free labeling agent migrates towards the anode.

5. The CZE method according to claim 4, wherein, The effective migration rate (µm) of the labeled glycan. eff The EOF mobility is less than the stated EOF mobility (µ). EOF Therefore, the negatively charged labeled glycan has a positive apparent mobility µ. app Migrate to the cathode, And due to the µ of the labeled dye eff Higher than the µ EOF The free labeled dye will have a negative µ value app Migrate towards the anode.

6. The method according to any one of claims 4 to 5, wherein, The alkaline background electrolyte has a pH > 7, preferably 7 to 9, and more preferably 7.5 to 8.

5.

7. The method according to any one of claims 4 to 6, wherein, The alkaline background electrolyte is a composition comprising a nitrogen-containing base and a carboxylic acid coion, wherein the nitrogen-containing base is preferably an amine buffer, and the carboxylic acid coion is preferably C. 4-10 Carboxylic acid, preferably C 4-10 Straight-chain carboxylic acids.

8. The method according to claim 7, wherein, The background electrolyte comprises 5 to 9 parts of an amine buffer and 1 to 5 parts of a carboxylic acid, preferably Tris and hexanoic acid; highly preferably, the alkaline background electrolyte is a composition comprising Tris and hexanoic acid, preferably 5 to 9 parts of Tris and 1 to 5 parts of hexanoic acid.

9. The method according to any one of claims 4 to 8, wherein, CZE capillary tubes are fused silica capillary tubes.

10. The method according to any one of claims 4 to 9, wherein, The labeling agent is a fluorescent dye.

11. The method according to claim 10, wherein, The fluorescent dye labeling agent is preferably a negatively charged sulfonate of an aromatic amine. Sulfonates of aromatic hydrocarbons having fused rings, preferably with two, three, or four benzene rings, preferably with one to four sulfonates, preferably with two to three sulfonates, and especially preferably with three sulfonates. Preferably, the fluorescent dye labeling agent has a primary amine group capable of forming a Schiff base with the carbohydrate.

12. The method according to any one of claims 1 to 9, wherein, - The carbohydrates are N-glycans cleaved from glycoproteins. - The buffer solution is an alkaline buffer solution with a pH of 7.5 to 8.

5. The labeling agent is an aromatic sulfonate having a primary amine group capable of forming a Schiff base with the carbohydrate, preferably aminopyrene trisulfonate (APTS) or aminonaphthalene trisulfonate (ANTS).

13. The capillary electrophoresis (CE) method according to any one of claims 1 to 4, for separating (e.g., purifying) labeled carbohydrates, preferably glycans. The carbohydrate is labeled with a labeling agent, which is positively charged in its free state. The method includes the following steps: - A reaction mixture containing labeled polysaccharides and an excess of free labeling agent (preferably dyes, especially fluorescent dyes) is applied into a CE capillary with positively charged walls. - The labeled glycan and the free labeling agent are separated by CE in a separation buffer with an acidic pH (under acidic background electrolyte conditions / using an acidic pH background electrolyte, wherein the pH is 2 to 7, preferably 3 to 6). In this process, an electroosmotic flow is driven from the cathode (negative electrode) to the anode (positive electrode), and the labeled carbohydrate migrates towards the anode, while the free labeling agent migrates towards the cathode.

14. The CZE method according to claim 13, wherein, The effective migration rate (µeff) of the labeled glycan is less than the EOF migration rate (µeff). EOF Therefore, the positively charged labeled glycan has a positive apparent mobility µ. app Migrate towards the anode, And due to the µ of the labeled dye eff Higher than the µ EOF The free labeled dye has a negative µ value. app Migrate towards the cathode.

15. The method according to any one of claims 1 to 14, wherein, No additional purification step is required for the mixture before it is applied to the CZE capillary.

16. The use of alkaline CE background electrolyte and carbohydrate labeling reagents for separating free labeling reagents from labeled carbohydrates by capillary electrophoresis (CE) under alkaline background electrolyte conditions. In methods for separating (e.g., purifying) labeled carbohydrates, preferably polysaccharides, wherein The carbohydrate is labeled with a marker, wherein the marker is negatively charged in its free form. The labeled carbohydrates, preferably labeled polysaccharides, are negatively charged. Preferably, the method includes the following steps: - A sample containing a reaction mixture of labeled polysaccharide and excess free labeling agent (preferably dye, especially fluorescent dye) is applied into a CZE capillary. - The labeled polysaccharide and the free labeling agent are separated by CZE in an alkaline pH separation buffer (under alkaline background electrolyte conditions / using alkaline pH background electrolyte). In this process, an electroosmotic flow is driven from the anode (positive polarity) to the cathode (negative polarity), and the labeled carbohydrate migrates towards the cathode, while the free labeling agent migrates towards the anode.

17. The use according to claim 11, wherein the pH is 7 to 10, preferably 7 to 9, and more preferably 7.5 to 8.

5.

18. The use of acidic background electrolytes and carbohydrate labeling agents for separating free labeling agents from labeled carbohydrates by capillary electrophoresis (CE) under acidic background electrolyte conditions. In methods for separating (e.g., purifying) labeled carbohydrates, preferably polysaccharides, wherein The carbohydrate is labeled with a marker, wherein the marker is positively charged in its free form. The labeled carbohydrates, preferably labeled polysaccharides, are positively charged. Preferably, the method includes the following steps: - A sample containing a reaction mixture of labeled polysaccharides and an excess of free labeling agent (preferably a dye, especially a fluorescent dye) is applied into a CZE capillary. - The labeled glycan and the free labeling agent are separated by CZE in an acidic pH separation buffer using the alkaline pH background electrolyte. In this process, an electroosmotic flow is driven from the cathode (negative polarity) to the anode (positive polarity), during which the labeled carbohydrate migrates toward the anode, while the free labeling agent migrates toward the cathode.

19. The use according to claim 19, wherein, The pH is 2 to 7, preferably 3 to 6.

20. The use according to any one of claims 16 to 19, for analyzing sample loss during carbohydrate purification, for example... - For example, HILIC columns used for HPLC analysis, or - Sample loss in the CE-MS method.

21. A kit for carbohydrate analysis, the kit comprising: - A negatively charged marker for free carbohydrates. - Alkaline electrolyte buffer, and - CZE capillaries with negatively charged walls, preferably exposed fused silica capillaries. - or - Positively charged free carbohydrate markers, - Acidic electrolyte buffer, and - CZE capillary with positively charged walls.

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