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WO2016068800A1 - Procédés de préparation d'échantillon, de détection et d'analyse relatifs aux glycanes - Google Patents

Procédés de préparation d'échantillon, de détection et d'analyse relatifs aux glycanes Download PDF

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Publication number
WO2016068800A1
WO2016068800A1 PCT/SG2015/050412 SG2015050412W WO2016068800A1 WO 2016068800 A1 WO2016068800 A1 WO 2016068800A1 SG 2015050412 W SG2015050412 W SG 2015050412W WO 2016068800 A1 WO2016068800 A1 WO 2016068800A1
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glycans
glycoproteins
beads
acid
glycan
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PCT/SG2015/050412
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English (en)
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Huatao FENG
Fong Yau Sam Li
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National University Of Singapore
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Publication of WO2016068800A1 publication Critical patent/WO2016068800A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • the present invention relates to biochemistry, in particular methods of identifying and analysing biochemistry molecules, in particular glycans.
  • Protein glycosylation a post-translational modification of proteins, involves enzymatic cross-linking of carbohydrate oligosaccharide groups, or glycan sequences, to polypeptide backbones. It is one of the most abundant post-translational modifications of proteins, with more than 50% of all mammalian proteins being glycosylated. Glycoproteins are involved in many biological processes, such as cell communication, cell proliferation and differentiation, host-pathogen interactions, cell recognition, signaling and adhesion, as well as the initiation of immune responses. Many diseases are often reflected in the glycosylation profiles of proteins. Cell surface proteins are responsive to the cell's changing environment in the biological matrix.
  • Protein domains on the cell surface are crucial for the communication among cells, the interaction with pathogens and the binding of chemical messengers. As cell surface proteins are easily accessible, analysis of glycosylation profiles of cell surface proteins can be very useful in the development of disease diagnostic methods, drug targets or biomarkers.
  • glycosylation also plays an important role in the development of new therapeutic drugs. About 70% of the recombinant proteins of interest for human therapy are glycoproteins. Glycosylation of the recombinant protein products affects critical product properties such as bio-safety and bio-activity. [0005] Due to the importance of protein glycosylation in the discovery and development of new biomarkers, therapeutic targets and drugs, glycoproteins are being widely studied and analyzed.
  • Glycosylation is a non-template driven process, thus the structure of glycoproteins cannot be directly predicted.
  • each glycosylation site contains a variety of glycan structures, leading to a highly complex mixture. This makes the analysis of glycans a big challenge.
  • most glycans are released at nano-gram quantities and cannot be easily amplified, research advances in the area of mapping glycan structure to function have been slow due to the lack of suitable methodologies for quantification.
  • several techniques have been developed to capture and enrich glycoproteins. However, so far no method is available for the analysis of glycans on those glycoproteins captured by as only tiny amounts of glycans can be obtained.
  • a method of capturing and/or enriching glycans from glycoproteins or biological materials carrying glycoproteins comprising:
  • kits for use in the capturing and/or enriching of glycans from glycoproteins or biological materials carrying glycoproteins comprising an oxidizing agent, magnetic, paramagnetic or polymeric beads, a chemical or an enzyme for separating glycans from glycoproteins, a glycan labeling agent and a glycan cleaving agent.
  • a method for detecting and analyzing glycans using multiplexed capillary electrophoresis with laser induced fluorescence comprising co-separating and analyzing a glycan sample in a multiplexed CE-LIF system, wherein the multiplexed CE-LIF comprises micellar electrokinetic chromatography (MEKC) in combination with capillary zone electrophoresis (CZE) or capillary gel electrophoresis (CGE).
  • MEKC micellar electrokinetic chromatography
  • CZE capillary zone electrophoresis
  • CGE capillary gel electrophoresis
  • Fig. 1 is a diagram showing the overall workflow of glycan capturing, enrichment, detection and analysis using the methods described in the present disclosure.
  • "X" represents a detectable label as defined herein.
  • Fig. 2 shows mass spectrometry results of glycoproteins obtained from matrix assisted laser desorption ionization coupled to time of flight (MALDI-TOF) using different sample oxidation incubations times.
  • MALDI-TOF matrix assisted laser desorption ionization coupled to time of flight
  • the oxidation times used in the different spectra are (from top to bottom): 10 minutes, 30 minutes, 15 hours and 24 hours.
  • the first spectrum shows that at shorter incubation time (10 minutes), the background signal was noisier compared to the others.
  • the second spectrum shows that at an incubation time of 30 minutes or longer, there is lower background interference.
  • Fig. 3 shows mass spectrometry results of glycoproteins obtained from MALDI- TOF using different ratios of cell numbers to fixed amount of beads used.
  • the number of beads used is fixed at 2.55x10 (1.5 mg beads of 1 ⁇ in diameter).
  • the numbers of cells used in the different spectra are (from top to bottom): 3 x 10 7 , 1 xlO 7 , 3 x 10 6 , 1 x 10 6 , 5 x 10 5 , 4 xlO 5 and 3 x 10 5 cells.
  • the last two spectra indicate that the signal intensity and peak resolution decreased significantly at 4x 10 5 cells, while at cell count of 5 x 10 5 and above, good peak signal and resolution could be obtained.
  • Fig. 4 shows mass spectrometry results of glycoproteins obtained from MALDI- TOF using different cell to beads coupling times.
  • the coupling times used in the different spectra are (from top to bottom): 1 hour, 5 hours, 15 hours, and 24 hours. From the results obtained, at first glance, there were not any significant differences between the four spectra. However, at the mass range of 10000 to 16000 m/z, the ratio of the peak intensity at m/z 11320 and m/z 15340 to that of m/z 13777 and m/z 14006 was increasing as the coupling time increased from 1 hour to 24 hours. Thus, in one example good detection signals were obtained for glycoproteins which are present in lower concentrations or glycoproteins that require more time to couple with the beads, using coupling time of about or at least 15 hours or overnight. 1 hour can be used when rapid screening is required.
  • Fig. 5 shows mass spectrometry results of glycoproteins obtained from MALDI- TOF using (a) chemical cell lysis method using SDS and (b) physical cell lysis method using a homogenizer.
  • the top spectrum corresponds to the chemical cell lysis method and the bottom spectrum corresponds to the physical cell lysis method.
  • the results indicate that using a homogenizer to lyse cells generated more fragments of smaller masses, while using SDS produced more fragments that were of higher mass. Both methods can be used for cell lysis.
  • Chemical cell lysis method for example one that uses SDS, can be used when rapid screening is required.
  • Fig. 6 shows mass spectrometry results of glycoproteins obtained from MALDI- TOF using different concentrations of SDS for cell lysis.
  • concentrations of SDS used in the different spectra are (from top to bottom): 0.05mM, 0.075mM, O. lOmM, 0.25mM and 0.50mM. The results indicate that the amount of glycoproteins obtained using five concentrations do not differ significantly.
  • Fig. 7 shows results of analysis of N-glycan sample obtained from bovine fetuin using the glycan capturing and/or enriching method as described herein. Peaks: 1, A3G(3,4,4)3S(3,6,6,6)4; 2, A3G(3,4,4)3S(3,3,6,6)4; 3, A3G(4,4,4)3S(6,6,6)3; 4, A3G(4,4,4)3S(3,6,6)3; 5, A3G(4,4,4)3S(3,3,6)3; 6, A3G(3,4,4)3S(3,6,6)3.
  • A3 triantennary with a GlcNAc linked ⁇ 1-2 to both mannose and the third GlcNAc linked ⁇ 1-4 to the al-3 linked mannose.
  • Galactoses beta linked (G) to the antenna the number in parentheses indicates the linkage position (3 indicated ⁇ 1 -3 ; 4 indicated ⁇ 1-4), the number following the G (or after the parentheses following the G) indicates the number of galactoses.
  • Sx, number (x) of sialic acids linked to galactose; the numbers 3 or 6 in parentheses after S indicate whether the sialic acid is in a a2-3 or a2-6 linkage.
  • N-glycans profile of bovine fetuin is consistent with what has been published earlier. This indicates that the glycan capturing and enriching method is effective for glycoproteins. As the fetuin N-glycans are highly sialylated, the results also confirmed that the method used would not damage the sialic acid content on N-glycans.
  • Fig. 8 shows the CE-LIF analysis of N-glycans obtained from enriched bovine fetuin sample.
  • the upper spectra shows the analysis of a 100 times diluted bovine fetuin glycans sample using online stacking method FASS.
  • the lower spectrum shows the analysis of an undiluted bovine fetuin glycans sample using the conventional CZE method.
  • the results indicate that the detection limit of the FASS method is slightly better than that of the conventional CZE method, but at the same time, a loss of resolution is observed in the FASS method compared to the conventional CZE method.
  • Fig. 9 shows the separation a mixture of eight glycan standards using the conventional CZE method. The result indicates that the CZE method provides good sensitivity for captured N-glycan analysis.
  • Fig. 10 shows results of CZE analysis of N-glycan sample obtained from a Chinese hamster ovary (CHO) cell clone using the method as described herein.
  • the inset graph shows the zoomed-in results from about 7 to about 11 minutes.
  • Fig. 11 shows the MEKC and CZE (shown as inset) electropherogram of separation of dextran ladder and C 0920 spiked.
  • MEKC buffer— 25mM SDS in 50mM Na 2 B 4 0 7 , pH 9.33; bare-fused silica capillary, effective length 20cm; voltage 20kV.
  • CZE buffer - 25mM NH 4 Ac, pH 4.75; PVA coated capillary, effective length 20cm; voltage -20kV.
  • the results show that separation of dextran ladder using MEKC gives better resolution and faster migration time compared to CZE.
  • the largest analyte migrates first and the smallest migrates last. This is different from the results obtained using CZE, and it allows MEKC to serve as a complementary method to cross-validate the results obtained from CZE.
  • Fig. 12 shows the structures of some of the glycans discussed in this disclosure.
  • Fig. 13 shows the separation and analysis of a mixture of eight glycan standards (each at 20 ⁇ g/L) using CZE, MEKC and capillary gel electrophoresis (CGE).
  • CZE buffer— 25mM NH 4 Ac, pH 4.75
  • PVA coated capillary effective length 20cm
  • voltage -30kV The following are used in MEKC: buffer - 25mM SDS in 50mM Na 2 B 4 0 7 , pH 9.33; bare-fused silica capillary, effective length 50cm; voltage 28kV.
  • CGE buffer - 1% HPC +1% HEC in 80mM MES and 40mM TRIS, pH6.11; coated capillary, effective length 50cm; voltage -30kV.
  • MEKC can be very useful for the anslysis of glycans with small GU (glucose unit) values, such as O-linked glycans and (highly) sialic glycans.
  • Fig. 14 shows plots used to visualize correlation coefficients of CZE combined with MEKC or CGE.
  • X-axis represents GU values from CZE;
  • Y-axis represents GU values of MEKC or CGE.
  • Fig. 14(a) shows the evaluation using GU values of eight glycan standards.
  • Fig. 14(b) shows the evaluation using the values of MT stan dard MTGU5-
  • CZE and CGE results showed high correlation coefficient, which indicates that the combination of these two mechanisms may not provide any significant cross validation information. A much lower correlation coefficient was observed from the combination of MEKC and CZE, suggesting that MEKC can be used as the complementary method to cross validate the results of CZE or CGE.
  • Fig. 14 shows plots used to visualize correlation coefficients of CZE combined with MEKC or CGE.
  • X-axis represents GU values from CZE
  • Y-axis represents GU values of MEKC or CGE.
  • Fig. 14(a) shows the evaluation using GU values of
  • Fig. 16 shows the separation and analysis of a mixture of cytidine monophosphate (CMP), cytidine diphosphate (CDP) and cytidine triphosphate (CTP).
  • Fig. 16(a) shows the result using CZE with the following conditions: 50mM sodium tetraborate buffer, total length of capillary: 49 cm. Sample loading: 0.50 psi for 15 s. Separation voltage: 30 kV (20 kV when pH values were lower than 9.0).
  • Fig. 16(b) shows the result using CZE with the following conditions: 40mM Citric Acid, 0.8mM cetyltrimethylammonium bromide (CTAB), Separation voltage: -20 kV.
  • CTAB cetyltrimethylammonium bromide
  • Fig. 17 is a diagram showing the workflow of glycan capturing, enrichment, detection and analysis using the methods described in some examples of the present disclosure.
  • the beads used are hydrazide functionalized, the glycans are separated from the glycoproteins using PNGase F, and the glycans are labeled with aminopyrene-3,6,8-trisulfonic acid (APTS).
  • APTS aminopyrene-3,6,8-trisulfonic acid
  • CE-LIF method is used for the detection and analysis of the glycans.
  • Such a method can comprise
  • the method can comprise
  • the method described herein can be used in the analysis of relinked glycans or O-linked glycans. In one exmaple, the method is used only for analysing N- linked glycans or N-glycans.
  • glycoprotein which can be an oligosaccharide. It can be homo- or heteropolymers of monosaccharide residues, and can be linear or branched. It can include N-, 0-, P- and C- linked glycans.
  • glycoprotein is used herein to refer to a protein that contains glycan(s) covalently attached to polypeptide side-chains. It can include N-, 0-, P- and C-glycoproteins.
  • N- glycoprotein is used herein to refer to a protein formed by N-linked glycosylation, which contains glycan attached to nitrogen, typically on the amide side-chain of asparagine residues of the glycoprotein.
  • N-glycan and “N-linked glycan” are used interchangeably to refer to glycan linked as such.
  • O-glycoprotein is used herein to refer to a protein formed by O-linked glycosylation, which contains glycan attached to oxygen, typically on serine or threonine residues of the glycoprotein but also on non-canonical amino acid residues such as hydroxylysine and hydroxyproline.
  • O-glycan and O-linked glycan are used interchangeably to refer to glycan linked as such.
  • biological materials is used herein to refer to chemical substances (elements or compounds) or mixture of substances, present or produced in a living organism. They can be organic or inorganic, molecular or non-molecular. They can be naturally occurring, or modified or produced using recombinant technologies.
  • the biological materials include but are not limited to microorganisms, tissues or intact cells.
  • Intact cells are cells that have not been lysed.
  • intact cells can be understood to refer to cells whose original cellular structure is intact and/or which retains full metabolic function.
  • tissue is used herein to refer to an ensemble of similar cells from the same origin.
  • the word "carry” and its derivatives such as carries, carrying, carried, etc., is used herein to refer to the presence of the substances, in particular glycoproteins in or on the biological materials.
  • the forces that allow the substances to be carried include but are not limited to force due to covalent bonds, force due to electrostatic interactions and van der Waals force.
  • Substances can also be present in the medium(s) that carries them due to enclosure, for example intracellular substances enclosed by cellular membranes.
  • the glycoproteins are surface glycoproteins.
  • glycoprotein is used herein to refer to glycoprotein at least a portion of which is on the surface of the biological materials.
  • the first step of the method can comprise (a) contacting the glycoproteins or biological materials carrying glycoproteins with an oxidizing agent. This may allow certain chemical group(s) in glycans to be converted to a form that allows the glycans to interact with other substances in the subsequent steps.
  • oxidizing agent is used herein to refer to chemical species that can remove electron(s) from other species.
  • the oxidizing agent used is periodate. In one specific example, the oxidizing agent used is sodium metaperiodate.
  • the amount of time that the glycoproteins or biological materials are in contact with the oxidizing agent can be any one of the following: about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 6 hours, about 12 hours, about 15 hours, about 24 hours, or from about 10 minutes to about 30 minutes, from about 30 minutes to about 1 hour, from about 1 hour to about 3 hours, from about 3 hours to about 6 hours, from about 6 hours to about 9 hours, from about 9 hours to about 12 hours, from about 12 hours to about 18 hours, from about 18 hours to about 24 hours.
  • step (a) after the glycoproteins or biological materials carrying glycoproteins are sufficiently oxidized in step (a), the oxidized products of step (a) can then put in contact with beads in step (b) to allow the oxidized glycoproteins in the oxidized products to bind to the beads.
  • the beads can be magnetic, paramagnetic, or polymeric beads.
  • the beads contain at least one functional group that binds to glycoproteins or oxidized glycoproteins.
  • the beads are hydrazide functionalized.
  • magnet is used herein to refer to small particles that can be used to bind target molecules.
  • magnetic bead is used herein to refer to bead that is at least partially made of or coated with a magnetic material.
  • parmagnetic bead is used herein to refer to bead that is at least partially made of or coated with a material that can become magnetic when being induced by external forces or substances.
  • polymeric bead is used herein to refer to bead that is at least partially made of or coated with a polymer.
  • the term "functional group” is used herein to refer to a portion of a molecule, which is recognizable or has been classified as having certain properties.
  • hydrozide is used herein to refer to a class of organic compounds sharing a common functional group characterized by a nitrogen to nitrogen covalent bond with 4 substituents with at least one of them being an acyl group.
  • acyl is used herein to refer to a-C(0)-R radical, wherein R is an alkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl group.
  • R is an alkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl group.
  • examples of acyl include acetyl and benzoyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon.
  • the amount of time that the oxidized products are in contact with the beads can be any one of the following: about 1 hour, about 2 hours, about 3 hours, about 5 hours, about 10 hours, about 15 hours, about 24 hours, or from about 1 hour to about 3 hours, from about 3 hours to about 6 hours, from about 6 hours to about 9 hours, from about 9 hours to about 12 hours, from about 12 hours to about 18 hours, from about 18 hours to about 24 hours.
  • the ratio of the number of intact cells, or cells isolated from the microorganisms or tissues to the number of beads can be any one of the following: about 1:700, about 1:600, about 1:500, about 1:400, about 1:300, about 1:200, about 1: 100, about 1:90, about 1:80, about 1:70, about 1:60, about 1:50, about 1:40, about 1:30, about 1:20, about 1: 10, about 1: 1, or from about 1:700 to about 1:600, from about 1:600 to about 1:500, from about 1:500 to about 1:400, from about 1:400 to about 1:300, from about 1:300 to about 1:200, from about 1:200 to about 1: 100, from about 1: 100 to about 1:90, from about 1:90 to about 1:80, from about 1:80 to about 1:70, from about 1:70 to abour 1:60
  • the method can further comprise (c) a cell lysis step after the oxidized glycoproteins are coupled to the beads after being in contact with the beads for a sufficient period of time.
  • the cell lysis step is carried out using a physical method.
  • the physical method involves the use of a homogenizer.
  • the cell lysis step is carried out using a chemical method.
  • SDS sodium dodecyl sulfate
  • the concentrations of SDS used can be any one of the following: about O.OlmM, about 0.05mM, about 0.075mM, about O.lmM, about 0.2mM, about 0.3mM, about 0.5mM, about 0.8mM, about ImM, or from about O.OlmM to about
  • 0.05mM from about 0.05mM to about O. lmM, from about O. lmM to about 0.5mM, from about 0.5mM to about ImM.
  • MALDI-TOF technique is used to measure the amount of glycoproteins captured.
  • beads were removed before the MALDI-TOF test.
  • beads were removed before the MALDI-TOF test via a chemical reaction or an enzymatic reaction.
  • an acid is used in the chemical reaction, such as formic acid, citric acid, acetic acid, phosphate acid, hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid or hydroiodic acid, or any combination thereof.
  • the concentration of the acid can be any one of the following: about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.8M, about 2.0M, about 2.5M, about 3.0M, or from about 0.5M to about 0.8M, from 0.8M to about 1.0M, from about 1.0M to about 1.2M, from about 1.2M to about 1.6M, from about 1.6M to about 2.0M, from about 2.0M to about 2.5M, from about 2.5M to about 3.0M.
  • the enzyme used can be PNGase F or endoglycosidase.
  • the glycans are separated from the protein backbones in the glycoproteins via a chemical reaction or an enzymatic reaction in step (d) to obtain glycans bound to the beads.
  • the glycans are separated from the glycoproteins via a chemical reaction. In another example, the glycans are separated from the glycoproteins via an enzymatic reaction. In one specific example, the enzyme used can be PNGase F or endoglycosidase.
  • the bead-bound glycans are labeled in a further step (e) with a detectable label.
  • the glycans are cleaved from the beads in a further step (f).
  • the labeling and the cleaving of the glycans are carried out simultaneously. In another example, the labeling and the cleaving of the glycans are carried out separately.
  • detectable label is used herein to refer to a label or a tag that can be detected with or without the aid of a detection apparatus or instrument. This may include fluorescent label, radioactive label and enzymatic label, etc.
  • the detectable label is a fluorescent label.
  • the fluorescent label can be any one or any combination of the following: 2- aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine (PA), 2- aminonaphthalene trisulfonic acid (ANTS), aminopyrene-3,6,8-trisulfonic acid (APTS), 2- Aminoacridone (AMAC) or 3-Aminoquinoline (3-AQ).
  • the labeling of glycans in step (e) is carried out in an acidic environment.
  • the cleaving of glycans from the beads in step (f) is also carried out in an acidic environment.
  • the simultaneous labeling and cleaving of glycans are carried out in an acidic environment.
  • the acidic environment is created by an acid, such as formic acid, citric acid, acetic acid, phosphate acid, hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid or hydroiodic acid, or any combination thereof.
  • an acid such as formic acid, citric acid, acetic acid, phosphate acid, hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid or hydroiodic acid, or any combination thereof.
  • the concentration of the acid can be any one of the following: about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.8M, about 2.0M, about 2.5M, about 3.0M, or from about 0.5M to about 0.8M, from 0.8M to about 1.0M, from about 1.0M to about 1.2M, from about 1.2M to about 1.6M, from about 1.6M to about 2.0M, from about 2.0M to about 2.5M, from about 2.5M to about 3.0M.
  • the labeling of glycans in step (e), the cleaving of glycans from the beads in step (f), separately or simultaneously can be carried out at any temperature of the following: about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, or from about 50°C to about 55°C, from about 55°C to about 60°C, from about 60°C to about 65°C, from about 65°C to about 70°C, from about 70°C to about 75°C, from about 75°C to about 80°C, from about 80°C to about 85°C, from about 85°C to about 90°C.
  • the present invention refers to a kit for use in the capturing and/or enriching of glycans from glycoproteins or biological materials carrying glycoproteins, comprising an oxidizing agent, magnetic, paramagnetic or polymeric beads, a chemical or an enzyme for separating glycans from glycoproteins, a glycan labeling agent and a glycan cleaving agent.
  • the kit further includes a cell lysis agent.
  • the cell lysis agent is a homogenizer. In another example, the cell lysis agent is a chemical. In one specific example, the cell lysis agent is SDS.
  • the oxidizing agent in the kit is periodate. In one specific example, the oxidizing agent in the kit is sodium metaperiodate.
  • the beads in the kit are hydrazide functionalized.
  • the enzyme for separating glycans from glycoproteins can be any enzyme for separating glycans from glycoproteins.
  • PNGase F or endoglycosidase are examples of PNGase F or endoglycosidase.
  • the glycans labeling agent in the kit is a fluorescent dye.
  • the fluorescent dye can be any one or any combination of the following: 2- aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine (PA), 2- aminonaphthalene trisulfonic acid (ANTS), aminopyrene-3,6,8-trisulfonic acid (APTS), 2- Aminoacridone (AMAC) or 3-Aminoquinoline (3-AQ).
  • the glycan cleaving agent in the kit can be any one or any combination of the following: formic acid, citric acid, acetic acid, phosphate acid,
  • hydrochloric acid nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid or PNGase F.
  • the present invention refers to a method for detecting and analyzing glycans using CE-LIF.
  • the CE-LIF method is a multiplexed method comprising the co- separation and co-analysis of a glycan sample.
  • the multiplexed CE-LIF comprises MEKC in combination with CZE or CGE.
  • the multiplexed CE-LIF comprises CZE in combination with MEKC, CZE or CGE.
  • the multiplexed CE-LIF analysis methods described herein can also be used for the separation and analysis of complex samples other than glycans. In some examples, the methods can be used for separation and analysis of nucleotides and/or nucleotide sugars. [0092] In some examples, the multiplexed CE-LIF analysis method described herein can be used for the detection of a small amount of sample.
  • the small amount of sample can be any of the following: about 0.1 ⁇ g, about 0.2 ⁇ g, about 0.3 ⁇ g, about 0.4 ⁇ g, about 0.5 ⁇ g, about 1 ⁇ g, or from about 0.01 ⁇ g to about 0.1 ⁇ g, from about 0.1 ⁇ g to about 0.2 ⁇ g, from about 0.2 ⁇ g to about 0.3 ⁇ g, from about 0.3 ⁇ g to about 0.4 ⁇ g, from about 0.4 ⁇ g to about 0.5 ⁇ g, from about 0.5 ⁇ g to about 1 ⁇ g.
  • CZE is commonly used for separating charged analytes.
  • PVA coated capillary is used for CZE separation.
  • acetate buffer is used for CZE separation.
  • an acid or neutral buffer is used for CZE separation.
  • the pH value of the buffer used for CZE separation can be any of the following: about 2.0, about 3.0, about 4.0, about 5.0, about 6.0, about 7.0, or from about 1.5 to about 2.5, from about 2.5 to about 3.5, from about 3.5 to about 4.5, from about 4.5 to about 5.5, from about 5.5 to about 6.5, from about 6.5 to about 7.0.
  • the buffer is CTAB free.
  • the buffer when an acidic or neutral buffer is used for CZE, the buffer contains CTAB.
  • the concentration of CTAB can be any one of the following: about O. lmM, about 0.2mM, about 0.3mM, about 0.4mM, about 0.5mM, about 0.6mM, about 0.7mM, about 0.8mM, about 0.9mM, about l.OmM, or from about OmM to about O.lmM, from about O.lmM to about 0.2mM, from about 0.2mM to about 0.3mM, from about 0.3mM to about 0.4mM, from about 0.4mM to about 0.5mM, from about 0.5mM to about 0.6mM, from about 0.6mM to about 0.7mM, from about 0.7mM to about 0.8mM, from about 0.8mM to about 0.9mM, from about 0.9mM to about l.OmM.
  • basic buffer is used for CZE separation.
  • the pH value of the buffer used for CZE separation can be any of the following: about 7.5, about 8.0, about 9.0, about 10.0, about 11.0, about 12.0, about 13.0, about 14.0, or from about 7.0 to about 7.5, from about 7.5 to about 8.5, from about 8.5 to about 9.5, from about 9.5 to about 10.5, from about 10.5 to about 11.5, from about 11.5 to about 12.5, from about 12.5 to about 13.5, from about 13.5 to about 14.0.
  • the buffer is SDS free.
  • the basic buffer used contains SDS.
  • the concentration of SDS is lower than 9mM.
  • the concentration of SDS can be any one of the following: about OmM, about lmM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, or from about OmM to about ImM, from about ImM to about 2mM, from about 2mM to about 3mM, from about 3mM to about 4mM, from about 4mM to about 5mM, from about 5mM to about 6mM, from about 6mM to about 7mM, from about 7mM to about 8mM, from about 8mM to about 9mM.
  • MEKC is the method used to achieve separation of neutral compounds based on differential partitioning of solutes between the hydrophobic interior of a charged micelle and the aqueous phase.
  • a neutral or alkaline buffer is used for MEKC separation.
  • the pH of the buffer used for MEKC separation can be any one of the following: about 7.0, about 8.0, about 9.0, about 10.0, about 11.0, about 12.0, or from about 7.0 to about 7.5, from about 7.5 to about 8.5, from about 8.5 to about 9.5, from about 9.5 to about 10.5, from about 10.5 to about 11.5, from about 11.5 to about 12.5.
  • borate buffers with SDS are used in MEKC separation.
  • the concentration of SDS is higher than 9mM.
  • the concentration of SDS can be any one of the following: about lOmM, about 25mM, about 50mM, about lOOmM, or from about 5mM to about 15mM, from about 15mM to about 25mM, from about 25mM to about 35mM, from about 35mM to about 45mM, from about 45mM to about 55mM, from about 55mM to about 65mM, from about 65mM to about 75mM, from about 75mM to about 85mM, from about 85mM to about 95mM, from about 95mM to about 105mM.
  • MEKC separation method can be used for samples containing glycans with small GU values, such as O-glycans or O-linked glycans, and highly sialic glycans.
  • sialic acid is used herein to refer to N- or O-substituted derivatives of neuraminic acid.
  • neuroaminic acid is used herein to refer to 5-amino-3,5-dideoxy- D-glycero-D-galacto-non-2-ulosonic acid, a monosaccharide with a nine-carbon backbone.
  • sialic glycans is used herein to refer to glycans with sialic acid bound to them.
  • highly sialic glycans is used herein to refer to glycans with at least 2 sialic acids bound to them.
  • CGE separates compounds based on their sizes and has been successfully applied in the preliminary experiments to analyze glycans in the present disclosure.
  • MES-TRIS buffer is used for CGE separation.
  • the pH of the buffer used for CGE separation can be any one of the following: about 4.5, about 5.5, about 6.5, about 7.5, about 8.5, or from about 4.0 to about 5.0, from about 5.0 to about 6.0, from about 6.0 to about 7.0, from about 7.0 to about 8.0, from about 8.0 to about 9.0.
  • the separation buffer systems can be the combination of any of the following: borate, phosphate, citrate, formate, tris-acetate, acetate, carbonate buffers and zwitterionic buffers (MES, Bis-Tris, PIPES, ACES, MOPS, TES, HEPPS, Tricine, TAPS, Bicine, CHES, CAPS, AMPD, TABS, AMPSO, CRES, CAPSO, AMP, CABS).
  • the glycans being detected and/or analyzed using the CE-LIF method described herein can be N-glycans or O-glycans.
  • the glycan sample being detected and/or analyzed using the CE-LIF method described herein is prepared by the glycan capturing and/or enriching method as described herein.
  • Example 1 With the aid of MALDI-TOF monitoring technique, a few parameters were studied for the capturing and enriching of cell surface glycoproteins.
  • Example 1.1 MALDI-TOF mass spectrometry
  • Example 1.2 Different oxidation incubation times (10 minutes, 30 minutes, 15 hours, and 24 hours) were tested. From the results, it was found at shorter incubation time (10 minutes), the background signal was noisier compared to the others (Fig. 2). 30 minutes is selected as one possible oxidation incubation time since it gave lower background interference and also shortened the experimental time, while 10 minutes can be used when rapid screening is required.
  • Example 1.3 By keeping the amount of beads fixed at about 2.55x10 , different cell numbers were tested to determine the number of cells required to produce a spectrum with desirable sensitivity and resolution on MALDI-TOF. The cell numbers tested were 3 x 10 7 , 1 xlO 7 , 3 x 10 6 , 1 x 10 6 , 5 x 10 5 , 4 xlO 5 and 3 x 10 5 cells. It was found that signal intensity and peak resolution decreased significantly at 4 x 10 5 cells (Fig. 3). At cell count of 5 x 10 5 and above, good peak signal and resolution were obtained. [00113] Example 1.4. Four different coupling times at 1 hour, 5 hours, 15 hours, and 24 hours were tested.
  • the ratio of the peak intensity at m/z 11320 and m/z 15340 to that of m/z 13777 and m/z 14006 was noted to be increasing as the coupling time increased from 1 hour to 24 hours (Fig. 4).
  • coupling time of 15 hours or overnight was chosen as one possible time used in the current method. 1 hour can be used when rapid screening is required.
  • Example 2 An example of the oxidizing agent used is periodate, such as sodium metaperiodate.
  • the oxidation reaction using periodate can convert cis-diols group of the carbohydrate moities in the glycoproteins to aldehydes.
  • the oxidized products are then put into contact with the beads, for example hydrazide functionalized beads. This allows the aldehydes formed from the oxidation reaction to couple to the beads by reacting with the hydrazide groups immobilized on the beads, forming covalent hydrazone bonds.
  • Example 3 When the biological materials carrying glycoproteins containing microorganisms, tissues or intact cells are used, the cells can be inspected under the microscope to ensure that they were not lysed in the oxidation and coupling processes.
  • Example 4 When the biological materials carrying glycoproteins contain microorganisms, tissues or intact cells, after the cell surface glycoproteins are bound to the beads, an additional cell lysis step is also carried out, leaving substantially only the cell surface glycoproteins attached to the beads.
  • the first technique was a physical method carried out using a homogenizer, such as a Heidolph Diax 900 homogenizer (Kelhaim, Germany), while the second technique was a chemical cell lysis method using reagents such as SDS.
  • Fig. 5 shows the results that were obtained from both lysis techniques. While using a homogenizer to lyse cells generated more fragments of smaller mass, using SDS produced more fragments that were of higher mass. However, using a homogenizer to lyse cells is more time consuming as only one sample can be processed at a time. In one instance, SDS method was therefore chosen for cell lysis.
  • Example 5 The concentration of SDS used for cell lysis was varied and tested. Five concentrations of SDS, 0.05 mM, 0.075 mM, 0.10 mM, 0.25 mM and 0.50 mM were studied (results see Fig. 6). About or at least 0.10 niM SDS was selected to ensure that the chemical cell lysis method also works on cells which are more difficult to be lysed.
  • Example 6 To validate the glycoprotein capture effect, the method was tested using a mixture of fetuin, a well-known glycoprotein standard bearing at least four N-linked glycosylation sites, and bovine serum albumin (BSA), a protein standard that does not contain any glycans.
  • BSA bovine serum albumin
  • Example 7 After the oxidized glycoproteins are coupled to the beads after being in contact with the beads for a sufficient period of time, or after the cells are lysed when the biological materials carrying glycoproteins contain microorganisms, tissues or intact cells, MALDI-TOF technique can be used to measure the amount of glycoproteins captured. When MALDI-TOF is used, beads can be removed before the MALDI-TOF test.
  • Two methods were tested for removing the beads from the glycoproteins before the MALDI-TOF test.
  • PNGase F targets the bond between the glycan and the peptide chain. Since the two reagents target the glycopeptide cleavage at different positions, it was expected that the MALDI spectrum obtained from both analyses to differ. However, the results showed that the two spectra did not differ significantly.
  • formic acid was chosen as the reagent used for glycopeptides cleavage. The reason was that formic acid is more cost effective and requires a shorter time for glycopeptides cleavage.
  • Example 8 Since an acidic environment is required for both the labeling and the releasing of glycans, the simultaneous hydrazone bond cleavage and APTs labeling were explored.
  • the protein backbones in the bead -bound glycoproteins can be released via an enzymatic reaction, using enzymes such as PNGase F or endoglycosidase. After the enzymatic reaction glycans are released from linked peptides or proteins. However, they are still coupled to the beads and it is necessary to release them from the beads before carrying out further analysis. To break the bonds between glycans and beads, acidic hydrolysis can be used.
  • An acid such as citric acid was used as an example and cleavage of glycans from the beads were achieved when acid concentrations ranging from 0.5 to 3 M, in particular 0.8 to 1.6 M were used.
  • Acidic hydrolysis method simplifies the sample processing steps by allowing glycan labeling and glycan cleaving from the beads to take place simultaneously.
  • the one-step glycan labeling and cleaving method also reduces the sample dilution times required when compared to the two-step method, thus making the lowest sample dilution times possible.
  • Example 9 The incubation temperature during glycan cleavage from beads was investigated at 60 °C, 70 °C, 80 °C and 90 °C, using fetuin sample. It was noted that the peak areas increased from 60 °C to 70 °C and started to drop slightly at 80 °C and 90 °C. As lower temperature may also be helpful to maintain N-glycan structure, temperature range of 65°C to 75°C was chosen as one possible temperature range, and specifically 70 °C was chosen as one possible temperature at which incubation can be carried out.
  • Beads with captured glycoproteins were treated by 0.01 unit of PNGase F enzyme. The mixture was incubated in a 37°C water bath, overnight. The supernatant was discarded and the beads were washed thrice with PBS. The sample was freeze-dried using a vacuum evaporator for 30 minutes. 2 ⁇ of APTS (100 mM in 1.2 M citric acid) and 2 ⁇ of sodium cyanoborohydride (1 M in THF) were added to the beads. The mixture was then incubated in a 70°C water bath for 1 hour. After incubation, ultra-pure water was added to the mixture. The beads were then removed and the solution was stored in a freezer for further use. HILIC uElution Plate from Waters (Milford, MA, USA) was used to purify glycans.
  • Bovine fetuin (1 mg) was dissolved into 1 ml of PBS solution. 50 ⁇ of the mixture was transferred into 2 ml centrifuge tube containing 400 ⁇ sodium periodate solution (1.6 mM in 0.1 M anhydrous sodium acetate, pH 5.6) and incubated at room temperature in the dark for 30 minutes. After incubation, protein was purified by ZipTip C4 protocol and reconstituted in 50 ⁇ ice-cold coupling buffer (0.1 M anhydrous sodium acetate, pH 5.6). 25 ⁇ of magnetic beads (0.75 mg) were transferred into a 2 ml centrifuge tube. An external magnet was used to attract the beads while discarding the supernatant.
  • the beads were washed thrice with ice-cold coupling buffer and resuspended in 50 ⁇ ice-cold coupling buffer. The beads were subsequently transferred to the oxidized fetuin sample and incubation was done at room temperature in the dark, overnight. The next day, the supernatant was discarded and the beads were washed thrice with 50 ⁇ 1 ammonium bicarbonate solution (50 mM, pH 8.5) and then reconstituted in ⁇ ammonium bicarbonate solution.
  • N-glycans profile of bovine fetuin as shown in Fig. 7 is consistent with what has been published earlier. This indicates that the glycan capturing and enriching method in this disclosure is effective for glycoproteins. As the fetuin N-glycans are highly sialylated, the results also confirmed that the method is this disclosure would not damage the sialic acid content on N-glycans.
  • a CHO clone was developed from CHO Kl cell line and it was used in the development of monoclonal antibody.
  • the monoclonal antibody generated contained N- glycans and MALDI-TOF results indicated that the dominated two N-glycans were GOF and GIF, with GOF at about 40.6% and GIF at about 32.6%, in term of their percentages in total N-glycans,
  • 1 x 10' CHO cells were removed from the cultured flask and washed twice with lx ice-cold PBS by centrifugation at 1000 rpm for 5 minutes at 4°C. The cells were then reconstituted in 100 ⁇ ⁇ of lx ice-cold PBS, transferred into a 2 mL amber tube containing 900 ⁇ ⁇ 1.6 mM sodium periodate solution and incubated at 4°C in the dark for 30 minutes.
  • the oxidation reaction was terminated by centrifuging the cell suspension at 1000 rpm for 5 min at 4°C to remove the solution and subsequently washing the cell pellet twice with lx ice-cold PBS and ice-cold coupling buffer respectively before re-suspending in 500 ⁇ L ⁇ o ⁇ the coupling buffer.
  • 50 ⁇ ⁇ (1.5 mg) of magnetic beads were washed thrice before re-suspending them in 50 ⁇ ⁇ of ice-cold coupling buffer.
  • the magnetic beads were then transferred to the oxidized cell sample and incubated overnight with gentle rotation at 4°C. After incubation, the cell suspension was removed using external magnet and the magnetic beads were washed once with ice-cold coupling buffer.
  • ice-cold blocking buffer (67 mM D-(+)- glyceraldehyde in coupling buffer) were added to the beads and incubated for 1 hour at 4 °C. Subsequently, the blocking buffer was removed and the beads were washed thrice with lx ice cold PBS. 200 ⁇ . of cell lysis buffer (0.10 mM SDS in 75% ethanol) were added to the beads and incubated on ice for 5 minutes before centrifuging at 13000 rpm for 10 minutes at 4°C. The lysis buffer was then removed and the beads were washed thrice with deionized water and reconstituted in 50 mM ammonia bicarbonate, pH 8.5.
  • Beads with captured glycoproteins were treated by 0.01 unit of PNGase F enzyme. The mixture was incubated in a 37°C water bath overnight. The supernatant was discarded and the beads were washed thrice with PBS. The sample was freeze-dried using a vacuum evaporator for 30 minutes. 2 ⁇ of APTS (100 mM in 1.2 M citric acid) and 2 ⁇ of sodium cyanoborohydride (1 M in THF) were added to the beads. The mixture was then incubated in a 70°C water bath for 1 hour. After incubation, ultra-pure water was added to the mixture. The beads were then removed and the solution was stored in a freezer for further use. HILIC uElution Plate from Waters (Milford, MA, USA) was used to purify glycans.
  • Borate buffers containing variable SDS concentrations could be used in MEKC.
  • 10 mM SDS in 50 mM borate buffer was prepared by adding 0.5 mL of 0.4 M SDS into 10 mL of 0.1M sodium tetraborate and diluted with pure water up to 20 mL. Subsequently, pH measurement was conducted and adjustment was made to reach pH 9.33.
  • the other two borate buffers containing SDS 25 mM and 50 mM were prepared by the same procedures with adjusted calculation in the total volume of borate and SDS used to obtain desired concentration.
  • Acetate buffers with SDS for MEKC were prepared by different concentrations of SDS (10 mM, 25 mM and 50 mM) in 25 mM acetate buffer, using similar method. The pH value was adjusted to 4.50 by 1M acetic acid. Acetate buffer could be used for CZE separation. The buffer was prepared by diluting 2.5 mL of 0.25 M ammonium acetate with water to 25 mL and adjusting the pH to 4.75. Buffer used for CGE was 1 % HPC (w/v), 1 % HEC (w/v), 80 mM MES and 40 mM TRIS. It was prepared by adding 9.6 mL of MES to 12 mL of TRIS stock solutions and diluted with water to 30 mL. The mixture was added by 0.3 g of HPC and HEC generating concentration of 1 % (w/v) for each polymer and desired pH was reached at 6.11.
  • the separation buffer systems can be the combination of any known buffers adapted to the desired separation, including borate, phosphate, citrate, formate, tris-acetate, acetate, carbonate buffers and zwitterionic buffers (MES, Bis-Tris, PIPES, ACES, MOPS, TES, HEPPS, Tricine, TAPS, Bicine, CHES, CAPS, AMPD, TABS, AMPSO, CRES, CAPSO, AMP, CABS).
  • CZE separation could use acetate buffers with pH in the range of 2 to 7, or in the range of 4-5.
  • the MEKC separation buffers could be borate buffers of basic pH values ranging from 8 to 12, or from 8.5 to 10.
  • the CGE separation could use buffers with pH values ranging from 5 to 9.
  • the concentrations of the buffers could be from 10 to 500 mM, or from 20 to 100 mM.
  • at least one surfactant/micelle is added into separation buffer.
  • the additive can be surfactants with at least one C8 to C24 alkyl chain, or tetramer, pentamer, and hexamer crown ethers, or ( ⁇ -, ⁇ -, ⁇ - or ⁇ ) cyclodextrins with or without functional groups like sulphonates, carboxylates, sulphates and phosphates.
  • the dodecylsulphate is used in one instance.
  • separation buffer such as HPC, HEC, PVA, PEO, LPA, PEG.
  • the mixture was stirred at 70 °C to facilitate the dissolution process and a transparent polymer solution was obtained.
  • a new 50 ⁇ ID fused capillary of 1.2 meters was rinsed by 0.1 M NaOH and deionized water respectively to activate the capillary wall prior to PVA coating.
  • the activated capillary was then injected by PVA solution and left at room temperature for 45 minutes.
  • the coating solution was removed out of the capillary and the capillary was heated at 140 °C for five hours under nitrogen gas flow.
  • the bare fused-silica capillary was used in MEKC separation.
  • the capillary was first washed by deionized water for five minutes, 0.1 M NaOH solution for ten minutes, deionized water for five minutes, and the running buffer for ten minutes. In between runs, the capillary was replenished with running buffer for two minutes and the running buffer was renewed after every five runs.
  • Sample injection was performed with a pressure of 0.5 psi for five seconds. The applied voltage was ranged from 20 kV - 30 kV and the experiments were carried out at room temperature of 25 °C.
  • PVA coated capillary was used for CZE separation. Prior to use, the coated capillary was rinsed with deionized water for ten minutes and running buffer for ten minutes. In between runs, the capillary was flushed by running buffer for five minutes and the running buffer was renewed after every five runs. Sample injection was performed with a pressure of 0.5 psi for five seconds. The applied voltage was ranged from -30 kV to - 15 kV and the experiments were carried out at room temperature of 25 °C.
  • the coated capillary was conditioned by deionized water for ten minutes and running buffer for 15 minutes. In between runs, the capillary was rinsed with running buffer for 12 minutes and the running buffer was renewed after every five runs. Sample injection was performed with a pressure of 0.5 psi for five seconds. The applied voltage was ranged from -30 kV to -20 kV and the experiments were carried out at room temperature of 25 °C.
  • Sensitivity is the crucial factor in the detection and analysis of glycans.
  • CE separation coupled with LIF detector provides a good solution for the analysis of small amount samples.
  • two types of online stacking methods - field- amplified sample stacking (FASS) and large-volume sample stacking using the electroosmotic flow pump (LVSEP). It was observed that FASS showed larger peaks, compared with LVSEP.
  • FASS field- amplified sample stacking
  • LVSEP electroosmotic flow pump
  • Bovine fetuin is a well-known protein standard with at least four N-linked glycoproteins.
  • a successfully enriched fetuin sample was analyzed.
  • the fetuin sample analysed utilizing FASS method was 100 times more diluted than the fetuin analysed utilizing conventional CZE.
  • the FASS method may prove to be more suitable due to its higher sensitivity as compared to that of conventional CZE.
  • the unique final sample matrix can minimize the error introduced by electrokinetic sample loading methods.
  • Example 15 CE-LIF for N-glycan captured from intact Chinese Hamster ovary (CHO) cells
  • Glycan sample from CHO cells was prepared using the capturing and enriching method described in this disclosure. The sample was subsequently analyzed using the conventional CZE method.
  • Fig. 10 shows the CE electropherograms of the N-glycans enriched from a lab-developed expressing clone.
  • Chromatography is the most popular technique in analytical chemistry. However, in cases where tens of target compounds and complex sample matrices are involved, it is difficult to achieve baseline separation for all the compounds in the samples.
  • the development of multiplexed chromatography methods provides a novel approach to enhance separation performance by the combination of two or more separation mechanisms simultaneously.
  • Example 16.1 The effect of concentration and pH of running buffer in the separation of dextran 'ladder' was examined.
  • Ammonia acetate buffers are generally used in glycan separation using CZE method. 25 mM ammonia acetate buffer was tested.
  • the separation of dextran ladder and a glycan standard is shown in Fig. 11. Borate buffer was used, and SDS was added to improve separation. SDS concentrations ranging from 10 mM to 50 mM were used in either 50 mM borate buffer (pH 9.3) or 25 mM acetate buffer (pH 4.5). Both acidic and basic buffer systems were able to separate the dextran ladder.
  • borate buffer containing SDS showed better resolution, faster migration time and better reproducibility as compared to acetate buffer. It may be due to the strong electroosmotic flow (EOF) at high pH value and additional selectivity achieved by borate complexation with carbohydrates. 25 mM SDS in 50 mM borate buffer provided the most efficient migration time and slightly better peak shapes and was used for following experiments.
  • EEF electroosmotic flow
  • the electropherogram of separation of the APTS-derivatized dextran ladder shows largest analyte migrates first and the smallest migrates last. This is different from CZE result due to the application of normal polarity separation mode.
  • SDS micelle in MEKC could trap excess labeling fluorophore simultaneously and simplified the sample preparation procedure.
  • Example 16.2 Commercial CGE analytical kits for DNA test have been applied in the separation of glycans. As shown in Fig. 13, the CGE analysis using MES-TRIS buffers containing polymers displayed similar separation result as the CZE electropherogram. Only small impurity peaks of excess fluorophore were observed at the beginning of electropherogram, indicating the polymer matrix may also trap the excess APTs.
  • Table 2 Linear range, correlation coefficients and LOD of three glycan standards by CZE,
  • Example 17 To apply the multiplexed glycan separation methods, it is necessary to evaluate the reproducibility of different mechanisms, in terms of migration time and peak area.
  • Glycan standards were spiked into dextran ladder solution at 1 mg/L respectively, like C0920 in Fig. 11. Six replicates of experiment for each spiked-standard were performed by CZE, MEKC and CGE. Usually, migration times of glycans are expressed in GU with reference to an oligosaccharide ladder. The relative standard deviations (RSDs) of migration time (MT), RSDs of peak area, and GU values obtained from those modes of CE were calculated and results are summarized in Table 3. In addition, relative migration times of MT standard / MTGU5 were also counted.
  • RSDs relative standard deviations
  • the second or third separation mechanism contributes in two ways to achieve accurate and precise determination of glycans. Firstly, it makes double- checking of target compounds possible and allows confirmation that the peak found is the correct one. Secondly, in cases that not all compounds can be separated by the first separation mechanism, or in cases where the separation of all target compounds can only be achieved with extremely long separation time, or in cases that need very tight control of the experiments conditions which are hard to maintain constant, the second or third mechanism can be designed to achieve the separation of specific target compounds which are difficult to be baseline separated by the first mechanism.
  • GU values used in N-glycan analysis are based on oligosaccharide ladder. As a kind of data normalization, it definitely can cause some loss in original information. To maintain as much as possible separation capacity and minimization of run to run peak fluctuations, other data process methods were explored. Except GU values, researchers also applied GU2 peak (such as maltose or sucrose) as internal standards to normalize results. However, in MEKC mechanism, GU2 peak appeared at the end of separation and the migration time was therefore very unstable. As a result, the data normalization showed unreliable results. In contrast, the internal standard with medium GU value is a good alternative. The internal standard can have a GU value ranging from about 4-8, or at around GU 5.
  • Example 19 The CE-LIF analysis of glycans on glycoprotein fetuin
  • Fetuin is a typical glycoprotein present in the circulation which is synthesized by hepatocytes. It exists in a variety of glycoforms containing bi-, tri-, and terra- antennary oligosaccharides with variable sialylation.
  • MEKC, CZE, and CGE were applied for the analysis of bovine fetuin. It was observed that CGE gave only two dominant peaks suggesting lack of sensitivity in accordance to LOD experimental part where CGE exhibited highest LOD among those three methods. In contrast, six dominant peaks were achieved in MEKC and CZE indicating better resolution (Fig. 15).
  • oligosaccharides with a greater proportion of a 2,6-linked sialic acids were less retained compared to the a 2,3 -linked ones.
  • the migration order is also coordinated with Glycobase databse (NIBRT).
  • NEBRT Glycobase databse
  • peak 1 possesses more of a 2,6-linked sialic acids than the other peaks and thus migrates first.
  • peak 6, having more a 2,6-linked sialic acid migrates after peak 5 due to the existence of Gal(P l,3)GlcNAc.
  • This migration order was assigned in reverse for MEKC as it employed normal polarity (Fig.
  • CZE allows for high sensitivity, rapid analysis times and ease of automation for routine analysis.
  • Combination of MEKC and CZE/CGE separations can provide powerful support for studies of complex samples by increasing resolution and capacity of chromatographic separation, not limited to glycan analysis.
  • Example 20 The CE-LIF analysis of nucleotides

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Abstract

La présente invention concerne un procédé amélioré pour la capture et/ou l'enrichissement de glycanes à partir de glycoprotéines ou de matériaux biologiques portant des glycoprotéines. Dans un mode de réalisation, le procédé pour la capture et/ou l'enrichissement de glycanes comprend les étapes consistant à : mettre en contact l'échantillon de glycoprotéines avec un agent oxydant, tel que le periodate, lier les produits oxydés à des billes magnétiques, paramagnétiques ou polymères, qui pourraient être à fonction hydrazide, séparer les glycanes des glycoprotéines à l'aide d'une réaction chimique impliquant des acides ou d'une réaction enzymatique, telle que l'utilisation d'une PNGase pour obtenir des glycanes liés à des billes, enfin étiqueter les glycanes liés aux billes et dissocier les glycanes des billes. Un autre mode de réalisation concerne un procédé pour détecter et analyser les glycanes à l'aide d'électrophorèse capillaire multiplexée avec un système de fluorescence induite par laser (CE-LIF) comprenant la chromatographie électrocinétique micellaire (MEKC) en combinaison avec l'électrophorèse capillaire de zone (CZE) ou l'électrophorèse capillaire en gel (CGE)
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