JP2006038674A - Analysis method of glycoprotein sugar chain, and manufacturing method of unlabeled sugar chain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis method which enables a simple and quick operation, excellent separability of a sugar chain mixture, and excellent sensitivity and quantitativeness, and has no problem in a device, concerning the analysis method of a glycoprotein sugar chain, and also to provide a manufacturing technique for manufacturing and supplying an unlabeled sugar chain having a known structure stably without any actual problem. <P>SOLUTION: This analysis method of the glycoprotein sugar chain is characterized by having processes for: liberating an N-bond type sugar chain from the glycoprotein in aqueous solution having pH of 6-9, and performing fluorescence labeling to an amino acid in the liberated glycosylamine type sugar chain; and analyzing the fluorescence-labeled glycosylamine type sugar chain by HPLC-FLD. This manufacturing method of the unlabeled sugar chain is characterized by performing analysis and isolation by HPLC-FLD, and eliminating a fluorescent functional group in the glycosylamine type sugar chain in the isolated prescribed fraction. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a technique for analyzing a glycoprotein sugar chain, which is a biopolymer, and a technique for producing an unlabeled sugar chain. More specifically, a method for analyzing a glycoprotein sugar chain, which enables simple and rapid profile analysis of sugar chains constituting the glycoprotein using high performance liquid chromatography (HPLC), and a high The present invention relates to a method for producing a pure unlabeled sugar chain.

  A sugar chain constituting an in vivo glycoprotein, particularly an N-linked sugar chain, is a component that greatly affects the biological activity and the like of the glycoprotein. Also, sugar chains often play an important role in the application of glycoproteins to biopharmaceuticals.

  On the other hand, the type of monosaccharide constituting the sugar chain, the branched structure of the sugar chain, and the like differ depending on the type of sugar protein from which the sugar chain is derived, and sugar chains having extremely diverse structures have been reported. Furthermore, even specific glycoproteins exhibit a diversity called microheterogeneity within the molecular population and within the molecule. In recombinant glycoproteins, it is also known that the sugar chain structure changes due to subtle changes in culture conditions. Therefore, in biochemical research of glycoproteins, and in the characterization and quality evaluation of pharmaceuticals to which glycoproteins are applied, techniques for analyzing sugar chains of glycoproteins having such diversity and heterogeneity. Is essential.

  Various analytical methods have been developed so far for the analysis of sugar chains of glycoproteins. In particular, after the sugar chain is chemically or enzymatically released from the sugar protein, (1) the released sugar chain is separated by high pH anion exchange HPLC analysis (HPAEC-PAD) using an electrochemical detector. Method of detection, (2) After the free sugar chain is fluorescently labeled (reductive amination reaction) with an aldehyde group at the reducing end in the presence of a reducing agent with a fluorescent reagent, HPLC with a fluorescence detector (HPLC-FLD) Alternatively, a method of analyzing by capillary electrophoresis with a laser-excited fluorescence detector (CE-LIF) or the like is widely used.

  Among these, (1) the method of separating and detecting free sugar chains by HPAEC-PAD without labeling has an advantage that a sugar chain profile can be obtained by a simple operation. However, expensive and special HPLC equipment and detectors are required, the detection principle is noisy, the sensitivity is low compared to the fluorescence detection method, maintenance to obtain reproducibility is difficult due to the structure of the equipment, and detection Since sensitivity depends on the structure of the sugar chain, there are problems such as low quantitativeness. These problems are a major obstacle when quality evaluation of pharmaceuticals to which glycoproteins are applied is based on sugar chains.

  On the other hand, (2) a method in which a released sugar chain is fluorescently labeled with various fluorescent reagents using a reductive amination reaction and then analyzed by HPLC-FLD or CE-LIF (fluorescence detection method) is a sugar chain. It is excellent in separation of the mixture and in terms of sensitivity and quantification. Therefore, it can be applied to the quality evaluation of pharmaceutical products to which sugar protein is applied based on sugar chains. However, the reductive amination reaction requires a plurality of steps in principle and is complicated and requires several days to obtain a sugar chain. In addition, a reducing agent such as sodium cyanoborohydride, which is a poisonous substance, is required, and there is a safety and health problem.

  Thus, conventionally, it is a method for analyzing a glycoprotein sugar chain, which allows easy and quick analysis, is excellent in separation of a sugar chain mixture, and is excellent in terms of sensitivity and quantification, and An analysis method free from problems on the apparatus has not been obtained, and the development of an analysis method capable of solving these problems has been desired.

  Furthermore, for biochemical studies of glycoproteins and the development of pharmaceuticals to which glycoproteins are applied, standard sugar chains with a known structure, particularly unlabeled standard sugar chains, that is, labeled with fluorescent functional groups. There is no need for free standard sugar chains. In other words, the structure is known for the study of sugar chain biology, such as analysis of the interaction between sugar-binding protein and sugar chain in vivo, and the practical application research for the development of new drugs involving sugar chains. Since the utility value of an unlabeled standard sugar chain is extremely high, its stable production and supply are desired.

  For example, sugar chains perform important functions such as information transfer between cells through interactions with sugar-binding proteins in vivo, and therefore interaction analysis using many complex carbohydrate sugar chains is necessary. In order to conduct this interaction analysis, the commercialization of a sugar chain array in which standard sugar chains are immobilized has been advancing, and an unlabeled standard sugar chain that can be immobilized on a sugar chain array, etc., is being sought. It has been. In addition, in a study for creating a database of relationships between various sugar chains and retention times in HPLC, a standard sugar chain having a known structure is required. However, conventionally, there are few standard sugar chains having a known structure that are stably supplied, and in particular, few unlabeled standard sugar chains are supplied.

  Examples of the technique for producing an unlabeled sugar chain include a method of chemically synthesizing and a method of biological synthesis using a cell into which a glycosyltransferase gene has been introduced. However, the method of chemically or biologically synthesizing requires high-level and special techniques, but is not practical and promising in terms of structure arbitraryness and completeness and recovery amount.

  A method for recovering unlabeled sugar chains from the fluorescently labeled sugar chains has also been reported. In other words, a standard sugar chain having a known structure can be obtained by releasing a sugar chain from a sugar protein, fluorescently labeling the released sugar chain by a reductive amination reaction, and then separating by HPLC. However, it is extremely difficult to remove a fluorescent functional group from a fluorescently labeled sugar chain by a reductive amination reaction. A method of returning to an unlabeled sugar chain using hydrogen peroxide or the like under slightly acidic conditions However, it was a method in which the yield was poor and sugar chain degradation was unavoidable. Therefore, it is extremely difficult to convert to an unlabeled sugar chain by this method. Similarly, sugar chains fluorescently labeled by other methods are extremely difficult to return to unlabeled sugar chains.

  Furthermore, the method by the above-mentioned HPAEC-PAD is also mentioned and is often used. According to this method, an unlabeled standard sugar chain can be separated and collected. However, this method has a problem in the performance of the apparatus as described above and requires a mobile phase containing a high concentration of sodium hydroxide. . Further, when the separated and recovered sugar chain is used as a standard sugar chain, an operation such as desalting is required, and there are significant practical problems such as complicated post-treatment.

  Thus, conventionally, a production technique for stably producing and supplying an unlabeled sugar chain having a known structure without practical problems has not been developed, and its development has been desired.

  The present invention is a method for analyzing a glycoprotein sugar chain, which is easy to operate and enables rapid analysis, is excellent in separation of a sugar chain mixture, is excellent in sensitivity and quantification, and further on the apparatus. The problem is to provide an analysis method that is free from problems. Another object of the present invention is to provide a production technique capable of stably producing and supplying an unlabeled sugar chain having a known structure without practical problems.

  The inventor of the present invention uses a variety of reagents used for fluorescent labeling of amino acid amino groups for glycosylamine amino groups obtained by releasing N-linked sugar chains of glycoproteins. Based on the idea that there is a possibility that can be done.

  As a result, if a sugar chain is released from a glycoprotein as a glycosylamine and fluorescent labeling of the released glycosylamine-type sugar chain is performed under predetermined conditions, a stable labeled glycosylamine can be obtained and analyzed by HPLC-FLD. In addition, if the amino group of the glycosylamine-type sugar chain obtained by releasing the glycoprotein is fluorescently labeled and the labeled glycosylamine-type sugar chain is analyzed by HPLC-FLD, The complexities of conventional fluorescent labeling by reductive amination reaction, problems that require time for analysis, safety and health problems, etc. are solved, and the analysis is simple and quick, and the glycan mixture It has been found that the advantage of HPLC-FLD that it is excellent in the separation of and excellent in sensitivity and quantification is also maintained.

  Further, the present inventor can easily and quantitatively remove a fluorescent functional group from a glycosylamine-type sugar chain in which an amino group is fluorescently labeled. After highly sensitive analysis of the type sugar chain by HPLC-FLD, by removing the fluorescent label from the glycosylamine type sugar chain separated by HPLC, an unlabeled sugar chain having a known structure can be easily and It was found that it can be obtained quantitatively. The present invention has been completed based on these findings.

  That is, the present invention comprises a step of releasing an N-linked sugar chain from a glycoprotein in an aqueous solution maintained at pH 6-9 and fluorescently labeling the amino group of the released glycosylamine sugar chain. The present invention provides a method for analyzing a glycoprotein sugar chain, comprising a step of analyzing a fluorescently labeled glycosylamine-type sugar chain by HPLC-FLD (claim 1).

  The present invention also releases an N-linked sugar chain from a glycoprotein in an aqueous solution maintained at pH 6-9, and performs fluorescent labeling on the amino group of the released glycosylamine sugar chain. Unlabeled, characterized in that a fluorescently labeled glycosylamine-type sugar chain is analyzed and separated by HPLC-FLD, and a fluorescent functional group of the glycosylamine-type sugar chain in the fractionated fraction is eliminated A method for producing a sugar chain is provided (claim 5).

  In the method for analyzing a sugar protein sugar chain and the method for producing an unlabeled sugar chain, as a method for releasing an N-linked sugar chain from a sugar protein, a method for obtaining a sugar chain as a glycosyl hydrazide using hydrazine Usually, it is carried out enzymatically in water using peptide N-glycosidase F (PNGase F). Claim 2 is the method for analyzing a glycoprotein sugar chain, wherein the release of the N-linked sugar chain from the glycoprotein is performed using peptide N-glycosidase F. It corresponds to. The reaction of liberating N-linked sugar chains from glycoproteins performed using this peptide N-glycosidase F is usually performed at about 37 ° C. for about 2 hours at which elimination of sialic acid is suppressed. It is desirable.

  Release of the N-linked sugar chain from the glycoprotein is carried out as shown in the following formula 1 (a) to obtain a glycosylamine sugar chain having an amino group at the reducing end. The released glycosylamine-type sugar chain is usually rapidly hydrolyzed with a rotatory rotation and converted to a free sugar chain having a hemiacetal structure as shown in the following formula 1 (b). Is done.

  As a result of detailed examination of reaction conditions for releasing N-linked sugar chains from glycoproteins, the inventors can prevent hydrolysis of glycosylamine in neutral to weakly alkaline water. The present inventors have found that fluorescent labeling of the lower glycosylamine is possible, and that the thus-labeled glycosylamine can be analyzed by HPLC-FLD. Therefore, in the method for analyzing a glycoprotein sugar chain and the method for producing an unlabeled sugar chain of the present invention, the release of the N-linked sugar chain from the glycoprotein and the fluorescent label are pH 6.0-9.0, Preferably, it is performed under the condition of pH 8.0-9.0.

  Fluorescent labeling of the released glycosylamine is performed using an amino group labeling reagent or amino group protecting reagent used for fluorescent labeling of ordinary amino acids and the like. More specifically, examples of the fluorescent labeling reagent to be used include orthophthalaldehyde (OPA), 9-fluorenylmethyl chloroformate (Fmoc-Cl), dansyl chloride, coumarin and the like. Fmoc-Cl, which is easy to desorb, is desirable to recover Fluorescent labeling can be easily performed by mixing such an organic solvent solution of an amino group protecting reagent with a sample solution after digestion with an enzyme.

  Claim 3 corresponds to this preferred embodiment, that is, the aforementioned glycoprotein sugar chain analysis method, wherein Fmoc-Cl is used for fluorescent labeling of glycosylamine. Formula 2 shows the labeling with Fmoc-Cl (Formula 2 (c)) and the elimination reaction (Formula 2 (d)).

  In the case of the conventional method using a reductive amination reaction, fluorescent labeling is performed on the hemiacetal group at the reducing end. As described above, the labeling reaction and purification of the labeled sugar chain are complicated and require a long operation. Requires time (several days). More specifically, (1) the labeling reaction needs to be performed at a high sugar chain concentration because the reaction does not proceed efficiently when a large amount of water is contained. Therefore, it is necessary to concentrate or dry the sample before the reaction, and it takes several hours to concentrate or dry. (2) The labeling reaction is usually performed by heating to 50 to 80 ° C. under acidic conditions. There is concern about the elimination of sialic acid in the sugar chain. On the other hand, in order to suppress the elimination of (3) sialic acid, the labeling reaction may be performed at about 37 ° C., but in this case, it is about overnight. There is a problem that it takes more time.

  On the other hand, the labeling of the amino group according to the present invention can be performed immediately by a simple operation such as mixing a fluorescent reagent dissolved in an organic solvent into the solution after enzymatic digestion, and the concentration and drying of the enzyme reaction solution can be performed. The labeling reaction when Fmoc-Cl is used is sufficient for non-acidic conditions at 37 ° C. for 1 hour or less, and sial is used because it is carried out at non-acidic conditions at a relatively low temperature. There is no concern about acid elimination.

  FIG. 2 shows the relationship between the labeling reaction time and the HPLC-FLD peak height (and reaction temperature) when Fmoc-Cl is used. At a reaction temperature of 37 ° C., the reaction time is about 1 hour, and the peak height reaches saturation, indicating that the reaction is complete within 1 hour.

  In addition, when a conventional reductive amination reaction is used, there is a problem in safety and health because sodium cyanoborohydride, which is a poisonous substance, is used as a reducing agent. In the method of the present invention, such a poisonous substance is used. Since it is not necessary, it is excellent in terms of safety and health.

  In analysis by HPLC-FLD, it is essential to remove excess fluorescent reagent and reducing agent before analysis by HPLC after fluorescent labeling. Conventionally, the removal of the excess fluorescent reagent and the reducing agent has been performed by a method involving gel filtration and the like, which is complicated and takes a long time.

  However, in the method of the present invention, excess reagent (Fmoc-Cl, etc.) after fluorescent labeling is removed by adding an organic solvent insoluble in water to an aqueous solution to which fluorescent labeling is performed, mixing, stirring, And this organic solvent layer can be separated and the separated organic solvent layer can be easily removed. That is, since excess reagent is distributed to the organic solvent layer and sugar chains remain in the aqueous layer, the fluorescently labeled sugar chain can be easily purified by collecting the aqueous layer. Examples of the water-insoluble organic solvent used here include chloroform, ether, ethyl acetate and the like, and one or more mixed solvents selected from these are preferably used.

  Normally, excess reagent can be completely removed from the chromatogram by repeating the above operation three times. This operation does not require any special pretreatment column or complicated operation, and the time required is 15 minutes or less. Claim 4 is this preferred embodiment, that is, the method for analyzing glycoprotein sugar chains, wherein an aqueous solution insoluble in water is added to an aqueous solvent insoluble in water, mixed, stirred, This embodiment corresponds to an aspect further comprising a step of separating the organic solvent layer and removing the separated organic solvent layer.

  As described above, the time required for preparing a sample for sugar chain analysis is about 2 hours for releasing N-linked sugar chains, about 1 hour for labeling reaction, about 15 minutes for purification with an organic solvent, about 3 in total. About half an hour is sufficient, and the conventional method using the reductive amination reaction usually requires one week (about two days at the shortest), but is extremely quick and simple.

  The sample prepared as described above is subjected to measurement by HPLC-FLD. The conditions similar to the conditions applied to the sugar chain profile analysis performed conventionally are applied for the conditions of HPLC-FLD. For example, a normal phase HPLC using an amino column and a separation detection method using a fluorescence detector can be used, and analysis can be performed with high resolution.

  The method for producing an unlabeled sugar chain of the present invention is used in the HPLC-FLD measurement as described above, and the glycosylamine fractionated and labeled by HPLC is fractionated and separated by HPLC from a predetermined fraction. The fluorescent functional group is eliminated to obtain an unlabeled sugar chain. The method for removing the fluorescent functional group from the labeled sugar chain may be carried out in the same manner as the deprotection method for amino acids in which the amino group is protected. For example, removal of the Fmoc group from a sugar chain labeled with Fmoc-Cl can be achieved by adding morpholine and dimethylformamide to an aqueous solution of the labeled sugar chain and in the range of 4 ° C. to 50 ° C. for about 5 minutes to 180 minutes. Just keep it. It can be easily carried out by keeping the temperature at 20 to 40 ° C., more preferably 37 ° C. for 20 minutes. When the temperature exceeds 50 ° C., sialic acid is likely to be eliminated. On the other hand, when the temperature is less than 4 ° C., a long reaction time is required.

  The present invention is characterized by comprising a reagent for fluorescently labeling an amino group and a reagent for desorption of a label in addition to the aforementioned method for analyzing a glycoprotein sugar chain and the method for producing an unlabeled sugar chain. A kit for analyzing a glycoprotein sugar chain is provided (claim 6). With this kit, the analysis of glycoprotein sugar chains and the production of unlabeled sugar chains by the method of the present invention described above can be carried out quickly and easily. Examples of the reagent for fluorescently labeling an amino group include orthophthalaldehyde (OPA), 9-fluorenylmethyl chloroformate (Fmoc-Cl), dansyl chloride, and coumarin. Reagents for elimination of the labeling group include morpholine and dimethylformamide.

The present invention further provides a kit for analyzing a glycoprotein sugar chain, which is a kit for analyzing a glycoprotein sugar chain as described above, and further comprises a standard glycoprotein (Claim 7). With this kit, an unlabeled standard sugar chain having a known structure, which is a component of the standard glycoprotein, can be easily produced by the method of the present invention. Standard glycoproteins include fetuin, α ₁ -acid glycoprotein, fibrinogen, transferrin, thyroglobulin, recombinant immunoglobulin G, ovalbumin, ribonuclease B, and the like.

  According to the method for analyzing a glycoprotein sugar chain of the present invention, the analysis of a glycoprotein sugar chain that takes several days in the conventional HPLC-FLD analysis method can be performed with a simple operation within a few hours. . In addition, quantitative and sensitivity (several times higher than the labeling method with 2-aminobenzoic acid) equal to or better than the conventional HPLC-FLD analysis is achieved, and a sugar chain profile having excellent resolution is obtained. be able to. Furthermore, the fluorescent functional group introduced into the sugar chain can be removed by a simple operation, and the conversion rate to an unlabeled product is almost 100%. Therefore, by the method for producing an unlabeled sugar chain of the present invention, a high-purity unlabeled sugar chain having a known structure can be easily obtained by converting the sugar chain in the fraction separated and collected by HPLC into an unlabeled product. Can be manufactured.

  Since the method for analyzing glycoprotein sugar chains and the method for producing unlabeled sugar chains of the present invention have such excellent characteristics, the analysis of sugar chain profiles and various structures in the characteristics analysis and quality control of glycoprotein pharmaceuticals. It can preferably be applied to the production of high-purity unlabeled sugar chains having the same. In addition, it can be expected as a new method for regulating stem cells in regenerative medicine, and further, it can be expected to be applied to clinical analysis and the like by utilizing speedup. Since the labeled sugar chain obtained by the method for analyzing a glycoprotein sugar chain of the present invention can be easily converted to an unlabeled sugar chain, the production method of the present invention is extremely useful as a method for producing a sugar chain standard product. It is a high value and original method.

  Next, the best mode of the present invention will be described by taking as an example the case where the Fmoc reagent is used as a fluorescent reagent, but the present invention is not limited to this example.

[Profile Analysis of Glycoprotein Sugar Chain] (Embodiment 1)
(1) Release of glycosylamine First, glycosylamine-type sugar chains are obtained from the glycoprotein by an enzymatic release method using N-glycosidase F (PNGase F). As an example of more specific conditions, a sample glycoprotein is dissolved in 20 mM phosphate buffer to be about 1 to 10 mg / mL, and 1 unit / μL of PNGaseF is added to 100 μL of this solution. By adding 10 μL (10 units) and incubating at 37 ° C., N-linked sugar chains are enzymatically released as glycosylamine-type sugar chains. Since the pH of the buffer is extremely important for the recovery amount of the labeled sugar chain finally obtained, a buffer having an appropriate pH of pH 6.0 to pH 9.0, preferably pH 8.0 to pH 9.0 is preferably used. Use to perform enzymatic reactions. The enzyme reaction time is 1 hour to 24 hours (more preferably about 2 to 5 hours), and good results are obtained.

(2) Labeling and purification of glycosylamine To the enzyme reaction solution containing free glycosylamine thus obtained, 20 mM phosphate buffer (pH 8.5) or purified water was added to make the total volume 400 μL, and then 5 mg / Add 200 μL of mL of Fmoc-Cl acetone solution, leave it at 37 ° C. for 1 hour, add 300 μL of chloroform, stir vigorously, separate the layers, and discard the chloroform layer containing excess Fmoc. This extraction operation with chloroform is further repeated twice, and the aqueous layer containing the labeled glycosylamine (N-linked sugar chain) is directly subjected to analysis by HPLC-FLD, or is dried under reduced pressure and dissolved in an appropriate amount of water. A part of the sample is subjected to analysis by HPLC-FLD.

(3) Analysis by HPLC-FLD Examples of the profile analysis method for Fmoc-labeled sugar chains by HPLC-FLD include the following conditions.

Apparatus: Shimadzu LC-10AD pump, Shimadzu DGU-12A degasser, Hitachi 655-A52 column oven, Jasco FP-920 fluorescence detector Analytical column: Showa Denko Shodex Asahipak NH2P-50 4E 4.6 mm × 250 mm
Mobile phase A: 2% acetic acid acetonitrile solution Mobile phase B: 5% acetic acid 3% triethylamine aqueous solution Elution condition: Gradient, 0 minute (20% B) → 10 minutes (20% B) → 90 minutes (90% B) → 90 1 minute (95% B) → 105 minutes (30% B) → 105.1 minutes (20% B) → 120 minutes (20% B)
Column temperature: 50 ° C
Detection: excitation wavelength 266 nm, fluorescence wavelength 310 nm

[Production of unlabeled sugar chain] (Embodiment 2)
Using the fluorescently labeled sugar chain (glycosylamine) released from the glycoprotein and fractionated by HPLC by the method of Embodiment 1, an unlabeled sugar chain standard product is produced by the following method. That is, 30 μL of dimethylformamide and 20 μL of morpholine are added to an aqueous solution (20 μL) containing a fluorescently labeled sugar chain, which is one fraction collected by HPLC, and left at 37 ° C. for 10 minutes or more. Thereafter, 500 μL of diethyl ether is added and vigorously stirred. After centrifugation, the aqueous layer is recovered and dried to quantitatively recover unlabeled sugar chains.

  Examples are described below, but the present invention is not limited to these examples.

EXAMPLE 1 Sugar chain profile analysis of fetuin Embodiment of fetuin (derived from bovine), which is known to have mainly three-branched complex sugar chains having a plurality of sialic acids at the reducing end, as a glycoprotein sample According to the method described in 1, sugar chain profile analysis was performed. The results are shown in FIG. FIG. 1 (a) shows the result of analysis using 5-fold amount of 2-aminobenzoic acid-labeled sugar chain obtained from the same fetuin by reductive amination. The method of the present invention (Embodiment 1) was several times more sensitive than the reductive amination method, and a similar separation pattern and similar quantitativeness were obtained.

Example 2 De-Fmocization of Fetwin Sugar Chain According to the method described in Embodiment 2, Fmocized sugar chain (HPLC eluate) obtained in Example 1 was de-Fmoced. The results are shown in FIG. Since no peak was observed in the Fmoc sugar chain elution range at the wavelength at which the Fmoc sugar chain was detected, it was shown that the de-Fmoc reaction was completely performed and an unlabeled sugar chain was obtained. Further, this unlabeled sugar chain was labeled with 2-aminobenzoic acid, and HPLC analysis was performed at a wavelength at which the 2-aminobenzoic acid labeled sugar chain was detected. The results are shown in FIG. Since the sugar chain profile of the 2-aminobenzoic acid-labeled sugar chain was obtained, it was shown that the unlabeled sugar chain was present in the sample of FIG. 1 (c).

Example 3 Production of an unlabeled sugar chain having the target sugar chain structure In the chromatogram obtained by the same operation as in Example 1, the fraction corresponding to the maximum peak was recovered (peak A in FIG. 1 (b)). . This peak is known to be a three-branched sugar chain having three residues of sialic acid and has a single structure. This Fmoc sugar chain was subjected to a de-Fmoc operation by the method of Embodiment 2. As a result, an unlabeled three-branched sugar chain having three sialic acid residues was obtained. In order to confirm this, this sugar chain was further labeled with 2-aminobenzoic acid and subjected to HPLC analysis at a wavelength at which the 2-aminobenzoic acid-labeled sugar chain was detected. As a result, a single peak was obtained at the same retention time as the peak in the chromatogram of the fetuin-derived 2-aminobenzoic acid-labeled sugar chain (FIG. 1 (d)) (FIG. 1 (e)). From this, it was shown that a three-branched sugar chain unlabeled body having three sialic acid residues contained in fetuin was prepared with high purity.

Example 4 a ₁ -Aglycoprotein Profile Analysis of Acid Glycoprotein a ₁ -Acid Glycoprotein known to have mainly four-branched complex type sugar chains having a plurality of sialic acids at the reducing end ( In accordance with the method described in Embodiment 1, sugar chain profile analysis was performed using human-derived samples as samples. The results are shown in FIG. A sugar chain profile characteristic of a ₁ -acidic glycoprotein was obtained.

Example 5 Analysis of transferrin sugar chain profile Transferrin (human-derived) known to have mainly two-branched complex sugar chains having a plurality of sialic acids at the reducing end was used as a glycoprotein sample. According to the method described in 1, sugar chain profile analysis was performed. The results are shown in FIG. A sugar chain profile characteristic of transferrin was obtained.

Example 6 Sugar chain profile analysis of fibrinogen As described in Embodiment 1, fibrinogen having a plurality of sialic acids at the reducing end and fibrinogen known to have mainly two-branched complex type sugar chains is used as a glycoprotein sample. According to the method, sugar chain profile analysis was performed. The results are shown in FIG. A sugar chain profile characteristic of fibrinogen was obtained.

Example 7 Sugar chain profile analysis of thyroglobulin It is known that it has a high mannose type sugar chain containing 5-9 residues of mannose and a mainly three-branched complex type sugar chain having multiple sialic acids at the reducing end. According to the method described in Embodiment 1, sugar chain profile analysis was performed using the obtained thyroglobulin as a glycoprotein sample. The results are shown in FIG. A sugar chain profile characteristic of thyroglobulin was obtained.

Example 8 Analysis of Sugar Chain Profile of Recombinant Immunoglobulin G Immunoglobulin G, a commercially available antibody drug that is known to have mainly double-branched complex sugar chains with little sialic acid at the reducing end As a protein sample, sugar chain profile analysis was performed according to the method described in Embodiment 1. The results are shown in FIG. A sugar chain profile characteristic of recombinant immunoglobulin G was obtained.

Example 9 Sugar chain profile analysis of ovalbumin A protein sample obtained by purifying egg albumin, which is known to have mainly high mannose type and mixed sugar chains without sialic acid at the reducing end, from chicken egg white, In accordance with the method described in Embodiment 1, a sugar chain profile analysis was performed. The results are shown in FIG. A sugar chain profile characteristic of ovalbumin was obtained.

Example 10 Sugar chain profile analysis of ribonuclease B According to the method described in Embodiment 1, a sugar chain profile was obtained using ribonuclease B, which is known to have a high mannose sugar chain containing 5 to 9 residues of mannose, as a sugar protein sample. Analysis was performed. The results are shown in FIG. A sugar chain profile characteristic of ribonuclease B was obtained.

It is a figure which shows the chromatogram of the HPLC-FLD of the sugar chain derived from fetuin. It is a graph which shows the relationship between the reaction time of a labeling reaction, and the peak height of HPLC-FLD. It is a figure which shows the chromatogram of HPLC-FLD of the sugar_chain | carbohydrate derived from various glycoproteins. It is a figure which shows the chromatogram of HPLC-FLD of the sugar_chain | carbohydrate derived from various glycoproteins.

Claims (7)

  a step of releasing an N-linked sugar chain from a glycoprotein in an aqueous solution maintained at pH 6-9 and fluorescently labeling the amino group of the released glycosylamine-type sugar chain; A method for analyzing a glycoprotein sugar chain, comprising a step of analyzing an amine-type sugar chain by HPLC-FLD.   The method for analyzing a glycoprotein sugar chain according to claim 1, wherein the N-linked sugar chain is released from the glycoprotein using peptide N-glycosidase F.   The method for analyzing a glycoprotein sugar chain according to claim 1 or 2, wherein the fluorescent labeling of the amino group of glycosylamine is performed using Fmoc-Cl.   The method further comprises a step of adding an organic solvent insoluble in water to an aqueous solution to be subjected to fluorescent labeling, mixing, stirring, separating the aqueous layer and the organic solvent layer, and removing the separated organic solvent layer. The method for analyzing a sugar protein sugar chain according to any one of claims 1 to 3.   In an aqueous solution maintained at pH 6-9, the N-linked sugar chain is released from the glycoprotein, and the amino group of the released glycosylamine sugar chain is fluorescently labeled. A method for producing an unlabeled sugar chain, comprising analyzing and separating a type sugar chain by HPLC-FLD, and desorbing a fluorescent functional group from a glycosylamine type sugar chain in the fractionated fraction.   A kit for analyzing a glycoprotein sugar chain, comprising a reagent for fluorescently labeling an amino group and a reagent for desorption of the label. The kit for analyzing a glycoprotein sugar chain according to claim 6, further comprising a standard glycoprotein.

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