Method for producing sialic acid-containing sugar chains

Abstract

[Problem] The purpose of the present invention is to provide a method for producing a sugar chain-containing composition having higher purity (homogeneity) from chicken eggs. [Solution] Provided is a method for producing a composition containing a high-purity sugar chain having alpha 2, 6-binding sialic acid at all non-reducing ends thereof from an egg-derived sugar chain extract. The method is characterized in that a treatment is carried out such that sugar chains having alpha 2, 6-binding sialic acids at all non-reducing ends thereof are enriched in a sugar chain extract of chicken egg origin.

Description

Technical Field

[0001] The present disclosure relates to a method for preparing a sugar chain containing sialic acid, and more specifically, to a method for preparing a composition containing a sugar chain having α2,6-bound sialic acid at all non-reducing ends in high purity from chicken eggs. The present disclosure also relates to a composition containing a sugar chain having α2,6-bound sialic acid at all non-reducing ends in high purity. Background Art

[0002] It has been clearly shown that sugar chains play various roles in living bodies and are used for modification of pharmaceutical active ingredients, preparation of drugs, and for assays, etc. Sugar chains used for such uses require a high degree of uniformity in terms of quality or accuracy.

[0003] Hitherto, as one of the methods for producing sugar chains, a method for isolating and purifying sugar chains from glycoproteins present in chicken eggs has been known (Non-Patent Document 1, Patent Documents 1 and 2).

[0004] Reference List

[0005] Patent Literature

[0006] [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2003-128703

[0007] [Patent Document 2] Japanese Unexamined Patent Application Publication No. 2011-162762 Non-Patent Document

[0008] [Non-patent document 1] Liu et al., "Improved isolation and characterization procedure of sialyglycopeptide from egg yolk powder", Carbohydrate Research 452 (2017) 122-128 Summary of the invention

[0009] Problems to be solved by the present invention

[0010] An object of the present disclosure is to provide a method for preparing a sugar chain-containing composition having higher purity (homogeneity) from chicken eggs.

[0011] Means used to solve problems

[0012] It has been hitherto believed that sialic acid on the non-reducing ends of sugar chains prepared from chicken eggs is all of the α2,6-bound type. The inventors of the present invention have recently discovered, by using analyses different from those conventionally and generally used, that a sugar chain mixture extracted from chicken eggs contains sugar chains having α2,3-bound sialic acid at some or all of the non-reducing ends as trace impurities, thereby completing the present invention.

[0013] The present invention encompasses the following features:

[0014] (1) A method for preparing a composition containing highly pure sugar chains having α2,6-linked sialic acid at all non-reducing ends from a sugar chain extract derived from chicken eggs, characterized in that a treatment is performed such that sugar chains having α2,6-linked sialic acid at all non-reducing ends in the sugar chain extract derived from chicken eggs are enriched.

[0015] (2) The production method according to (1), wherein the sugar chain is a disialyl sugar chain.

[0016] (3) The preparation method according to (1), wherein the sugar chain is in the form of the following compound: [Chemical Formula 1]

[0017]

[0018] (4) The production method according to (1), wherein the enrichment treatment includes separating sugar chains having α2,6-bound sialic acid at all non-reducing termini from sugar chains having α2,3-bound sialic acid at any non-reducing termini.

[0019] (5) The production method according to (4), wherein the separation is performed by liquid chromatography.

[0020] (6) The production method according to (1), wherein the enrichment treatment comprises enzymatically treating the sugar chains having α2,3-linked sialic acid at any non-reducing terminus or treating them by acid hydrolysis.

[0021] (7) The production method according to (6), wherein the enzyme treatment comprises treatment with α2,3-sialidase.

[0022] (8) The production method according to (1), wherein the enrichment treatment includes derivatizing sugar chains having α2,6-linked sialic acid at all non-reducing ends or sugar chains having α2,3-linked sialic acid at any non-reducing end.

[0023] (9) The production method according to (1), further comprising introducing a lipophilic substituent into the sugar chain.

[0024] (10) The preparation method according to (9), wherein the method for introducing the lipophilic substituent is to esterify, thioesterify, amidate or hydrazide the carboxyl group of sialic acid.

[0025] (11) The production method according to (9), wherein the lipophilic substituent is a benzyl group introduced into the carboxylic acid of sialic acid.

[0026] (12) A composition produced by the production method according to any one of (1) to (11).

[0027] (13) A composition consisting essentially of sugar chains having α2,6-linked sialic acid at all non-reducing termini.

[0028] (14) The composition according to (13), wherein the sugar chain is a disialyl sugar chain.

[0029] (15) The production method according to (13), wherein the sugar chain is in the form of the following compound:

[0030] [Chemical formula 2]

[0031]

[0032] (16) The composition according to (13), wherein at least 97% of all sugar chains are sugar chains having α2,6-linked sialic acid at all non-reducing ends.

[0033] (17) The composition according to (13), wherein at least 98% of all sugar chains are sugar chains having α2,6-linked sialic acid at all non-reducing ends.

[0034] (18) The composition according to (13), wherein at least 99% of all sugar chains are sugar chains having α2,6-linked sialic acid at all non-reducing ends.

[0035] (19) The composition according to (13), wherein:

[0036] At least 95% of all sugar chains are sugar chains with α2,6-linked sialic acid at all non-reducing termini, and

[0037] The composition comprises less than 2% of all sugar chains having α2,3-linked sialic acid at any non-reducing terminus.

[0038] (20) A method for preparing a composition containing sugar chains having α2,6-bound sialic acid at all non-reducing terminals in high purity from a sugar chain mixture containing sugar chains having α2,6-bound sialic acid at all non-reducing terminals and sugar chains having α2,3-bound sialic acid at any non-reducing terminal, the method being characterized by purifying the sugar chains in the sugar chain mixture by:

[0039] (a) Liquid chromatography using a HILIC column;

[0040] (b) degrading or denaturing sugar chains having α2,3-linked sialic acid at any non-reducing terminus, followed by liquid chromatography; or

[0041] (c) Derivatizing sugar chains having α2,6-bound sialic acid at all non-reducing ends, followed by liquid chromatography.

[0042] (21) The production method according to (20), wherein the sugar chain is a disialyl sugar chain.

[0043] (22) The method according to (20), wherein the sugar chain is in the form of the following compound:

[0044] [Chemical formula 3]

[0045]

[0046] (23) The production method according to (20), wherein the degradation comprises enzymatic treatment or acid hydrolysis treatment of the sugar chain having α2,3-linked sialic acid at any non-reducing terminus.

[0047] (24) The production method according to (23), wherein the enzyme treatment comprises treatment with α2,3-sialidase.

[0048] (25) The production method according to (20), wherein the denaturation includes derivatizing either or both of a sugar chain having α2,6-binding sialic acid at all non-reducing ends and a sugar chain having α2,3-binding sialic acid at any non-reducing end.

[0049] Those skilled in the art will be able to appreciate that inventions with any combination of one or more features of the present invention described above are also encompassed within the scope of the present invention.

[0050] Effects of the Invention

[0051] According to the present disclosure, a method for preparing a sugar chain-containing composition having higher purity (homogeneity) from chicken eggs is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The results of the analysis of the disialyl sugar chain asparagine mixture are shown. The upper figure shows the results of the analysis of the disialyl sugar chain asparagine-Fmoc mixture obtained and prepared from chicken eggs according to Reference Example 2 by HPLC in HILIC mode. The lower figure shows the mass spectrometry results (ESI-MS) of the secondary peak (3.7%) found by HPLC in HILIC mode.

[0053] Figure 2 Shown in Figure 1 Results of HPLC analysis of the secondary peaks found in .

[0054] Figure 3 Shown are the HPLC results in HILIC mode of the disialyl sugar chain asparagine-Fmoc mixture obtained in Reference Example 2 and the impurities obtained in Example 2 (left figure), and the HPLC results in ODS mode of the impurities found by the aforementioned HPLC (right figure).

[0055] Figure 4 Each shows the result of HPLC analysis of the impurities obtained in Example 2. In the figure, the vertical axis is absorbance (mAU), and the horizontal axis is time (minutes).

[0056] Figure 5 The results of HPLC analysis of the reaction products of enzyme treatment (α2,3-sialidase or α2,3-2,6-2,8-sialidase) of impurities obtained in Example 2 are shown.

[0057] Figure 6 The results of HPLC analysis of the reaction products of the enzyme treatment (α2,3-sialidase or α2,3-2,6-2,8-sialidase) of GT-25001 are shown.

[0058] Figure 7 The results of HPLC analysis of the reaction products of the enzyme treatment (α2,3-sialidase or α2,3-2,6-2,8-sialidase) of GT-25025 are shown.

[0059] Figure 8 Shown is a comparison of the amount of impurities between batches of a disialylglycosylated asparagine-Fmoc mixture prepared from defatted egg yolk.

[0060] Fig. 9 The HPLC results of the enzyme treatment (α2,3-sialidase) reaction product of the disialyl sugar chain asparagine-Fmoc mixture obtained and prepared from chicken eggs according to Reference Example 2 are shown.

[0061] Fig.10Shown are the enzyme treatment (α2,3-sialidase) reaction products of the disialyl sugar chain asparagine-Fmoc mixture obtained and prepared from chicken eggs according to Reference Example 2 and the HPLC results after purification thereof.

[0062] Fig.11 HPLC results after the condensation reaction of the disialyl sugar chain asparagine-Fmoc mixture obtained and prepared from chicken eggs according to Reference Example 2 are shown.

[0063] Fig.12 HPLC results of the reaction product of the disialyl sugar chain asparagine-Fmoc mixture obtained and prepared from chicken eggs according to Reference Example 2 after Bn treatment, deprotection of Bn and Fmoc groups, and subsequent re-Fmoc treatment are shown.

[0064] Fig.13 Shown are HPLC results of the enzyme treatment (α2,3-sialidase) reaction products of commercially available SGP having cleaved and labeled sugar chains. DETAILED DESCRIPTION

[0065] 1. Introduction

[0066] In one aspect, the present disclosure relates to a method for preparing a composition containing highly pure sugar chains having α2,6-linked sialic acid at all non-reducing ends from a chicken egg-derived sugar chain extract (hereinafter also referred to as "the preparation method (1) of the present disclosure").

[0067] In another aspect, the present disclosure relates to a method for preparing a composition containing sugar chains having α2,6-bound sialic acid at all non-reducing ends with high purity from a sugar chain mixture containing sugar chains having α2,6-bound sialic acid at all non-reducing ends and sugar chains having α2,3-bound sialic acid at any non-reducing end (hereinafter also referred to as "the preparation method (2) of the present disclosure") .

[0068] In yet another aspect, the present disclosure relates to a composition prepared by the method according to the preparation method (1) or (2) of the present invention.

[0069] In yet another aspect, the disclosure relates to a composition consisting essentially of sugar chains having α2,6-linked sialic acid at all non-reducing termini.

[0070] In the present disclosure, the "chicken egg-derived sugar chain extract" is a fraction composed of specific sialic acid sugar chains extracted / isolated from chicken eggs, preferably yolks thereof. The "chicken egg-derived sugar chain extract" is generally a fraction composed of disialyl sugar chains.

[0071] The sugar chains contained in the “hen egg-derived sugar chain extract” may be extracted in any form such as free sugar chains, sugar amino acids, and glycopeptides, and a person skilled in the art may appropriately select a desired form.

[0072] In one embodiment of the present disclosure, the "chicken egg-derived sugar chain extract" is obtained mainly in the form of sialic acid glycopeptide (SGP). In such an embodiment, in the process of sugar chain extraction from chicken eggs, treatment by peptidase is not performed.

[0073] In another embodiment of the present disclosure, the "chicken egg-derived sugar chain extract" is obtained mainly in the form of sugar chain asparagine. In such an embodiment, during the sugar chain extraction process from chicken eggs, treatment with peptidase is performed.

[0074] In the present disclosure, "chicken egg-derived sugar chain extract" refers to a sugar chain-containing composition obtained by any means that can be used to obtain sugar chain compounds from chicken eggs (preferably egg yolks). The means that can be used to obtain sugar chain compounds from chicken eggs (preferably egg yolks) may include, but are not limited to, the means described in, for example, the above-mentioned Patent Documents 1 and 2 and Non-Patent Document 1 (which are incorporated herein by reference).

[0075] "Glycolic acid extract derived from chicken eggs" can be obtained, for example, by phenol extraction of raw egg yolk and subsequent purification using 6 size exclusion and ion exchange columns (A. Seko et al., Biochim. Biophys. Acta-Gen. Subj. 1335 (1997) 23.). In the above process, porous graphite carbon column (PGC) column chromatography can also be used instead of size exclusion chromatography (Y. Zou et al., J. Carbohydr. Chem. 31 (2012) 436.). In addition, the above process can be optimized by lipid removal via diethyl ether extraction (B. Sun et al., Carbohydr. Res. 396 (2014) 62).

[0076] In addition, it is also possible to use extraction with an acetone aqueous solution of raw egg yolk, followed by purification with an activated carbon column to obtain a "chicken egg-derived sugar chain extract".

[0077] In addition, the "chicken egg-derived sugar chain extract" can be obtained by washing the egg yolk powder with diethyl ether and extracting it with an ethanol aqueous solution and then purifying it with an activated carbon column (L. Liu et al., Carbohydrate. Res. 452 (2017) 122).

[0078] Furthermore, extraction with an aqueous solution of defatted egg yolk powder and subsequent ODS purification to obtain a “hen egg-derived sugar chain extract” (Japanese Unexamined Patent Application Publication No. 2011-162762) can also be used.

[0079] 2. Preparation method (1)

[0080] The production method (1) of the present disclosure is characterized by performing a treatment that allows the sugar chains having α2,6-binding sialic acid at all non-reducing ends in the chicken egg-derived sugar chain extract to be enriched.

[0081] In one embodiment, the sugar chains enriched by the preparation method (1) of the present disclosure are in the following form:

[0082] [Chemical formula 4]

[0083]

[0084] In the production method (1) of the present disclosure, the enrichment of sugar chains having α2,6-bound sialic acid at all non-reducing ends is carried out by separation from sugar chains other than the sugar chains. The separation is not particularly limited as long as it is a means that can separate substances based on molecular weight, charge and other chemical properties, but preferably uses liquid column chromatography.

[0085] In one embodiment of the present disclosure, the enrichment of sugar chains having α2,6-bound sialic acid at all non-reducing ends is performed by a treatment comprising separating sugar chains having α2,6-bound sialic acid at all non-reducing ends from sugar chains having α2,3-bound sialic acid at any non-reducing end. Sugar chains having α2,6-bound sialic acid at all non-reducing ends and sugar chains having α2,3-bound sialic acid at any non-reducing end differ only in the binding mode of the sugar chains at the non-reducing ends, and the molecular weight is also the same. The inventors of the present invention recently discovered that a mixture of the two sugar chains can be separated by liquid chromatography using a HILIC column. Therefore, in one embodiment, chromatography using a liquid HILIC column is used to separate sugar chains having α2,6-bound sialic acid at all non-reducing ends and sugar chains having α2,3-bound sialic acid at any non-reducing end.

[0086] In another embodiment, prior to the treatment for separation, a treatment is performed to degrade sugar chains having α2,3-bound sialic acid at any non-reducing end. The degradation includes, for example, a treatment to specifically degrade α2,3-bound sialic acid by enzyme or acid hydrolysis. Enzymes that can be used for the enzyme treatment may include, but are not limited to, α2,3-sialidase, nonspecific sialidase, sialyltransferase with sialidase activity, and the like.

[0087] In another embodiment, prior to the treatment for separation, a treatment is performed to denature the sugar chain having α2,3-bound sialic acid at any non-reducing end. The denaturation may include, for example, the introduction of substituents by, for example, lactonization of α2,3-bound sialic acid and esterification, thioesterification, amidation, hydrazide, etc. It is known that α2,3-bound sialic acid forms a lactone in the molecule when the carboxylic acid at position 1 of sialic acid is activated (Glycoconj. J. (2021) 38: 157-166), and lactonization takes advantage of this property.

[0088] In another embodiment, prior to the treatment for separation, a treatment is performed to derivatize the sugar chain having α2,6-bound sialic acid at all non-reducing ends. The derivatization may include, for example, esterification, thioesterification, amidation, hydrazide, etc. of the sialic acid carboxyl group. The derivatized sugar chain may be used as it is, or may include a step of removing a substituent.

[0089] The advantages of pretreatment before separation such as described above are that it facilitates the separation of α2,6-conjugated sialic acid and that it can use a common column suitable for large-scale production.

[0090] 3. Preparation method (2)

[0091] The preparation method (2) of the present disclosure is characterized in that the sugar chains in the sugar chain mixture are purified by:

[0092] (a) Liquid chromatography using a HILIC column;

[0093] (b) degrading or denaturing sugar chains having α2,3-linked sialic acid at any non-reducing terminus, followed by liquid chromatography; or

[0094] (c) Derivatizing sugar chains having α2,6-bound sialic acid at all non-reducing ends, followed by liquid chromatography.

[0095] The sugar chain mixture in the aforementioned embodiment is not particularly limited as long as it is a sugar chain mixture containing sugar chains having α2,6-binding sialic acid at all non-reducing ends and sugar chains having α2,3-binding sialic acid at any non-reducing end, and includes any sugar chain extract extracted from chicken eggs or their yolks, as well as a mixture of sugar chains prepared by chemical synthesis, a mixture of sugar chains cultured by genetic recombination technology, and the like.

[0096] In one embodiment, the sugar chain purified by the preparation method (2) of the present disclosure is in the following form:

[0097] [Chemical formula 5]

[0098]

[0099] The degradation includes, for example, a treatment that preferentially and specifically degrades α2,3-bound sialic acid by an enzyme. The enzyme that can be used for the enzyme treatment may include, but is not limited to, α2,3-sialidase, non-selective sialidase, sialyltransferase having sialidase activity, and the like.

[0100] In another embodiment, a treatment for denaturing sugar chains having α2,3-bound sialic acid at any non-reducing terminus is performed before the treatment for separation. The denaturation may include, for example, lactonization, esterification, thioesterification, amidation, hydrazide, etc. of α2,3-bound sialic acid.

[0101] In another embodiment, prior to the treatment for separation, a treatment is performed to derivatize the sugar chain having α2,6-bound sialic acid at the non-reducing end. The derivatization may include, for example, esterification, thioesterification, amidation, hydrazide, etc. of the sialic acid carboxyl group. The derivatized sugar chain may be used as it is, or may include a step of removing a substituent.

[0102] In one embodiment of the present disclosure, the preparation method (1) or (2) of the present disclosure further comprises introducing a lipophilic substituent into the enriched or purified sugar chain. The introduction of the substituent is usually introduced into the carboxylic acid group of sialic acid. Therefore, the sugar chain can be stabilized. The introduction of the substituent into the carboxylic acid group of sialic acid is within the technical scope of those skilled in the art.

[0103] In one embodiment, the method for introducing the lipophilic substituent useful in the present disclosure may include esterification, thioesterification, amidation, hydrazide formation, etc. of the sialic acid carboxyl group.

[0104] In another embodiment, lipophilic substituents useful in the present disclosure may include, but are not limited to, benzyl and phenacyl.

[0105] 4. Composition

[0106] The composition prepared by the above preparation method is a composition consisting essentially of sugar chains having α2,6-binding sialic acid at all non-reducing ends.

[0107] In one embodiment of the present disclosure, "consisting essentially of" may mean that of all sugar chains contained in the composition, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% are sugar chains with α2,6-linked sialic acid at all non-reducing termini.

[0108] In another embodiment of the present disclosure, "consisting essentially of" may mean that of all sugar chains contained in the composition, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% are sugar chains having α2,6-binding sialic acid at all non-reducing termini, and of all sugar chains having α2,6-binding sialic acid at any non-reducing termini The amount of glycans with α2,3-bound sialic acid at the target site is less than 2.5%, less than 2.4%, less than 2.3%, less than 2.2%, less than 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%.

[0109] Note that the terms used herein will be used to describe some specific embodiments and are not intended to limit the present invention.

[0110] Furthermore, unless the content clearly indicates otherwise, the term “comprising” used herein means the presence of described items (such as components, steps, elements and numbers) and does not exclude the presence of other items (such as components, steps, elements and numbers).

[0111] Unless otherwise defined, all terms (including technical and scientific terms) used in this article have the same meaning as those widely recognized by technicians in the technical field to which the present invention belongs. Unless otherwise explicitly defined, the terms used in this article should be interpreted as having the same meaning as in this article and in the relevant technical field, and should not be interpreted as having an idealized or overly formal meaning.

[0112] Although sometimes use the term such as first and second to represent multiple elements, it should be recognized that these elements are not limited by these terms. These terms are only used for the purpose of distinguishing one element from another element, and for example, the first element can be described as the second element, and similarly, the first element can be described as the second element without departing from the scope of the present invention.

[0113] The present invention will now be described in more detail by way of examples. However, the present invention can be embodied through a plurality of embodiments and should not be construed as being limited to some of the examples described herein.

[0114] Example

[0115] (Reference Example 1) Synthesis of a mixture of disialyl sugar chains and asparagine

[0116] One egg yolk was cracked and added to ethanol (EtOH, 67 mL) under stirring. After stirring for about 5 hours, it was filtered and further washed with EtOH (30 mL). EtOH (83 mL) was added to the obtained precipitate again, and it was stirred overnight, and then filtered, and then the precipitate was washed with EtOH (30 mL). Next, the precipitate was dried to obtain 3 g of defatted egg yolk.

[0117] After dissolving the obtained defatted egg yolk in a phosphate buffer (pH=7.0, 30 mL), sodium azide (10 mg) was added. Furthermore, Orientase ONS (from HBI, 1.0 g) was added and allowed to stand at 50°C for about 24 hours. After confirming the completion of the reaction by thin layer chromatography (TLC), the reaction solution was purified by Celite TM (Celite Corporation). After the filtrate was concentrated, it was purified by gel filtration column chromatography (Sephadex G-25, 2.5×100 cm, water). The fractions containing the target sugar were collected and concentrated, and then freeze-dried.

[0118] Tris-HCl / calcium chloride buffer (pH=7.5, 43mL) and sodium azide (21mg) were added to the obtained dry powder (about 430mg) to dissolve the powder. In addition, pronase E (43mg) was added and allowed to stand for 24 hours while checking pH=7.5 every 12 hours. When the pH change was confirmed, the pH was adjusted to pH=7.5 by tris(hydroxymethyl)aminomethane aqueous solution or hydrochloric acid aqueous solution. After standing for 24 hours, pronase E (21.5mg) was added to the reaction solution again and allowed to react again for about 48 hours while checking pH=7.5. When the pH change was confirmed, the pH was adjusted to pH=7.5 by tris(hydroxymethyl)aminomethane aqueous solution or hydrochloric acid aqueous solution. After confirming the completion of the reaction with TLC, Celite filtration was performed. After the filtrate was concentrated, it was purified by gel filtration column chromatography (Sephadex G-25, 2.5×100cm, water). The fractions containing the target disialyl sugar chain compound are collected and concentrated, followed by freeze-drying to obtain a disialyl sugar chain asparagine mixture.

[0119] (Reference Example 2) Disialyl sugar chain in which the amino nitrogen of asparagine is protected by an Fmoc group Asparagine-Fmoc synthesis

[0120] The disialyl sugar chain asparagine mixture (120mg) obtained in reference example 1 was dissolved in water (1.5mL), and sodium bicarbonate (26mg) was added. In addition, Fmoc-OSu [N- (9-fluorenylmethoxycarbonyl) oxysuccinimide] (68mg) dissolved in dimethylformamide (DMF, 2.5mL) was added, and then it was reacted at room temperature for 2 hours. After confirming the disappearance of the source material with TLC, it was concentrated with an evaporator. Water (15mL) and diethyl ether (25mL) were added to the residue, and it was stirred for 10 minutes. Next, after removing the diethyl ether layer, the aqueous layer was washed with diethyl ether (15mL), and further freeze-dried after concentration. Next, the residue obtained was purified on a gradient with an ODS column (Wakogel 100C18). The fractions containing sugar chains were collected and freeze-dried after concentration. The obtained residue was purified by a preparative HPLC column (YMC-Pack R&D ODS, D-ODS-5-A, 20×250 mm, acetonitrile / 25 mM aqueous ammonium acetate solution=20 / 80, 7.5 mL / min). After the main peak appeared after aliquoting for about 15 minutes, it was concentrated. Next, desalting was performed with an ODS column. It was freeze-dried, and 13.3 mg of the target disialyl sugar chain asparagine-Fmoc was obtained, which is a sugar chain asparagine compound in which the amino nitrogen of asparagine is protected by an Fmoc group.

[0121] (Reference Example 3) (1S, 2S (3)) Disialyl sugar chain asparagine-Fmoc (GT-25034) preparation

[0122] [Chemical formula 6]

[0123]

[0124] To GT-25002 (50 mg, MW: 2267.8, 22 μmol, GlyTech, Inc.), protease inhibitors (0.5 mL, 1 tablet of cOmplete, Roche, 11836170001 dissolved in water (0.5 mL)), 2 mM PMSF / DMSO (40 μL), bacterial alkaline phosphatase (40 μL, Roche, 03359123001, 14 Unit / μL), 1 M MOPS buffer (1.0 mL, pH=7.0, NACALAI TESQUE, INC., 23442-81), BSA (50 mg, SIGMA, A3159-50G) and water (9.4 mL) were added, and 50 μL was dispensed for HPLC analysis. To the sugar chain aqueous solution (about 11mL) after distribution, α2,3-sialyltransferase (291mUnit, 40Unit / mL, JT, ish-467) and cytidine 5'-monophosphoric acid-bDN-acetylneuraminic acid, disodium salt (CMP-sialic acid, 247mg, from SANYO FINE CO., LTD.) were added and shaken at 30°C. After 6 hours, 8μL of the reaction solution was diluted in water (32μL) and 5μL was injected for HPLC analysis. The attenuation of the source material peak eluted at 17.5 minutes in the HPLC analysis and the generation of a new peak eluted at 15.5 minutes were confirmed. The mass spectrometer confirmed that the new peak generated was GT-25034, and the reaction was terminated. After terminating the reaction, and after purification by HPLC; concentration; and desalting by ODS support, (1S,2S(3)) disialyl sugar chain asparagine-Fmoc (MW: 2560.4, 15 μmol, yield 66%) was obtained in a yield of 37 mg.

[0125] HPLC analysis conditions (ODS): (CAPCELL PAK C18 UG120 250×4.6 mm, flow rate 0.70 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, gradient A85%→A75% (20 minutes)→A5% (25 minutes)→A85% (45 minutes))

[0126] HPLC purification conditions: (CAPCELLPAK C18 UG120 250×20 mm, flow rate 7.0 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, gradient A80%→A80% (5 min)→A75% (25 min)→A10% (30 min)→A80% (45 min))

[0127] (Reference Example 4) ((1S(3),2S) disialyl sugar chain asparagine-Fmoc (GT-25035) preparation

[0128] [Chemical formula 7]

[0129]

[0130] To GT-25006 (50 mg, MW: 2267.8, 22 μmol, GlyTech, Inc.), protease inhibitors (0.5 mL, 1 tablet of cOmplete, Roche, 11836170001 dissolved in water (0.5 mL)), 2 mM PMSF / DMSO (0.04 mL), bacterial alkaline phosphatase (0.04 mL, Roche, 03359123001, 14 Unit / μL), 1 M MOPS buffer (1.0 mL, pH=7.0, NACALAI TESQUE, INC. 23442-81), BSA (50 mg, SIGMA, A3159-50G) and water (9.4 mL) were added, and 50 μL was dispensed for HPLC analysis. To the sugar chain aqueous solution (about 11 mL) after distribution, α2,3-sialyltransferase (291 mUnit, 40 Unit / mL, JT, ish-467) and cytidine 5 '-monophosphoric acid-bDN-acetylneuraminic acid, disodium salt (CMP-sialic acid, 247 mg, from SANYO FINE CO., LTD.) were added and shaken at 30 ° C. After 7 hours, 8 μL of the reaction solution was diluted in water (32 μL) and 5 μL was injected for HPLC analysis (analysis conditions). The attenuation of the source material peak eluted at 17.0 minutes in the HPLC analysis and the generation of a new peak eluted at 14.5 minutes were confirmed. As a result of mass spectrometry, it was confirmed that the new peak generated was GT-25035, and the reaction was terminated. After termination of the reaction, and after purification by HPLC; concentration; and desalting by ODS support, (1S,2S(3)) disialyl sugar chain asparagine-Fmoc (MW: 2560.35, 13.4 μmol, yield 61%) was obtained in a yield of 34.3 mg.

[0131] HPLC analysis conditions (ODS): (CAPCELL PAK C18 UG120 250×4.6 mm, flow rate 0.70 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, gradient A85%→A75% (20 minutes)→A5% (25 minutes)→A85% (45 minutes))

[0132] HPLC purification conditions: (CAPCELLPAK C18 UG120 250×20 mm, flow rate 7.0 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, gradient A80%→A80% (5 min)→A75% (25 min)→A10% (30 min)→A80% (45 min))

[0133] (Reference Example 5) (1S(3), 2S(3)) Disialyl sugar chain asparagine-Fmoc (GT-25025) Preparation

[0134] [Chemical formula 8]

[0135]

[0136] GT-25022 (5 g, MW: 1977.84, 2.5 mmol, GlyTech, Inc.) was dissolved in water (45 mL), and MOPS buffer (5.0 mL, pH = 7.0, NACALAI TESQUE, INC. 23442-81) was added, and then 200 μL was distributed for HPLC analysis. Bacterial alkaline phosphatase (0.3 mL, Roche, 03359123001, 14 Unit / μL), α2,3-sialyltransferase (500 mg) and cytidine 5'-monophosphoryl-bDN-acetylneuraminic acid, disodium salt (CMP-sialic acid, 5 g, from SANYO FINE CO., LTD.) were added to the sugar chain aqueous solution (about 50 mL) after distribution, and it was shaken at 4 ° C. After 27 hours, 2 μL of the reaction solution was diluted in water (198 μL), and 10 μL was injected for HPLC analysis. The attenuation of the source material peak eluted at 12.5 minutes in the HPLC analysis and the generation of a new peak eluted at 8.0 minutes were confirmed. The mass spectrometer confirmed that the generated new peak was GT-25025, and the reaction was terminated. After the reaction was terminated, and after purification by HPLC; concentration; and desalting by electrodialysis, (1S(3), 2S(3)) disialyl sugar chain asparagine-Fmoc (MW: 2560.35, 2.1 mmol, yield 83%) was obtained in a yield of 5.4 g.

[0137] HPLC analysis conditions (ODS): (CAPCELL PAK C18 UG120 250×4.6 mm, flow rate 0.70 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, isocratic (A: 80%, B: 20%)

[0138] HPLC purification conditions: (SP-300-5-ODS-BIO 250×30 mm, flow rate 25 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, gradient A95%→A95% (10 minutes)→A85% (10.1 minutes)→A82% (40 minutes)

[0139] (Example 1) Analysis of disialyl sugar chain asparagine-Fmoc

[0140] The disialyl sugar chain asparagine-Fmoc (5 mg, 2.0 μmol) obtained in Reference Example 2 was dissolved in water and supplemented to 10.0 mL. 10 μL of the supplemented sugar chain aqueous solution was injected into HPLC and analyzed. As a result, after the disialyl sugar chain asparagine-Fmoc peak (94.9%) eluted at about 23 minutes, another peak (3.7%) showing the same mass eluted at about 24 minutes was confirmed ( Figure 1 As a result of comparing the retention time on HPLC with the disialyl sugar chains containing α2,3-sialic acid prepared in Reference Examples 3, 4, and 5, the peak eluted at about 24 minutes was consistent with that of GT-25034 and GT-25035 ( Figure 2 ).

[0141] HPLC analysis conditions: (Asahipak NH2P-50 4E 5μm 250×4.6mm, flow rate 0.70mL / min, mobile phase A: 0.5% phosphoric acid aqueous solution, B: 90% acetonitrile aqueous solution containing 0.5% phosphoric acid, gradient A25%→A75% (25 minutes)→A90% (26 minutes)→A90% (28 minutes)→A25% (29 minutes)→A25% (45 minutes))

[0142] ESI-MS of the peak eluting at about 24 minutes: For C 103 H 154 N 8 O 66 [M+H] + The calculated value of [M+2H] is 2559.89. +2 1280.45, [M+3H] +3 853.96, the measured values ​​are 1280.44, 853.95.

[0143] (Example 2) Impurity Analysis of Disialyl Sugar Chain Asparagine-Fmoc

[0144] After dissolving the disialyl sugar chain asparagine-Fmoc (200 mg) obtained in Reference Example 2 in water (4 mL), it was diluted with acetonitrile (12 mL). After dilution, the slightly turbid sugar chain aqueous solution was purified by HPLC and freeze-dried. (HPLC purification conditions) After freeze-drying, the impurity purified product (2.4 mg) was obtained by desalting with an ODS support ( Figure 3 ).

[0145] As a result of comparative analysis of the obtained impurity-purified products and the sugar chains prepared by Reference Examples 2, 3, 4, and 5, GT-25034 and GT-25035 were each consistent ( Figure 4 ).

[0146] HPLC purification conditions: (Asahipak NH2P-90 20F 300×20mm, flow rate 7mL / min, mobile phase A: 0.5% phosphoric acid aqueous solution, B: 90% acetonitrile aqueous solution containing 0.5% phosphoric acid, gradient A25%→A25% (5 minutes)→A75% (60 minutes)

[0147] HPLC analysis conditions (ODS(1)): (CAPCELL PAK C18 UG120 250×4.6 mm, flow rate 0.70 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, isocratic (A: 80%, B: 20%)

[0148] HPLC analysis conditions (ODS(2)): (CAPCELL PAK C18 UG120 250×4.6 mm, flow rate 0.70 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, isocratic (A: 82%, B: 18%)

[0149] HPLC analysis conditions (HILIC): (Asahipak NH2P-50 4E 5μm 250×4.6mm, flow rate 0.70mL / min, mobile phase A: 0.5% phosphoric acid aqueous solution, B: 90% acetonitrile aqueous solution containing 0.5% phosphoric acid, gradient A25%→A75% (25 minutes)→A90% (26 minutes)→A90% (28 minutes)→A25% (29 minutes)→A25% (45 minutes))

[0150] (Example 3) Estimation of impurity structure

[0151] The impurities prepared in Example 2 were degraded by sialidase.

[0152] 100mM sodium acetate aqueous solution (44μL) and α2,3-sialidase (5mUnit, TAKARA, 4455) were added to the impurities (50μg) dissolved in water (5μL), and it was reacted at 37°C for 1.5 hours. After 1.5 hours, the reaction solution (10μL) was diluted in water (90μL), and 10μL was injected for HPLC analysis. As a result of HPLC, the source material peaks at 10.5 minutes and 11.5 minutes had completely disappeared, and the generation of new peaks was confirmed at 14 minutes. As a result of mass spectrometry, it was confirmed that the new peak generated was a monosialic acid form with a sialic acid residue cut from the impurity.

[0153] On the other hand, 100mM sodium acetate aqueous solution (44μL) and α2,3-2,6-2,8-sialidase (10mUnit, neuraminidase, SANYO FINE CO., LTD.) were added to the impurities (50μg) dissolved in water (5μL) and allowed to react for 1.5 hours at 37°C. After 1.5 hours, the reaction solution (10μL) was diluted in water (90μL) and 10μL was injected for HPLC analysis. As a result of HPLC analysis, the source material peaks at 10.5 minutes and 11.5 minutes had completely disappeared, and the generation of new peaks was confirmed at 17.5 minutes. As a result of mass spectrometry, it was confirmed that the new peaks generated were desialylated forms ( Figure 5 ).

[0154] HPLC analysis conditions (ODS): (CAPCELL PAK C18 UG120 250×4.6 mm, flow rate 0.70 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, isocratic (A: 80%, B: 20%)

[0155] (Example 4) Analysis of Enzyme Digestion Products of GT-25001

[0156] Degradation of GT-25001 by sialyl hydrolases.

[0157] 100 mM sodium acetate aqueous solution (44 μL) and α2,3-sialidase (5 mUnit, TAKARA, 4455) were added to GT-25001 (50 μg) dissolved in water (5 μL), and allowed to react at 37°C for 1.5 hours. After 1.5 hours, the reaction solution (10 μL) was diluted in water (90 μL), and 10 μL was injected for HPLC analysis. As a result of HPLC analysis, it was confirmed that there was no change relative to the source material peak at 11 minutes ( Figure 6 ).

[0158] On the other hand, 100mM sodium acetate aqueous solution (44μL) and α2,3-2,6-2,8-sialidase (10mUnit, neuraminidase, SANYO FINE CO., LTD.) were added to GT-25001 (50μg) also dissolved in water (5μL), and it was reacted at 37°C. After 1.5 hours, the reaction solution (10μL) was diluted in water (90μL), and 10μL was injected for HPLC analysis. As a result of HPLC analysis, the source material peak at 11 minutes had completely disappeared, and the generation of a new peak was confirmed at 17.5 minutes. As a result of mass spectrometry, it was confirmed that the new peak generated was a desialylated form ( Figure 6 ).

[0159] HPLC analysis conditions (ODS): (CAPCELL PAK C18 UG120 250×4.6 mm, flow rate 0.70 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, isocratic (A: 80%, B: 20%)

[0160] (Example 5) Analysis of Enzyme Digestion Products of GT-25025

[0161] Degradation of GT-25025 by sialyl hydrolases.

[0162] 100mM sodium acetate aqueous solution (44μL) and α2,3-sialidase (5mUnit, TAKARA, 4455) were added to GT-25025 (50μg) dissolved in water (5μL), and allowed to react at 37°C for 1.5 hours. After 1.5 hours, the reaction solution (10μL) was diluted in water (90μL), and 10μL was injected for HPLC analysis. As a result of HPLC analysis, the source material peak at 11 minutes had completely disappeared, and the generation of a new peak was confirmed at 17.5 minutes. As a result of mass spectrometry, it was confirmed that the new peak generated was a desialylated form ( Figure 7 ).

[0163] On the other hand, 100mM sodium acetate aqueous solution (44μL) and α2,3-2,6-2,8-sialidase (10mUnit, neuraminidase, SANYO FINE CO., LTD.) were added to GT-25025 (50μg) also dissolved in water (5μL), and it was reacted at 37°C for 1.5 hours. After 1.5 hours, the reaction solution (10μL) was diluted in water (90μL), and 10μL was injected for HPLC analysis. As a result of HPLC analysis, the source material peak at 11 minutes had completely disappeared, and the generation of a new peak was confirmed at 17.5 minutes. As a result of mass spectrometry, it was confirmed that the new peak generated was a desialylated form ( Figure 7 ).

[0164] HPLC analysis conditions (ODS): (CAPCELL PAK C18 UG120 250×4.6 mm, flow rate 0.70 mL / min, mobile phase A: 25 mM ammonium acetate aqueous solution, B: acetonitrile, isocratic (A: 80%, B: 20%)

[0165] (Example 6) Analysis of differences between batches of disialyl sugar chain asparagine mixtures obtained from chicken eggs

[0166] Using defatted egg yolk from multiple production batches as raw materials, disialyl sugar chain asparagine-Fmoc was prepared according to Reference Examples 1 and 2. The obtained disialyl sugar chain asparagine-Fmoc (5 mg, 2.0 μmol) was dissolved in water and supplemented to 10.0 mL. 10 μL of the supplemented sugar chain aqueous solution was injected into HPLC and analyzed. As a result, it was confirmed that all batches contained a peak eluting at about 24 minutes, which corresponded to about 3.5% to 5.0% of impurities GT-25034 and GT-25035 ( Figure 8 ).

[0167] HPLC analysis conditions: (Asahipak NH2P-50 4E 5μm 250×4.6mm, flow rate 0.70mL / min, mobile phase A: 0.5% phosphoric acid aqueous solution, B: 90% acetonitrile aqueous solution containing 0.5% phosphoric acid, gradient A25%→A75% (25 minutes)→A90% (26 minutes)→A90% (28 minutes)→A25% (29 minutes)→A25% (45 minutes))

[0168] (Example 7) Purification of disialyl sugar chain asparagine-Fmoc

[0169] The disialyl sugar chain asparagine-Fmoc (50 mg, 20 μmol) obtained in Reference Example 2 was dissolved in a 75% acetonitrile solution (2.5 mL). It was injected into an HPLC column and purified so as not to be contaminated by impurities GT-25034 and GT-25035. By desalting with a fraction containing GT-25001, GT-25001 (36 mg, 14 μmol, yield 70%) with 0.2% contamination of GT-25034 and GT-25035 was obtained.

[0170] Preparative HPLC conditions: (Asahipak NH2P-90 20F 300×20 mm, flow rate 8 mL / min, mobile phase A: aqueous solution containing 5% acetic acid / 3% triethylamine, B: acetonitrile solution containing 2% acetic acid, gradient A25%→A25% (5 minutes)→A65% (5.1 to 65 minutes))

[0171] (Example 8) Removal of impurities contained in a disialyl sugar chain asparagine-Fmoc mixture (1)

[0172] The disialyl sugar chain asparagine-Fmoc (2g) obtained in reference example 2 was dissolved in a buffer solution (pH=5.5, 20mL) containing 100mM sodium acetate and 10mM calcium chloride, α2,3-sialidase (2Unit, TAKARA, 4455) was added, and it was reacted at 37°C. After 16 hours, the reaction solution (1μL) was diluted in water (50μL), and acetonitrile (50μL) was subsequently added, and 20μL was injected for HPLC analysis. As a result of HPLC analysis, the peak at 32 minutes (GT-25001) did not change, the peak at 27 minutes (GT-25034 / GT-25035 mixture) had completely disappeared, and the generation of a new peak was confirmed at 11 minutes. As a result of mass spectrometry, it was confirmed that the new peak generated was a monosialic acid form ( Fig. 9 ).

[0173] After confirming the completion of the reaction, the reaction solution was heated at 80° C. for 10 minutes. The obtained enzyme-treated solution was purified by HPLC column, and GT-25001 (1.4 g, yield 70%) having a GT-25034 / GT-25035 mixture of 0.3% or less was obtained by desalting the fraction containing GT-25001 with an ODS support ( Fig.10 ).

[0174] HPLC analysis conditions: (Asahipak NH2P-50 4E 5μm 250×4.6mm, flow rate 0.70mL / min, mobile phase A: aqueous solution containing 5% acetic acid and 3% triethylamine, B: acetonitrile containing 2% acetic acid, isocratic (A: 60%, B: 40%))

[0175] Preparative HPLC conditions: (SP-300-5-ODS-BIO 5μm 250×30mm, flow rate 25mL / min, mobile phase A: 25mM ammonium acetate aqueous solution, B: acetonitrile, gradient A90%→A80% (30 minutes)

[0176] ESI-MS: For C 103 H 154 N 8 O 66 [M+H] + The calculated value of [M+2H] is 2559.89. +2 1280.45, [M+3H] +3 853.96, the measured values ​​are 1280.44, 853.95.

[0177] (Example 9) Removal of impurities contained in disialyl sugar chain asparagine-Fmoc (2) (by lactonization) Derivatization

[0178] 500mM HOBt and 500mM DIC were added to the disialyl sugar chain asparagine-Fmoc obtained in Reference Example 2 (50mg), and it was reacted at room temperature for 1 hour. After 1 hour, the reaction solution (10μL) was diluted with an acetonitrile / water mixed solution (7:3, 90μL), and then 5μL was injected for HPLC analysis. As a result of HPLC analysis, it was confirmed that the peak at 28 minutes (GT-25001) did not change much, and the peak at 24 minutes (GT-25034 / GT-25035 mixture) had completely disappeared ( Fig.11 ).

[0179] Preparative HPLC conditions: (CAPCELLPAK UG120 C18 5μm 250×4.6mm, flow rate 0.7mL / min, developing solvent A: water / trifluoroacetic acid (1000:1), B: acetonitrile / trifluoroacetic acid (1000:1), gradient A99%→A70% (30 minutes))

[0180] HPLC analysis conditions: (ACQUITY UPLC Polysaccharide BEH Amide, 1.7 μm 150×2.1 mm, flow rate 0.35 mL / min, mobile phase A: 50 mM ammonium formate (pH 4.4), B: acetonitrile, gradient A30%→A30% (66 min)

[0181] (Example 10) Removal of impurities contained in disialyl sugar chain asparagine-Fmoc (3) (by derivatization of benzyl groups) Biochemical (esterification)

[0182] To the disialyl sugar chain asparagine-Fmoc (25 mg) obtained in Reference Example 2, NMP / DMF / water (84:15:1) (250 μL) was added, and it was stirred at 40°C for 1 hour to dissolve. After 1 hour, benzyl bromide (8.6 μL, 3.6 equivalents) was added, and it was stirred at 40°C. After 6 hours, 1M sodium carbonate (50 μL) was added, and it was stirred at room temperature for 2 hours, and then purified by ODS column. After desalting by solid phase extraction, freeze drying was performed, and GT-25001 with sialic acid Bn was obtained with 2.5 mg. In order to confirm the removal of impurities, 100mM sodium hydroxide (10 μL) was added to the obtained GT-25001 with sialic acid Bn (1 mg), so that it was reacted at 40°C for 1 hour, and the Bn and Fmoc groups were deprotected. Subsequently, 1M sodium bicarbonate (10 μL), acetonitrile (20 μL) and Fmoc-OSu (2.4 mg) were added for re-Fmocization. After the solution was subjected to solid phase extraction to remove contaminants, it was concentrated under reduced pressure. Water was added to the concentrated solution to obtain a sample solution. As a result of analyzing the sample solution, it was confirmed that the peak at 24 minutes (GT-25034 / GT-25035 mixture) had completely disappeared ( Fig.12 ).

[0183] Preparative HPLC conditions: (CAPCELLPAK UG120 C18 5μm 250×20mm, flow rate 7.0mL / min, developing solvent A: 25mM ammonium acetate, B: acetonitrile, gradient A75%→A70% (30 minutes)

[0184] HPLC analysis conditions: (ACQUITY UPLC Polysaccharide BEH Amide, 1.7 μm 150×2.1 mm, flow rate 0.35 mL / min, mobile phase A: 50 mM ammonium formate (pH 4.4), B: acetonitrile, gradient A30%→A30% (66 min)

[0185] (Example 11) Removal of impurities contained in SGP (derivatization by 2,3-sialidase)SGP (from TCI, S0523, 2mg, 1.6μmol) was dissolved in 100mM sodium acetate buffer (pH 5.5), 2μL of α2,3-sialidase (0.5U / mU, TAKARA, 4455) was added, and it was reacted at 37°C for 2 hours and purified to obtain SGP (0.6mg, yield 33%) from which impurities were removed. Water was added to the SGP from which impurities were removed to adjust to 2g / mL to obtain an SGP aqueous solution. Water (15.3μL) and 5% RapiGest solution (6μL) were added to the SGP aqueous solution (7.5μL), and it was incubated at 90°C for 3 minutes. The solution was returned to room temperature, fast PNGase (1.2μL) was added, and it was incubated at 50°C for 3 minutes. GlycoWorks RapiFluo-MS labeled reagent (12μL) was added to the solution, and it was allowed to stand at room temperature for 5 minutes. Acetonitrile (358 μL) was added to the solution for dilution, and the sugar chains labeled by RFMS were subjected to solid phase extraction using a GlycoWorks HILIC μ elution plate, freeze-dried, and then redissolved in a solvent to obtain a sample solution. As a result of analyzing the sample solution, the disappearance of impurities derived from α2,6 / α2,3 and α2,3 / α2,6 was confirmed ( Fig.13 ).

[0186] Preparative HPLC conditions: (CAPCELLPAK UG120 C18 5μm 250×4.6mm, flow rate 0.7mL / min, developing solvent A: water / trifluoroacetic acid (1000:1), B: acetonitrile / trifluoroacetic acid (1000:1), gradient A99%→A70% (30 minutes)

[0187] HPLC analysis conditions: (ACQUITY UPLC Polysaccharide BEH Amide, 1.7 μm 150×2.1 mm, flow rate 0.35 mL / min, mobile phase A: 50 mM ammonium formate (pH 4.4), B: acetonitrile, gradient A25%→A31% (66 min).

Claims

1. A method for preparing a composition containing highly pure sugar chains having α2,6-linked sialic acid at all non-reducing ends from a sugar chain extract derived from chicken eggs, characterized in that The treatment is performed so that sugar chains having α2,6-binding sialic acid at all non-reducing ends in the chicken egg-derived sugar chain extract are enriched.

2. The preparation method according to claim 1, wherein the sugar chain is a disialyl sugar chain.

3. The preparation method according to claim 1, wherein the sugar chain is in the form of the following compound: [Chemical formula 1] 4 . The preparation method according to claim 1 , wherein the enrichment treatment comprises separating sugar chains having α2,6-bound sialic acid at all non-reducing termini from sugar chains having α2,3-bound sialic acid at any non-reducing termini. The preparation method according to claim 4 , wherein the separation is performed by liquid chromatography. 6 . The production method according to claim 1 , wherein the enrichment treatment comprises enzymatic treatment or treatment by acid hydrolysis of sugar chains having α2,3-binding sialic acid at any non-reducing terminus. The production method according to claim 6 , wherein the enzyme treatment comprises treatment with α2,3-sialidase. 8 . The preparation method according to claim 1 , wherein the enrichment treatment comprises derivatizing sugar chains having α2,6-bound sialic acid at all non-reducing termini or sugar chains having α2,3-bound sialic acid at any non-reducing termini. 9 . The preparation method according to claim 1 , further comprising introducing a lipophilic substituent into the sugar chain.

10. The preparation method according to claim 9, wherein the method for introducing the lipophilic substituent is to esterify, thioesterify, amidate or hydrazide the carboxyl group of sialic acid.

11. The preparation method according to claim 9, wherein the lipophilic substituent is a benzyl group introduced into the carboxylic acid of sialic acid.

12. A composition prepared by the preparation method according to any one of claims 1 to 11.

13. A composition consisting essentially of sugar chains having α2,6-bound sialic acid at all non-reducing termini. The composition according to claim 13 , wherein the sugar chain is a disialyl sugar chain.

15. The preparation method according to claim 13, wherein the sugar chain is in the form of the following compound: [Chemical formula 2] 16 . The composition according to claim 13 , wherein at least 97% of all sugar chains are sugar chains having α2,6-bound sialic acid at all non-reducing termini. 17 . The composition according to claim 13 , wherein at least 98% of all sugar chains are sugar chains having α2,6-bound sialic acid at all non-reducing termini. 18 . The composition according to claim 13 , wherein at least 99% of all sugar chains are sugar chains having α2,6-bound sialic acid at all non-reducing termini.

19. The composition of claim 13, wherein: At least 95% of all sugar chains are sugar chains with α2,6-linked sialic acid at all non-reducing termini, and The composition comprises less than 2% of all sugar chains having α2,3-linked sialic acid at any non-reducing terminus.

20. A method for preparing a composition comprising sugar chains having α2,6-bound sialic acid at all non-reducing ends in high purity from a sugar chain mixture comprising sugar chains having α2,6-bound sialic acid at all non-reducing ends and sugar chains having α2,3-bound sialic acid at any non-reducing end, the method being characterized by purifying the sugar chains in the sugar chain mixture by: (a) Liquid chromatography using a HILIC column; (b) degrading or denaturing sugar chains having α2,3-linked sialic acid at any non-reducing terminus, followed by liquid chromatography; or (c) Derivatizing sugar chains having α2,6-bound sialic acid at all non-reducing termini, followed by liquid chromatography. The preparation method according to claim 20 , wherein the sugar chain is a disialyl sugar chain.

22. The preparation method according to claim 20, wherein the sugar chain is in the form of the following compound: [Chemical formula 3] 23 . The production method according to claim 20 , wherein the degradation comprises enzymatic treatment or treatment by acid hydrolysis of a sugar chain having α2,3-binding sialic acid at any non-reducing terminus. The production method according to claim 23 , wherein the enzyme treatment comprises treatment with α2,3-sialidase. The production method according to claim 20 , wherein the denaturation comprises derivatizing either or both of sugar chains having α2,6-bound sialic acid at all non-reducing termini and sugar chains having α2,3-bound sialic acid at any of the non-reducing termini.

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