JP5709157B2 - Production method of sugar chain - Google Patents

Description

  The present invention relates to a method for producing a sugar chain.

  In recent years, protein preparations such as erythropoietin (EPO) and IgG have been put into practical use. Protein preparations are mainly produced by a production method using genetic recombination (Patent Document 1), organic chemical synthesis (Non-Patent Document 1), or the like.

  In the case of the production method using gene recombination, it is known that the type of sugar chain that binds to the recombinant protein differs depending on the host in which the recombinant protein is expressed, and thus the activity on mammals differs ( Non-patent document 2). For example, when expressed in E. coli, the EPO has no sugar chain and therefore has no activity on mammals and is degraded in blood. In addition, when expressed in yeast or insect cells, it has a sugar chain different from that of the mammalian type, so that it does not have the aforementioned activity and is degraded in the liver or the like. In contrast, when expressed in mammalian cells, the EPO has a mammalian sugar chain to which sialic acid is bound, and has the aforementioned activity. However, the production method using the mammal-derived cells has a problem that the cost is high and sugar chains are not uniform.

  On the other hand, in the case of a production method utilizing organic chemical synthesis, at present, sugar chains that can be produced are limited to double-chain sugar chains. Also, in nature, pheasant bird eggs have double-stranded sugar chains to which sialic acid is bound, but sugar chains having three or more branched chains in which sialic acid is bound to each branched chain. Does not exist (Non-Patent Documents 3 and 4). For this reason, there is a need for a means for supplying a cheap and large amount of a protein preparation bound with a three- or four-chain sugar chain bound with sialic acid.

Japanese Examined Patent Publication No. 7-68272

Hirano et al., Angew. Chem. Int. Ed. 2009, Vol. 48, p. 9557-9560 Takeuchi et al., Proc. Natl. Acad. Sci. USA, 1989, Vol. 86, p. 7819-7822 Sumiyoshi et al., Biosci. Biotechnol. Biochem. 2009, Vol. 73, no. 3, p. 543-551 Sumiyoshi et al., Biosci. Biotechnol. Biochem. 2010, Vol. 74, no. 3, p. 606-613

  An object of this invention is to provide the manufacturing method of the sugar chain which can supply the sugar chain required for manufacture of a protein formulation cheaply and in large quantities. Another object of the present invention is to provide a method for producing a glycoprotein preparation capable of supplying a large amount of the glycoprotein preparation at low cost.

The method for producing a sugar chain of the present invention is a method for producing a sugar chain,
It is characterized by having a sugar chain separation step of separating a sugar chain to which sialic acid is bound from an egg of an Emu ( Dromaius novaehollandiae ).

The method for producing a glycoprotein preparation of the present invention is a method for producing a glycoprotein preparation,
Having a sugar chain binding step of binding a sugar chain to a protein;
As the sugar chain, a sugar chain produced by the sugar chain production method of the present invention is used.

  According to the method for producing a sugar chain of the present invention, it is possible to supply a large amount of sugar chains necessary for producing a protein preparation at a low cost. Moreover, according to the method for producing a protein preparation of the present invention, since the sugar chain produced by the method for producing a sugar chain of the present invention is used, a protein preparation can be supplied in a large amount at a low cost.

FIG. 1 is a gel filtration chromatogram of the dried solid in Example 1. FIG. 2 is an anion exchange HPLC profile of the PA-glycan mixture in Example 1. FIG. 3 is a reverse phase HPLC profile of the sugar chain fraction in Example 1. FIG. 4 is a reverse phase HPLC profile of a sugar chain fraction and a standard product in Example 1. FIG. 5 is a reverse-phase HPLC profile of the sugar chain fraction and the standard enzyme-treated product in Example 1. 6 is a mass spectrum of the sugar chain fraction in Example 1. FIG. FIG. 7 is a reverse phase HPLC profile of the sugar chain fraction in Example 2. FIG. 8 is a reverse phase HPLC profile of a sugar chain fraction and a standard product in Example 2. FIG. 9 is a reversed-phase HPLC profile of the sugar chain fraction and the standard enzyme-treated product in Example 2. FIG. 10 is a mass spectrum of the sugar chain fraction in Example 2.

In the method for producing a sugar chain of the present invention, the sugar chain separation step comprises:
A glycoprotein fractionation step of fractionating a glycoprotein fraction from the egg of the emu ( Dromaius novaehollandiae );
A sugar chain fractionation step of fractionating the fractionated glycoprotein fraction into a sugar chain fraction and a protein fraction;
It is preferable to include a sialic acid-binding sugar chain fractionation step of fractionating the sugar chain fraction to which the sialic acid is bound from the sugar chain fraction.

  In the method for producing a sugar chain of the present invention, it is preferable that the sugar chain has three or four branched chains, and sialic acid is bonded to all the three or four branched chains.

In the method for producing a sugar chain of the present invention, the sugar chain is preferably represented by the following chemical formula (1) or (2).

  In the method for producing a glycoprotein preparation of the present invention, the glycoprotein preparation is preferably erythropoietin (EPO), IgG, interleukin or interferon.

<Method for producing sugar chain>
As described above, the method for producing a sugar chain of the present invention is a method for producing a sugar chain, and includes a sugar chain separation step of separating a sugar chain to which sialic acid is bound from an egg of an Emu ( Dromaius novae Hollandiae ). It is characterized by. The sugar chain production method of the present invention is characterized in that in the sugar chain separation step, a sugar chain to which sialic acid is bound is separated from the emu egg, and other steps are not limited at all.

  Examples of eggs of the emu include whole egg, egg yolk, egg white, defatted egg yolk, powdered egg yolk, powdered egg white, and the like, preferably egg white. As the eggs of the emu, for example, commercially available products may be used, or eggs obtained by breeding the emu and laying eggs may be used.

  The type of the sugar chain is not particularly limited, and examples thereof include monosaccharides, disaccharides, oligosaccharides, and polysaccharides, and oligosaccharides and polysaccharides are preferable. In the case of the oligosaccharide or polysaccharide, examples of the sugar chain include an N-linked sugar chain and an O-linked sugar chain, and an N-linked sugar chain is preferable. The sugar chain produced by the production method of the present invention is, for example, a glycoprotein sugar chain that can be bound to a protein to produce a glycoprotein.

  The sugar residue constituting the sugar chain is not particularly limited. For example, glucose, galactose, mannose, glucosamine, N-acetylglucosamine, galactosamine, N-acetylgalactosamine, fucose, xylose, N-acetylneuraminic acid, N -Glycolylneuraminic acid etc. are mentioned. The sugar residue may be modified, for example. The modification is not particularly limited, and examples thereof include modification by esterification, acylation, amination, etherification, nitration, hydrolysis, dehydration reaction, oxidation-reduction, and the like.

  As described above, sialic acid is bound to the sugar chain. Sialic acid is a substance in which the functional group of neuraminic acid ((4S, 5R, 6R, 7S, 8R) -5-amino-4,6,7,8,9-pentahydroxy-2-oxononanoic acid) is substituted. Specifically, for example, the aforementioned N-acetylneuraminic acid, N-glycolylneuraminic acid and the like can be mentioned. The sugar chain to which the sialic acid is bound is generally called a mammalian sugar chain, and it is reported that the sialic acid is involved in influenza infection even in birds. In the sugar chain, the number of the sialic acids is not particularly limited, and may be one, for example, or two or more. The binding site of sialic acid in the sugar chain is not particularly limited, but for example, the non-reducing end of the sugar chain is preferable.

  The sugar chain may be, for example, a straight chain type or a branched chain type, but a branched chain type is preferable. When the sugar chain is a branched chain type, the number of branched chains is, for example, 2 to 5, preferably 3 to 4, and more preferably 4. In the present invention, the branched chain refers to, for example, a sugar chain branched from the main chain. When the sugar chain is a branched chain type, the sialic acid may be bonded to all branched chains or may be bonded to some branched chains. Examples of the sugar chain include a four-chain sugar chain in which 1 to 4 sialic acids are bonded, a three-chain sugar chain in which 1 to 3 sialic acids are bonded, and a double-chain sugar in which one or two sialic acids are bonded. A chain. In the sugar chain, for example, the sialic acid is preferably bonded to all the branched chains, and more preferably bonded to the non-reducing ends of all the branched chains. The sugar chain preferably has 3 or 4 branched chains, and sialic acid is preferably bonded to all the 3 or 4 branched chains. By binding the sialic acid, degradation (metabolism) of the sugar chain in vivo is suppressed. In addition, as the number of sialic acids bound increases, degradation of the glycoprotein to which the sugar chains are bound in vivo is further suppressed.

Specific examples of the sugar chain to which the sialic acid is bonded include sugar chains of the following chemical formula (1) or (2).

  The molecular formula of the sugar chain of the chemical formula (1) is Siaα2-3Galβ1-4GlcNAcβ1-2 (Siaα2-3Galβ1-4GlcNAcβ1-4) Manα1-3 (Siaα2-3Galβ1-4GlcNAcβ1-2 (Siaα2-3Galβ1-4GlcNAcβ1-6) 6) It is represented by Manβ1-4GlcNAcβ1-4GlcNAc. The sugar chain of the chemical formula (1) has four uniform branched chains, and sialic acid is bonded to the non-reducing end of each branched chain. The molecular formula of the sugar chain of the chemical formula (2) is Siaα2-3Galβ1-4GlcNAcβ1-2 (Siaα2-3Galβ1-4GlcNAcβ1-4) Manα1-3 (Siaα2-3Galβ1-4GlcNAcβ1-2Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAcβ1-4 Represented. The sugar chain of the chemical formula (2) has three uniform branched chains, and sialic acid is bonded to the non-reducing end of each branched chain.

  Below, one Embodiment of the manufacturing method of the sugar_chain | carbohydrate of this invention is described. In addition, the manufacturing method of the sugar chain of this invention is not restrict | limited to the following embodiment.

(Embodiment 1)
In the present embodiment, the method for producing a sugar chain includes a glycoprotein fractionation step for fractionating a glycoprotein fraction from the eggs of the emu, and the fractionated glycoprotein fraction, the sugar chain fraction and the protein fraction. And a sialic acid-binding sugar chain fractionation step of fractionating the sugar chain fraction bound with the sialic acid from the sugar chain fraction.

[Glycoprotein fractionation process]
Prepare the emu egg and divide it into egg white and egg yolk. On the other hand, a mixed solvent of methanol and chloroform is prepared as an extraction solvent. The egg white and an equal volume of the mixed solvent are mixed and homogenized. This mixed solution is centrifuged, and the interface layer formed at the interface between the aqueous phase and the organic phase is recovered. An equal weight of acetone as an extraction solvent is added to the interface layer and mixed. The mixture is centrifuged and the precipitate is collected. The precipitate is dried under reduced pressure to obtain a glycoprotein fraction.

  The glycoprotein fraction contains, for example, glycoprotein, glycopeptide and glycoamino acid. The glycoprotein fraction may contain components other than glycoprotein, glycopeptide and glycoamino acid, for example. The component is not particularly limited.

  The extraction solvent is not limited to the above-mentioned mixed solvent of methanol and chloroform, acetone or the like, and may be any solvent that can fractionate the glycoprotein fraction. Examples of the extraction solvent include an aqueous solvent and an organic solvent. Examples of the aqueous solvent include water. Examples of the organic solvent include methanol, chloroform, acetone, dichloromethane, dichloroethane, and the like. The extraction solvent may be one type or a mixed solvent in which two or more types are combined. Examples of the mixed solvent include a mixed solvent of water-saturated phenol and chloroform other than the above-described mixed solvent of methanol and chloroform. In the mixed solvent, the volume ratio of each solvent can be appropriately set according to the polarity of the glycoprotein and is not particularly limited.

  The extraction conditions are not limited to the above-described method, and conventionally known conditions can be adopted. Moreover, the said collection method is not restrict | limited to the above-mentioned method, A conventionally well-known method is employable.

[Sugar chain fractionation process]
Next, the glycoprotein fraction and the hydrazine compound are put into a sufficiently dried test tube and stirred to dissolve the glycoprotein fraction in the hydrazine compound. The test tube is sealed and heated to release sugar chains from the glycoprotein in the glycoprotein fraction. Further, the test tube is allowed to stand until it reaches room temperature, and then the hydrazine compound is distilled off under reduced pressure. The residue is suspended in toluene, and the process of distilling off the hydrazine compound under reduced pressure is repeated to obtain a sugar chain fraction.

  Examples of the hydrazine compound include hydrazine hydrates such as hydrazine monohydrate, anhydrous hydrazine and the like, preferably anhydrous hydrazine. The hydrazine hydrate has a lower risk of explosion and the like and is less expensive than the anhydrous hydrazine. For this reason, when the hydrazine hydrate is used as the hydrazine compound, the sugar chain can be produced more safely and inexpensively. The hydrazine compound is preferably used in an amount of, for example, 30 to 1000 gram equivalent, more preferably 30 to 100 gram equivalent, with respect to 1 gram equivalent of glycoprotein.

  In the sugar chain fractionation step, the conditions such as dissolution, heating, distillation under reduced pressure and the like are not particularly limited and can be appropriately set according to the amount of glycoprotein and the like.

  The sugar chain contained in the sugar chain fraction has an acetyl group eliminated by the aforementioned hydrazine decomposition. For this reason, the sugar chain fraction is treated with an acetylating agent to reacetylate the sugar chain. The acetylating agent is not particularly limited, and examples thereof include acetyl halides such as acetyl chloride and acetyl bromide, acetic anhydride, and the like, and preferably acetic anhydride. The acetylating agent is preferably used in an amount of, for example, 100 to 1000 molar equivalents, and more preferably 100 to 200 molar equivalents with respect to 1 molar equivalent of the amino group.

  Furthermore, the protein contained in the glycoprotein fractionated in the glycoprotein fractionation step may be enzymatically decomposed before the sugar chain fractionation step. The enzyme is not particularly limited, and examples thereof include trypsin, pepsin, thermolysin, and pronase. The decomposition conditions can be appropriately set according to conditions such as the type of enzyme and the amount of treatment. By the enzymatic decomposition, for example, unnecessary peptides and the like are decomposed and removed, so that the glycoprotein is reduced in molecular weight. For this reason, the production efficiency of sugar chains can be improved.

[Sialic acid-binding sugar chain fractionation process]
Then, water is added to the sugar chain fraction to dissolve, and freeze-dried. This lyophilizate is dissolved in a pyridylamino (PA) reagent and reacted. A reducing reagent is added to the PA reaction solution and reacted. To this reduction reaction liquid, a mixed solvent of water and an organic solvent is added and centrifuged to remove the lower organic phase. After this removal process is repeated several times, the liquid part is removed and the dry solid is recovered. The dried solid is fractionated and purified by gel filtration chromatography, anion exchange column chromatography, and reverse phase column chromatography to obtain sialic acid-bonded sugar chains.

  The PA reagent is not particularly limited, and examples thereof include an acetic acid solution containing 2-aminopyridine. The reducing reagent is not particularly limited, and examples thereof include an acetic acid solution containing a methylamine borane complex. The organic solvent is not particularly limited, and examples thereof include phenol and chloroform.

  In the sialic acid-binding sugar chain fractionation step, conditions for various reactions, centrifugation, and various chromatography are not particularly limited and can be set as appropriate. The fractionation and purification method is not particularly limited, and a conventionally known method can be appropriately employed.

<Method for producing glycoprotein preparation>
As described above, the method for producing a glycoprotein preparation of the present invention is a method for producing a glycoprotein preparation, which includes a sugar chain binding step for binding a sugar chain to a protein, and the sugar chain of the present invention is used as the sugar chain. A sugar chain produced by the method for producing a chain is used.

  The method for producing the sugar chain protein preparation is not limited at all, except that the sugar chain binding step is used, and the sugar chain produced by the method for producing a sugar chain of the present invention is used as the sugar chain. A conventionally known method can be employed. The glycoprotein preparation includes a sugar chain to which sialic acid is bound, produced by the sugar chain production method of the present invention. For this reason, the said glycoprotein formulation has high activity with respect to a biological body, for example.

  Below, one Embodiment of the manufacturing method of the glycoprotein formulation of this invention is described. In addition, the manufacturing method of the glycoprotein formulation of this invention is not restrict | limited to the following embodiment.

(Embodiment 2)
The method for producing a glycoprotein preparation in the present embodiment includes a sugar chain production step for producing a sugar chain to which sialic acid is bound, a protein production step for producing a protein, and a binding step for binding the protein and the sugar chain. including.

[Sugar chain production process]
First, the sialic acid-bonded sugar chain is obtained using the sugar chain production method of Embodiment 1. The sialic acid-bonded sugar chain is hydrolyzed with acid, mixed with ammonium hydrogen carbonate and then reacted with halogenoacetic acid to form a halogenoacetamidation (—NH—COCH ₂ X, X = halogeno group). The halogeno group is not particularly limited, and examples thereof include a chloro group and a bromo group. The halogenoacetamidation method is not particularly limited, and for example, a conventionally known method can be appropriately employed.

[Protein production process]
Next, a recombinant protein to be bound to the sugar chain is prepared. Specifically, a DNA fragment encoding a desired protein is introduced into a host to produce a transformant, the transformant is cultured, the culture is recovered, and the culture is purified.

  The protein is not particularly limited, and examples thereof include erythropoietin (EPO), IgG, interleukin, and proteins constituting interferon. The interferon is not particularly limited, and examples thereof include IFN-α, IFN-β, IFN-ω, IFN-ε, IFN-κ, IFN-γ, and IFN-λ. Although it does not restrict | limit especially as said interleukin, For example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 and the like.

  The host is not particularly limited, and examples thereof include Escherichia coli, yeast, filamentous fungi, actinomycetes, COS cells, CHO cells, and Sf9 cells. The E. coli is not particularly limited, and examples thereof include strains such as S17-1, BL21 (DE3), BL21-CodonPlus (DE3) -RIL, Rosetta2 (DE3), Rosetta-gami2 (DE3).

  In the protein production process, methods for introducing a DNA fragment into the host, culturing the transformant, recovering and purifying the culture are not particularly limited, and for example, conventionally known methods can be appropriately employed. In the present invention, the method for producing the protein is not limited to the above-described method for producing a recombinant protein, and for example, a conventionally known method can be appropriately employed.

[Bonding process]
A crude glycoprotein is obtained by adding a condensing agent and condensing the halogenoacetamidoglycan and the protein. The crude glycoprotein is purified to obtain a glycoprotein preparation.

  The condensing agent is not particularly limited, and examples thereof include 4-mercaptophenylacetic acid (MPAA). The condensation conditions are not particularly limited, and for example, conventionally known conditions can be appropriately set. The purification method is not particularly limited, and for example, a conventionally known method can be adopted. In the bonding step, each functional group may be appropriately protected with a protective agent, for example. In the bonding step, the protected functional group may be appropriately deprotected with a deprotecting agent, for example. The protective agent and deprotecting agent are not particularly limited.

  Examples of the glycoprotein preparation include glycoproteins that can be used as drugs, physiologically active substances, and the like, and are not particularly limited. Specific examples of the glycoprotein include the aforementioned erythropoietin (EPO), IgG, interleukin, interferon and the like to which a sugar chain is bound. The interferon is not particularly limited, and examples thereof include the aforementioned interferon. It does not restrict | limit especially as said interleukin, For example, the above-mentioned interleukin is mentioned.

  In the coupling step, as the sugar chain, in addition to the sugar chain produced by the sugar chain production method of the present invention, other sugar chains may be used.

  The type of sugar chain, sugar residue, modification, number of sugar chains, and the like are not particularly limited, but are the same as, for example, the sugar chain in the sugar chain production method. For example, sialic acid may or may not be bound to the other sugar chain, but the former is preferably the former. The method for producing the other sugar chain is not particularly limited, and for example, a conventionally known method can be appropriately employed.

(Embodiment 3)
The method for producing a glycoprotein preparation in the present embodiment includes a peptide production step and a glycopeptide production step in addition to the sugar chain production step, the protein production step and the binding step. And combine.

[Sugar chain production process]
First, in the same manner as in Embodiment 2, a halogenoacetamidated sugar chain is obtained.

[Peptide production process]
Next, a peptide is produced by a solid phase synthesis method as follows.

  First, the column is packed with a carrier. A condensation reagent containing an amino group is added to the column to introduce an amino group into the carrier. Next, a washing reagent is added to the column to wash the carrier into which the amino group has been introduced. After washing, a deprotecting reagent is added to deprotect the amino group. Then, the peptide is elongated by adding a condensation reagent containing the amino group to the column. After removing the deprotecting reagent, the cleavage reagent is added to the column, and the elongated peptide is cut out from the carrier.

  The carrier is not particularly limited, and examples thereof include trityl chloride resin, HMPB (4- (4-hydroxymethyl-3-methoxyphenoxy) butyric acid) -PEGA (poly (ethylene glycol) -poly (dimethylacrylamide) copolymer) resin, and the like. Is mentioned. Examples of the column include a synthesis column widely used for peptide synthesis, and are not particularly limited. The method for packing the carrier into the column is not particularly limited and can be set as appropriate.

  The condensing reagent containing an amino group is prepared, for example, by adding an amino acid protected with a protecting group and a condensing agent to a solvent. The protective group is not particularly limited, and examples thereof include a fluorenylmethoxycarbonyl (Fmoc) group and a tert-butoxycarbonyl (Boc) group. The condensing agent is not particularly limited. For example, 2- (1H-benzotriazol-1-yl) 1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU), 1-hydroxy-1H -Benzotriazole (HOBt), N, N-diisopropylethylamine (DIPEA), etc. are mentioned. The solvent is not particularly limited, and examples thereof include dimethylformamide (DMF) and dichloromethane (DCM). The conditions for the introduction reaction are not particularly limited and can be set as appropriate.

  The cleaning reagent is not particularly limited, and examples thereof include the aforementioned solvents. The cleaning conditions are not particularly limited and can be set as appropriate.

  The deprotecting reagent is prepared, for example, by adding a deprotecting agent to a solvent. The deprotecting agent is not particularly limited, and examples thereof include piperidine, 2-mercaptoethanol, TFA (acetonitrile), TIS (triisopropylsilane), EDT (ethanedithiol), and the like. Although the said solvent is not restrict | limited in particular, For example, the above-mentioned solvent etc. are mentioned. The conditions for the deprotection reaction are not particularly limited and can be set as appropriate.

  The conditions for the extension reaction are not particularly limited and can be set as appropriate. The washing, deprotection and extension reactions are repeated to further extend the peptide. Although the length in particular of the said peptide is not restrict | limited, For example, it is 1-100 amino acid residues, Preferably, it is 5-50 amino acid residues, More preferably, it is 10-20 amino acid residues.

  The cleavage reagent is prepared, for example, by adding a cleavage agent to a solvent. The cleavage agent is not particularly limited, and examples thereof include an acetic acid-trifluoroethanol solution. Although the said solvent is not restrict | limited in particular, For example, the above-mentioned solvent etc. are mentioned. The cutout conditions are not particularly limited and can be set as appropriate.

  In the present invention, the method for producing the peptide is not limited to the solid phase synthesis method, and for example, a conventionally known method can be appropriately employed.

[Glycopeptide manufacturing process]
A condensing reagent is added to the peptide cleaved from the carrier and the halogenoacetamidated sugar chain and reacted to obtain a glycopeptide.

  Although the condensing reagent of the said peptide and sugar_chain | carbohydrate is not restrict | limited in particular, 4-mercaptophenylacetic acid (MPAA) etc. are mentioned. The conditions for the condensation reaction are not particularly limited and can be set as appropriate.

[Protein production process]
A recombinant protein is obtained in the same manner as in Embodiment 2.

[Bonding process]
The glycopeptide is mixed with a thioesterification reagent, and the C-terminal α-carboxylic acid of the glycopeptide is thioesterified. The thioesterified glycopeptide and the recombinant protein are added to a buffer and reacted to obtain a crude glycoprotein. The crude glycoprotein is stirred for several hours with the addition of a deprotection reagent to remove the protecting group. The crude glycoprotein that has been deprotected is purified to obtain a glycoprotein preparation.

  The thioesterification reagent is not particularly limited, and examples thereof include a mixed liquid of thiophenol, PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) and DIPEA. The conditions for the thioesterification are not particularly limited and can be appropriately set.

  The buffer solution is not particularly limited, and examples thereof include a phosphate buffer solution (PBS). The buffer solution may contain additives such as thiophenol (PhSH), benzyl mercaptan (BnSH), and ethyl mercaptan (EtSH). The reaction conditions are not particularly limited and can be set as appropriate.

  Examples of the deprotection reagent include the aforementioned deprotection reagent. The conditions for the deprotection reaction are not particularly limited and can be set as appropriate.

  The method for purifying the crude protein is not particularly limited, and for example, a conventionally known method can be adopted.

  In the bonding step, each functional group may be appropriately protected with a protective agent, for example. In the bonding step, the protected functional group may be appropriately deprotected with a deprotecting agent, for example.

  Examples of the glycoprotein preparation include the aforementioned glycoprotein preparation.

  In the present invention, the coupling method is not limited to the above-described method, and for example, a conventionally known method can be appropriately employed.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

<Example 1>
In this example, a sugar chain was prepared from an egg of Emu ( Dromaius novaehollandiae ), and a glycoprotein bound with this sugar chain was synthesized.

[Fractionation of glycoprotein fraction]
Five Emu eggs (manufactured by Changnan Green System) were separated into egg yolk and egg white. Extracts obtained by mixing methanol and chloroform in 1: 1 (volume ratio) were mixed in an equal volume to five egg whites and homogenized. The mixture was centrifuged at 3000 × g (g is gravitational acceleration, hereinafter the same) for 20 minutes, and the interface layer formed at the interface between the aqueous phase and the organic phase was recovered. An equal weight of acetone was added to the interface layer, mixed, and centrifuged under the same conditions as described above. After centrifugation, the precipitate was collected and dried under reduced pressure for 3 days to obtain a powdered glycoprotein fraction.

[Fractionation of sugar chain fraction]
In a well-dried test tube with a screw cap (for example, inner diameter 15 mm × length 150 mm), 50 mg of the glycoprotein fraction was added, and hydrazine monohydrate (H ₂ NNH ₂ .H ₂ O, Nacalai) 5 mL of Tesque) was added. The screw cap of the test tube was tightened and stirred using a vortex mixer (registered trademark) to dissolve the glycoprotein fraction in the hydrazine monohydrate. The boundary between the screw cap and the test tube body was lined with nylon tape, and the inside of the test tube was sealed. This test tube was heated at 90 ° C. for 10 hours to release sugar chains from the glycoprotein fraction. After heating, the test tube was allowed to stand until it reached room temperature. After removing the nylon tape from the test tube and opening the screw cap, the hydrazine monohydrate was distilled off under reduced pressure using a vacuum pump with a cooling trap at -70 ° C. To this test tube, 0.2 mL of toluene was added, stirred and suspended, and distilled under reduced pressure in the same manner as described above. The distillation of hydrazine monohydrate using toluene was repeated three times to obtain a sugar chain fraction.

[Reacetylation of sugar chains]
In the test tube, 5 mL of an ice-cooled saturated aqueous sodium bicarbonate solution and 0.2 mL of acetic anhydride were added and mixed with 2 mg of the sugar chain fraction for 5 minutes. Furthermore, 5 mL of an ice-cooled saturated sodium hydrogen carbonate aqueous solution and 0.2 mL of acetic anhydride were added to the test tube, and the mixture was reacted for 30 minutes while mixing once every about 5 minutes under ice-cooling. The obtained reaction solution is put into a 100 mL beaker, and about 25 g of a cation exchange resin (trade name: DOWEX 50WX8 (200 to 400 mesh, H ⁺ type), manufactured by Dow Chemical Co.) is added, and the pH of the reaction solution is about Added until 3. The reaction solution and cation exchange resin were packed in a glass column, and the eluate was collected from the lower part of the column. Further, the glass column was washed with 5 times the amount of the cation exchange resin volume, and the washing liquid was collected. The eluate and the washing solution were placed in an eggplant-shaped flask, and distilled off using a rotary evaporator, and dried.

[Fractionation and purification of sialic acid-linked sugar chain]
A small amount of water was added to the eggplant-shaped flask to dissolve 2 mg of the dry solid content. This lysate was put into a screw cap test tube with a conical Teflon (registered trademark) seal (inner diameter 25 mm × length 90 mm) and freeze-dried. After the freeze-drying, 0.5 mL of a PA-forming reagent in which 2760 mg of 2-aminopyridine (manufactured by Nacalai Tesque) is dissolved in 1 mL of acetic acid is added to the test tube, and the remaining in the test tube is heated with warm air from a dryer. The test was completely dissolved and reacted at 90 ° C. for 1 hour. In this test tube, 1.75 mL of a reducing reagent prepared by dissolving 6 g of dimethylamine borane complex in 2.4 mL of acetic acid and 1.5 mL of water was added and mixed, and reacted at 80 ° C. for 35 minutes. To this reaction solution, a mixed solvent of 5 mL of a water saturated phenol / chloroform solution in which equal amounts of phenol saturated with water and chloroform were mixed and 3.75 mL of water was added and mixed. This mixture was centrifuged at 1000 × g for 2 minutes to remove the lower organic phase. To the upper aqueous phase, 5 mL of a water saturated phenol / chloroform solution was again added and mixed. This mixed solution was centrifuged under the same conditions as described above to remove the lower layer. This removal process was repeated twice. Then, 5 mL of chloroform was added to the upper aqueous phase and mixed, and centrifuged under the same conditions as described above to remove the lower organic phase. Using the concentrator, the liquid part of the upper aqueous phase was removed and dried.

  The dried solid was subjected to gel filtration under the following conditions.

(Analysis conditions for gel filtration)
(1) Detection equipment: HPLC system with fluorescence detector (manufactured by Shimadzu Corporation)
(2) Column: TOYOPARL HW-40F
(Inner diameter 2.6 mm x 40 cm, manufactured by Tosoh Corporation)
(3) Buffer for buffer equilibration:
10 mmol / L acetic acid-ammonia buffer (pH 6.0)
(4) Mobile phase: Solvent 10 mmol / L acetic acid-ammonia buffer (pH 6.0)
Flow rate 50mL / h
(5) Preparative volume: 3.8 mL / fraction

  FIG. 1 shows the gel filtration chromatogram of the dried solid. In FIG. 1, the vertical axis represents the fluorescence intensity, and the horizontal axis represents the fraction number. The eluent having a peak (P1) near Vo in FIG. 1 was collected.

  The eluate was concentrated, and a mixture of fluorescently labeled PA-glycans was recovered. This mixture was subjected to anion exchange HPLC under the following conditions.

(Analysis conditions for anion exchange HPLC)
(1) Analytical instrument: HPLC system with fluorescence detector (manufactured by Shimadzu Corporation)
(2) Column: Trade name: Q-Sepharose Fast Flow
(Inner diameter 20 mm x 10 cm, manufactured by GE Healthcare)
(3) Buffer for buffer equilibration: ammonia water (pH 9.0)
(4) Mobile phase: solvent A: aqueous ammonia (pH 9.0)
B: 0.5 mol / L acetic acid-ammonia buffer (pH 9.0)
Gradient condition: time (solvent ratio (A: B))
0 minutes (A: B = 100: 0)
10 minutes (A: B = 100: 0)
40 minutes (A: B = 60: 40)
Flow rate 3mL / min

  FIG. 2 shows the anion exchange HPLC profile of the PA sugar chain mixture. In FIG. 2, the vertical axis represents fluorescence intensity, and the horizontal axis represents retention time (minutes). The eluent of the peak indicated by S4 in FIG. 2 (tetrasialylated sugar chain fraction with four sialic acids bound thereto, A4) was collected.

  The tetrasialylated sugar chain fraction was subjected to reverse phase HPLC under the following conditions.

(Reverse phase HPLC analysis conditions)
(1) Analytical instrument: HPLC system with fluorescence detector (manufactured by Shimadzu Corporation)
(2) Column: Trade name: Cosmosil 5C18-P
(Inner diameter 10 mm x 25 cm, manufactured by Nacalai Tesque)
(3) Column equilibration buffer: 20 mmol / L acetic acid-ammonia buffer
+ 0.35% n-butanol buffer (pH 4.0)
(4) Mobile phase: Solvent 20 mmol / L acetic acid-ammonia buffer
+ 0.35% n-butanol buffer (pH 4.0)
Flow rate 3mL / min

  FIG. 3 shows the reverse phase HPLC profile of the tetrasialylated sugar chain fraction. In FIG. 3, the vertical axis represents fluorescence intensity, and the horizontal axis represents retention time (minutes). The eluent (sugar chain fraction) of the peak (P2) indicated by the arrow in FIG. 3 was collected.

  The sugar chain fraction separated by the reverse phase HPLC was subjected to size fractionation HPLC under the following conditions.

(Analysis conditions for size fractionation HPLC)
(1) Analytical instrument: HPLC system with fluorescence detector (manufactured by Shimadzu Corporation)
(2) Column: Product name: TSK gel Amide 80
(Inner diameter 2.0 mm x 25 cm, manufactured by Tosoh Corporation)
(3) Buffer solution for column equilibration: mixed solvent of A and B (solvent ratio (A: B) = 8: 2)
A: Acetonitrile
B: 50 mmol / L formic acid-ammonia buffer (pH 4.4)
(4) Mobile phase: solvent A: acetonitrile
B: 50 mmol / L formic acid-ammonia buffer (pH 4.4)
Gradient condition: time (solvent ratio (A: B))
0 minutes (A: B = 80: 20)
10 minutes (A: B = 70: 30)
35 minutes (A: B = 30: 70)
Flow rate 0.18mL / min

[Confirmation of sugar chain]
A standard product of a four-chain sugar chain to which sialic acid represented by the chemical formula (1) was bound was subjected to the anion exchange HPLC, reverse phase HPLC and size fractionation HPLC described above. Note that the standard product is a four-chain sugar chain to which sialic acid represented by the chemical formula (1) is bound. org / tools / glycomod /). The elution positions of the peaks in the anion exchange HPLC, reverse phase HPLC, and size fraction HPLC of each of the fractions and the standard product were compared to confirm the sugar chain structure.

The yield of the fraction obtained by the size fraction HPLC was calculated as follows based on the method of Hase et al. (Methods Mol., 1993, Vol. 14, p.69-80). First, PA-GluNAc in which PA was bonded to N-acetylglucosamine was measured as Xpmol (X = 10 to 100) as a fluorescently labeled sugar, and subjected to the size fractionation HPLC in the same manner as described above, and the fluorescence intensity was measured. And the peak area (A) was computed from the obtained chromatogram. On the other hand, the peak area (B) was calculated for the fraction and the standard product in the same manner as the fluorescently labeled sugar. The calculated peak area was substituted into the following formula 1, and the yield of the fraction and the standard product was calculated.
Yield = ((A / B) × X) / Factor value (1)
A = peak area of the fluorescently labeled sugar
B = peak area of the fraction or the standard product
X = the amount of the fluorescence-labeled sugar subjected to HPLC (pmol)
Factor value = 1.3

  The tetrasialylated glycan fraction, the glycan fraction separated by the reverse-phase HPLC, and the standard are each added to an acetic acid-ammonia buffer containing 50 mU exoglycosidase and reacted at 37 ° C. for 10 minutes. I let you. As the exoglycosidase, β-galactosidase, β-N-acetylhexosaminidase (manufactured by Seikagaku Corporation) and α2,3-sialidase (manufactured by Takara Bio Inc.) were used. Each reaction solution was subjected to the aforementioned reverse phase HPLC and size fractionation HPLC. The elution positions of the peaks in the reverse phase HPLC and the size fraction HPLC of the enzyme-treated product of each fraction and the standard enzyme-treated product were compared, and the behavior by enzyme treatment was compared.

  FIG. 4 shows the reverse-phase HPLC profile of the tetrasialylated sugar chain fraction and the standard product, and FIG. 5 shows the sialidase-treated product of the tetrasialylated sugar chain fraction and the standard product of sialidase. And shows the reverse phase HPLC profile. 4 and 5, the vertical axis represents fluorescence intensity, and the horizontal axis represents retention time (minutes). In both figures, the profile of the tetrasialylated sugar chain fraction is shown in the upper row and the profile of the standard product is shown in the lower row so that the peaks can be easily compared. As shown in FIG. 4, the peak of the tetrasialylated sugar chain fraction coincided with the peak of the standard product. Moreover, as shown in FIG. 5, both peaks after the enzyme treatment also coincided. From this result, it was shown that the tetrasialylated sugar chain fraction was a four-chain sugar chain to which sialic acid was bound.

  Each fraction obtained by the reverse phase HPLC was subjected to structural analysis by a MALDI-TOF method (negative mode) using a mass spectrometer (device name: AUTOFLEX II, manufactured by BRUKER) equipped with a 337 nm nitrogen laser. The molecular mass was measured. In the measurement, the extraction voltage is 20 kV, 30 v / v% ethanol containing 1 mg / mL 2,5-dihydroxybenzoic acid is used as a matrix, and the mass range is m / z 280 to 4,080. And m / z 80 to 3,300 in negative ion mode reflector mode. Each spectrum was measured by 150 laser shots.

FIG. 6 shows a mass spectrum obtained by the MALDI-TOF method (negative mode). In FIG. 6, the vertical axis represents ionic strength (au), and the horizontal axis represents m / z. As shown in FIG. 6, a peak of [M−H] ⁻ was detected at m / z 3612.003. Further, as described above, this sugar chain is a four-chain sugar chain to which the sialic acid is bound. From these results, the four-chain sugar chain to which the sialic acid is bonded is represented by the chemical formula (1). -4GlcNAcβ1-2 (Siaα2-3Galβ1-4GlcNAcβ1-6) Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAc. The yield of the four-chain sugar chain was 30 mg per egg of the Emu.

<Example 2>
In this example, in the anion exchange HPLC, the eluent of the peak shown in S3 of FIG. 2 (trisialylated sugar chain fraction bound with three sialic acids, A3) was collected, and the standard product was The sugar chain was fractionated from the emu egg and the structure of the sugar chain was confirmed in the same manner as described above except that the three-chain sugar chain to which sialic acid represented by the chemical formula (2) was bound was used.

  FIG. 7 shows the reverse phase HPLC profile of the trisialylated sugar chain fraction. In FIG. 7, the vertical axis represents fluorescence intensity, and the horizontal axis represents retention time (minutes). The eluent (sugar chain fraction) of the peak (P3) indicated by the arrow in FIG. 7 was collected.

  FIG. 8 shows the reversed-phase HPLC profile of the trisialylated glycan fraction and the standard product, and FIG. 9 shows the sialidase-treated product of the trisialylated glycan fraction and the sialidase-treated product of the standard product. The reverse phase HPLC profile is shown. 8 and 9, the vertical axis represents fluorescence intensity, and the horizontal axis represents retention time (minutes). In both figures, the profile of the trisialylated sugar chain fraction is shown in the upper row and the profile of the standard product is shown in the lower row so that the peaks can be easily compared. As shown in FIG. 8, the peak of the trisialylated sugar chain fraction coincided with the peak of the standard product. Moreover, as shown in FIG. 9, both peaks after the enzyme treatment also coincided. From this result, it was shown that the trisialylated sugar chain fraction was a three-chain sugar chain to which sialic acid was bound.

FIG. 10 shows a mass spectrum obtained by the MALDI-TOF method (negative mode). In FIG. 10, the vertical axis represents ionic strength (au), and the horizontal axis represents m / z. As shown in FIG. 10, a peak of [M−H] ⁻ was detected at m / z 2955.230. Further, as described above, this sugar chain is a three-chain sugar chain to which the sialic acid is bound. From these results, the sugar chain to which the three sialic acids are bonded is represented by the chemical formula (2). 4GlcNAcβ1-2Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAc. The yield of the three-chain sugar chain was 60 mg per egg of the Emu.

  As described above, from the emu egg, sialic acid is bonded to each branched chain represented by the chemical formula (2) and the four-chain sugar chain having sialic acid bonded to each branched chain represented by the chemical formula (1). A three-chain sugar chain could be produced. In living organisms other than humans and their products, there are hardly any ones containing three-chain sugar chains or four-chain sugar chains in which sialic acid is bound to each branched chain, other than the emu confirmed by the present inventors. It has not been confirmed in the eggs of other birds. For this reason, by using the eggs of the emu, a three-chain sugar chain or a four-chain sugar chain to which sialic acid is bound can be supplied at a low cost and in large quantities.

  According to the present invention, sugar chains necessary for the production of a protein preparation can be supplied at low cost and in large quantities. For this reason, according to the present invention, protein preparations can be supplied at low cost and in large quantities. Therefore, the present invention is applicable to a wide range of fields such as the medical field, and the field of application is not limited.

Claims (4)

A method for producing a sugar chain, comprising:
From egg emu (Dromaius novaehollandiae), it has a sugar chain separation step for separating a sugar chain has sialic acid attached,
Production method wherein the sugar chain, characterized in der Rukoto those represented by the following chemical formula (1) or (2).

The sugar chain separation step comprises
A glycoprotein fractionation step of fractionating a glycoprotein fraction from the egg of the emu ( Dromaius novaehollandiae );
A sugar chain fractionation step of fractionating the fractionated glycoprotein fraction into a sugar chain fraction and a protein fraction;
The method for producing a sugar chain according to claim 1, further comprising a sialic acid-binding sugar chain fractionation step of fractionating the sugar chain fraction bound to the sialic acid from the sugar chain fraction. A method for producing a glycoprotein formulation comprising:
Having a sugar chain binding step of binding a sugar chain to a protein;
A production method using the sugar chain produced by the production method according to claim 1 or 2 as the sugar chain. The method for producing a glycoprotein preparation according to claim 3 , wherein the glycoprotein preparation is erythropoietin (EPO), IgG, interleukin or interferon.

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