JPWO2016076440A1 - Method for producing glycopeptide or glycoprotein - Google Patents

Abstract

An embodiment of the present invention is a mutant endo-β-N-acetylglucosaminidase (endoenzyme variant) having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine, or An endoenzyme mutant having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is glutamine or alanine, and other than the 175th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 In the presence of an endoenzyme variant having an amino acid sequence modified within a homology of 80% or more to the amino acid sequence by deletion, addition and / or substitution of one or more amino acid residues , A sugar chain donor represented by the following general formula (1) (X1-X6: carbohydrate-derived group, H; Z1: H, GlcNAc If, to provide a method for producing a glycopeptide or glycoprotein by reacting a sugar receptor, a.

Description

  The present invention relates to a method for producing a glycopeptide or glycoprotein.

Many of the peptides and proteins in the preparation containing protein are compounds having sugar chains (hereinafter referred to as glycopeptides or glycoproteins). N-linked sugar chains and O-linked sugar chains are known as sugar chains possessed by glycoproteins. Among them, N-linked sugar chains have an important effect on the maintenance of protein function and structure. In particular, complex sugar chains called human-type sugar chains are often important.
Against this background, large-scale preparation of glycoproteins has been required, and preparation has been carried out by a method of culturing cells produced by genetic engineering methods. However, the N-linked sugar chain of the glycoprotein obtained by such a method is not a constant structure but a glycoprotein in which several to several tens or more kinds of sugar chains are bound and has a uniform structure. In recent years, it has been one of the problems to convert a sugar chain part into a complex sugar chain or the like.

  In glycoprotein production, as a method for controlling a sugar chain, a method using cells into which a glycosyltransferase gene has been introduced is known (see, for example, JP-A-2014-54263). As a result, a glycoprotein having a high content of sugar chains of mammals, that is, complex sugar chains, is obtained.

Moreover, an antibody is mentioned as a glycoprotein which has a high function.
Antibodies have been used not only for diagnosis and prevention of various human diseases but also for treatment because of their high binding activity, high binding specificity, and stability in blood. In particular, due to advances in cell engineering technology, antibodies can be prepared in large quantities by cultured cells and have been used as the main component of antibody drugs.
Among immunoglobulins (IgG) used in antibody pharmaceuticals, IgG1 has a high antibody-dependent cytotoxic activity (hereinafter referred to as ADCC activity) and complement-dependent cytotoxic activity (hereinafter referred to as CDC activity). It is known to have an effector function. In recent years, antibodies such as Rituxan and Herceptin, which have been used for treatment, have an antitumor effect due to such activity as a major effect.

It is known that two N-linked sugar chains possessed by IgG strongly affect ADCC activity and CDC activity. Particularly, regarding two N-linked sugar chains that bind to the 297th asparagine of IgG1, IgG1 having an N-linked sugar chain having a structure lacking fucose has been reported to cause a significant increase in ADCC activity (eg, Shitara et al., [J. Biol. Chem. Vol. 278, 3466-3473). , (2003)]). As N-linked sugar chains, complex sugar chains, high mannose sugar chains, and hybrid sugar chains are known. Of these, specific complex sugar chains are important for ADCC activity and CDC activity. It has been reported (see, for example, Pablo et al. [Nat. Biotechnol. Vol. 17, 176-180, (1999)]).
However, most of the IgG obtained from the above cultured cells or the like is a mixture having fucose added, and a mixture having a very wide variety of N-linked sugar chains.
On the other hand, regarding IgG1 having an N-linked sugar chain having a structure lacking fucose, the cultured cells derived from Chinese hamsters lacking an enzyme (fucosyltransferase) that adds fucose to the N-linked sugar chain It has been shown that it can be produced (see, for example, JP-A-2008-530243).

On the other hand, a method of introducing a new sugar chain into a glycoprotein in a test tube is also conceivable.
As a method for converting a sugar chain of a glycoprotein with an enzyme, a method using a sugar chain transfer activity of a carbohydrate hydrolase (endo-β-N acetylglucosaminidase, hereinafter referred to as “endo enzyme”) has been known for a long time. (See, for example, JP-A-7-59587). In the presence of an endoenzyme, a complex N-linked sugar chain serving as a sugar chain donor and a glycoprotein having only N acetylglucosamine as an asparagine residue as a sugar chain acceptor are reacted to form a complex A glycopeptide or glycoprotein having a type sugar chain can be obtained.

In addition, in order to improve the transglycosylation activity of endoenzymes, by creating mutants in which amino acid residues essential for enzyme activity are mutated (hereinafter referred to as endoM mutants), It has been reported that transglycosylation can be efficiently carried out with a small amount of sugar chain donor (for example, Umekawa et al. [J. Biol. Chem. Vol. 285 (1), 511-521, (2010 )]reference).
Furthermore, regarding the introduction of a complex type sugar chain into IgG, an oxazoline derivative of a complex type sugar chain chemically synthesized to an IgG1 polypeptide (hereinafter referred to as GlcNAc-IgG1) having N acetylglucosamine as an asparagine residue is used as a sugar donor. In addition, a method for preparing IgG1 having a complex sugar chain at a high content by the action of a variant of an endoenzyme different from the above has been reported (see, for example, International Publication No. 2013120066).

  In the above method, a method for obtaining glycopeptides or glycoproteins having complex sugar chains using cells (JP 2014-54263 A and JP 2008-530243 A, Shitara et al., [J. Biol. Chem. Vol.278, 3466-3473, (2003)] and Pablo et al., [Nat. Biotechnol. Vol.17, 176-180, (1999)]) are non-uniform sugar chains and other sugar chains. Mixing such as (high mannose type sugar chain) is inevitable.

  In addition, since hair mold-derived endo-β-N acetylglucosaminidase (hereinafter referred to as “endo M”) used in the method described in JP-A-7-59587 is a hydrolase, Once glycosylated or glycoproteins are produced, they are inevitably hydrolyzed, resulting in a low glycosylation yield.

On the other hand, Umekawa et al. [J. Biol. Chem. Vol. 285 (1), 511-521, (2010)], which has reduced hydrolysis activity as much as possible, and the end described in WO2013120066. In enzyme mutants, oxazoline derivatives that are unstable at room temperature are used as sugar chain donors, so that the conditions that can be used are limited, and depending on the type of sugar chain, preparation of the sugar chain donor itself is difficult. is there.
Thus, there has been a demand for a method for easily producing a complex glycoprotein using an endo M mutant.

  The present disclosure has been made in view of the above, and in the present disclosure, a production capable of easily producing a glycoprotein or glycopeptide having a high content of a specific complex type sugar chain using a stable sugar chain donor. It is an object to provide a method.

Specific means for solving the problems include the following aspects.
<1> Mutant endo-β-N-acetylglucosaminidase having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine, or the amino acid sequence represented by SEQ ID NO: 1 A mutant endo-β-N-acetylglucosaminidase having an amino acid sequence in which the 175th amino acid residue is glutamine or alanine, and one other than the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 Or a mutant endo-β-N-acetylglucosaminidase having an amino acid sequence modified within a range of homology of 80% or more to the amino acid sequence by deletion, addition and / or substitution of a plurality of amino acid residues In the presence of glycan, a sugar chain donor represented by the following general formula (1) is reacted with a sugar chain acceptor To produce a glycopeptide or glycoprotein.

(In General Formula (1), X ¹ and X ² each represent an independent saccharide-derived group or a hydrogen atom, and at least one of X ¹ and X ² contains one or more GlcNAc or GlcNAcA, GlcNAc or GlcNAcA contained in ¹ and X ² is linked to Man bound to Man at α1-3 or Man bound to Man at α1-6 at β1-2, X ³ , X ⁴ , X ⁵ and X ⁶ each independently represents a hydrogen atom or a saccharide-derived group, Z ¹ represents a hydrogen atom or GlcNAc, and GlcNAc contained in Z ¹ is β1-4 to Man bound to GlcNAc at β1-4 bonded to that .Y ¹ is .GlcNAc representing a monovalent substituent represents N- acetylglucosaminyl group, GlcNAcA the hydroxymethyl group at the 6-position of GlcNAc carboxylase Β1-4 represents a β-glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc or β-glycoside bond between position 1 of Man and position 4 of GlcNAc, where Man represents a mannosyl group. Α1-6 represents an α glycoside bond between the 1-position of Man and the 6-position of Man, and α1-3 represents an α-glycoside bond between the 1-position of Man and the 3-position of Man.)
In the present disclosure, GlcNAc bonded to Y ¹ does not include a substituent atom other than a hydrogen atom bonded to the carbon at the 1-position reducing end of GlcNAc.
<2> The method for producing a glycopeptide or glycoprotein according to <1>, wherein the sugar chain receptor is represented by the following general formula (2).

^{GlcNAc-R 2} Formula (2)
(In general formula (2), R ² represents a monovalent substituent. GlcNAc represents an N-acetylglucosaminyl group.)
<3> The method for producing a glycopeptide or glycoprotein according to <1> or <2>, wherein the sugar chain receptor is an IgG polypeptide in which an N-acetylglucosaminyl group is bound to a constant region.
<4> In the general formula (1), at least one of X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ is a saccharide-derived group, and the saccharide-derived group is an oligo <1> to << which is a group having a structure in which a sugar residue and any one group selected from an azide group, an alkynyl group, an epoxy group, an amino group, a thiol group, and an isocyanate group are bonded via a linker 3> The method for producing a glycopeptide or glycoprotein according to any one of 3>.
<5> In the general formula (1), Z ¹ , X ³ , X ⁴ , X ⁵ and X ⁶ are hydrogen atoms, X ¹ is α1-6 and Man bound to Man is β1-2 and GlcNAcA is GlcNAcA a group derived from a carbohydrate binding to have a group derived from ^{carbohydrates X 2} are bonded GlcNAcA with β1-2 to Man bound to Man in [alpha] 1-3 <1> ~ the <4> It is a manufacturing method of the glycopeptide or glycoprotein as described in any one.
<6> The immunoglobulin G is produced by carrying out the above reaction at a molar ratio of the sugar chain donor to the sugar chain acceptor (number of moles of sugar chain donor / number of moles of sugar chain acceptor) of 5 or more. It is a manufacturing method of the glycopeptide or glycoprotein as described in any one of>-<4>.
<7> The method for producing a glycopeptide or glycoprotein according to <6>, wherein the molar ratio is 1 to 4.

  According to the present disclosure, it is possible to provide a production method capable of easily producing a glycoprotein or glycopeptide having a specific complex-type sugar chain at a high content using a stable sugar chain donor.

FIG. 1 is a view of CE measurement after reducing a sample at each treatment time with endo M of mogamulizumab (IgG). FIG. 2 is a view of CE measurement without reduction treatment of a sample at each treatment time by endo M of mogamulizumab (IgG). FIG. 3 is a graph showing a CE measurement after a reduction treatment of a sample after a reaction (sugar chain transfer, sugar chain donor; SGP) to mogamulizumab (GlcNAc-IgG) after endo M treatment. In the figure, the results according to the reaction time from 0 to 24 hours are shown. FIG. 4 is a view of CE measurement after reduction treatment of a sample after a reaction (glycosyl transfer, sugar chain donor; SGP) to mogamulizumab (GlcNAc-IgG) after endo M treatment. In the figure, the results according to the reaction time from 45 hours to 70 hours are shown. The lowermost figure shows the result of measuring the sample after purification of GlcNAc-IgG from the reaction solution with protein A-Sepharose after 70 hours. FIG. 5 shows the results of SDS-PAGE of the sample after the reaction (sugar chain transfer, sugar chain donor; SGP) to mogamulizumab (GlcNAc-IgG) after endo M treatment. The results according to the reaction time from 0 to 70 hours are shown at the top of each lane in the figure. FIG. 6 shows a lectin blot with SSA lectin for Mogamulizumab (GlcNAc-IgG) before and after End M treatment, and for a sample after 70 hours reaction (glycosyl transfer, sugar chain donor; SGP) to Mogamulizumab. The results are shown. M is a marker, lane 1 is a sample before endo-M treatment, lane 2 is a sample after endo-M treatment, and lane 3 is a measurement result for a sample after sugar chain transfer. FIG. 7 is a graph of CE measurement after reduction treatment of a sample after reaction (sugar chain transfer, sugar chain donor; compound A-1) to mogamulizumab (GlcNAc-IgG) after endo M treatment; a) is a sample after 0 hour reaction, and (b) is a sample after 44 hours. FIG. 8 is a diagram of MALDI-TOF MS measurement after reduction treatment of a sample after reaction (sugar chain transfer, sugar chain donor; compound A-1) to mogamulizumab (GlcNAc-IgG) after End M treatment. Yes, (a) is a sample after 0 hour reaction, and (b) is a sample after 44 hours. FIG. 9 shows that SDS-PAGE was performed on a sample before and after treatment with Endo M, and after the reaction with Mogamulizumab after treatment (glycosylation, 44 hours, sugar chain donor; Compound A-1). It is a result. M is a marker, lane 1 is mogamulizumab before endo-M treatment, lane 2 is mogamulizumab after endo-M treatment, lane 3 is a result obtained by reacting compound A-1 which is a sugar chain donor with mogamulizumab It is. FIG. 10 shows the results of high-performance liquid chromatography (HPLC) analysis of a sample after 0 hour reaction after reaction to GlcNAc1-β-O-ethylazide (glycosyl transfer, sugar chain donor; compound 110). FIG. 11 shows the result of high-performance liquid chromatography (HPLC) analysis of a sample after reaction for 1 hour after reaction to GlcNAc1-β-O-ethylazide (glycosyl transfer, sugar chain donor; compound 110).

Terms used throughout this specification and claims will be explained.
“˜” representing a numerical range represents a range including upper and lower numerical values. Furthermore, in this specification, unless otherwise specified, the amount of each component such as sugar chain donor, sugar chain acceptor, IgG, and mutant enzyme is the total amount of a plurality of substances present in the reaction solution. means.

“Homology” is defined as the percentage of residues that are identical after aligning the sequence, introducing gaps, if necessary, to achieve maximum homology (percent) in amino acid sequence variants. Is done. Methods and computer programs for alignment are well known in the art, such as “Align 2”, authored by Gentec Corporation, published on 10 December 1991 in the United States Copyright Office, washington, DC 20559. Submitted with user documentation.
In addition, amino acid residues are represented by either a three letter code or a single letter code.

  “Transglycosylation” is to bind a part of the sugar chain of the sugar chain donor to a sugar chain acceptor. In the present disclosure, the left-side part (hereinafter sometimes referred to as a sugar chain donating part) after hydrolysis at the position of the arrow in the following general formula (1) is transferred to a sugar chain acceptor. Means.

In the general formula (1), X ¹ and X ² each represent an independent saccharide-derived group or a hydrogen atom, and at least one of X ¹ and X ² contains one or more GlcNAc or GlcNAcA, and X ¹ and GlcNAc or GlcNAcA contained in X ² is linked in β1-2 to Man bound to Man in Man or α1-6 bound to Man in [alpha] 1-3. X ³ , X ⁴ , X ⁵ and X ⁶ each independently represent a hydrogen atom or a saccharide-derived group. Z ¹ represents a hydrogen atom or GlcNAc, and GlcNAc contained in Z ¹ is bonded to Man bound to GlcNAc at β1-4 at β1-4. Y ¹ represents a monovalent substituent. GlcNAc represents an N-acetylglucosaminyl group, and GlcNAcA represents a sugar residue in which the hydroxymethyl group at position 6 of GlcNAc is a carboxyl group. β1-4 represents a β-glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc, or β-glycoside bond between position 1 of Man and position 4 of GlcNAc. Man represents a mannosyl group, α1-6 represents an α-glycoside bond between the 1-position of Man and the 6-position of Man, and α1-3 represents an α-glycoside bond between the 1-position of Man and the 3-position of Man.
In the present disclosure, GlcNAc bonded to Y ¹ does not include a substituent atom other than a hydrogen atom bonded to the carbon at the 1-position reducing end of GlcNAc.

  Further, “6SO3” and “3SO3” described later represent an acidic group bonded to a specific position of a sugar residue. For example, (6SO3) GlcNAc indicates that a sulfonic acid group is bonded to position 6 of GlcNAc. The sugar residue refers to a monosaccharide, which will be described later, forms a substituent group by dehydration condensation or the like, and is a generic term for a mannosyl group (Man), a glucosaminyl group (GlcNAc), and the like. In addition, sugar residues containing acidic groups as described above, and acidic sugar residues such as GlcA, GlcNAcA, NeuAc, and NeuGc may be collectively referred to as acidic sugar residues.

  In the present specification, GlcA, GlcNAcA, NeuAc, and NeuGc mean that the carboxyl group in these sugar residues includes those in which an ester bond or an amide bond is formed with other compounds. For example, when GlcA is an esterified product in which the carboxyl group at the 6-position is ester-bonded, the esterified product includes up to the carbonyl group at the 6-position of GlcA as shown in the broken line part of the following formula (a). It means containing sugar residues. In addition, when GlcA is an amidated product in which the carboxyl group at the 6-position is an amide bond, as shown in the broken line part of the following formula (b), the carbonyl group at the 6-position of GlcA is removed. It means containing sugar residues.

  In formula (1), * indicates the position of bonding with the compound that forms an ester bond and an amide bond.

  In the present specification, the sugar residue in the sugar chain donor means that the hydroxyl group in the sugar residue includes an ether bond with another compound or the like. For example, when Man is an etherified product in which the hydroxyl group at the 2-position is ether-bonded, it means that the ether residue contains a sugar residue containing up to the 2-position oxygen atom that is ether-bonded.

  The saccharide-derived group means not only a natural or non-natural sugar chain but also a complex in which the sugar chain and a compound other than the sugar chain are bound.

In addition, the sugar chain transfer reaction may be simply referred to as “reaction”. In addition, the activity of the endo M mutant or endo M mutant homologue in sugar chain transfer may be simply referred to as sugar chain transfer activity. The sugar chain transfer activity refers to the ability of endoenzyme mutants and endoenzyme mutant homologues to transfer sugar chains, and specifically, the amount of product produced by sugar chain transfer in a certain time (mol / min). It is represented by In the transglycosylation reaction, the enzymatic activity in hydrolysis and the enzymatic activity in transglycosylation cannot be measured separately, but the endo-M mutant used in the present disclosure is compared with the transglycosylation activity. Since the hydrolysis activity is remarkably suppressed, the transglycosylation activity can be estimated by measuring the amount of the product with respect to the time generated after the transglycosylation.
“Transglycosylation yield” refers to the amount of glycoprotein to which a complex type glycan generated after the reaction is transferred, unless otherwise specified. “Transglycosylation yield” refers to the molarity of the product of glycan transfer. The ratio of the number to the number of moles of sugar chain receptor used in the reaction is shown.

  “Endo-β-N acetylglucosaminidase” means an enzyme that hydrolyzes between GlcNAc and GlcNAc as indicated by an arrow in the general formula (1), and is sometimes referred to as “endo enzyme”. In addition, “End M” is an endoenzyme derived from hair mold Mucor Himaris (GenBank Accession No. BAB43869), and “End A” is an endoenzyme derived from Arthrobacter protoformier (GenBank Accession No. AAD10851). ), “End S” means Streptococcus pyogenes-derived endo enzyme (GenBank Accession No. AAK00850). In addition, as described below, an endoenzyme in which an amino acid is substituted is referred to as “endoenzyme mutant”.

  Regarding the notation indicating a specific amino acid residue in the amino acid sequence of the protein, for example, in the amino acid sequence of endo M, the 175th asparagine residue is indicated as N175. In addition, regarding the notation of an amino acid residue substituted, that is, an amino acid residue substituted with another amino acid, for example, a glutamine residue in which the 175th asparagine residue of End M is substituted with a glutamine residue is represented as N175Q, Endo M containing this is referred to as Endo M N175Q variant, or simply N175Q variant. In addition, the N175Q mutant and the N175A mutant of End M may be collectively referred to as End M mutant. In SEQ ID NO: 1, those having a homology of 80% or more (relative to SEQ ID NO: 1) introduced with mutations other than the 175th amino acid residue are referred to as endo M mutant homologs.

“Immunoglobulin G” refers to IgG among IgM, IgA, IgD, IgE, and IgG which are antibodies. In this specification, it may be called IgG. Moreover, when it describes with immunoglobulin G or IgG, it means that it is a polypeptide containing both the two heavy chains and two light chains which IgG has. Further, as will be described later, when it is simply expressed as immunoglobulin G or IgG, its sugar chain structure is not limited unless otherwise specified. That is, it means a mixture of IgG containing various types of N-linked sugar chains.
The “constant region” means a C-terminal region of a heavy chain possessed by IgG and may be referred to as an Fc region. The Fc region in the present disclosure may be an Fc region having a natural sequence or a mutated Fc region. Although the boundary between the Fc region and the non-Fc region may change, in human IgG, it is defined as the region from the position of 226th cysteine (C226) or 230th proline (P230) to the C-terminus.

  As used herein, carbohydrate moieties are described with reference to nomenclature commonly used to describe oligosaccharides. These nomenclatures are found, for example, in Hubbard et al. [Ann. Rev. Biochem., 50, 555 (1981)]. According to this method, Mannose is represented by Man, 2-N-acetylglucosamine is represented by GlcNAc, galactose is Gal, GlcA is glucuronic acid, fucose is Fuc, and glucose is Glc. Sialic acid is represented by the abbreviated notation NeuAc for 5-N-acetylneuraminic acid and NeuGc for 5-glycolylneuraminic acid. Further, the N-acetylglucosaminyl group may be referred to as a GlcNAc residue, and the mannosyl group may be referred to as a Man residue. A sugar residue obtained by oxidizing the 6-position hydroxymethyl group of GlcNAc into a carboxyl group is referred to as GlcNAcA.

“Monosaccharide” means a compound that forms one sugar, for example, a compound in which sugars such as Gal, GlcNAc, and Glc are not bound to each other.
“Glycosidic bond” refers to a bond in which the hydroxyl group at the 1-position of a sugar or monosaccharide in a sugar chain and the hydroxyl group of another sugar are dehydrated and condensing with each other via an oxygen atom. A glycosidic bond refers to a glycosidic bond in which the 1-position of a saccharide and the 6-position of another saccharide are linked in an α-type. In addition, the hydroxyl group at the 1-position of the sugar has α type and β type.
In addition, as an expression for explaining a sugar chain, a sugar chain in which three monosaccharides are bonded may be referred to as three sugars, and in a case where five sugar chains are bonded, they may be referred to as five sugars.
A plurality of monosaccharides having glycosidic bonds, that is, sugar residues connected by glycosidic bonds may be referred to as oligosaccharide residues. In addition, “oligosaccharide residue” means that a monosaccharide includes a sugar residue such as GlcNAcβ1-2, which forms a sugar residue.

  The N-linked sugar chain bound to IgG described in this specification and the like may be a human type as shown in, for example, Kabat et al. [“Sequence of Proteins of Immunological Interest”, (1999)]. For IgG1, IgG2, IgG3 and IgG4, and mouse type IgG1, IgG2a, IgG2b and IgG3, it means that an N-linked sugar chain binds to the 297th asparagine residue in these amino acid sequences.

The “complex sugar chain” is a sugar chain having a specific sugar chain structure among N-linked sugar chains, having a structure derived from at least a core sugar chain, and a non-reducing end of a trimannosyl group Refers to a sugar chain having at least one β1-2 glycosidic linked GlcNAc residue.
The “core sugar chain” refers to a sugar chain moiety represented by C-1 below in the N-linked sugar chain, and “trimannosyl” refers to three mannose moieties in the core sugar chain. Say. As shown in the broken line part of C-1, among the trimannosyl sites, Man that binds with α1-6 on the nonreducing end side is Man2, Man2 that binds α1-3 on the nonreducing end side is Man3 and the reducing end The Man on the side is called Man1.

“GlcNAc-IgG” refers to an IgG polypeptide in which one GlcNAc is bound to at least one constant region. That is, it refers to an IgG polypeptide in which only GlcNAc at the reducing end of at least one core sugar chain is bound to the constant region of the IgG polypeptide among the two N-linked sugar chains of IgG. As for IgG, the non-reducing end side of the N-linked sugar chain [left side of the arrow in the general formula (1)] is referred to as GlcNAc-IgG. Similarly, for glycopeptides and glycoproteins, It is called GlcNAc-peptide and GlcNAc-protein.

“Peptide” and “protein” are generally referred to as peptides having a small number of amino acid residues, and proteins having a large number of amino acid residues, and there is no clear difference in the number of amino acid residues. Those having 50 residues or more are called proteins. A protein may also be referred to as a polypeptide.
“1 unit” refers to a non-reducing end sugar chain portion of a sialoglycopeptide (hereinafter referred to as SGP), which is a complex N-linked sugar chain, as paranitrophenyl GlcNAc (GlcNAcβ1-O-pNP). The amount is 1 μmole transition in 1 minute.

In the present disclosure, in the preparation of enzymatic glycoprotein, a stable natural-type substrate can be used, and a glycopeptide or glycoprotein having a transglycosylation yield and a transglycosylation yield that could not be achieved by the prior art Can be manufactured. The reason for having such an effect in the present disclosure is not clear, but the applicant considers as follows.
The endo M mutant used in the present disclosure is a mutant that has a significantly low hydrolysis activity while maintaining the transglycosylation activity. The endo M mutant is a mutant prepared in consideration of the reaction process of endo M.

  Endo M, when hydrolyzing a chitobiosyl site (GlcNAcβ1-4GlcNAc), which is a natural substrate, first generates an oxazolinium ion intermediate of GlcNAc, which is assumed to be an intermediate generated during hydrolysis. Next, before water is added to the oxazolinium ion intermediate, a sugar chain transfer reaction is performed by adding a hydroxyl group of GlcNAc different from the intermediate. For this reason, by combining a compound obtained by oxazolinizing GlcNAc, which is a pseudo substance of the oxazolinium ion intermediate (hereinafter referred to as an oxazoline derivative), with the above-mentioned mutant, GlcNAc-peptide or GlcNAc-glycoprotein can be obtained. It can be presumed that effective sugar chain transfer occurs.

Conventionally, an oxazoline derivative is effective as a substrate for an endo M mutant, and a substrate having a natural type chitobiosyl structure has not been used. However, it is considered that the endo M mutant also has the ability to hydrolyze a natural type substrate (substrate having a chitobiosyl structure) to transfer sugar chains, that is, to form an oxazolinium ion intermediate. Furthermore, when the intermediate is formed, the presence of an appropriate sugar chain acceptor results in a characteristic of the endo M mutant, that is, the rate of sugar chain transfer is greater than the rate of hydrolysis. It is considered that the reaction takes place preferentially and the product after sugar chain transfer can be obtained in a sufficient yield.
In particular, for glycosylation receptors such as GlcNAc-IgG where GlcNAc is located inside the Fc region and where endoenzymes are unlikely to be accessible from the outside, a long time is required to obtain a sufficient transglycosylation yield. In this case, it is considered that the presence of a stable sugar chain donor in the reaction solution, such as a natural substrate, is effective.

≪Glycopeptide or glycoprotein production process≫
The production method for producing a glycopeptide or glycoprotein according to the present disclosure includes a mutant endo-β-N-acetylglucosaminidase having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine Or deletion, addition or substitution of one or a plurality of amino acid residues other than the 175th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 has a homology of 80% or more with respect to the amino acid sequence By reacting a sugar chain donor represented by the general formula (1) with a sugar chain acceptor in the presence of a mutant endo-β-N-acetylglucosaminidase having an amino acid sequence modified within the range. A step of producing a glycopeptide or glycoprotein.
Details will be described below.

[Glycopeptide or glycoprotein]
The glycopeptide or glycoprotein produced in the present disclosure includes those found in nature and those synthesized. In addition, it is known that the second amino acid residue from the asparagine is always a threonine residue or a serine residue on the C-terminal side of the asparagine to which the N-linked sugar chain binds in a naturally occurring glycoprotein. However, the same applies to the present disclosure.

The glycopeptide or glycoprotein produced by the production method of the present disclosure is not particularly limited. For example, in the case of animal cells, examples include glycoproteins and glycopeptides that are secreted extracellularly or on the cell surface and have an N-linked sugar chain. Examples include various hormones, cell adhesion factors, various receptors, and cells. Examples include an outer matrix, various enzymes, various cytokines, antibacterial or antiviral peptides, and antibodies.
Among these, from the viewpoint of sugar transfer yield, antibacterial or antiviral peptides and bioactive peptides, hormones, cytokines or antibodies are preferred.

[Immunoglobulin G]
The immunoglobulin G (IgG) in the present disclosure means a molecule including at least a constant region (hereinafter referred to as Fc region) and a variable region (hereinafter referred to as Fab region). Not only IgG but also IgG variants and fusion proteins containing these are included. Specifically, natural IgG such as human IgG and mouse IgG such as mouse IgG, and derivatives thereof, for example, chimeric IgG, recombinant IgG capable of inducing antigen-antibody reaction of humanized IgG, etc. included. These may be either monoclonal IgG or polyclonal IgG, and include single or a combination of two or more. The derivative of the natural IgG means that IgG having a mutation such as deletion, addition, substitution, etc. introduced into a part of the amino acid sequence of the natural IgG is included, and these mutations occurred naturally. Not only things but also artificial things are included.

Presence of IgG in the solution is confirmed, for example, by measuring with a capillary electrophoresis (CE) apparatus, an SDS-polyacrylamide electrophoresis (SDS-PAGE) apparatus, or a MALDI-TOF MS.
The measurement by CE can be performed using ProteomeLab PA800 manufactured by Beckman Coulter Inc. equipped with a capillary column (30.2 cm × 50 μm, manufactured by Beckman Coulter Inc., paired fused silica) under the following analysis conditions.
・ Analysis conditions: after sample injection, measured at 25 ° C. and 15 kv for 30 minutes ・ Detection wavelength: 200 nm
The measurement by SDS-PAGE was performed using a 15% polyacrylamide gel in a commercially available slab gel apparatus (manufactured by Atto Corporation, e-PAGEEL), and after electrophoresis at 20 mA for 70 minutes, the gel was stained with Coomassie brilliant blue, The resulting gel can be evaluated. As a marker, DynaMarker Multi Color III (manufactured by Biodynamics Laboratory) is used.

Examples of the MALDI-TOF MS include the following method.
That is, a certain amount of acetone was added to the solution after the reaction, the dissolved portion was dried, and then a certain amount of DHBA solution (20 mg / mL 2,5-dihydroxybenzoic acid dissolved in 50% aqueous methanol solution) was added. Dissolve. Thereafter, a part of the dissolved solution is spotted on a plate for MALDI-TOF MS analysis, dried, and measured under the following conditions by an autoflex speed-tko1 reflector system manufactured by Bruker Daltonics, Inc. The mass of the product can be confirmed.
<Condition>
Measurement mode: positive ion mode and reflector mode
・ Measurement voltage: 1.5 kv to 2.5 kv
Measurement molecular weight range: 0 to 3000 (m / z) and 45000 to 60000 (m / z)
・ Total number of times: 1000-8000

(Endoenzyme and mutant endoenzyme)
In the present disclosure, a mutant in which a mutation is introduced into the amino acid of endo M is used. The endoenzyme shown by SEQ ID NO: 1 is an endo βN acetylglucosaminidase (GenBank Accession No. BAB43869) derived from Mucor Himalis.
In one embodiment of the present disclosure, a mutant endo-β-N-acetylglucosaminidase (endo M mutant) having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 is glutamine or alanine, Alternatively, it is a mutant endo-β-N-acetylglucosaminidase having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is glutamine or alanine, and the amino acid sequence shown in SEQ ID NO: 1 It has an amino acid sequence modified within a range of homology of 80% or more to the amino acid sequence by deletion, addition and / or substitution of one or more amino acid residues other than the 175th amino acid residue In the presence of mutant endo-β-N-acetylglucosaminidase (endo M mutant homolog) By performing the response, producing a glycopeptide or glycoprotein.
In the present disclosure, the endo M mutant is N175Q or N175A, so that hydrolysis of the product after the transglycosylation reaction can be sufficiently suppressed. In addition, from the viewpoint of sugar chain transfer yield and sugar chain transfer yield, N175Q mutant is preferable.

  The endo M mutant of the present disclosure can be prepared by a normal genetic engineering technique, and can be prepared using various host types and corresponding appropriate protein expression vectors. Examples of the host include Escherichia coli, Brevibacillus, cyanobacteria, lactic acid bacteria, yeast, insect cells and animal cells. Among these, from the viewpoint of ease of preparation and expression level, it is preferable to prepare by a method using E. coli as a host or a method using yeast as a host. The specific production method is described in detail in the literature by Umekawa et al. [J. Biol. Chem. Vol. 285 (1), 511-521, (2010)] for Escherichia coli. This is described in detail in Japanese Utility Model Publication No. 5959587.

  The disclosed endo M mutant and endo M mutant homolog are fused endoenzyme mutants or fused endoenzyme mutants fused with other peptides or proteins on the C-terminal side or N-terminal side in the reaction. It can also be used as a homolog. The peptide or protein that can be fused is not particularly limited as long as it does not inhibit the transglycosylation reaction. For example, hexahistidine peptide (amino acid sequence is from N-terminal to HHHHHH), flag peptide (amino acid sequence is N-terminal) To DYKDDDDK), influenza HA polypeptide (amino acid sequence is YPYDVPDYA from the N-terminus), glutathione-S-transferase, luciferase, avidin, chitin-binding protein, c-myc, thioredoxin, disulfide isomerase (DsbA), maltose-binding protein ( Green fluorescent protein (GFP) such as MBP). Among these, hexahistidine peptides and flag peptides are particularly preferable from the viewpoint of ease of preparation of endo M mutant or endo M mutant homolog.

  In the above-mentioned fusion-type endo M mutant or fusion-type endo M mutant homolog, there is a gap between the polypeptide part of the peptide or protein to be fused and the polypeptide part of the endo-M mutant or endo-M mutant homolog. Includes a peptide portion as a linker peptide region of several to 50 residues. In addition, it does not specifically limit as a kind of amino acid residue which comprises a linker peptide area | region. The linker peptide region may include an amino acid sequence portion (protease site) that is hydrolyzed by a protease. Although it does not specifically limit as a protease site, For example, a factor Xa site, a thrombin site, an enterokinase site, a precision protease site is mentioned.

As shown in the publicly known information (http://www.cazy.org/), saccharide hydrolase found in nature has a family of hundreds of glycosylhydrases from the homology of amino acid sequences. (GH family). Endo M is a glycosyl hydrolase belonging to the GH family 85, and other proteins belonging to the GH family 85 are known to be widely distributed from humans to bacteria. When a homology search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) that can be generally used is performed based on the amino acid sequence of End M (SEQ ID NO: 1), End M and other GH families 85 It can be seen that the protein belonging to the group has high homology within the region including the catalytic region from the amino terminal side to the vicinity of the 500th amino acid.
From the above viewpoint, if the endoenzyme mutant homologue in the present disclosure has an amino acid sequence length of about 500 residues including the catalytic region of endoM, it has transglycosylation activity as endoM. Can be estimated.
The endo M mutant homologue in the present disclosure is any amino acid other than the 175th amino acid residue as long as it has 80% homology with the N175Q mutant or N175A mutant and has transglycosylation activity. Those prepared by substituting, deleting and adding residues to other amino acids may also be used.

  From the above viewpoint, in the present disclosure, an endoenzyme mutant homolog resulting from deletion, addition and / or substitution of one or more amino acid residues other than the 175th amino acid residue of endo M (SEQ ID NO: 1) As long as the homology to the endo M (SEQ ID NO: 1) is 80% or more, the transglycosylation activity can be sufficiently maintained. Further, if it is 90% or more, it is preferable because the sugar chain transfer yield and the sugar chain transfer yield are easily improved, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99%. That's it.

  In the present disclosure, the concentration of the endo M mutant or endo M mutant homolog in the reaction solution is preferably 0.5 U to 20 U / ml. By being 0.5 U or more, the sugar chain transfer yield can be increased, and by being 20 U or less, the sugar chain transfer yield can be increased. Here, 1 unit (U) indicates the amount of enzyme that transfers 1 μmol of sialoglycopeptide (hereinafter referred to as SGP) to β-paranitrophenyl GlcNAc (GlcNAcβ1-O-pNP) in 1 minute. Furthermore, as a density | concentration, it is more preferable that it is 1U-10U / ml, and it is especially preferable that it is 2U-5U / ml.

  In addition, regarding the degree of purification of the endo M mutant or endo M mutant homolog used in the reaction, the results of SDS-polyacrylamide electrophoresis, etc. from the viewpoints of shortening the reaction time, sugar chain transfer yield and sugar chain transfer yield, etc. Therefore, the degree of purification is preferably 50% or more, more preferably 70% or more, particularly preferably 80% or more, and most preferably 90% or more. When the degree of purification is 50% or more, the reaction time can be shortened and the sugar chain transfer yield is easily increased.

(Sugar chain donor)
The sugar chain donor in the present disclosure is not particularly limited as long as it is a sugar chain donor represented by the following general formula (1). Since it has a structure including a β1-4 bond between GlcNAc and GlcNAc (chitobiosyl structure), it is stable as a substrate and can exist stably in a solution for a long time, so that it can be applied to a sugar chain receptor that requires a long reaction time. It can respond enough.

In the general formula (1), X ¹ and X ² each represent an independent saccharide-derived group or a hydrogen atom, and at least one of X ¹ and X ² contains one or more GlcNAc or GlcNAcA, and X ¹ and GlcNAc or GlcNAcA contained in X ² is linked in β1-2 to Man bound to Man in Man or α1-6 bound to Man in [alpha] 1-3. X ³ , X ⁴ , X ⁵ and X ⁶ each independently represent a hydrogen atom or a saccharide-derived group. Z ¹ represents a hydrogen atom or GlcNAc, and GlcNAc contained in Z ¹ is bonded to Man bound to GlcNAc at β1-4 at β1-4. Y ¹ represents a monovalent substituent. GlcNAc represents an N-acetylglucosaminyl group, and GlcNAcA represents a sugar residue in which the hydroxymethyl group at position 6 of GlcNAc is a carboxyl group. β1-4 represents a β-glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc, or β-glycoside bond between position 1 of Man and position 4 of GlcNAc. Man represents a mannosyl group, α1-6 represents an α-glycoside bond between the 1-position of Man and the 6-position of Man, and α1-3 represents an α-glycoside bond between the 1-position of Man and the 3-position of Man.
X ¹ and X ² have at least one GlcNAc, which means that the GlcNAc has at least a residue that is glycoside-bonded at β1-2 to either Man2 or Man3 of the core sugar chain moiety.

The carbohydrate-derived groups in X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ include GlcNAcβ1-2, Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2-3Galβ1-4GlcNAc 2, NeuGcα2-6Galβ1-4GlcNAcβ1-2 and NeuGcα2-3Galβ1-4GlcNAcβ1-2, heterologous antigen: Galα1-3Galβ1-4GlcNAcβ1-2, polylactosamine: [Galβ1-4GlcNAcβ1-3] nGalc1-4GlcNA Number), keratan sulfate [Galβ1-4GlcNAc (6SO3) β1-3] nGalβ1-4GlcNAcβ1-2 (n is an arbitrary number), [Gal (6SO3) β1-4GlcNAc (6SO3 β1-3] nGalβ1-4GlcNAcβ1-2 (n is an arbitrary number), LacDiNAc: GalNAcβ1-4GlcNAcβ1-2, sulfated LacDiNAc: GalNAcβ1-4GlcNAcβ1-2, terminal sulfate modified (3SO3) Galβ1-4GlcNAcβ1-2 : Galβ1-4 (Fucα1-3) GlcNAcβ1-2, Fucα1-2Galβ1-4 (Fucα1-3) GlcNAcβ1-2, Galβ1-3 (Fucα1-4) GlcNAcβ1-2, Fucα1-2Galβ1-3 (Fucα1β) GlcNAc1β -2, blood group antigens: Fucα1-2Galβ1-4GlcNAcβ1-2, Galα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1-2, GalNAcα1-3 (Fucα1-2) Galβ1- GlcNAcbeta1-2, HNK1 antigen: (3SO3) GlcAβ1-3Galβ1-4GlcNAcβ1-2, GlcAβ1-2, GlcNAcAβ1-2 the like, either any one of ^{X 1} and X ² of these groups, or both You may have.
When X ^{1 to} X ⁶ are as described above, Z ¹ may be a hydrogen atom or GlcNAcβ1-4.

Among the above-mentioned saccharide-derived groups, X ³ , X ⁴ , X ⁵ and X ⁶ from the viewpoint of ADCC activity and transglycosylation yield of IgG produced by the method for producing a glycopeptide or glycoprotein of the present disclosure. Is preferably a hydrogen atom.
In this case, both or one of ^{X 1} and X ^2, GlcNAcβ1-2, Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, a one of NeuAcarufa2-3Galbeta1-4GlcNAcbeta1-2, and Z ¹ is a hydrogen atom, or one or both of X ¹ and X ² is GlcNAcβ1-2, Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2-3Galβ1-4GlcNAcβ1-2 And Z ¹ is preferably GlcNAc. Still, both of ^{X 1} and X ² are GlcNAcβ1-2, Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, a one of NeuAcarufa2-3Galbeta1-4GlcNAcbeta1-2, and ^{Z 1} is hydrogen Or both X ¹ and X ² are any one of GlcNAcβ1-2, Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2-3Galβ1-4GlcNAcβ1-2, and Z ¹ Is more preferably GlcNAc.
Both X ¹ and X ² are Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2-3Galβ1-4GlcNAcβ1-2, and Z ¹ is a hydrogen atom, Or X ¹ and X ² are both Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2-3Galβ1-4GlcNAcβ1-2, and more preferably Z ¹ is GlcNAc. , X ¹ and X ² are either one of NeuAcα2-6Galβ1-4GlcNAcβ1-2 or Galβ1-4GlcNAcβ1-2, and Z ¹ is a hydrogen atom, or both X ¹ and X ² are , NeuAcα2 A one of 6Galβ1-4GlcNAcβ1-2 or Galbeta1-4GlcNAcbeta1-2, and particularly preferred that ^{Z 1} is GlcNAc, both ^{X 1} and X ² are, NeuAcarufa2-6Galbeta1-4GlcNAcbeta1-2 or Galβ1- Most preferably, it is any one of 4GlcNAcβ1-2, and Z ¹ is GlcNAc.

In addition, at least one of X ^{1 to} X ^{6 in} the general formula (1) is a saccharide-derived group, and the saccharide-derived group includes an oligosaccharide residue, an azide group, an alkynyl group, and an epoxy. It may be a group having a structure in which any one group selected from a group, an amino group, a thiol group and an isocyanate group is bonded via a linker.
Here, an azide group, an alkynyl group, an epoxy group, an amino group, a thiol group, and an isocyanate group may be referred to as a click reaction group. That is, the saccharide-derived group in the sugar chain donor may be bonded to the oligosaccharide residue constituting the saccharide-derived group and the click reaction group via a linker.
The sugar chain donor that is represented by the general formula (1) and to which the linker is not bonded is referred to as a linker-free sugar chain donor, and is represented by the general formula (1), and at least ^one of X ^{1 to} X ⁶ . One is a sugar-derived group, and the sugar-derived group is a sugar chain donor having a structure in which an oligosaccharide residue and a click reaction group are bonded via the linker. Sometimes called body. In addition, the click reaction group, the linker, and the oligosaccharide residue may be referred to as each block. Furthermore, the linker refers to a portion other than the click reactive group among those formed by bonding a compound used for the linker described later and a compound containing a click reactive group described later.

  It does not specifically limit in the coupling | bonding ratio of the said click reactive group and a linker, and arrangement | positioning of the click reactive group with respect to a linker. However, in terms of ease of preparation as a sugar chain donor, the bond ratio is preferably 1 or more, more preferably 1 to 10, and most preferably 1. Further, in the arrangement of the click reactive group with respect to the linker, it is preferably arranged at the end of the linker excluding the position where the oligosaccharide residue and the linker are bonded.

  The number of click reactive groups in the click reactive group-containing sugar chain donor is not particularly limited, but is preferably 1 or more from the viewpoint of ease of preparation and sugar chain transfer yield. It is preferable that it is, and it is especially preferable that it is 1-2.

  Examples of oligosaccharide residues bound to the linker include GlcNAcβ1-2, Galβ1-4GlcNAcβ1-2, NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2-3Galβ1-4GlcNAcβ1-2, NeuGcα2-6Galβ1-4GlcNc1-6Gc 3Galβ1-4GlcNAcβ1-2, heterologous antigen: Galα1-3Galβ1-4GlcNAcβ1-2, polylactosamine: [Galβ1-4GlcNAcβ1-3] nGalβ1-4GlcNAcβ1-2 (n is an arbitrary number), keratan sulfate [Galβ1-4GlcNAc (6) β1-3] nGalβ1-4GlcNAcβ1-2 (n is an arbitrary number), [(6SO3) Galβ1-4GlcNAc (6SO3) β1-3] nGalβ 1-4GlcNAcβ1-2 (n is an arbitrary number), LacDiNAc: GalNAcβ1-4GlcNAcβ1-2, sulfated LacDiNAc: GalNAcβ1-4GlcNAcβ1-2, (3SO3) Galβ1-4GlcNAcβ1-2, Lewis sugar chain: Galβ1-4 (u 3) GlcNAcβ1-2, Fucα1-2Galβ1-4 (Fucα1-3) GlcNAcβ1-2, Galβ1-3 (Fucα1-4) GlcNAcβ1-2, Fucα1-2Galβ1-3 (Fucα1-4) GlcNAcβ1-2, blood group antigens: Fucα1-2Galβ1-4GlcNAcβ1-2, Galα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1-2, GalNAcα1-3 (Fucα1-2) Galβ1-4GlcNAcβ1-2, HNK1 Original: (3SO3) GlcAβ1-3Galβ1-4GlcNAcβ1-2, GlcAβ1-2, GlcNAcAβ1-2 and the like.

Moreover, from the viewpoint of ease of preparation of the click-reactive group-containing sugar chain donor and sugar chain transfer yield, X ³ , X ⁴ , X ⁵ , X ⁶ and Z ^{1 in the} general formula (1) are It is preferably a hydrogen atom, and both or one of X ¹ and X ² contains at least one acidic sugar residue selected from NeuAc, NeuGc, GlcA and GlcNAcA.
Further, in the general formula (1), X ³ , X ⁴ , X ⁵ , X ⁶ and Z ¹ are hydrogen atoms, and both or one of X ¹ and X ² is NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2- 3Galβ1-4GlcNAcβ1-2, NeuGcα2-6Galβ1-4GlcNAcβ1-2, NeuGcα2-3Galβ1-4GlcNAcβ1-2, GlcAβ1-2 and GlcNAcAβ1-2 are more preferred. Further, X ³ , X ⁴ , X ⁵ , X ⁶ and Z ^{1 in the} general formula (1) are hydrogen atoms, and both or one of X ¹ and X ² is NeuAcα2-6Galβ1-4GlcNAcβ1-2, NeuAcα2 -3Galβ1-4GlcNAcβ1-2, NeuGcα2-6Galβ1-4GlcNAcβ1-2, NeuGcα2-3Galβ1-4GlcNAcβ1-2, GlcAβ1-2 and GlcNAcAβ1-2 are particularly preferred. Furthermore, it is most preferable that X ³ , X ⁴ , X ⁵ , X ⁶ and Z ^{1 in the} general formula (1) are hydrogen atoms, and that both X ¹ and X ² are GlcNAcAβ1-2.

  The click reactive group is preferably any one of an azido group, an alkynyl group, an epoxy group, and an amino group from the viewpoint of stability in aqueous solution and reaction specificity with other compounds. More preferably, it is any group. Further, in the reaction between the click reactive group and another compound, a bond can be easily and specifically formed by selecting an optimal combination.

The linker is not particularly limited as long as it binds both the click reactive group and the oligosaccharide residue and does not impair the effect of the production method of the present disclosure. For example, polyethylene glycol (PEG) Branched PEG, PEG derivative, hydroxyethyl cellulose (HEC), dextrin, polyoxazoline, carbohydrate, polysaccharide, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide ( PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone Polyphosphazenes, polyoxazolines, polyethylene maleic anhydride copolymers, polystyrene maleic anhydride copolymers, poly (1-hydroxymethylethylenehydroxymethyl formal) (PHF) and 2-methacryloyloxy-2′-ethyltrimethylammonium phosphate Examples include structures derived from water-soluble polymers such as (MPC), polyethylene, polypropylene, polyacrylate, polyphenylene, polyacrylonitrile, and the like. Among these, from the viewpoint of ease of preparation as a sugar chain donor and the yield of sugar chain transfer, the linker preferably has a structure derived from a water-soluble polymer, and further derived from polyethylene glycol or polypropylene glycol. More preferably, the structure has
Moreover, it is preferable to use said compound (polymer) for formation of the said linker.

The weight average molecular weight of the linker is not particularly limited, but is preferably from 100 to 10,000 from the viewpoint of ease of preparation as a sugar chain donor and sugar chain transfer yield. Furthermore, 100 to 7000 is more preferable, and 100 to 4000 is particularly preferable.
In addition, the preferred number of repeating units of polyethylene glycol and polypropylene glycol used for the formation of the linker is preferably 1 to 50, from the viewpoint of ease of preparation as a sugar chain donor and sugar chain transfer yield, and preferably 1 to 20 Is more preferable, and 3 to 10 is particularly preferable.

  The construction method of each block in the above-mentioned click reactive group-containing sugar chain donor is, for example, a linker-free sugar chain to a compound obtained by binding a compound containing a click reactive group and a compound used for forming a bond between the linker and the compound. Examples include a method of binding an oligosaccharide residue such as a donor. Moreover, the method of combining the compound used for formation of a linker and the compound containing a click reaction group with the compound obtained by couple | bonding the said oligosaccharide residue is also mentioned. As a method for constructing each block, any method may be used, and it is preferable to select as appropriate.

  The click reaction group in the click-reactive group-containing sugar chain donor and the linker method, and the linker and the oligosaccharide residue such as a linker-free sugar chain donor are combined in a known general synthetic chemistry. For example, a synthesis method described in JP 2010-119320 A, JP 2010-5028240 A and the like.

  In the case where the click reactive group is an azide group and an alkynyl group, the compound containing the click reactive group to be bonded to the linker is not particularly limited and is preferably selected as appropriate. From the viewpoint of the ease of preparation and sugar chain transfer yield, it is preferably a compound represented by the following general formula (3-1) or (3-2).

  A in general formula (3-1) and K in general formula (3-2) each represent a monovalent substituent. The monovalent substituent is not particularly limited as long as it can bind to the oligosaccharide residue. For example, an amino group, a hydroxyl group, a carboxyl group, a bromine group, a chlorine group, a sulfonesan group, a phosphate group, And substituents represented by the structural formula (L-1) or (L-2).

  * Represents the binding position to the terminal of polyethylene glycol in the above general formula (3-1) or (3-2).

  Some of the compounds represented by the general formula (3-1) are commercially available. For example, as a compound containing an azide group and having a functional group introduced, 11-azido-3,6,9-trioxa Undecane-1-amine (manufactured by Tokyo Chemical Industry Co., Ltd.) is known, and 2- [2- (2-propynyloxy) ethoxy] ethylamine and propargyl-PEG5-NHS (Tokyo Kasei) are compounds in which an alkynyl group is introduced. Manufactured by Kogyo Co., Ltd.).

In the preparation of the click reactive group-containing sugar chain donor, the compound used for forming the linker may be used as it is, or a compound in which a functional group is introduced into the compound used for forming the linker may be used. In the case of forming a bond between the linker and the linker-free sugar chain donor, for example, at least one saccharide-derived group of X ^{1 to} X ^{6 in} the general formula (1) is an acidic sugar. If it has a residue, the linker-free sugar chain donor can be used as it is for binding to the linker, which is preferable. On the other hand, when it does not have an acidic sugar residue, a product obtained by introducing a functional group such as an acidic residue into an oligosaccharide residue in a saccharide-derived group can be used. Examples of the functional group include an amino group, a carboxyl group, a thiol group, an isocyanate group, a hydroxyl group, and an epoxy group.

  In addition, the bond species between the oligosaccharide residue of the saccharide-derived group and the compound used for the linker, and the bond species between the compound having a click reaction group and the compound used for the linker may be any, for example, Amide bond, ester bond, ether bond, sulfide bond, and bond via sulfo group.

In the general formula (1), Y ¹ includes a substituent including a structure in which an oxygen atom, a nitrogen atom, a carbon atom, and a sulfur atom are directly bonded to the 1-position carbon of GlcNAc.
Y ¹ is not particularly limited as long as it does not reduce the sugar chain transfer yield and the sugar chain transfer yield. For example, hydroxyl group, alkoxy group, aryloxy group, alkenyloxy group, acyloxy group, alkyl group, alkenyl group, alkynyl group, aralkyl group, alkyl ester group, aryl ester group, amino group, amide group, imide group, azide group A carboxyl group, an alkylsulfinyl group, an arylsulfinyl group, an alkylsulfonyl group, an arylsulfonyl group, an alkylsulfonyloxy group, an arylsulfonyloxy group, or a halogen group. Among these groups, an alkoxy group, an alkyl ester group, The aryl ester group, amide group, alkylsulfinyl group, arylsulfinyl group, alkylsulfonyl group, arylsulfonyl group, alkylsulfonyloxy group and arylsulfonyloxy group further have a substituent. It can have.

Among the above, from the ease of preparation of a sugar chain donor, Y ¹ is a hydroxyl group, an optionally substituted alkoxy group having 1 to 30 carbon atoms, which may have a substituent A C6-C30 aryloxy group, the C1-C30 alkenyloxy group which may have a substituent, and the amide group which may have a substituent are preferable.

Furthermore, from the viewpoint of ease of preparation as a sugar chain donor and a yield of sugar chain transfer, Y ¹ is an alkoxy group optionally having a substituent having 1 to 10 carbon atoms, or having 2 to 10 carbon atoms. An alkenyloxy group which may have a substituent and an aryloxy group which may have a substituent having 6 to 24 carbon atoms are preferred, and further an alkoxy group having 1 to 8 carbon atoms and an alkyl group having 2 to 6 carbon atoms. An alkenyloxy group, an aryloxy group which may have a substituent having 6 to 12 carbon atoms, and an amide group which may have a substituent are particularly preferable.
Specific examples include a methoxy group, an ethoxy group, a phenoxy group, a paramethoxyphenoxy group, and a paranitrophenoxy group.

  In addition, the amide group which may have a substituent is preferably an amide group to which an amino group of the side chain of asparagine of a peptide or protein is bonded, and the solubility in the reaction solution and the sugar chain transfer yield. From the above viewpoint, an amide group to which an amino group of the side chain of the asparagine of the peptide is bonded is more preferable, and an amide group as shown in the following N-1 and N-2 is most preferable.

(N-1 is an asparagine bound to GlcNAc, N-2 is a hexapeptide containing asparagine bound to GlcNAc (the sequence of the peptide portion is Lys-Val-Ala-Asn-Lys-Thr). * Is 1 of GlcNAc Indicates the position of binding to the position.)

  In addition, the substituent which may be substituted includes an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 12 carbon atoms, an aryl group having 6 to 12 carbon atoms, a halogen atom, a cyano group, a nitro group, When it is at least one selected from an amino group, a carboxyl group and a pyridyl group and there are two or more substituents, each substituent may be the same or different.

As a method for preparing the sugar chain as described above, a method of extracting from sugar or a method of chemically preparing it can be considered. Among complex sugar chains, a sugar chain donor having a large number of sugar residues, for example, a sugar chain donor having an oligosaccharide residue of 8 or more sugars is extracted from egg yolk for ease of preparation (for example, international publication). 96/2255 pamphlet and JP 2011-231293) and these methods and exoglycosidase, for example, commercially available sialidase, galactosidase or N-acetyl β-glucosaminidase, to prepare a sugar chain donor relatively easily. be able to.
The sugar chain donor obtained directly by the method of extraction from nature is a peptide in which Y ¹ in the general formula (1) contains asparagine. However, in a sugar chain donor containing an alkoxy group, for ease of preparation, The sugar chain donor obtained by the above-mentioned method of extraction from nature can be obtained by sugar chain transfer to a GlcNAc derivative having an alkoxy group serving as a sugar chain acceptor by the action of the N175Q mutant. A method of preparing by such sugar chain transfer is described in, for example, JP-A-10-45788.

  On the other hand, for example, a sugar chain donor having 7 or less oligosaccharide residues may be prepared by the above-described method or by a chemical synthesis method. As a chemical preparation method, for example, it can be prepared by the method described in Wang et al. [J. Am. Chem. Soc., Vol. 134 (29), 12308 (2012)].

  In addition, as a method for producing IgG using the above sugar chain donor, the above sugar chain donor and GlcNAc-peptide or GlcNAc-protein are reacted in the presence of an endo M mutant or an endo M mutant homolog. Thus, IgG can also be produced. Furthermore, after the glycan transfer, a desired complex type glycan is obtained by combining with an exo-type sugar hydrolase or glycosyltransferase, and removing or adding a sugar residue or oligosaccharide residue on the non-reducing end side. Can also be obtained.

As the click-reactive group-containing sugar chain donor, from the viewpoint of ease of preparation and sugar chain transfer yield, the click-reactive group is either an azide group or an alkynyl group, and the linker is a water-soluble polymer. The click-reactive group-containing sugar chain donor has 1 to 4 click reaction groups, and Y ¹ in the general formula (1) has a hydroxyl group and a substituent. An optionally substituted alkoxy group having 1 to 30 carbon atoms, an aryloxy group having 6 to 30 carbon atoms which may have a substituent, and an alkenyloxy having 1 to 30 carbon atoms which may have a substituent. It is preferably any one selected from the group of an amide group which may have a group and a substituent.
Further, the click reactive group is either an azide group or an alkynyl group, the linker has a structure derived from polyethylene glycol or polypropylene glycol, and the click reactive group in the click reactive group-containing sugar chain donor The number is 1 or 2, and Y ¹ in the general formula (1) has an alkoxy group having 1 to 8 carbon atoms, an alkenyloxy group having 2 to 6 carbon atoms, and a substituent having 6 to 12 carbon atoms. It is more preferably any one selected from the group of an aryloxy group which may be present and an amide group which may have a substituent.
Furthermore, the click reactive group is either an azide group or an alkynyl group, the linker has a structure derived from polyethylene glycol, and the number of the click reactive group in the click reactive group-containing sugar chain donor is one. Or X ³ , X ⁴ , X ⁵ , X ⁶ and Z ^{1 in the} general formula (1) are hydrogen atoms, and both or one of X ¹ and X ² is NeuAc, NeuGc, GlcA and Including at least one acidic sugar residue selected from GlcNAcA, wherein Y ¹ in the general formula (1) is a methoxy group, an ethoxy group, a phenoxy group, a paramethoxyphenoxy group, a paranitrophenoxy group, or a peptide asparagine. Particularly preferred is any one selected from the group of amide groups to which an amino group in the side chain is bonded.
Furthermore, the click reactive group is either an azide group or an alkynyl group, the linker has a structure derived from polyethylene glycol, and the number of the click reactive group in the click reactive group-containing sugar chain donor is one. Or X ³ , X ⁴ , X ⁵ , X ⁶ and Z ^{1 in the} general formula (1) are hydrogen atoms, and both or one of X ¹ and X ² is NeuAcα2-6Galβ1-4GlcNAcβ1-2 , NeuAcα2-3Galβ1-4GlcNAcβ1-2, NeuGcα2-6Galβ1-4GlcNAcβ1-2, NeuGcα2-3Galβ1-4GlcNAcβ1-2, GlcAβ1-2 and GlcNAcAβ1-2, Y ¹ ethoxy group, Phenoxy group, paramethoxyphenoxy group, paranitroph Most preferably, it is any one selected from the group of amide groups to which an amino group in the side chain of an asparagine of the peptide is bonded.

  Specific examples of the click reactive group-containing sugar chain donor include the following, but the production method of the present disclosure is not limited to these.

  In the above general formulas (4-1-1) to (4-4-2), n each independently represents an integer of 1 or more, and preferably 1 to 20.

(Sugar chain receptor)
The sugar chain receptor in the present disclosure is not particularly limited as long as it contains GlcNAc and 1-position of GlcNAc is bound to a peptide or protein, as long as it does not inhibit the sugar chain transfer activity of the endo M mutant.
However, from the viewpoint of ease of preparation as a sugar chain receptor and the yield of sugar chain transfer, a sugar chain receptor represented by the following general formula (2) is preferable.
^{GlcNAc-R 2} Formula (2)
(In general formula (2), R ² represents a peptide or protein containing an asparagine residue. GlcNAc represents an N-acetylglucosaminyl group.)

  Examples of the GlcNAc-peptide and GlcNAc-protein include those obtained by removing the non-reducing terminal site of the N-linked sugar chain of the glycopeptide or glycoprotein described above. It is also known that the second amino acid residue from asparagine is always a threonine residue or a serine residue on the C-terminal side of asparagine to which an N-linked sugar chain in a naturally occurring glycoprotein binds. However, the same applies to the sugar chain receptor in the present disclosure.

  The GlcNAc-peptide or GlcNAc-protein as a sugar chain receptor of the present disclosure can be prepared from the above-mentioned glycopeptide or glycoprotein by hydrolysis treatment of a commercially available endoenzyme or a self-prepared endoenzyme, or asparagine. Glycopeptides can also be prepared by a method of sequentially extending amino acid sites in a sugar amino acid derivative to which GlcNAc is bound (Patent Document, JP-A-10-45788).

  As commercially available endoenzymes, End M (manufactured by Tokyo Chemical Industry Co., Ltd.), End H (manufactured by New England Biolabs), End S (manufactured by Sigma-Aldrich), End D (manufactured by Cosmo Bio), End F1 to F3 (Sigma-Aldrich). Non-commercial enzymes include glycosyl hydrolases belonging to GH family 85, or enzymes that belong to GH family 18 and have been shown to hydrolyze N-linked sugar chains. Can do.

~ GlcNAc-IgG ~
In one embodiment of the present disclosure, IgG having a GlcNAc group bound to a constant region (GlcNAc-IgG) can be used as a sugar chain receptor. Among these, GlcNAc-IgG1 is preferable.
GlcNAc-IgG in the present disclosure is a sugar chain receptor used to produce immunoglobulin G (IgG), and is an immunity in which one N-acetylglucosaminyl group (GlcNAc) is bound to one constant region. Globulin G (IgG). Therefore, there are two types of GlcNAc-IgG, one having two GlcNAc bound to two constant regions, one constant region having one GlcNAc bound thereto, and one having a constant region to which no GlcNAc is bound. In the present disclosure, both IgGs are included.

As a method for preparing IgG used for the preparation of a sugar chain receptor, IgG that is a component of a commercially available antibody drug may be used, or a method prepared by a method using cells into which an antibody gene has been introduced is used. Alternatively, those prepared with yeast may be used.
Further, the content of IgG fucose used for the preparation of the sugar chain receptor is preferably 50% or less, more preferably 30% or less, particularly 10% or less from the viewpoint of ADCC activity. Preferably there is.

As a method for producing GlcNAc-IgG, it can be obtained by treating IgG with an endoenzyme or an exo-type carbohydrate hydrolase as described in WO2013120066. Examples of endoenzymes include commercially available enzymes, End M (manufactured by Tokyo Chemical Industry Co., Ltd.), End H (manufactured by New England Biolabs), End S (manufactured by Sigma-Aldrich), End D (manufactured by Cosmo Bio), End F1-F3 (Sigma-Aldrich) etc. are mentioned. As enzymes that are not commercially available, those prepared from glycosyl hydrolase belonging to GH family 85 and endococcal faecalis-derived endoenzyme belonging to GH family 18 (Genbank AAR20477) may be used. These endoenzymes may be used alone or in combination depending on the type of N-linked sugar chain contained in IgG.
Among the above endoenzymes, endo M is most preferable in that various types of sugar chains possessed by IgG can be hydrolyzed.

  A preferred method for producing the IgG of the present disclosure is a mutant endo-β-N-acetylglucosaminidase having an amino acid sequence in which the amino acid residue at position 175 of the amino acid sequence represented by SEQ ID NO: 1 is glutamine, or SEQ ID NO: 1. The amino acid sequence was modified within a range of homology of 90% or more by deletion, addition and / or substitution of one or more amino acid residues other than the 175th amino acid residue. In the presence of a mutant endo-β-N-acetylglucosaminidase having an amino acid sequence, the molar ratio of the sugar chain donor represented by the general formula (1) and IgG1 which is a sugar chain acceptor (of the sugar chain donor) (Number of moles / number of moles of sugar chain receptor) at 50 or more.

(reaction)
The reaction of the present disclosure is performed in a solution in which the End M mutant or End M mutant homolog, sugar chain donor, and sugar chain acceptor are dissolved. The solution used for the reaction is not particularly limited as long as it does not inhibit the transglycosylation activity of the endo M mutant or the endo M mutant homolog. For example, phosphate buffer, citrate buffer, carbonate buffer, Examples include Tris-HCl buffer, MOPS buffer, HEPES buffer, borate buffer, and tartrate buffer. These buffers may be used alone or in combination.
Among the above, from the viewpoint of the transglycosylation activity of the endo M mutant or endo M mutant homolog, a phosphate buffer is preferable, and specifically, sodium phosphate, potassium phosphate, and magnesium phosphate are preferable, and more preferable. Are sodium phosphate and potassium phosphate.
The concentration of the phosphate buffer is preferably 10 mM to 250 mM, more preferably 20 mM to 150 mM, more preferably 50 mM to 100 mM, from the viewpoint of glycosyl transfer activity of the endo M mutant or endo M mutant homolog. . By increasing the buffering capacity to 10 mM or more, by increasing the buffering capacity to 250 mM or less, the transglycosylation activity of the endo M mutant or endo M mutant homolog can be enhanced, and the transglycosylation yield and transglycosylation yield can be increased it can.

  In addition, the pH of the solution in the reaction is preferably 5.5 to 8.5, more preferably 6.0 to 8.0, and more preferably 6.5 to 7 from the viewpoint of increasing the sugar chain transfer yield and the sugar chain transfer yield. .5 is particularly preferred.

  In the method for producing a glycopeptide or glycoprotein, the molar ratio of the sugar chain donor to the sugar chain acceptor (molar concentration of the sugar chain donor / molar concentration of the sugar chain acceptor) is determined from the viewpoint of the yield of sugar chain transfer. 0.1 or more, more preferably 0.2 or more, particularly preferably 1 or more, and most preferably 3 or more. When the molar ratio is 0.1 or more, the transglycosylation activity and the transglycosylation yield can be shortened by increasing the transglycosylation activity of the endo M mutant or the endo M mutant homologue. Can be increased.

  In addition, in the method for producing IgG, which is a glycoprotein, from the viewpoint of sugar chain transfer yield, the molar ratio of sugar chain donor to sugar chain acceptor (number of moles of sugar chain donor / number of moles of sugar chain acceptor). ) Is preferably 5 or more, more preferably 15 or more, and particularly preferably 25 or more. By setting the molar ratio to 5 or more, the transglycosylation activity of endo M mutant or endo M mutant homolog can be increased to shorten the reaction time, and the transglycosylation yield and transglycosylation yield can be reduced. Can be increased.

  On the other hand, from the economical viewpoint in sugar chain donor manufacture, the molar ratio is preferably 0.5 to 20, more preferably 1 to 10, and more preferably 1 to 4. When it is 0.5 or more, the sugar chain transfer yield is further increased, and when it is 20 or less, it is more economical in sugar chain donor production.

The temperature in the reaction includes the viewpoint of the transglycosylation activity of the endo M mutant or endo M mutant homolog, the transglycosylation yield, the viewpoint of increasing the transglycosylation yield, and the stability of the sugar chain receptor, endo M mutant, etc. From the viewpoint of properties, it is preferably 4 ° C to 45 ° C, more preferably 20 ° C to 40 ° C, and particularly preferably 25 ° C to 35 ° C. By setting it to 4 degreeC or more, glycan transfer activities, such as an endo M variant, can be improved, and when it is 35 degrees C or less, glycan transfer yield and glycan transfer yield can be increased.
In addition, when performing a sugar chain transfer reaction to an unstable sugar chain receptor, a reaction at a temperature as low as possible within the above temperature range, that is, a reaction at 4 ° C. to 10 ° C. is preferable, and particularly at 4 ° C. The reaction is preferred.

  When IgG is used as a sugar chain receptor, the reaction time is 5 hours to 100 hours from the viewpoint of the sugar chain transfer activity of the endo M mutant or endo M mutant homolog and the sugar chain transfer yield. It is preferably 20 hours to 70 hours, and particularly preferably 35 hours to 50 hours. When the reaction time is 5 hours or longer, the sugar chain transfer yield is increased, and when the reaction time is 100 hours or shorter, the sugar chain transfer yield can be increased. In addition, as a reaction method, from the viewpoint of further increasing the sugar chain transfer yield of the reaction, a sugar chain donor and an endo M mutant or an endo M mutant homolog are added to the reaction solution again after a certain time of reaction. Thus, the reaction is preferably performed for a certain time.

  When IgG is used as the sugar chain acceptor, the concentration of the sugar chain donor in the reaction solution in the reaction is preferably 1 μM to 150 μM, more preferably 1 μM to 50 μM, and more preferably 10 μM to 10 μM from the viewpoint of the sugar chain transfer yield. 50 μM is particularly preferred.

(Pharmaceutical composition containing glycoprotein)
Since the glycopeptide or glycoprotein produced in the present disclosure does not use solids, animal tissues, cells, or the like as a production method, it is extremely unlikely to be contaminated with foreign contaminants or viruses. It can be provided as a component of a maintained pharmaceutical composition or drug. In addition to the glycopeptide or glycoprotein produced according to one embodiment of the present disclosure, the pharmaceutical composition includes a pharmaceutically acceptable stabilizer, buffer, excipient, binder, disintegrant, and taste agent. An agent, a colorant, a fragrance, and the like can be appropriately added to form a dosage form such as an injection, tablet, capsule, granule, fine granule, and powder.
Moreover, since IgG produced in one embodiment of the present disclosure is an IgG having a specific complex-type sugar chain at a high content rate, it is extremely useful as a pharmaceutical composition. Since the production method according to the present disclosure can prepare IgG having only the desired sugar chain structure, it contributes to the preparation of antibody drugs that can predict the degree of ADCC activity, CDC activity, and blood stability in advance. it can.

  Hereinafter, the production method of the present disclosure will be described in detail by way of examples. However, the scope of the production method of the present disclosure is not limited to the embodiments described in these examples.

<Sugar chain receptor>
A commercially available mogamulizumab preparation (manufactured by Kyowa Hakko Kirin Co., Ltd.) used for the preparation of the sugar chain receptor used in this example is IgG1 and has the following properties.
・ Molecular weight: 149000
・ Glycan structure: Conjugated sugar chains containing mainly Fuc with 0 to 2 Gal added and not containing Fuc, and other N-linked sugars containing Fuc with 0 to 2 Gal added Containing chain ・ Structure: Human IgG1 in which a part of the variable region is replaced with mouse
-Function: CC chemokine receptor 4 as an antigen-Preparation: Prepared by introducing a gene expression construct encoding mogamulizumab into Chinese hamster ovary cells, and then inducing expression
<End M mutant>
The N175Q mutant (manufactured by Tokyo Chemical Industry Co., Ltd., glycosynthase recombinant) was used as the endo M mutant used in this example.

<Various reagents>
Unless otherwise specified, preparation of sugar chain donor, preparation of sugar chain acceptor, and reagents used for various measurements were used as they were made by Tokyo Chemical Industry Co., Ltd.

<Various measurement methods>
Chromatography for purification of IgG and the like was performed at a flow rate of 0.5 ml / min by column operation using Protein A-Sepharose manufactured by GE Healthcare. The column was used after filling a simple column (manufactured by Bio-Rad) having a pore diameter of 10 mm and a length of 90 mm and equilibrating with a predetermined buffer solution.

The measurement by CE was performed using ProteomeLab PA800 manufactured by Beckman Coulter Inc. equipped with a capillary column (length 30.2 cm × pore diameter 50 μm, manufactured by Beckman Coulter Inc., paired fused silica) under the following analysis conditions.
・ Analysis conditions: after sample injection, measured at 25 ° C. and 15 kv for 30 minutes ・ Detection wavelength: 200 nm
In the CE measurement, the sample was pre-treated in advance under reducing or non-reducing conditions before the measurement. As shown in FIG. 1, the reason for performing the reduction treatment is that the S—S bond of IgG is cleaved to produce a heavy chain having a large molecular weight and a light chain having a small molecular weight, and then CE measurement is performed. This is because the peak separation between the heavy chain that has undergone glycan transfer and the heavy chain that has not undergone glycan transfer in the sample is better, and the confirmation of the reaction becomes easier. The sample after the reduction treatment was obtained by adding 50 mM dithiotriitol (DTT) to the sample solution and incubating at 100 ° C. for 10 minutes.

The measurement by SDS-PAGE was carried out using a 15% polyacrylamide gel (e-PAGE made by Ato) in a commercially available slab gel apparatus (AE-6530 made by Ato), and after electrophoresis at 20 mA for 70 minutes. The gel was stained with Coomassie brilliant blue and the resulting gel was evaluated. As a marker, DynaMarker Multi Color III (manufactured by Biodynamics Laboratory) was used. The detection was performed by a method using commercially available Coomassie brilliant blue.
The concentration of the protein in the solution was estimated by measuring the absorbance obtained by irradiating the solution with UV transmitted light of 280 nm using a general UV detector.

For lectin blotting, SDS-PAGE was performed with a commercially available slab gel apparatus (AE-6687 manufactured by Atto), and then blotted on a PVDF membrane (Millipore, Immobilon-P) (Atosorb, manufactured by Atto). The gel after electrophoresis was transferred by blotting at 1 mA / cm ² for 60 minutes using Paper CB-09A), and the resulting membrane was subjected to subsequent lectin staining. The same marker as above was used.

  In NMR measurement, ECA-400 manufactured by JEOL Ltd. was used.

MALDI-TOF MS measurement was performed using an autoflex speed-tko1 reflector system manufactured by Bruker Daltonics.

  In HPLC measurement, a column MightysilRP-18 (manufactured by Kanto Chemical Co., Inc.) was connected to a LaChrom Elite L2000 series system (manufactured by Hitachi High-Technologies Corporation), and acetonitrile and 0.1% TFA containing 0.1% TFA were used as eluents. A solution having a gradient with water was used, and the detection wavelength was 210 nm.

Example 1
<Preparation of sugar chain receptor>
18 mg of the above-mentioned commercially available mogamulizumab preparation was dissolved in a sodium phosphate buffer (50 mM, pH 6.25, 15 ml), and 4 ° C. with a membrane concentrator [fraction molecular weight 10,000, Amicon Ultra 15 (Merck Millipore)]. Then, the buffer solution was exchanged. 18 mg of mogamulizumab (IgG1) obtained above was dissolved in sodium phosphate buffer (50 mM, pH 6.25, 15 ml), and 1.5 units (U) of endo M (about 0.600 mg) was added at 37 ° C. Incubation was performed in a thermostatic chamber (EYELA, THERISTOR TEMPPET T-80) with shaking. The reaction solution was analyzed by CE over time, and detection by CE was continued until it was confirmed from IgG that hydrolysis of N-linked sugar chain by endo-M was complete as shown in FIGS. It was.

  After 48 hours, the reaction solution was subjected to affinity chromatography using a column packed with Protein A-Sepharose carrier (0.7 ml) manufactured by GE. In the chromatography operation, after the reaction solution was added to the column, it was first washed with 7 ml of TBS [Tris-HCl buffer (containing 50 mM, pH 7.5, 50 mM NaCl)], and then citrate buffer (50 mM, pH 3). The bound IgG was eluted with 7 ml (containing 0.50 mM NaCl). The eluate was immediately neutralized with Tris-hydrochloric acid buffer (1.0 M, pH 8.0) and then a sodium phosphate buffer using a membrane concentrator [fraction molecular weight 10,000, Amicon Ultra 15 (Merck Millipore)]. In the solution (containing 50 mM, pH 7.4, 50 mM NaCl), the addition of the buffer and the centrifugal concentration were repeated many times until the pH became neutral. In addition, as FIG. 1 shows, the sample for the following CE measurement was obtained by the same operation also for the sample after 5 hours and 24 hours after the reaction time.

By the above operation, GlcNAc-IgG (sugar chain receptor) obtained when the reaction time was 48 hours was 14.6 mg.
The progress of the reaction is shown in FIGS. From these results, both the results of the CE measurement after the reduction treatment (FIG. 1) and the results of the CE measurement after the non-reduction treatment (FIG. 2) are completely extinguished after 24 hours. It was shown that a new peak was generated in a place with a short retention time. From the above, it was shown that the raw material IgG was hydrolyzed by endo M, and GlcNAc-IgG was produced almost quantitatively.

<Sugar chain donor>
The sugar chain donor used in this example is a sialoglycopeptide (SGP, manufactured by Tokyo Chemical Industry Co., Ltd.) and has the following structure. In the peptide site, H represents amino-terminal hydrogen and OH represents carboxyl-terminal hydroxyl group.

<Glycosyl transfer reaction>
GlcNAc-IgG (sugar chain receptor) 12.0 mg obtained above was dissolved in sodium phosphate buffer (50 mM, pH 7.0, 1.2 ml), and N175Q mutant (endo M mutant) 3 .6U (3.6 mg) and 69.7 mg of SGP (sugar chain donor) were added (final concentration: 24.6 μM), and the mixture was incubated at 30 ° C. in a thermostatic bath. The progress of the transglycosylation reaction was confirmed by collecting a part of the solution from the reaction solution after the incubation for the predetermined time and directly performing CE measurement. 48 hours after the start of incubation, N175Q mutant 3.6U (3.6 mg) and SGP (manufactured by Tokyo Chemical Industry Co., Ltd.) 69.7 mg (24 μM) were added again, followed by further incubation for 22 hours.

  1 ml of the solution containing IgG obtained above (the content of IgG was 12.0 mg) was subjected to affinity chromatography using a column packed with protein A-agarose carrier (0.7 ml). Chromatography was performed in the same manner as in the preparation of the sugar chain receptor of Example 1. The obtained IgG with a uniform sugar chain added was 6.6 mg.

<Measurement after transglycosylation>
The results of the CE measurement at each reaction time performed above are shown in FIGS. Moreover, the result of SDS-PAGE in each reaction time is shown in FIG. Further, a 10 kda reference marker manufactured by Beckman Coulter, Inc. was used as a standard substance for CE measurement. This is shown as “10 kD” in FIGS. 3 and 4.

From the results of FIG. 3 and FIG. 4, it was shown that the peak of GlcNAc-IgG decreased with the passage of time, while the peak of IgG having a complex type sugar chain increased. Particularly, the peak of IgG having a complex type sugar chain did not decrease so much, and after 70 hours, it decreased until almost no peak of GlcNAc-IgG was observed.
In addition, from the result of FIG. 5, the heavy chain band of IgG at 0 hour (molecular weight of 45 kDa to 50 kDa) disappeared considerably in the lane after 24 hours, and a new band appeared on the high molecular weight side instead of about 2 kDa, After 70 hours, the IgG heavy chain band almost disappeared.

  A fixed amount of the solution containing IgG obtained above after the reaction was transferred (blotted) to the PVDF membrane by the method described in the above lectin blot. The membrane was washed with TBS (TBST) containing 0.05% TWEEN 20, and then blocked with TBST containing 1% bovine serum albumin (1% BSA) for 1 hour at room temperature. After blocking, the membrane was washed with TBST three times for 5 minutes, and then biotinylated SSA lectin (manufactured by J-Oil Mills) was added at a concentration of 2 μg / ml and incubated at room temperature for 2 hours. After incubation, the membrane was washed with TBST for 5 minutes at room temperature three times, and then horseradish peroxidized streptavidin (manufactured by Vector) was incubated at a concentration of 2 μg / ml for 2 hours at room temperature. After incubation, the membrane was washed 3 times with TBST for 5 minutes at room temperature, DAB HRP substrate (manufactured by Vector) was added, and the colored band was detected visually to detect α2-6-bound NeuAc or NeuGc. The presence or absence of inclusion was judged. The results are shown in FIG.

From the results, no band was observed in Lane 1 and Lane 2 before End M treatment and after End M treatment, and in Lane 3 (arrow portion) after the transglycosylation reaction, a weight of around 45 to 48 kDa was observed. A clear band appeared at the top of the chain band several kDa. From this, it was shown that IgG (mogamulizumab) after the sugar chain transfer reaction has a complex type sugar chain containing the structure of sialic acid linked to α2-6.
Based on the above results, the sugar chain donor portion of the sugar chain donor is efficiently transferred to GlcNAc-IgG, which is a sugar chain acceptor, by the action of the endo M mutant, and IgG having a high content of complex type sugar chains. Was shown to be obtained. In addition, even when the reaction is carried out for a relatively long time, GlcNAc-IgG gradually decreases, so that the sugar chain donor itself is stably present in the solution and plays a role as a substrate for the endo M mutant. It was shown to have.

(Example 2)
<Sugar chain receptor>
As the sugar chain receptor, the GlcNAc-IgG that is the sugar chain receptor used in Example 1 (enzyme-treated N-linked sugar chain of a commercially available mogamulizumab preparation) was used as it was.

<Preparation of sugar chain donor (Compound A-1)>
50 ml of DMF was added to 1.00 g (0.426 mmol) of dicialononasaccharide-β-O-pNP (manufactured by Tokyo Chemical Industry Co., Ltd., product number N0913) having the following structure, and dissolved by heating to 40 ° C. Then, 11-azido-3,6,9-trioxaundecan-1-amine (MW = 218.24) 2.54 ml (12.8 mmol), N, N-diisopropylethylamine 2.20 ml (12.8 mmol), 1-Cyano-2-ethoxy-2- (oxoethylideneaminoxy) dimethylamino-morpholinocarbenium hexafluorophosphate (5.48 g, 12.8 mmol) was added and stirred for 20 hours. -O-pNP represents a paranitrophenoxy group.

The reaction was completed using thin layer chromatography [developing solution; ethyl acetate / methanol / water / acetic acid = 8/6/4/1, v / v], and disappearance of starting materials having an Rf value of around 0.18. Confirmed by. The crude product obtained by concentrating the solution after the reaction under reduced pressure was separated by gel filtration column chromatography (filler: Sephadex LH-20 (manufactured by GE Healthcare), eluent: methanol), and then the solvent was removed. The product (Compound A-1 (642 mg)) was obtained. The product was subjected to NMR measurement and mass measurement, and the following results were obtained.
・ NMR measurement
¹ H-NMR (400 MHz, D ₂ O) δ _H : 8.12 (2H, d, Ph), 7.04 (2H, d, Ph), 5.18 (1H, d, H-1 (GlcN)), 5.00 (1H , s, H-1 (Man)), 4.81 (1H, s, H-1 (Man)), 4.50 (1H, d, H-1 (GlcN)), 4.46 (2H, d, H-1 x2 ( GlcN)), 4.31 (2H, d, H-1 x2 (Gal)), 4.12 (1H, s, H-2 (Man)), 4.06 (1H, s, H-2 (Man)), 3.98 (1H , s, H-2 (Man)), 3.93 (2H, t), 2.57 (2H, dd, H-3eq x2 (Neu)), 1.97 (3H, s, CH3), 1.92 (6H, s, CH3 x2 ), 1.90 (6H, s, CH3 x2), 1.88 (3H, s).
Mass measurement _{MALDI-TOF MS: m / z} calcd for C 106 H 173 N 15 NaO 68 +: 2767.04; found: 2767.55
From the above measurement results, it was confirmed that Compound A-1, which is a sugar chain donor, was obtained in a yield of 55%.

<Glycosyl transfer reaction>
The sugar chain receptor GlcNAc-IgG (4.0 mg) was dissolved in sodium phosphate buffer (50 mM, pH 7.0, 0.4 ml), and N175Q mutant (endo M mutant) 1.2 U (1.2 mg) And 22 mg of compound A-1 (sugar chain donor) was added (final concentration 16 μM), and the mixture was incubated while shaking at 30 ° C. in a thermostatic bath. After a predetermined time, a part of the solution was collected from the reaction solution, and direct CE measurement was performed to confirm the progress of the transglycosylation reaction. Twenty-two hours after the start of incubation, N175Q mutant 1.2U (1.2 mg) and 22 mg (24 μM) of compound A-1 (sugar chain donor) were added again, followed by further incubation for 22 hours. In addition, a portion corresponding to a linker having a structure derived from polyethylene glycol (PEG) containing an azide group in compound A-1 is simply referred to as N3-PEG, and is attached to dicialoglycan (SG) to which the linker containing the azide group is bound. The corresponding part may be simply referred to as N3-PEG-SG. SG represents a portion other than the paranitrophenoxy group in the above-mentioned dicialononasaccharide-β-pNP.
In the examples, a portion having a sugar chain structure in the sugar chain donor, for example, a portion corresponding to dicialoglycan (SG) in SGP may be simply referred to as a sugar chain portion.

  About 0.4 ml of the solution obtained after the incubation obtained above (with an IgG content of 4.0 mg), the protein A-agarose carrier (0.2 ml) was filled in the same manner as in the sugar chain receptor of Example 1. Purification by affinity chromatography using the obtained column was performed to obtain 1.7 mg as an IgG fraction.

<Measurement after transglycosylation>
FIG. 7 shows the result of the CE measurement performed on the product obtained by reducing the product after the transglycosylation reaction performed as described above. (A) in FIG. 7 shows the result of CE measurement of the product immediately after the start of incubation (after 0 hours), and (b) in FIG. 7 shows the result after the transglycosylation reaction performed as described above (44). The result by CE measurement of the product after time (22 hours after adding a sugar-chain donor and N175Q variant) is shown. Further, a 10 kda reference marker manufactured by Beckman Coulter, Inc. was used as a standard substance for CE measurement. This is shown as “10 kD” in FIG.
Moreover, the result of having performed the MALDI-TOF MS measurement about what reduced the product after the transglycosylation reaction performed above is shown in FIG. Furthermore, FIG. 9 shows the results of SDS-PAGE for the sugar chain receptor and the product after the sugar chain transfer reaction.

From the result of FIG. 7, the peak of GlcNAc-IgG (sugar chain receptor) at the reaction time of 0 hour ((a) in FIG. 7) is almost after the sugar chain transfer reaction ((b) in FIG. 7). It disappeared, and instead, it was shown that a new peak was generated at the position on the high retention time side indicated by the arrow from the position of the GlcNAc-IgG peak indicated by the broken line.
Further, from the result of FIG. 8, at the reaction time of 0 hour ((a) in FIG. 7), the mass peak corresponding to the heavy chain after reduction of GlcNAc-IgG (sugar chain receptor) is observed after the sugar chain transfer reaction ( In FIG. 7, (b)) almost disappeared. Instead, a mass peak appeared on the high mass side, that is, at the position where the molecular weight of the complex type sugar chain (N3-PEG-SG) to which N3-PEG was bound increased.
In addition, from the results shown in FIG. 9, the band (lane 1) in the vicinity of the molecular weight of 45 kDa to 50 kDa before End M treatment shifts to the low molecular weight side after End M treatment (Lane 2). After the reaction (lane 3), a new high concentration band appeared on the high molecular weight side.

  From the results of FIGS. 7 to 9 described above, the compound A-1 (N3-PEG-SG-PNP) which is a sugar chain donor in the presence of the endo M mutant is compared with the GlcNAc-IgG which is a sugar chain acceptor. It was shown that IgG (mogamulizumab) having two N3-PEG-SGs can be obtained in a high yield by using the sugar chain transfer.

  From the results of Examples 1 and 2 above, by using a stable sugar chain donor having a chitobiosyl structure, the sugar chain transfer reaction efficiently proceeds with respect to the sugar chain acceptor, and the desired sugar chain donating moiety is obtained. It was shown that it is possible to produce a glycoprotein having a high yield.

Example 3
<Sugar chain receptor>
GlcNAc1-β-O-pNP (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as it was.

<Preparation of sugar chain donor (compound 110)>
GlcNAcA-M3GN2-MP (compound 110) used as a sugar donor is shown in the figure with reference to the synthesis method described in Ishida et al. [SFG-JSCR, Joint Meeting November, 16-19 (2014)]. It was synthesized by the synthetic route shown. That is, a known compound 102 and a commercially available compound 101 are reacted to construct a hexasaccharide, followed by deprotection, and 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-phthalimide-β-D. -The heptasaccharide was constructed by glycosylation with glucopyranoside. Subsequently, the heptasaccharide is deprotected, further acetylated and induced to an acetamide body, and the benzylidene acetal of the non-reducing terminal GlcNAc of the acetamide body is selectively deprotected, and then the 6-position is selectively converted to a carboxylic acid. did. Then, the compound 110 which has GlcNAcA in the non-reducing terminal was obtained by deprotecting the benzyl ether group by the catalytic hydrogenation method. MP represents a paramethoxyphenyl group.

(Synthesis of Compound 103)
500 mg (0.28 mmol) of the known compound 102 which can be synthesized with reference to Shigaraki et al. [J. Biol. Chem. Vol. 278, 3466-3473 (2003)] and 4-methoxyphenyl which is a commercially available compound 700 mL (1.1 mmol) of -3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimide-β-D-glucopyranoside (manufactured by Tokyo Chemical Industry Co., Ltd., product number M1609) and 12 mL of methylene chloride Then, molecular sieve was further added to dehydrate and dry the solution. The solution was cooled to −20 ° C., and 10 μL (0.056 mmol) of trimethylsilyltrifluoromethanesulfonic acid was added and reacted. After confirming the product by thin layer chromatography (developing solvent: hexane / ethyl acetate = 1/1, v / v), a crude product was obtained by adding triethylamine. The resulting crude product was purified by column chromatography [filler: PSQ100B (manufactured by Fuji Silysia), developing solvent: hexane / ethyl acetate = 2/1] to obtain the desired compound 103 in a yield of 46% ( Yield 350 mg).

(Synthesis of Compound 104)
350 mg (0.12 mmol) of Compound 103 was dissolved in a mixed solution of 5.3 mL of toluene, 7.0 mL of acetonitrile, and 3.5 mL of ion-exchanged water, and the system was cooled to 0 ° C. To this, 0.7 g (1.28 mmol) of ammonium cerium (IV) nitrate was added and reacted. The progress of the reaction was confirmed by thin layer chromatography (developing solvent: hexane / ethyl acetate = 1/1, v / v). After confirming the completion of the reaction, the organic layer was recovered by performing a liquid separation treatment to obtain a crude product. The resulting crude product was purified by column chromatography (PSQ100B (manufactured by Fuji Silysia), developing solvent: hexane / ethyl acetate = 2/1) to obtain the target compound 104 in a yield of 57% (a yield of 192 mg). It was.

(Synthesis of Compound 105)
To 192 mg (0.073 mmol) of the compound 104, 1.9 mL of methylene chloride was added and dissolved. This was cooled to 0 ° C., and reacted by adding 74 μL (0.73 mmol) of trichloroacetonitrile and 1 μL (0.007 mmol) of 1,8-diazabicyclo [5.4.0] -7-undecene. The reaction progress was confirmed by thin layer chromatography (developing solvent: toluene / ethyl acetate = 2/1, v / v). After confirming the completion of the reaction, silica gel (PSQ100B (manufactured by Fuji Silysia)) was used, washed with ethyl acetate and filtered, and the filtrate was concentrated to give crude compound 105 in 98% yield (200 mg yield). Obtained.

(Synthesis of Compound 106)
Compound (5) 200 mg (0.0719 mmol) and 4-methoxyphenyl 3,6-di-O-benzyl-2-deoxy-2-phthalimide-β-D-glucopyranoside (manufactured by Tokyo Chemical Industry Co., Ltd., product number M1615) 171 mg (0.29 mmol) was dissolved in 3.7 mL of methylene chloride, and molecular sieves were added to dry the solution. Thereafter, the solution was cooled to −20 ° C., 0.7 μL (0.0036 mmol) of trifluoromethanesulfone sun trimethylsilyl was added, and the mixture was allowed to react. The progress of the reaction was confirmed by thin layer chromatography (developing solvent: toluene / ethyl acetate = 3/1, v / v). After confirming the completion of the reaction, neutralization was performed by adding triethylamine. After the reaction mixture was filtered through Celite, the filtrate was concentrated and purified by column chromatography (filler: PSQ100B (manufactured by Fuji Silysia), developing solvent: toluene / ethyl acetate = 6/1). Compound 106 was obtained in a yield of 65% (a yield of 150 mg).

(Synthesis of Compound 107)
150 mg (0.047 mmol) of Compound 106 was dissolved in 3 mL of n-butanol, and 190 μL (2.8 mmol) of anhydrous ethylenediamine was added and reacted by refluxing. The progress of the reaction was confirmed by thin layer chromatography (developing solvent: methylene chloride / methanol = 20/1, v / v). After confirming the product and confirming that the reaction was completed, acetylation was performed by adding 0.9 mL (9.3 mmol) of acetic anhydride and stirring. The progress of the acetylation reaction was confirmed by thin layer chromatography (developing solvent: methylene chloride / methanol = 20/1, v / v). After confirming the completion of the reaction, triethylamine was added for neutralization, and the organic layer was recovered by a liquid separation treatment to obtain a crude product. The obtained crude product was purified by column chromatography (filler: PSQ100B (manufactured by Fuji Silysia), developing solvent: methylene chloride / methanol = 50/1) to obtain the target compound 107 in a yield of 80% (yield 100 mg).

(Synthesis of Compound 108)
To 100 mg (0.035 mmol) of compound 107, 2 mL of methylene chloride was added and dissolved. The reaction was carried out by adding 80% aqueous trifluoromethanesulfonic acid solution at room temperature to such an extent that the reaction solution did not become cloudy and stirring at room temperature. The reaction progress was confirmed by thin layer chromatography (developing solvent: methylene chloride / methanol = 20/1, v / v). After confirming the completion of the reaction, the reaction solution was subjected to liquid separation treatment, and the organic layer was recovered to obtain a crude product. The obtained crude product was purified by column chromatography [filler: PSQ100B (manufactured by Fuji Silysia), developing solvent: methylene chloride / methanol = 30/1] to obtain the desired compound 108 in a yield of 52% ( Yield 48 mg).

(Synthesis of Compound 109)
24 mg (0.0089 mmol) of Compound 108 was dissolved by adding 0.5 mL of acetonitrile, and then 0.12 mL of phosphate buffer prepared to 0.74 M was added. To this, 1.6 mg (0.018 mmol) of sodium perchlorate, 2,2,6,6-tetramethylpiperidine 1-oxy free radical 0.2 mg (0.0018 mmol), 5 μL of 12% aqueous solution of sodium hypochlorite (0.0018 mmol) was sequentially added, and the reaction was performed by stirring at room temperature. The progress of the reaction was confirmed by thin layer chromatography (developing solvent: methylene chloride / methanol = 3/1, v / v). After confirming the completion of the reaction, the reaction solution was subjected to liquid separation treatment, and the organic layer was recovered. The obtained crude product was purified by column chromatography [filler: PSQ100B (manufactured by Fuji Silysia), developing solvent: methylene chloride / methanol = 10/1] to obtain the target compound 109 in a yield of 55% ( Yield 13 mg).

(Synthesis of Compound 110)
To 13 mg (0.0047 mmol) of Compound 109, 1.6 mL of ethyl acetate, 1.6 mL of ethanol and 0.8 mL of ion exchange water were added and dissolved. To this, 13 mg of palladium hydroxide was added, and the reaction was carried out by stirring overnight at room temperature in a hydrogen atmosphere. The progress of the reaction was confirmed by thin layer chromatography (developing solvent: ethyl acetate / methanol / water / acetic acid = 2/2/1 / 0.1, v / v). After confirming the completion of the reaction, the reagent was filtered off, and the resulting crude product was purified by gel filtration chromatography (filler: Sephadex G-10 manufactured by GE Healthcare, eluent: ion-exchanged water). The target compound 110 was obtained in 93% yield (yield 6.5 mg).
・ NMR measurement
1H-NMR (D2O) δ: 7.03 (2H, dd, J = 8.9, 2.1 Hz, Ph), 6.95 (2H, dd, J = 9.2, 1.8 Hz, Ph), 5.11 (1H, s, H-1 ( Man)), 5.03 (1H, d, J = 8.2 Hz, H-1 (GlcN)), 4.90 (1H, s, H-1 (Man)), 4.62 (1H, d, J = 6.0 Hz, H- 1 (GlcN)), 4.57 (2H, d, J = 8.7 Hz, H-1 (GlcN x2)), 4.24 (1H, s, H-1 (Man)), 4.17 (1H, s, H-2 ( Man)), 4.10 (1H, s, H-2 (Man)), 2.07 (3H, s, CH3), 2.05 (6H, s, CH3 x2), 2.03 (3H, s, CH3).
-Mass measurement MALDI-TOF MS: m / z calcd for C64H92N4O39: 1450.53; found: 1450.19
From the above measurement results, it was confirmed that the target compound 110 was obtained with a yield of 55%.

<Glycosyl transfer reaction>
0.625 μmol of GlcNAc-β-ethyl azide (manufactured by Tokyo Chemical Industry Co., Ltd.), which is a sugar chain receptor, is dissolved in sodium phosphate buffer (50 mM, pH 7.0, 0.01 ml), and N175Q mutant (End M) (Mutant) 10 mU (1.0 mg) and 0.79 μmol of Compound 10 as a sugar chain donor were added (molar ratio: number of moles of sugar chain donor / number of moles of sugar chain acceptor = 1.264), Incubation was performed at 30 ° C. in a thermostatic bath. One hour later, a part of the solution was collected from the reaction solution and subjected to HPLC measurement to confirm the progress of the sugar chain transfer reaction.

<Measurement after transglycosylation>
FIG. 10 and FIG. 11 show the results of HPLC measurement of the product obtained by reducing the product after the transglycosylation reaction performed as described above. FIG. 10 shows the result after 0 hour reaction time, and FIG. 11 shows the result after 1 hour reaction time. The “sugar chain acceptor” in FIGS. 10 and 11 represents GlcNAc1-β-O-ethylazide, and the “sugar chain donor” is Compound 110. “GlcNAc1-β-O-MP” in FIG. 11 indicates a product after hydrolysis of compound 110 after 1 hour, and “transfer product” indicates that the reducing end of compound 110 is 1 A product in which the position is β-O-ethylazide, that is, a compound in which β-O-MP of compound 110 is changed to β-O-ethylazide.

  From the results of FIG. 10 and FIG. 11, the peak areas of the sugar chain acceptor and the sugar chain donor at the reaction time of 0 hour (FIG. 10) decreased after the sugar chain transfer reaction (FIG. 11). It was shown that a new peak occurs at a position on the high retention time side indicated by an arrow from the position of the acceptor peak. In addition, a new peak of GlcNAc1-β-O-MP was also generated as part of the donor hydrolyzate.

As described above, due to the action of the endo M mutant, GnNAc1-β-O-ethylazide, which is a sugar chain acceptor, allows the compound 110, which is a sugar chain donor, to be more effective in a short time and at a low molar ratio. It was shown to transfer sugar chains to
Moreover, since the sugar chain part of the compound 110 which is a sugar chain donor is smaller in structure than the sugar chain part (sialoglycan (SG)) of the compound represented by the general formula 4-1-1, the endo M mutant It is presumed that reaction inhibition is unlikely to occur as a substrate for the above. From this, the compound represented by the general formula 4-2-1 having the sugar chain part of the compound 110 also has two N3-PEGs, like the compound represented by the general formula 4-1-1. It was suggested that it can be an effective sugar chain donor for obtaining IgG (mogamulizumab).

The disclosure of Japanese application 2014-232200 is incorporated herein by reference in its entirety.
All references, patent applications, and technical standards mentioned in this specification are to the same extent as if individual references, patent applications, and technical standards were specifically and individually stated to be incorporated by reference. Incorporated herein by reference.

Claims (7)

Mutant endo-β-N-acetylglucosaminidase having an amino acid sequence in which the 175th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is glutamine or alanine, or the 175th amino acid of the amino acid sequence shown in SEQ ID NO: 1 One or a plurality of mutant endo-β-N-acetylglucosaminidase having an amino acid sequence in which the residue is glutamine or alanine and other than the 175th amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 In the presence of a mutant endo-β-N-acetylglucosaminidase having an amino acid sequence modified within a homology of 80% or more to the amino acid sequence by deletion, addition or substitution of the amino acid residues of
A sugar chain donor represented by the following general formula (1);
A sugar chain receptor;
A process for producing glycopeptides or glycoproteins, wherein glycopeptides or glycoproteins are produced by reacting.


(In the general formula (1), X ¹ and X ² each represent an independent saccharide-derived group or a hydrogen atom, and at least one of X ¹ and X ² contains one or more GlcNAc or GlcNAcA, GlcNAc or GlcNAcA contained in ¹ and X ² is linked to Man bound to Man at α1-3 or Man bound to Man at α1-6 at β1-2, X ³ , X ⁴ , X ⁵ and X ⁶ each independently represents a hydrogen atom or a saccharide-derived group, Z ¹ represents a hydrogen atom or GlcNAc, and GlcNAc contained in Z ¹ is β1-4 to Man bound to GlcNAc at β1-4 bonded to that .Y ¹ is .GlcNAc representing a monovalent substituent represents N- acetylglucosaminyl group, GlcNAcA the hydroxymethyl group at the 6-position of GlcNAc-carbonitrile A sugar residue which is a syl group, β1-4 represents a β-glycoside bond between position 1 of GlcNAc and position 4 of GlcNAc, or β-glycoside bond between position 1 of Man and position 4 of GlcNAc, where Man is a mannosyl group Α1-6 represents an α glycoside bond between the 1-position of Man and the 6-position of Man, and α1-3 represents an α-glycoside bond between the 1-position of Man and the 3-position of Man.) The method for producing a glycopeptide or glycoprotein according to claim 1, wherein the sugar chain receptor is represented by the following general formula (2).
^{GlcNAc-R 2} Formula (2)
(In general formula (2), R ² represents a peptide or protein containing an asparagine residue. GlcNAc represents an N-acetylglucosaminyl group.)   The method for producing a glycopeptide or glycoprotein according to claim 1 or 2, wherein the sugar chain receptor is an IgG polypeptide in which an N-acetylglucosaminyl group is bound to a constant region. In the general formula (1), at least one of X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ is a saccharide-derived group, and the saccharide-derived group is an oligosaccharide residue. And a group having a structure in which any one group selected from an azide group, an alkynyl group, an epoxy group, an amino group, a thiol group, and an isocyanate group is bonded via a linker. A method for producing the glycopeptide or glycoprotein according to any one of the above. In the general formula (1), Z ¹ , X ³ , X ⁴ , X ⁵ and X ⁶ are hydrogen atoms, and X ¹ is α1-6 and Man bound to Man, and βlc-2 binds GlcNAcA. 5. A saccharide-derived group, wherein X ² is a saccharide-derived group in which GlcNAcA is bound by β1-2 to Man linked to Man by α1-3. 5. A method for producing the glycopeptide or glycoprotein according to Item.   The immunoglobulin G is produced by performing the reaction at a molar ratio of the sugar chain donor to the sugar chain acceptor (number of moles of sugar chain donor / number of moles of sugar chain acceptor) of 5 or more. 6. The method for producing a glycopeptide or glycoprotein according to any one of items 5.   The method for producing a glycopeptide or glycoprotein according to claim 6, wherein the molar ratio is 1 to 4.