WO2016076440A1 - Procédé de production de glycopeptide ou de glycoprotéine - Google Patents

Procédé de production de glycopeptide ou de glycoprotéine Download PDF

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WO2016076440A1
WO2016076440A1 PCT/JP2015/082170 JP2015082170W WO2016076440A1 WO 2016076440 A1 WO2016076440 A1 WO 2016076440A1 JP 2015082170 W JP2015082170 W JP 2015082170W WO 2016076440 A1 WO2016076440 A1 WO 2016076440A1
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group
sugar chain
glcnac
amino acid
man
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憲二 山本
松崎　祐二
正人 羽生
隼 岩城
徳行 湯浅
純一 熊田
幹生 石原
宜秀 西川
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東京化成工業株式会社
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Priority to JP2016559131A priority Critical patent/JP6888207B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for producing a glycopeptide or glycoprotein.
  • glycopeptides 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 of glycoproteins.
  • N-linked sugar chains have an important effect on the maintenance of protein function and structure.
  • complex sugar chains called human-type sugar chains are often important.
  • 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.
  • the N-linked sugar chain of glycoprotein obtained by such a method is not a constant structure but a glycoprotein in which several to several tens of 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.
  • 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.
  • antibodies can be prepared in large quantities by cultured cells and have been used as the main component of antibody drugs.
  • 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.
  • ADCC activity antibody-dependent cytotoxic activity
  • CDC activity complement-dependent cytotoxic activity
  • N-linked sugar chains possessed by IgG strongly affect ADCC activity and CDC activity. Particularly, for 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)]).
  • N-linked glycans complex glycans, high mannose glycans and hybrid glycans are known. Among them, specific glycans are important for ADCC activity and CDC activity.
  • IgG1 having an N-linked sugar chain having a structure lacking fucose is obtained by culturing 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).
  • a method of introducing a new sugar chain into a glycoprotein in a test tube is also conceivable.
  • 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- ⁇ -N acetylglucosaminidase, hereinafter referred to as “endo enzyme”) has been known for a long time. (See, for example, JP-A-7-59587).
  • 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.
  • endoM mutants mutants in which amino acid residues essential for enzyme activity are mutated. 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).
  • an oxazoline derivative of a complex sugar chain chemically synthesized to an IgG1 polypeptide having N-acetylglucosamine as an asparagine residue (hereinafter referred to as GlcNAc-IgG1) is a sugar donor.
  • GlcNAc-IgG1 an oxazoline derivative of a complex sugar chain chemically synthesized to an IgG1 polypeptide having N-acetylglucosamine as an asparagine residue
  • endo M hair mold-derived endo- ⁇ -N acetylglucosaminidase
  • 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.
  • 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 ⁇ 1-3 or Man bound to Man at ⁇ 1-6 at ⁇ 1-2, X 3 , X 4 , X 5 and X 6 each independently represents a hydrogen atom or a sugar-derived group, Z 1 represents a hydrogen atom or GlcNAc, and GlcNAc contained in Z 1 is ⁇ 1-4 to Man bound to GlcNAc at ⁇ 1-4 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 ⁇ 1-4 represents a ⁇ -glycoside
  • GlcNAc-R 2 general 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 having an N-acetylglucosaminyl group bound to a constant region.
  • X 1 , X 2 , X 3 , X 4 , X 5 and X 6 is a saccharide-derived group
  • the saccharide-derived group is an oligo ⁇ 1> to ⁇ 1> are groups 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>
  • Z 1 , X 3 , X 4 , X 5 and X 6 are hydrogen atoms
  • X 1 is ⁇ 1-6 and Man bonded to Man is ⁇ 1-2
  • GlcNAcA is GlcNAcA a group derived from a carbohydrate binding to have a group derived from carbohydrates
  • X 2 are bonded GlcNAcA with ⁇ 1-2 to Man bound to Man in [alpha] 1-3 ⁇ 1>
  • 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.
  • ⁇ 7> The method for producing a glycopeptide or glycoprotein according to ⁇ 6>, wherein the molar ratio is 1 to 4.
  • 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 diagram obtained by performing CE measurement after reducing the sample after the reaction (sugar chain transfer, sugar chain donor; SGP) to mogamulizumab (GlcNAc-IgG) after End M treatment. In the figure, the results according to the reaction time from 0 to 24 hours are shown.
  • 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 diagram obtained by performing CE measurement after reducing the
  • FIG. 4 is a diagram obtained by performing CE measurement after reducing the sample after reaction (glycosyl transfer, sugar chain donor; SGP) to mogamulizumab (GlcNAc-IgG) after End M treatment.
  • the results according to the reaction time from 45 hours to 70 hours are shown.
  • the lowermost figure shows the result of measuring a 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.
  • 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 of reaction (glycosylation, sugar chain donor; SGP) to mogamulizumab.
  • M is a marker
  • lane 1 is a sample before endo-M treatment
  • lane 2 is a sample after endo-M treatment
  • lane 3 is a measurement result for a sample after sugar chain transfer.
  • FIG. 7 is a view 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 endo M treatment. Yes, (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 endo M treatment. Yes, (a) is a sample
  • FIG. 10 shows the result of high performance liquid chromatography (HPLC) analysis of a sample after 0 hour reaction after reaction to GlcNAc1- ⁇ -O-ethylazide (glycosyl transfer, sugar chain donor; compound 110).
  • FIG. 11 shows the results of high-performance liquid chromatography (HPLC) analysis of a sample after 1 hour reaction after reaction to GlcNAc1- ⁇ -O-ethylazide (glycosyl transfer, sugar chain donor; compound 110).
  • 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 means that a part of the sugar chain of the sugar chain donor is bound to the sugar chain acceptor.
  • the left-side part hereinafter sometimes referred to as a sugar chain donating part
  • hydrolysis at the position of the arrow in the following general formula (1) is transferred to a sugar chain acceptor.
  • 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 to Man linked to Man by ⁇ 1-3 or Man linked to Man by ⁇ 1-6 at ⁇ 1-2.
  • 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 linked to GlcNAc at ⁇ 1-4 at ⁇ 1-4.
  • Y 1 represents a monovalent substituent.
  • GlcNAc represents an N-acetylglucosaminyl group
  • GlcNAcA represents a sugar residue in which the hydroxymethyl group at the 6-position of GlcNAc is a carboxyl group.
  • ⁇ 1-4 represents a ⁇ glycosidic bond between position 1 of GlcNAc and position 4 of GlcNAc or ⁇ glycoside bond between position 1 of Man and position 4 of GlcNAc.
  • Man represents a mannosyl group
  • ⁇ 1-6 represents an ⁇ glycoside bond between the 1-position of Man and the 6-position of Man
  • ⁇ 1-3 represents an ⁇ -glycoside bond between the 1-position of Man and the 3-position of Man.
  • 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.
  • 6SO3 and “3SO3” described later represent an acidic group bonded to a specific position of a sugar residue.
  • (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.
  • 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.
  • 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 has formed with other compounds.
  • 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.
  • 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.
  • * indicates a bonding position with a compound that forms an ester bond or an amide bond.
  • 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.
  • 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.
  • sugar chain transfer reaction may be simply referred to as “reaction”.
  • 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.
  • 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- ⁇ -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”.
  • End M is an endoenzyme derived from hair mold Mucor Himaris (GenBank Accession No. BAB43869)
  • 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).
  • an endoenzyme in which an amino acid is substituted is referred to as an “endoenzyme variant”.
  • the 175th asparagine residue is indicated as N175.
  • an amino acid residue substituted that is, an amino acid residue substituted with another amino acid
  • 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.
  • the N175Q mutant and the N175A mutant of End M may be collectively referred to as End M mutant.
  • 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.
  • N-acetylglucosaminyl group may be referred to as a GlcNAc residue
  • mannosyl group may be referred to as a Man residue
  • GlcNAcA 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, for example ⁇ 1-6
  • 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 ⁇ -type.
  • the hydroxyl group at the 1-position of the sugar has ⁇ type and ⁇ type.
  • 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.
  • the “oligosaccharide residue” means that a monosaccharide includes a sugar residue such as GlcNAc ⁇ 1-2-, for example, which forms a sugar residue.
  • the N-linked sugar chain bound to IgG described in this specification and the like can be any human type as shown in, for example, Kabat et al. [“Sequence of Proteins of Immunological Interest”, (1999)].
  • 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.
  • a “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 ⁇ 1-2 glycosidic linked GlcNAc residue.
  • Core sugar chain refers to the sugar chain moiety represented by C-1 below in the N-linked sugar chain, and “trimannosyl” refers to the three mannose moieties in the core sugar chain.
  • Man2 that binds ⁇ 1-3 on the non-reducing 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 of two N-linked sugar chains possessed by IgG is bound to the constant region of the IgG polypeptide.
  • 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.
  • glycopeptides and glycoproteins It is referred to as GlcNAc-peptide and GlcNAc-protein.
  • Protein 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 terminal sugar chain part of a sialoglycopeptide (hereinafter referred to as SGP), which is a complex N-linked sugar chain, as paranitrophenyl GlcNAc (GlcNAc ⁇ 1-O-pNP). The amount is 1 ⁇ mole transition in 1 minute.
  • a stable natural-type substrate 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 the chitobiosyl site (GlcNAc ⁇ 1-4GlcNAc), 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.
  • GlcNAc-peptide or GlcNAc-glycoprotein can be obtained. It can be presumed that effective sugar chain transfer occurs.
  • 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.
  • 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.
  • 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.
  • the production method for producing a glycopeptide or glycoprotein according to the present disclosure includes a mutant endo- ⁇ -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
  • a sugar chain donor represented by the general formula (1) reacting a sugar chain donor represented by the general formula (1) with a sugar chain acceptor in the presence of a mutant endo- ⁇ -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.
  • 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.
  • glycopeptide or glycoprotein produced by the production method of the present disclosure is not particularly limited.
  • examples include glycoproteins and glycopeptides that are secreted extracellularly or on the cell surface and have N-linked sugar chains, such as various hormones, cell adhesion factors, various receptors, cells
  • examples include an outer matrix, various enzymes, various cytokines, antibacterial or antiviral peptides, and antibodies.
  • antibacterial or antiviral peptides and bioactive peptides, hormones, cytokines or antibodies are preferred.
  • the immunoglobulin G 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.
  • CE capillary electrophoresis
  • SDS-PAGE SDS-polyacrylamide electrophoresis
  • MALDI-TOF MS 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 ⁇ m, manufactured by Beckman Coulter Inc., paired fused silica) under the following analysis conditions.
  • ⁇ Analysis conditions after sample injection, measured at 25 ° C.
  • 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, and 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 by the following conditions under an autoflex speed-tko1 reflector system manufactured by Bruker Daltonics. The mass of the product can be confirmed.
  • Measurement mode positive ion mode and reflector mode
  • Measurement voltage 1.5kv ⁇ 2.5kv
  • Measurement molecular weight range 0 to 3000 (m / z) and 45000 to 60000 (m / z)
  • Accumulation 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 ⁇ N acetylglucosaminidase (GenBank Accession No. BAB43869) derived from Mucor Himalis.
  • a mutant endo- ⁇ -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
  • a mutant endo- ⁇ -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- ⁇ -N-acetylglucosaminidase (endo M mutant homolog) By performing the response, producing a glycopeptide or glycoprotein.
  • the endo M mutant is N175Q or N175A, so that hydrolysis of the product after the transglycosylation reaction can be sufficiently suppressed.
  • 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 types of hosts and corresponding appropriate protein expression vectors.
  • the host include Escherichia coli, Brevibacillus, cyanobacteria, lactic acid bacteria, yeast, insect cells and animal cells.
  • E. coli Escherichia coli
  • Brevibacillus cyanobacteria
  • lactic acid bacteria yeast
  • insect cells insect cells and animal cells.
  • a specific production method is described in detail in the literature of Umekawa et al. [J. Biol. Chem. Vol.285 (1), 511-521, (2010)] for Escherichia coli, and specially for yeast. 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.
  • 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).
  • hexahistidine peptides and flag peptides are particularly preferable from the viewpoint of ease of preparation of endo M mutant or endo M mutant homolog.
  • 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.
  • a peptide portion as a linker peptide region of several to 50 residues.
  • the linker peptide region may include an amino acid sequence portion (protease site) that is hydrolyzed by a protease.
  • protease site for example, a factor Xa site, a thrombin site, an enterokinase site, a precision protease site is mentioned.
  • 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.
  • 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.
  • 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)
  • the homology to endo M (SEQ ID NO: 1) is 80% or more, the transglycosylation activity can be sufficiently maintained.
  • 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.
  • 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.
  • the sugar chain transfer yield can be increased, and by being 20 U or less, the sugar chain transfer yield can be increased.
  • 1 unit (U) indicates the amount of enzyme that transfers 1 ⁇ mol of sialoglycopeptide (hereinafter referred to as SGP) to ⁇ -paranitrophenyl GlcNAc (GlcNAc ⁇ 1-O-pNP) in 1 minute.
  • the concentration is more preferably 1 U to 10 U / ml, and particularly preferably 2 U to 5 U / ml.
  • 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.
  • 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 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 containing a ⁇ 1-4 bond between GlcNAc and GlcNAc (chitobiosyl structure), it is stable as a substrate and can exist stably in solution for a long time, so that it can be used as a sugar chain receptor that requires a long reaction time. It can respond enough.
  • 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 to Man linked to Man by ⁇ 1-3 or Man linked to Man by ⁇ 1-6 at ⁇ 1-2.
  • 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 linked to GlcNAc at ⁇ 1-4 at ⁇ 1-4.
  • Y 1 represents a monovalent substituent.
  • GlcNAc represents an N-acetylglucosaminyl group
  • GlcNAcA represents a sugar residue in which the hydroxymethyl group at the 6-position of GlcNAc is a carboxyl group.
  • ⁇ 1-4 represents a ⁇ glycosidic bond between position 1 of GlcNAc and position 4 of GlcNAc or ⁇ glycoside bond between position 1 of Man and position 4 of GlcNAc.
  • Man represents a mannosyl group
  • ⁇ 1-6 represents an ⁇ glycoside bond between the 1-position of Man and the 6-position of Man
  • ⁇ 1-3 represents an ⁇ -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 ⁇ 1-2 to either Man2 or Man3 of the core sugar chain moiety.
  • the saccharide-derived groups in X 1 , X 2 , X 3 , X 4 , X 5 and X 6 include GlcNAc ⁇ 1-2, Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc 2, NeuGc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2 and NeuGc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, heterologous antigens: Gal ⁇ 1-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, polylactosamine: [Gal ⁇ 1-4GlcNAc ⁇ 1-3] nGalc1 ⁇ GcNA Number), keratan sulfate [Gal ⁇ 1-4GlcNAc (6SO3) ⁇ 1-3] nGal ⁇ 1-4GlcNAc ⁇ 1-2 (n is an arbitrary number), [Gal (6SO3) ⁇ 1-4GlcNAc (6SO3 ⁇ 1-3] nGal ⁇ 1-4Gl
  • 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.
  • both or one of X 1 and X 2 is any one of GlcNAc ⁇ 1-2, Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, and Zc 1 is a hydrogen atom, or both or one of X 1 and X 2 is any one of GlcNAc ⁇ 1-2, Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2 And Z 1 is preferably GlcNAc.
  • both X 1 and X 2 are any one of GlcNAc ⁇ 1-2, Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, and Z 1 is hydrogen.
  • X 1 and X 2 are either one of GlcNAc ⁇ 1-2, Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, and Z 1 Is more preferably GlcNAc.
  • X 1 and X 2 are both Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, and Z 1 is a hydrogen atom, More preferably, both X 1 and X 2 are any one of Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, and Z 1 is GlcNAc.
  • X 1 and X 2 are either one of NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2 or Gal ⁇ 1-4GlcNAc ⁇ 1-2, and Z 1 is a hydrogen atom, or both X 1 and X 2 are , NeuAc ⁇ 2 A one of 6Gal ⁇ 1-4GlcNAc ⁇ 1-2 or Galbeta1-4GlcNAcbeta1-2, and particularly preferred that Z 1 is GlcNAc, both X 1 and X 2 are, NeuAcarufa2-6Galbeta1-4GlcNAcbeta1-2 or Gal ⁇ 1- Most preferably, it is any one of 4GlcNAc ⁇ 1-2 and Z 1 is GlcNAc.
  • 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 azido group, an alkynyl group, 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.
  • 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.
  • 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 includes at least one of X 1 to X 6 .
  • the click reaction group, the linker, and the oligosaccharide residue may be referred to as each block.
  • 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.
  • the binding ratio between the click reactive group and the linker and the arrangement of the click reactive group with respect to the linker are not particularly limited. However, from the standpoint of easy 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 1 or 2 is particularly preferable.
  • oligosaccharide residues bound to the linker include GlcNAc ⁇ 1-2, Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-6Gal ⁇ 1-4Glc1 3Gal ⁇ 1-4GlcNAc ⁇ 1-2, heterologous antigen: Gal ⁇ 1-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, polylactosamine: [Gal ⁇ 1-4GlcNAc ⁇ 1-3] nGal ⁇ 1-4GlcNAc ⁇ 1-2 (n is an arbitrary number), keratan sulfate [Gal ⁇ 1-4GlcNAc (6) ⁇ 1-3] nGal ⁇ 1-4GlcNAc ⁇ 1-2 (n is an arbitrary number), [(6SO3) Gal ⁇ 1-4GlcNAc (6SO3) ⁇ 1-3] nGa ⁇ 1-4GlcNAc ⁇ 1-2 (n is an arbitrary number), LacDiNAc: Gal ⁇
  • 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.
  • X 3 , X 4 , X 5 , X 6 and Z 1 are hydrogen atoms, and one or both of X 1 and X 2 are NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2- More preferably, it is any one of 3Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, GlcA ⁇ 1-2, and GlcNAcA ⁇ 1-2.
  • 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 NeuAc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuAc ⁇ 2 -3Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, GlcA ⁇ 1-2 and GlcNAcA ⁇ 1-2 are particularly preferred.
  • X 3 , X 4 , X 5 , X 6 and Z 1 in the general formula (1) are hydrogen atoms, and both X 1 and X 2 are GlcNAcA ⁇ 1-2.
  • 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.
  • polyethylene glycol (PEG) Branched PEG, PEG derivative, hydroxyethyl cellulose (HEC), dextrin, polyoxazoline, saccharide, 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 anhydr
  • 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 100 to 10,000 from the viewpoint of ease of preparation as a sugar chain donor and sugar chain transfer yield. Further, 100 to 7000 is more preferable, and 100 to 4000 is particularly preferable.
  • 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 the yield of sugar chain transfer. 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.
  • bonding the said oligosaccharide residue is also mentioned.
  • 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.
  • 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 viewpoints of ease of preparation and sugar chain transfer yield, compounds represented by the following general formula (3-1) or (3-2) are preferred.
  • 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.
  • 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).
  • Some of the compounds represented by the general formula (3-1) are commercially available.
  • 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 Chemical Industry Co., Ltd.) are compounds in which an alkynyl group is introduced. Manufactured by Kogyo Co., Ltd.).
  • 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.
  • 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.
  • 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.
  • the functional group include an amino group, a carboxyl group, a thiol group, an isocyanate group, a hydroxyl group, and an epoxy group.
  • 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.
  • 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.
  • 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.
  • Y 1 may have a hydroxyl group, an optionally substituted alkoxy group having 1 to 30 carbon atoms, or a substituted group.
  • An aryloxy group having 6 to 30 carbon atoms, an alkenyloxy group having 1 to 30 carbon atoms which may have a substituent, and an amide group which may have a substituent are preferable.
  • Y 1 is an alkoxy group optionally having a substituent having 1 to 10 carbon atoms, or a C 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 preferable, 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.
  • 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 this viewpoint, an amide group to which the 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.)
  • substituents which may be substituted include 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.
  • a method for extracting from sugar or a method of chemically preparing it can be considered.
  • 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 ⁇ -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.
  • Y 1 in the general formula (1) contains asparagine.
  • 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.
  • a sugar chain donor having 7 or less oligosaccharide residues may be prepared by the above-mentioned method or by a chemical synthesis method.
  • 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)].
  • 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.
  • IgG can also be produced.
  • 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.
  • the click-reactive group-containing sugar chain donor is either an azide group or an alkynyl group, and the linker is a water-soluble polymer.
  • the number of the click reactive groups in the click reactive group-containing sugar chain donor is 1 to 4, 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 optionally substituted aryloxy group having 6 to 30 carbon atoms, and an optionally substituted alkenyloxy having 1 to 30 carbon atoms It is preferably any one selected from the group of an amide group which may have a group and a substituent.
  • 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
  • the click reactive group in the click reactive group-containing sugar chain donor 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.
  • the click reactive group is either an azide group or an alkynyl group
  • the linker has a structure derived from polyethylene glycol
  • the number of the click reactive group in the click reactive group-containing sugar chain donor is one.
  • 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.
  • 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.
  • 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 ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2 , NeuAc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-6Gal ⁇ 1-4GlcNAc ⁇ 1-2, NeuGc ⁇ 2-3Gal ⁇ 1-4GlcNAc ⁇ 1-2, GlcA ⁇ 1-2 and GlcNAcA ⁇ 1-2, and the methoxy group Y 1 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.
  • click reactive group-containing sugar chain donor examples include the following, but the production method of the present disclosure is not limited to these.
  • 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.
  • a sugar chain receptor represented by the following general formula (2) is preferable.
  • GlcNAc-R 2 general formula (2) (In general formula (2), R 2 represents a peptide or protein containing an asparagine residue. GlcNAc represents an N-acetylglucosaminyl group.)
  • GlcNAc-peptide and GlcNAc-protein examples 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 of a commercially available endoenzyme or a self-prepared endoenzyme, or asparagine.
  • a glycopeptide 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).
  • 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 IgG having a GlcNAc group bound to a constant region
  • GlcNAc-IgG can be used as a sugar chain receptor.
  • GlcNAc-IgG1 is preferable.
  • GlcNAc-IgG in the present disclosure is a sugar chain receptor used for producing immunoglobulin G (IgG), and is an immunity in which one N-acetylglucosaminyl group (GlcNAc) is bound to one constant region.
  • GlcNAc-IgG 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.
  • both IgGs are included.
  • IgG 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.
  • GlcNAc-IgG 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.
  • the 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 to F3 (manufactured by Sigma-Aldrich) and the like.
  • endoenzymes 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 production method of the IgG of the present disclosure is a mutant endo- ⁇ -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 represented by 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.
  • the molar ratio of the sugar chain donor represented by the general formula (1) to 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.
  • 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.
  • a phosphate buffer is preferable, and specifically, sodium phosphate, potassium phosphate, and magnesium phosphate are preferable, and more preferable.
  • the concentration of the phosphate buffer is preferably 10 mM to 250 mM, more preferably 20 mM to 150 mM, and more preferably 50 mM to 100 mM, from the viewpoint of the transglycosylation activity of the endo M mutant or endo M mutant homolog. .
  • 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.
  • 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.
  • the molar ratio of the sugar chain donor to 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.
  • 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.
  • 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.
  • the molar ratio is preferably 0.5 to 20, more preferably 1 to 10, and more preferably 1 to 4.
  • 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.
  • 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.
  • 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, more 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.
  • 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.
  • the concentration of the sugar chain donor in the reaction in the reaction is preferably 1 ⁇ M to 150 ⁇ M, more preferably 1 ⁇ M to 50 ⁇ M, more preferably 10 ⁇ M 50 ⁇ M is particularly preferred.
  • 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.
  • 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.
  • 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.
  • ⁇ 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 Conjugate glycan containing Fuc with 0 to 2 Gal added and containing Fuc with 0 to 2 Gal added, and other N-linked sugars 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.
  • 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 ⁇ m, 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 sample was pre-treated in advance under reducing or non-reducing conditions before the measurement. As shown in FIG.
  • the reason for performing the reduction treatment is that the S—S bond of IgG is cleaved to generate 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 performed using a 15% polyacrylamide gel (e-PAGE made by Ato) on 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.
  • AE-6530 made by Ato
  • DynaMarker Multi Color III manufactured by Biodynamics Laboratory
  • 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.
  • SDS-PAGE was performed with a commercially available slab gel apparatus (AE-6687 manufactured by Ato), and then blotted on a PVDF membrane (Millipore, Immobilon-P) (Atosorb, manufactured by Ato). Using Paper CB-09A), the gel after electrophoresis was transferred by blotting at 1 mA / cm 2 for 60 minutes, and the resulting membrane was subjected to subsequent lectin staining. The same marker as above was used.
  • MALDI-TOF MS measurement was performed using an autoflex speed-tko1 reflector system manufactured by Bruker Daltonics.
  • a column MightysilRP-18 manufactured by Kanto Chemical Co., Inc.
  • a LaChrom Elite L2000 series system manufactured by Hitachi High-Technologies Corporation
  • acetonitrile containing 0.1% TFA and 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.
  • a sodium phosphate buffer 50 mM, pH 6.25, 15 ml
  • U units
  • reaction solution was subjected to affinity chromatography using a column packed with Protein A-Sepharose carrier (0.7 ml) manufactured by GE.
  • Protein A-Sepharose carrier 0.7 ml
  • TBS Tris-HCl buffer (containing 50 mM, pH 7.5, 50 mM NaCl)]
  • 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)].
  • Tris-hydrochloric acid buffer 1.0 M, pH 8.0
  • a sodium phosphate buffer using a membrane concentrator [fraction molecular weight 10,000, Amicon Ultra 15 (Merck Millipore)].
  • the addition of the buffer and the centrifugal concentration were repeated many times until the pH became neutral.
  • 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.
  • the sugar chain donor used in this example is a sialoglycopeptide (SGP, manufactured by Tokyo Chemical Industry Co., Ltd.) and has the following structure.
  • H represents amino-terminal hydrogen
  • OH represents carboxyl-terminal hydroxyl group.
  • ⁇ Glycosyl transfer reaction 12.0 mg of GlcNAc-IgG (sugar chain receptor) 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 ⁇ M), 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.
  • N175Q mutant 3.6U (3.6 mg) and SGP (manufactured by Tokyo Chemical Industry Co., Ltd.) 69.7 mg (24 ⁇ M) were added again, followed by further incubation for 22 hours.
  • FIGS. 5 shows the results of SDS-PAGE at each reaction time.
  • 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.
  • 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, biotinylated SSA lectin (manufactured by J-Oil Mills) was added at a concentration of 2 ⁇ g / ml, and incubated at room temperature for 2 hours.
  • the membrane was washed 3 times with TBST for 5 minutes at room temperature, and then horseradish peroxidized streptavidin (manufactured by Vector) was incubated at a concentration of 2 ⁇ g / ml for 2 hours at room temperature. After incubation, the membrane was washed with TBST for 5 minutes at room temperature three times, DAB HRP substrate (manufactured by Vector) was added, and the colored band was visually detected to detect ⁇ 2-6-bound NeuAc or NeuGc. The presence or absence of inclusion was judged. The results are shown in FIG.
  • Example 2 ⁇ Sugar chain receptor>
  • 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.
  • —O-pNP represents a paranitrophenoxy group.
  • 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.
  • sodium phosphate buffer 50 mM, pH 7.0, 0.4 ml
  • N175Q mutant endo M mutant
  • N175Q mutant 1.2U (1.2 mg) and 22 mg (24 ⁇ M) of compound A-1 (sugar chain donor) were added again, followed by further incubation for 22 hours.
  • 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 bonded.
  • 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- ⁇ -pNP.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 8 shows the result of MALDI-TOF MS measurement performed on the product obtained by reducing the product after the sugar chain transfer reaction performed as described above.
  • FIG. 9 shows the results of SDS-PAGE for the sugar chain receptor and the product after the sugar chain transfer reaction.
  • Example 3 ⁇ Sugar chain receptor> GlcNAc1- ⁇ -O-pNP (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as it was.
  • 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.
  • 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.
  • the organic layer was recovered by performing a liquid separation treatment to obtain a crude product.
  • the reagent was filtered off, and the resulting crude product was purified by gel filtration chromatography (filler: GE Healthcare Sephadex G-10, eluent: ion-exchanged water).
  • the target compound 110 was obtained in 93% yield (yield 6.5 mg).
  • ⁇ Glycosyl transfer reaction> 0.6175 ⁇ mol of GlcNAc- ⁇ -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 ⁇ mol 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.
  • 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
  • FIG. 11 shows the result after 1 hour reaction time.
  • the “sugar chain acceptor” in FIGS. 10 and 11 represents GlcNAc1- ⁇ -O-ethylazide, and the “sugar chain donor” is Compound 110.
  • GnNAc1- ⁇ -O-ethylazide which is a sugar chain acceptor
  • the compound 110 which is a sugar chain donor
  • the sugar chain portion of compound 110 which is a sugar chain donor
  • formula 4-1-1 sialoglycan (SG)
  • 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).

Abstract

Un mode de réalisation de la présente invention concerne un procédé de production pour produire des glycopeptides ou des glycoprotéines par la réaction d'un récepteur de chaîne de sucre avec un donneur de chaîne de sucre représenté par la formule générale (1) (dans laquelle X1 à X6 sont des groupes dérivés d'un sucre ou H et Z1 représente H ou GlcNAc) en présence d'une endo-β-N-acétylglucosaminidase mutante (variant d'endoenzyme) qui présente une séquence d'acides aminés dans laquelle le résidu d'acide aminé 175 de la séquence d'acides aminés représentée par la séquence SEQ ID NO:1 est la glutamine ou l'alanine ou qui présente une séquence d'acides aminés dans laquelle le résidu d'acide aminé 175 de la séquence d'acides aminés représentée par la séquence SEQ ID NO: 1 est la glutamine ou l'alanine et qui présente une séquence d'acides aminés qui a été modifiée par délétion, addition ou substitution d'un ou d'une pluralité de résidus d'acides aminés autres que le résidu d'acide aminé 175 de la séquence d'acides aminés représentée par la séquence SEQ ID NO:1 de façon à être homologue à raison d'au moins 80 % par rapport à la séquence d'acides aminés susmentionnée.
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JP2018027078A (ja) * 2016-08-10 2018-02-22 公益財団法人野口研究所 ポリエチレングリコール鎖が導入された化合物の製造方法
WO2024053574A1 (fr) * 2022-09-09 2024-03-14 第一三共株式会社 Nouvel oligosaccharide, intermédiaire de production pour un nouvel oligosaccharide, procédé de production d'un nouvel oligosaccharide et procédé de production d'un intermédiaire de production pour un nouvel oligosaccharide

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WO1999061591A1 (fr) * 1998-05-22 1999-12-02 Kirin Beer Kabushiki Kaisha GENE D'ENDO-β-N-ACETYLGLUCOSAMINIDASE
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
JP2018027078A (ja) * 2016-08-10 2018-02-22 公益財団法人野口研究所 ポリエチレングリコール鎖が導入された化合物の製造方法
WO2024053574A1 (fr) * 2022-09-09 2024-03-14 第一三共株式会社 Nouvel oligosaccharide, intermédiaire de production pour un nouvel oligosaccharide, procédé de production d'un nouvel oligosaccharide et procédé de production d'un intermédiaire de production pour un nouvel oligosaccharide

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