Highly sialylated immunoglobulin

Abstract

Methods for preparing hypersialylated IgG have been described.

Description

[Technical Field]

[0001] (Claiming priority) This application claims the interests of U.S. Provisional Patent Application No. 63 / 026,826, filed on 19 May 2020, and U.S. Provisional Patent Application No. 63 / 108,741, filed on 2 November 2020. All of the foregoing is incorporated herein by reference.

[0002] (Field of Invention) This disclosure relates to a method for preparing hypersialylated IgG. [Background technology]

[0003] Intravenous immunoglobulin (IVIg), prepared from pooled plasma from human donors (e.g., plasma pooled from at least 1,000 donors), is used to treat a variety of inflammatory disorders. However, IVIg preparations have clear limitations, including variability in efficacy, clinical risks, high costs, and limited supply. While different IVIg preparations are often treated as clinically interchangeable products, it is well known that significant differences exist in product preparations that may affect tolerability and activity in selected clinical applications. Current maximum dose regimens yield only partial and non-sustained responses in many cases. In addition, the long infusion times (4–6 hours) associated with high-dose IVIg treatment consume significant resources at infusion centers, negatively impacting patient-reported outcomes such as convenience and quality of life.

[0004] The identification of the crucial anti-inflammatory role of Fc domain sialylation has presented an opportunity to develop more potent immunoglobulin therapies. Commercial IVIg preparations generally exhibit low levels of sialylation on the Fc domain of the present antibody. Specifically, this indicates low levels of disyalylation of branched glycans in the Fc region.

[0005] Washburn et al. (Proceedings of the National Academy of Sciences, USA 112:E1297-E1306 (2015)) described a controlled sialylation process for generating highly tetra-Fc-sialylated IVIg, which was shown to yield a product with consistently enhanced anti-inflammatory activity. [Overview of the project] [Means for solving the problem]

[0006] Sialization reactions driven by ST6Ga1 using CMP-NANA as a substrate have properties that make improvement of the reaction difficult, whether evaluated by the overall disialylation level, the time to reach a specific disialylation level, or the amount of enzyme and substrate required to reach a specific overall disialylation level. For example, (a) CMP-NANA is not completely stable and hydrolyzes spontaneously even in the absence of enzymes. (b) ST6Gal1 is thought to catalyze the hydrolysis of CMP-NANA without productive addition to Gal in branched glycans. (c) Cytidine monophosphate (CMP), a byproduct generated by either enzyme addition or CMP-NANA hydrolysis, can act as a competitive inhibitor of ST6Gal1. (d) CMP has been observed to catalyze a reverse enzymatic reaction to remove NeuAc from the newly formed glycan. Thus, the level of byproducts increases over time, which can lead to the deceleration or even reversal of the desired sialylation reaction.

[0007] This disclosure is at least in part based on the finding that using BIS-TRIS as a buffer results in a far less favorable reverse reaction compared, for example, with MOPS as a buffer.

[0008] Accordingly, this specification describes a method for producing hypersialylated IgG (hsIgG), comprising: (a) providing a pooled IgG antibody; (b) generating a galactosylated IgG antibody by incubating the pooled IgG antibody in a reaction mixture comprising β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion, UDP-Gal or a salt thereof, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl2; and (c) generating hsIgG by incubating the galactosylated IgG antibody in a reaction mixture comprising ST6Gal or its enzymatically active portion, CMP-NANA or a salt thereof, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl2.

[0009] Accordingly, this specification also describes a method for preparing hypersialylated IgG (hsIgG), comprising (a) providing a pooled IgG antibody, and (b) incubating the pooled IgG antibody in a reaction mixture containing β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion, UDP-Gal or a salt thereof, ST6Gal or its enzymatically active portion, CMP-NANA or a salt thereof, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl2 to produce an hsIgG preparation.

[0010] This specification also describes a method for preparing hypersialylated IgG (hsIgG), comprising: (a) providing a pooled IgG antibody; (b) incubating the pooled IgG antibody in a galactosylation reaction mixture containing β1,4-galactosyltransferase (B4GalT) or its enzymatically active moiety, UDP-Gal or a salt thereof, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl2 to generate a galactosylated IgG antibody; (c) adding ST6Gal or its enzymatically active moiety and CMP-NANA or a salt thereof to the galactosylation reaction mixture to generate a sialylation reaction mixture; and (d) incubating the sialylation reaction mixture to generate hsIgG.

[0011] In some embodiments, B4GalT or its enzymatically active portion is at least 85% identical to SEQ ID NO: 13.

[0012] In some embodiments, ST6Gal or its enzymatically active portion includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 19.

[0013] In some embodiments, the total incubation time is less than 72 hours.

[0014] In some embodiments, the incubation time for the reaction mixture containing ST6Gal or its enzymatically active portion is less than 40 hours.

[0015] In some embodiments, each of the reaction mixtures independently contains BIS-TRIS at approximately 10 to approximately 500 mM and approximately pH 5.5 to approximately pH 8.5.

[0016] In some embodiments, the reaction mixture each independently contains a BIS-TRIS buffer at approximately 50 mM and pH 7.3.

[0017] In some embodiments, the pooled IgG antibody is provided as a composition further comprising a BIS-TRIS buffer at about pH 7.2.

[0018] In some embodiments, each of the reaction mixtures independently comprises from about 1 to about 20 mM of MnCl2.

[0019] In some embodiments, each of the reaction mixtures independently comprises from about 4.5 to about 5.5 mM of MnCl2.

[0020] In some embodiments, the reaction mixture comprises from about 0.038 to about 0.046 of UDP-Gal or a salt thereof per gram of the pooled IgG antibody.

[0021] In some embodiments, the reaction mixture comprises from about 0.1425 to about 0.1575 of CMP-NANA or a salt thereof per gram of the IgG antibody.

[0022] In some embodiments, the reaction mixture containing CMP-NANA is supplemented with additional CMP-NANA or a salt thereof during the incubation.

[0023] In some embodiments, the total amount of CMP-NANA or a salt thereof added to the reaction mixture containing CMP-NANA is from about 0.1425 to about 0.1575.

[0024] In some embodiments, the total amount of CMP-NANA is added to the sialylation reaction mixture in less than 7 portions.

[0025] In some embodiments, the reaction mixture containing B4GalT or an enzymatically active portion thereof comprises from about 7.2 to about 8.8 U of B4GalT or an enzymatically active portion thereof per gram of the pooled IgG1.

[0026] In some embodiments, the reaction mixture containing ST6Gal1 or an enzymatically active portion thereof contains from about 17.1 to about 18.9 U of ST6Gal1 or an enzymatically active portion thereof per gram of pooled IgG1.

[0027] In some embodiments, the incubation is carried out at from about 20 to about 50 °C.

[0028] In some embodiments, the incubation is carried out at about 37 °C.

[0029] In some embodiments, the IgG antibody comprises IgG antibodies isolated from at least 1000 donors.

[0030] In some embodiments, at least 50%, 55%, 60%, 65% or 70% w / w of the IgG antibody is an IgG1 antibody.

[0031] In some embodiments, at least 90% of the donor subjects have been exposed to a virus.

[0032] In some embodiments, about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in hsIgG have sialic acid on both α1,3 branches and α1,6 branches.

[0033] In some embodiments, about 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans in hsIgG have sialic acid on both α1,3 branches and α1,6 branches.

[0034] In some embodiments, at least 60%, 65%, 70%, 75%, 80% or 85% of the branched glycans in the Fab domain of hsIgG have sialic acid on both α1,3 arms and α1,6 arms connected through NeuAc-α2,6-Gal terminal linkages.

[0035] In some embodiments, at least 80% of the branched Fc glycans in hsIIgG have sialic acid in both the α1,3 branching and the α1,6 branching.

[0036] In some embodiments, at least 60%, 65%, and 70% of the branched glycans in the Fab domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal ligatures.

[0037] In some embodiments, at least 85% of the Fc glycans in hsIIgG have sialic acid in both the α1,3 branching and the α1,6 branching.

[0038] In some embodiments, at least 60%, 65%, and 70% of the branched glycans in the Fab domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal ligatures.

[0039] In some embodiments, at least 90% of the branched Fc glycans in hsIIgG have sialic acid in both the α1,3 branching and the α1,6 branching.

[0040] In some embodiments, at least 60%, 65%, and 70% of the branched glycans in the Fab domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal ligatures.

[0041] Furthermore, this specification describes a method for preparing immunoglobulin G (IgG) having a very high level of Fc sialylation, particularly disialylation (sialylation in both the alpha-1,3 and alpha-1,6 branching of the glycan as defined in Asn297 (EU numbering)). The method described herein can provide hypersialylated IgG (hsIgG) in which more than 70% of the branched glycan in the Fc domain is sialylated in both branchings (i.e., alpha-1,3 and alpha-1,6 branching). hsIgG contains a diverse mixture of IgG antibodies, mainly IgG1 antibodies. Antibody diversity is high. The immunoglobulin used to prepare hsIgG can be obtained, for example, from pooled human plasma (e.g., plasma pooled from at least 1,000 to 30,000 donors). The immunoglobulin can be obtained from IVIg, including commercially available IVIg. hsIgG has a much higher level of sialic acid in the branched glycan in the Fc region than IVIg. This yields compositions that differ from IVIg in both structure and activity. hsIgG can be prepared as described in International Publication No. 2014 / 179601 or Washburn et al. (Proceedings of the National Academy of Sciences, USA 112:E1297-E1306 (2015)), both of which are incorporated herein by reference.

[0042] The methods described herein are improved methods for preparing hsIgG.

[0043] This specification describes a method for preparing hypersialylated (hsIgG), comprising: (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture containing β1,4-galactosyltransferase I (B4GalT) and UDP-Gal to produce galactosylated IgG antibodies; and (c) incubating the galactosylated IgG antibodies in a reaction mixture containing ST6Gal1 and CMP-NANA to produce an hsIgG preparation, wherein the galactosylation reaction mixture and the sialylation reaction mixture contain bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer.

[0044] The present invention also describes a method for preparing hypersialylated (hsIgG), comprising: (a) providing a mixture of IgG antibodies; and (b) incubating the mixture of IgG antibodies in a reaction mixture containing β1,4-galactosyltransferase I (B4GalT), UDP-Gal, ST6Gal1, and CMP-NANA in bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer for at least 24 hours to produce an hsIgG preparation.

[0045] In various embodiments, B4GalT comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 13, ST6Gal1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, step (b) is run for at least 8, 12, 18, 24, 30, or 40 hours, step (c) is run for at least 8, 12, 18, 24, 30, or 40 hours, step (c) comprises adding ST6Gal1 and CMP-NANA to the reaction mixture of step (a), the reaction is carried out in BIS-TRIS at pH 5.5-8.5 with a concentration of 10-500 mM, the reaction mixture comprises 1-20 mM MnCl2, UDP-Gal is present in 5 μM UDP-Gal / g IgG antibody, and CMP-NANA is present in 5 μM CMP-NANA / g The IgG antibodies are present, incubation is performed at 20-50°C, incubation is performed at 30-45°C, the IgG antibodies include IgG antibodies isolated from at least 1000 donors, at least 50%, 55%, 60%, 65%, or 70% w / w of the IgG antibodies are IgG1 antibodies, at least 90% of the donor subjects have been exposed to the virus, approximately 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in the hsIgG preparation have sialic acid in both α1,3 and α1,6 branching, approximately 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans in the hsIgG preparation have α1,3 Both the branched and α1,6 branched segments have sialic acid, and at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in the Fab domain have sialic acid in both the α1,3 arms and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages, and at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in the Fc domain have sialic acid in both the α1,3 arms and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages, and the incubation in step (a) is 12 to 30 hours, and the incubation in step (a) is 20 to 40 hours.

[0046] In hypersialylated IgG, at least 60% of the branched glycans in the Fc region (e.g., 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98%, including up to 100%) are disialylated via NeuAc-α2,6-Gal terminal ligatures (i.e., in both the α1,3 branching and α1,6 arms). In some embodiments, less than 50% of the branched glycans in the Fc region (e.g., less than 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) are monosialylated via NeuAc-α2,6-Gal terminal ligatures (i.e., sialylated in either the α1,3 branching or the α1,6 branching).

[0047] In some embodiments, the polypeptides are derived from plasma, for example, human plasma. In certain embodiments, the polypeptides are overwhelmingly IgG polypeptides (e.g., IgG1, IgG2, IgG3, or IgG4, or mixtures thereof), but trace amounts of other polypeptides containing trace amounts of other immunoglobulin subclasses may also be present.

[0048] As used herein, the term “antibody” means a polypeptide comprising at least one immunoglobulin variable region, e.g., an immunoglobulin variable domain or an amino acid sequence providing an immunoglobulin variable domain sequence. For example, an antibody may have a heavy (H) chain variable region (V in this specification). H (abbreviated as) and light (L) chain variable region (V in this specification) L It may include (abbreviated as ). In another example, an antibody contains two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab, F(ab')2, Fd, Fv, and dAb fragments), as well as intact immunoglobulins of complete antibodies, e.g., types IgA, IgG, IgE, IgD, IgM (and their subtypes). The light chain of an immunoglobulin may be of kappa or lambda type.

[0049] As used herein, the term “constant region” refers to a polypeptide corresponding to or derived from one or more constant region immunoglobulin domains of an antibody. A constant region may include any or all of the following immunoglobulin domains: H 1 domain, hinge region, C H 2 domains, C H 3 domains (derived from IgA, IgD, IgG, IgE, or IgM), and C H 4 domains (derived from IgE or IgM).

[0050] As used herein, the term “Fc region” refers to a dimer of two “Fc polypeptides,” where each “Fc polypeptide” contains the constant region of the antibody excluding the first constant region immunoglobulin domain. In some embodiments, the “Fc region” comprises two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. The “Fc polypeptide” refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may include some or all of the flexible hinge present at the N-terminus of these domains. In the case of IgG, the “Fc polypeptide” includes the immunoglobulin domains Cgamma2 (Cgamma2, Cγ2) and Cgamma3 (Cgamma3, Cγ3), and the lower part of the hinge between Cgamma1 (Cgamma1, Cγ1) and Cγ2. While the boundaries of Fc polypeptides can vary, human IgG heavy chain Fc polypeptides are typically defined to include residues starting at P232 and extending to their carboxyl terminus, following the EU system (Edelman et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)). In the case of IgA, the Fc polypeptide includes the immunoglobulin domains C alpha 2 (Cα2) and C alpha 3 (Cα3), as well as the lower part of the hinge between C alpha 1 (Cα1) and Cα2. The Fc region may be synthetic, recombinant, or generated from natural sources such as IVIg.

[0051] As used herein, “glycan” is a sugar and may be a monomer or polymer of sugar residues, such as at least three sugars, and may be linear or branched. “Glycan” may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo N-acetylglucosamine, etc.). The term “glycan” includes homopolymers and heteropolymers of sugar residues. The term “glycan” also includes the glycan component of complex carbohydrates (e.g., polypeptides, glycolipids, proteoglycans, etc.). The term also includes free glycans, which include glycans that have been cleaved or otherwise released from complex carbohydrates.

[0052] As used herein, the term “glycoprotein” refers to a protein containing a peptide backbone covalently linked to one or more sugar moieties (i.e., glycans). Sugar moieties may be in the form of monosaccharides, disaccharides, oligosaccharides, and / or polysaccharides. Sugar moieties may contain a single unbranched sugar residue or one or more branched chains. Glycoproteins may contain O-linked sugar moieties and / or N-linked sugar moieties.

[0053] As used herein, “IVIg” is a pooled polyvalent IgG preparation containing all four IgG subgroups, extracted from the plasma of at least 1,000 human donors. IVIg is approved as a plasma protein replacement therapy for immunocompromised patients. The level of IVIg Fc glycansialylation varies among IVIg preparations but is generally less than 20%. The level of disialylation is generally much lower than 20%. As used herein, the term “derived from IVIg” refers to polypeptides obtained from the manipulation of IVIg. For example, polypeptides purified from IVIg (e.g., enriched with sialylated IgG or modified IVIg (e.g., enzymatically sialylated IVIg IgG)).

[0054] As used herein, “N-glycosylation site of Fc polypeptide” refers to an amino acid residue within the Fc polypeptide to which a glycan is N-linked. In some embodiments, the Fc region contains a dimer of the Fc polypeptide, and the Fc region contains one or two N-glycosylation sites on each Fc polypeptide.

[0055] As used herein, "percentage of branched glycans (%)" refers to the number of moles of glycan X relative to the total number of moles of glycans present, where X represents the glycan in question.

[0056] The terms “pharmaceutically effective amount” or “therapeutably effective amount” refer to an amount (e.g., dose) that is effective in treating a patient with any of the disorders or conditions described herein. It should also be understood that “pharmaceutically effective amount” may be interpreted herein as an amount that, when administered alone or in combination with other therapeutic agents, or when taken in any dose or route, produces the desired therapeutic effect.

[0057] "Pharmaceutical preparations" and "pharmaceutical products" may be included in a kit containing a preparation or product and instructions for use.

[0058] "Pharmaceutical formulations" and "pharmaceutical products" generally refer to compositions in which a final predetermined level of sialylation is achieved and which are free of process impurities. Therefore, "pharmaceutical formulations" and "pharmaceutical products" are substantially free of ST6Gal1 and / or sialic acid donors (e.g., cytidine 5'-monophosphone-N-acetylneuraminic acid) or their by-products (e.g., cytidine 5'-monophosphate).

[0059] "Pharmaceutical preparations" and "pharmaceutical products" generally do not substantially contain cells that produce glycoproteins (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA) in the case of recombinants.

[0060] "Purified" (or "isolated") refers to a polynucleotide or polypeptide that is removed or separated from other components present in the natural environment. For example, an isolated polypeptide is one that has been isolated from other components of the cell in which it was produced (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA). An isolated polynucleotide is one that has been isolated from other nuclear components (e.g., histones) and / or upstream or downstream nucleic acids. An isolated polynucleotide or polypeptide does not have to contain 60%, or at least 75%, or at least 90%, or at least 95% of the other components present in the natural environment of the indicated polynucleotide or polypeptide.

[0061] As used herein, the term "sialylated" refers to a glycan having a terminal sialic acid. The term "monosialized" refers to a branched glycan having, for example, one terminal sialic acid in the α1,3 branch or α1,6 branch. The term "disialized" refers to a branched glycan having terminal sialic acids in both arms, for example, the α1,3 arm and the α1,6 arm. [Brief explanation of the drawing]

[0062] [Figure 1] This shows a short branched core oligosaccharide containing two N-acetylglucosamine and three mannose residues. One of the branches is referred to as the "α1,3 arm" in the art, and the second branch is referred to as the "α1,6 arm." Square: N-acetylglucosamine, dark gray circle: mannose, light gray circle: galactose, rhombus: N-acetylneuraminic acid, triangle: fucose. [Figure 2] This diagram shows common Fc glycans found in IVIg. Squares: N-acetylglucosamine, dark gray circles: mannose, light gray circles: galactose, diamonds: N-acetylneuraminic acid, triangles: fucose. [Figure 3]This diagram illustrates how immunoglobulins, such as IgG antibodies, can be sialylated by performing a galactosylation step followed by a sialylation step. Squares: N-acetylglucosamine, dark gray circles: mannose, light gray circles: galactose, diamonds: N-acetylneuraminic acid, triangles: fucose. [Figure 4] The reaction products of typical IgG-Fc glycan profiles from reactions starting with IVIg are shown. The left figure is a schematic diagram of the enzymatic sialylation reaction to convert IgG to hsIgG, and the right figure shows the IgG Fc glycan profiles of the starting IVIg and hsIgG. The bars, from left to right, correspond to IgG1, IgG2 / 3, and IgG3 / 4, respectively. [Figure 5] This shows the level of A2F formation resulting from the sialylation of Fc-containing proteins using various buffers at different pH levels. [Figure 6] This shows the level of 1,6-A1F formation resulting from the sialylation of Fc-containing proteins using various buffers at different pH levels. [Figure 7A] This shows the effect of high MnCl2 concentrations on the galactosylation and sialylation of IVIg. The bars, from left to right, represent G1F+NeuAc;G1+NeuAc. [Figure 7B] This graph shows the effect of high MnCl2 concentrations on the galactosylation and sialylation of IVIg. The bars, from left to right, represent 5 mM; 10 mM; 20 mM; 40 mM; and 61 mM. [Figure 8] This demonstrates the effect of high MnCl2 concentrations on the disialization of IVIg. [Figure 9] This shows the effect of MnCl2 concentrations below 10 mM on the galactosylation of IVIg (grouped by MnCl2 concentration). [Figure 10] This shows the effect of MnCl2 concentrations below 10 mM on the galactosylation of IVIg, grouped by time. [Figure 11] This shows the effect of salt on IgG1 galactosylation by glycopeptide LC-MS. [Figure 12]This shows the effect of salt on IgG1 sialylation by glycopeptide LC-MS. [Figure 13] This study demonstrates the effect of salt on IgG2 / 3 galactosylation by glycopeptide LC-MS. [Figure 14] This study demonstrates the effect of salt on IgG2 / 3 sialylation by glycopeptide LC-MS. [Figure 15] This shows the effect of salt on IgG3 / 4 galactosylation by glycopeptide LC-MS. [Figure 16] This shows the effect of salt on IgG3 / 4 sialylation by glycopeptide LC-MS. [Figure 17] This describes a method for converting UDP-Gal to UMP and UDP. [Figure 18] This study demonstrates that nonspecific degradation of UDP-Gal can be detected during galactosylation of IVIg. [Figure 19] This study demonstrates that nonspecific degradation of UDP-Gal can be detected during galactosylation of IVIg. [Modes for carrying out the invention]

[0063] Antibodies are glycosylated within the constant region of the heavy chain and at conserved locations in the Fab domain. For example, human IgG antibodies have a single N-linked glycosylation site at Asn297 in the CH2 domain. Each antibody isotype has different N-linked carbohydrate structures in the constant region. In the case of human IgG, the core oligosaccharide typically consists of GlcNAc2Man3GlcNAc with a different number of outer residues. Variations between individual IgGs can arise through the binding of galactose and / or galactose-sialic acid to one or both of the terminal GlcNAc, or through the binding of a third GlcNAc arm (bisected GlcNAc).

[0064] This disclosure partially encompasses a method for preparing an immunoglobulin (e.g., human IgG) having an Fc region having a specific level of branched glycans sialylated in both arms of the branched glycan (e.g., via NeuAc-α2,6-Gal terminal linkage). The level may be measured for individual Fc regions (e.g., the number of branched glycans sialylated in the α1,3 arms, α1,6 arms, or both of the branched glycan in the Fc region) or for the overall composition of the polypeptide preparation (e.g., the number or percentage of branched glycans sialylated in the α1,3 arms, α1,6 arms, or both of the branched glycan in the Fc region of the polypeptide preparation).

[0065] Naturally derived polypeptides that can be used to prepare hypersialylated IgG include, for example, IgG in human serum (specific human serum pooled from more than 1,000 donors), intravenous immunoglobulin (IVIg), polypeptides derived from IVIg (e.g., polypeptides purified from IVIg with concentrated sialylated IgG), or modified IVIg (e.g., enzymatically sialylated IVIg IgG).

[0066] N-linked oligosaccharide chains are added to proteins within the lumen of the endoplasmic reticulum. Specifically, an initial oligosaccharide (typically 14-saccharide) is added to the amino group on the side chain of an asparagine residue contained within the Asn-X-Ser / Thr target consensus sequence, where X can be any amino acid other than proline. The structure of this initial oligosaccharide is common to most eukaryotes and contains three glucose, nine mannose, and two N-acetylglucosamine residues. This initial oligosaccharide chain can be trimmed by a specific glycosidase enzyme in the endoplasmic reticulum to obtain a short branched core oligosaccharide consisting of two N-acetylglucosamine and three mannose residues. One of the branches is referred to in the art as the "α1,3 arm," as shown in Figure 1, and the second branch is referred to as the "α1,6 arm."

[0067] N-glycans can be subdivided into three distinct groups called "high-mannose type," "hybrid type," and "complex type," and a common pentasaccharide core (Man(α1,6)-(Man(α1,3))-Man(β1,4)-GlcpNAc(β1,4)-GlcpNAc(β1,N)-Asn) arises in all three groups.

[0068] Figure 2 shows more common Fc glycans present in IVIg.

[0069] Additionally or alternatively, one or more monosaccharide units of N-acetylglucosamine may be added to the coremannose subunit to form a "complex glycan." Adding galactose to the N-acetylglucosamine subunit, and adding a sialic acid subunit to the galactose subunit, may result in a chain terminated by either sialic acid, galactose, or an N-acetylglucosamine residue. Additionally, a fucose residue may be added to the N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by a specific glycosyltransferase.

[0070] "Hybrid glycans" possess characteristics of both high-mannose and complex glycans. For example, one branch of a hybrid glycan may contain primarily or exclusively mannose residues, while another branch may contain N-acetylglucosamine, sialic acid, galactose sugars, and / or fucose sugars.

[0071] Sialic acids are a family of nine-carbon monosaccharides with heterocyclic structures. They possess a negative charge via a carboxylic acid group attached to the ring, as well as other chemical embellishments including N-acetyl and N-glycolyl groups. The two main types of sialic acid residues found in polypeptides produced in mammalian expression systems are N-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). These typically arise as terminal structures attached to galactose (Gal) residues at the non-reducing ends of both N- and O-linked glycans. The glycosidic bond configuration for these sialic acid groups can be either α2,3 or α2,6.

[0072] The Fc region is glycosylated at a conserved N-linked glycosylation site. For example, each heavy chain of an IgG antibody has a single N-linked glycosylation site at Asn297 in the CH2 domain. IgA antibodies have N-linked glycosylation sites in the CH2 and CH3 domains, IgE antibodies have N-linked glycosylation sites in the CH3 domain, and IgM antibodies have N-linked glycosylation sites in the CH1, CH2, CH3, and CH4 domains.

[0073] Each antibody isotype has different N-linked carbohydrate structures in its constant region. For example, IgG has a single N-linked bisected carbohydrate at Asn297 of the CH2 domain in each Fc polypeptide of the Fc region, which also contains the C1q and FcγR binding sites. In human IgG, the core oligosaccharide typically consists of GlcNAc2Man3GlcNAc with a different number of outer residues. Variations between individual IgGs can arise via galactose and / or galactose-sialic acid binding in one or both of the terminal GlcNAc, or via the binding of a third GlcNAc arm (bisected GlcNAc).

[0074] Immunoglobulins, such as IgG antibodies, can be sialylated by a galactosylation step followed by a sialylation step. β-1,4-galactosyltransferase 1 (B4GalT) is a type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5'-diphosphoseglactone ([[(2)R,3S,4R,5R)-5-(2,4-dioxapyrimidine-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]hydrogen phosphate, UDP-Gal) to GlcNAc as a β-1,4 linkage. Alpha-2,6-sialyltransferase 1 (ST6) is a type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5'-monophospho-Nacetylneuraminic acid (((2R,4S,5R,6R)-5-acetamido-2-[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidine-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-4-hydroxy-6-(1,2,3-trihydroxypropyl)oxan-2-carboxylic acid, CMP-NANA, or CMP-sialic acid) to Gal via an α-2,6 linkage. The reaction generally proceeds as shown in Figure 3.

[0075] The glycans of polypeptides can be evaluated using any method known in the art. For example, the sialylation of a glycan composition (e.g., the level of sialylated branched glycans at α1,3 branching and / or α1,6 branching) can be characterized using the method described in International Publication No. 2014 / 179601.

[0076] In some embodiments of the hsIgG compositions prepared by the methods described herein, at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans in the Fc domain have sialic acid in both the α1,3 arms and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages. Furthermore, in some embodiments, at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in the Fab domain have sialic acid in both the α1,3 arms and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages. Overall, in some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans have sialic acid in both the α1,3 arms and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages.

[0077] In some embodiments, the hsIgG composition prepared by the method described herein has at least 50%, 55%, 60%, 65%, 70%, or 75% of the branched glycans in the Fc domain having sialic acid in both the α1,3 and α1,6 arms.

[0078] enzyme Enzyme activity As described herein, 1 U of B4GlaT is equivalent to the formation of 1 nmol of Gal-GlcNAc (also called LacNAc) by the transfer of UDP-Gal to GlcNAc per minute.

[0079] As described herein, 1 U of ST6Gal1 is equivalent to the formation of 1 nmol of NeuAc-Gal-GlcNAc (also known as Sa-LacNAc) by the transfer of NeuAc from CMP-NANA to Gal-GlcNAc (LacNAc) per minute.

[0080] Galactosyltransferase Beta-1,4-galactosyltransferase (B4GalT), such as human B4GalT, such as human B4Galt1, and its orthologues, mutants, and variants are suitable for use in the methods herein, together with fusion proteins and polypeptides containing them. Beta-1,4-galactosyltransferase 1 (B4GalT) is a type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5'-diphosphosegalactose (UDP-Gal) to GlcNAc as a β-1,4 linkage. B4Galt1 is one of seven β-1,4-galactosyltransferase (β4GalT) genes, each encoding a type II membrane-bound glycoprotein that appears to have exclusive specificity to the donor substrate UDP-galactose. It is all the translocated galactoses in the β1,4 linkage to the analogous acceptor sugars: GlcNAc, Glc, and Xyl. B4Galt1 adds galactose to an N-acetylglucosamine residue at either the non-reducing end of a monosaccharide or glycoprotein carbohydrate chain. B4GalT1 is also called GGTB2. Table 1 lists four alternative transcripts (NCBI gene ID 2683) encoding the four isoforms of B4GALT1.

[0081] [Table 1]

[0082] [Table 2]

[0083] [Table 3]

[0084] [Table 4]

[0085] The soluble form of B4GalT1 is induced from the membrane form by proteolytic treatment. The cleavage site is located at positions 77-78 of B4GALT1 isoform 1 (SEQ ID NO: 5).

[0086] In some embodiments, one or more amino acids of B4GALT1 corresponding to amino acids 113, 130, 172, 243, 250, 262, 310, 343, or 355 of B4GALT1 isoform 1 (SEQ ID NO: 5) are conserved with respect to (SEQ ID NO: 5).

[0087] In some embodiments, the enzyme is, for example, the enzymatically active portion of B4GalT1. In some embodiments, the enzyme is the enzymatically active portion of B4GALT1 isoform 1 (SEQ ID NO: 5), or an ortholog, mutant, or variant of SEQ ID NO: 5. In some embodiments, the enzyme is the enzymatically active portion of B4GALT1 isoform 2 (SEQ ID NO: 6), or an ortholog, mutant, or variant of SEQ ID NO: 6. In some embodiments, the enzyme is the enzymatically active portion of B4GALT1 isoform 3 (SEQ ID NO: 7), or an ortholog, mutant, or variant of SEQ ID NO: 7. In some embodiments, the enzyme is the enzymatically active portion of B4GALT1 isoform 4 (SEQ ID NO: 8), or an ortholog, mutant, or variant of SEQ ID NO: 8.

[0088] In some embodiments, the enzymatically active portion of B4GalT1 does not include a cytoplasmic domain, e.g., SEQ ID NO: 9. In some embodiments, the enzymatically active portion of B4GalT1 does not include a transmembrane domain, e.g., SEQ ID NO: 10. In some embodiments, the enzymatically active portion of B4GalT1 does not include a cytoplasmic domain, e.g., SEQ ID NO: 9, nor a transmembrane domain, e.g., SEQ ID NO: 10.

[0089] In some embodiments, the enzymatically active portion of B4GalT1 includes all or part of a luminal domain, such as SEQ ID NO: 11, or its ortholog, variant, or variant.

[0090] In some embodiments, the enzymatically active portion of B4GalT1 comprises amino acids 109-398 of SEQ ID NO: 5, or its orthologue, mutant, or variant. In some embodiments, the enzymatically active portion of B4GalT1 consists of SEQ ID NO: 5, or its orthologue, mutant, or variant.

[0091] A preferred functional portion of B4GalT1 may contain, or consist of, an amino acid sequence that is at least 80% (85%, 90%, 95%, 98%, or 100%) identical to that of SEQ ID NO: 12.

[0092] Furthermore, amino acid sequences that are at least 80% (85%, 90%, 95%, 98%, or 100%) identical to SEQ ID NO: 13, or that consist of such amino acid sequences, are also suitable for use in the method described herein.

[0093] sialylation enzymes ST6, e.g., ST6Gal1, e.g., the enzymatically active portion of human ST6Gal1, as well as its orthologues, mutants, and variants, are suitable for use in the methods described herein, together with fusion proteins and polypeptides containing it. Alpha-2,6-sialyltransferase 1 (ST6) is a type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) to Gal as an α-2,6 linkage. ST6Gal1 is also called ST6N or SIAT1. Four alternative transcripts (NCBI gene ID 6480) encoding two isoforms of ST6GAL1 are listed in Table 1.

[0094] [Table 5]

[0095] [Table 6]

[0096] [Table 7]

[0097] [Table 8]

[0098] The soluble form of ST6Gal1 is induced from the membrane form by proteolytic treatment.

[0099] In some embodiments, one or more amino acids of ST6Gal1 corresponding to amino acids 142, 149, 161, 184, 189, 212, 233, 335, 353, 354, 364, 365, 369, 370, 376, or 406 of ST6Gal1 isoform a (SEQ ID NO: 14) are conserved with respect to SEQ ID NO: 14.

[0100] Furthermore, for example, the enzymatically active portion of ST6Gal1 is also provided herein. In some embodiments, the enzyme is the enzymatically active portion of ST6Gal1 isoform a (SEQ ID NO: 14), or an ortholog, variant, or variant of SEQ ID NO: 14. In some embodiments, the enzyme is the enzymatically active portion of ST6Gal1 isoform b (SEQ ID NO: 15), or an ortholog, variant, or variant of SEQ ID NO: 15.

[0101] In some embodiments, the enzymatically active portion of ST6Gal1 does not include a cytoplasmic domain, e.g., SEQ ID NO: 16. In some embodiments, the enzymatically active portion of ST6Gal1 does not include a transmembrane domain, e.g., SEQ ID NO: 17. In some embodiments, the enzymatically active portion of ST6Gal1 does not include either a cytoplasmic domain, e.g., SEQ ID NO: 16, or a transmembrane domain, e.g., SEQ ID NO: 17.

[0102] In some embodiments, the enzymatically active portion of ST6Gal1 includes all or part of a luminal domain, such as SEQ ID NO: 18, or its orthologue, variant, or variant.

[0103] In some embodiments, the enzymatically active portion of ST6Gal1 comprises amino acids 87-406 of SEQ ID NO: 14 (SEQ ID NO: 19), or its orthologue, mutant, or variant. In some embodiments, the enzymatically active portion of ST6Gal1 consists of SEQ ID NO: 19, or its orthologue, mutant, or variant.

[0104] A preferred functional portion of ST6Gal1 may include, or consist of, an amino acid sequence that is at least 80% (85%, 90%, 95%, 98%, or 100%) identical to SEQ ID NO: 19.

[0105] In some embodiments, ST6Gal1 includes or consists of SEQ ID NO: 19, the portion of SEQ ID NO: 19 from amino acids 4 to 320, or the portion of SEQ ID NO: 19 from amino acids 5 to 320.

[0106] Furthermore, amino acid sequences that are at least 80% (85%, 90%, 95%, 98%, or 100%) identical to SEQ ID NO: 20, or derived from such amino acid sequences, are also suitable for use in the method described herein.

[0107] antibody The methods described herein include galactosylation and sialylation of antibodies. Suitable antibodies include, for example, IgG antibodies. Antibodies, such as IgG antibodies, can be pooled. For example, pooled IgG antibodies include IVIg.

[0108] In some embodiments, the IgG antibody comprises IgG antibodies isolated from at least 1000 donors.

[0109] In some embodiments, at least 50%, 55%, 60%, 65%, or 70% w / w of the IgG antibody is IgG1 antibody.

[0110] In some embodiments, at least 90% of the donor subjects have been exposed to the virus.

[0111] In some embodiments, the methods described herein include providing a mixture of IgG antibodies. In some embodiments, providing a mixture of IgG antibodies includes (a) providing pooled plasma from at least 1000 human subjects, and (b) isolating a mixture of IgG antibodies from the pooled plasma. In some embodiments, the mixture of IgG antibodies is isolated from intravenous immunoglobulin. In some embodiments, the mixture of IgG antibodies is intravenous immunoglobulin. In some embodiments, the step of isolating a mixture of IgG antibodies from pooled plasma includes ethanol precipitation or caprylic acid (also called octanoic acid) precipitation. In some embodiments, the step of isolating a mixture of IgG antibodies from pooled plasma includes binding the IgG antibodies to an ion-exchange column and eluting the IgG antibodies from the ion-exchange column.

[0112] In some embodiments, an antibody, such as the antibody described herein, such as IgG, such as pooled IgG, such as IVIg, is provided as part of the solution. In some embodiments, the concentration of the antibody, such as the antibody described herein, such as pooled IgG, such as IVIg, is about 100 mg / mL to about 200 mg / mL. In some embodiments, the concentration of the antibody is 150 mg / mL or about 150 mg / mL.

[0113] In some embodiments, the solution consists of or includes an antibody, such as the antibody described herein, such as IgG, such as pooled IgG, such as IVIg, and a buffer. In some embodiments, the buffer is selected from the group consisting of BIS-TRIS, MOPS, MES, PIPES, BES, MOPSO, TEA, POPSO, EPPS, and combinations thereof.

[0114] In some embodiments, the solution consists of or includes an antibody, for example, the antibody described herein, for example, IgG, for example, pooled IgG, for example, IVIg, and BIS-TRIS buffer. In some embodiments, the solution consists of or includes an antibody in 50 mM BIS-TRIS buffer.

[0115] In some embodiments, the solution consists of or includes an antibody, for example, an antibody described herein, for example, IgG, for example, pooled IgG, for example, IVIg, and a BIS-TRIS buffer, for example, 50 mM BIS-TRIS buffer at approximately pH 6.8 to approximately pH 7.4. In some embodiments, the solution consists of an antibody, for example, an antibody described herein, for example, IgG, for example, pooled IgG, for example, IVIg, and a BIS-TRIS buffer, for example, at approximately pH 6.8 to approximately pH 7.3, approximately pH 6.8 to approximately pH 7.2, approximately pH 6.8 to approximately pH 7.1, approximately pH 6.8 to approximately pH 7.0, approximately pH 6.8 to approximately pH 6.9, approximately pH 6.9 to approximately pH 7.4, approximately pH 7.3 to approximately pH 7.2, approximately pH 6. The solution consists of or includes 50 mM BIS-TRIS buffer at pH 9 to approximately pH 7.1, approximately pH 6.9 to approximately pH 7.0, approximately pH 7.0 to approximately pH 7.4, approximately pH 7.0 to approximately pH 7.3, approximately pH 7.0 to approximately pH 7.2, approximately pH 7.0 to approximately pH 7.1, approximately pH 7.1 to approximately pH 7.4, approximately pH 7.1 to approximately pH 7.3, approximately pH 7.1 to approximately pH 7.2, approximately pH 7.2 to approximately pH 7.4, or approximately pH 7.3 to approximately pH 7.4. In some embodiments, the solution consists of or includes an antibody, for example, the antibody described herein, for example, IgG, for example, pooled IgG, for example, IVIg, and a BIS-TRIS buffer, for example, 50 mM BIS-TRIS buffer at approximately pH 7.3.

[0116] Enzymatic galactosylation and sialylation The methods described herein may include a galactosylation step. An exemplary galactosylation reaction is shown in Figure 3. Accordingly, this specification provides a composition (galactosylation mixture) comprising an antibody, for example the antibody described herein; a galactosylating enzyme, for example the galactosylating enzyme described herein, for example B4GalT or its enzymatically active portion or variant; UDP-gal or a salt thereof; a buffer, for example the buffer described herein, for example BIS-TRIS buffer; and optionally MnCl2; and a method for galactosylating an antibody, for example the antibody described herein, by incubating the composition under conditions effective for galactosylating the antibody, as described herein, for example, to produce a galactosylated antibody.

[0117] The methods described herein may include a sialylation step. An exemplary sialylation reaction is shown in Figure 3. Accordingly, this specification provides a composition (sialylation reaction mixture) comprising, for example, a galactosylated antibody as described herein, a sialylation enzyme, for example, a sialylation enzyme as described herein, for example, ST6Gal1, or an enzymatically active portion or variant thereof, CMP-NANA or a salt thereof, a buffer, for example, a buffer as described herein, for example, BIS-TRIS buffer, and optionally MnCl2, and a method for sialylation, for example, of an antibody as described herein, e.g., hypersialylation, of an antibody.

[0118] In some embodiments, the galactosylation and sialylation steps are carried out sequentially in the same reaction mixture; that is, the galactosylation reaction mixture becomes a sialylation reaction mixture upon addition of sialylation enzyme and CMP-NANA or a salt thereof. In some embodiments, the galactosylation reaction mixture is not filtered, fractionated, or purified before the sialylation step. In some embodiments, the galactosylation and sialylation steps are carried out separately, for example, by providing a pre-galactosylated antibody, but these antibodies may be processed (e.g., filtered, fractionated, or purified) and / or stored before the sialylation step.

[0119] Therefore, the methods described herein may also include sequential galactosylation and sialylation steps. An exemplary galactosylation and sialylation reaction is shown in Figure 3. Accordingly, this specification provides a composition (galactosylation reaction mixture) comprising, for example, an antibody as described herein, a galactosylating enzyme, for example, a galactosylating enzyme as described herein, for example, B4GalT or its enzymatically active portion or variant, UDP-gal or a salt thereof, a buffer, for example, a buffer as described herein, for example, BIS-TRIS buffer, and optionally MnCl2; b) incubating the composition under conditions effective for galactosylating the antibody, as described herein, for example; c) adding a sialing enzyme, for example, a sialing enzyme as described herein, for example, ST6Gal1 or its enzymatically active portion or variant and CMP-NANA or a salt thereof to the galactosylation reaction mixture to generate a sialing reaction reaction; and d) incubating the composition under conditions effective for sialing the galactosylating antibody, as described herein, for example.

[0120] This specification also provides a composition comprising, for example, a highly sialylated antibody, for example, an antibody as described herein, for example, an antibody as described herein, a galactosylase, for example, a galactosylase as described herein, for example, B4GalT or its enzymatically active moiety or variant, UDP-gal or a salt thereof, a buffer, for example, a buffer as described herein, for example, BIS-TRIS buffer, a sialyase, for example, a sialyase as described herein, for example, ST6Gal1 or its enzymatically active moiety or variant, CMP-NANA or a salt thereof, and optionally MnCl2; and d) a method for galactosylating and sialylation by incubating the composition under conditions effective for galactosylating and acyling the antibody, for example, as described herein.

[0121] In some embodiments, one or more components of one or more reaction mixtures are replenished during incubation. That is, the reaction mixture may contain a certain amount of components at the start of the reaction (which may change during the reaction), but additional amounts of components may be replenished during the reaction.

[0122] In some embodiments, the galactosylation reaction mixture contains about 50 to about 200 mg / mL of antibody, for example, the antibody described herein, for example, IgG, for example, pooled IgG, for example, IVIg. In some embodiments, the galactosylation reaction mixture contains about 50 to about 200, about 50 to about 150, about 50 to about 100, about 100 to about 200, about 100 to about 200, about 100 to about 150, or about 150 to about 200 mg / mL of antibody, for example, the antibody described herein, for example, IgG, for example, pooled IgG, for example, IVIg.

[0123] In some embodiments, the galactosylation reaction mixture includes an antibody at a concentration of 50 mg / mL or more, 75 mg / mL or more, 100 mg / mL or more, 125 mg / mL or more, 150 mg / mL or more, or 200 mg / mL or more, such as the antibodies described herein, such as IgG, such as pooled IgG, such as IVIg.

[0124] In some embodiments, the galactosylation reaction mixture contains about 6.0 to about 15.0 U of galactosylase per gram of antibody. In some embodiments, the galactosylation reaction mixture contains about 7.0 to about 9.0 U of galactosylase per gram of antibody. In some embodiments, the galactosylation reaction mixture contains about 7.2 to about 8.8 U of galactosylase per gram of antibody. In some embodiments, the galactosylation reaction mixture contains 7.5 or about 7.5 U of galactosylase per gram of antibody. In some embodiments, the galactosylation reaction mixture contains 8.0 or about 8.0 U of galactosylase per gram of antibody.

[0125] In some embodiments, the galactosylation reaction mixture is supplemented with about 6.0 to about 15.0 U of galactosylase per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with about 7.0 to about 9.0 U of galactosylase per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with about 7.2 to about 8.8 U of galactosylase per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with 7.5 or about 7.5 U of galactosylase per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with 8.0 or about 8.0 U of galactosylase per gram of antibody.

[0126] In some embodiments, the galactosylation reaction mixture contains about 0.030 to about 0.050 mmol of UDP-gal or its salt per gram of antibody. In some embodiments, the galactosylation reaction mixture contains about 0.038 to about 0.046 mmol of UDP-gal or its salt per gram of antibody. In some embodiments, the galactosylation reaction mixture contains 0.038 or about 0.038 mmol of UDP-gal or its salt per gram of antibody. In some embodiments, the galactosylation reaction mixture contains 0.042 or about 0.042 mmol of UDP-gal or its salt per gram of antibody.

[0127] In some embodiments, the galactosylation reaction mixture is supplemented with about 0.030 to about 0.050 mmol of UDP-gal or its salt per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with about 0.038 to about 0.046 mmol of UDP-gal or its salt per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with 0.038 or about 0.038 mmol of UDP-gal or its salt per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with 0.042 or about 0.042 mmol of UDP-gal per gram of antibody.

[0128] In some embodiments, the sialylation reaction mixture contains about 14.0 to about 20.0 U of sialyase per gram of antibody. In some embodiments, the sialylation reaction mixture contains about 17.1 to about 18.9 U of sialyase per gram of antibody. In some embodiments, the sialylation reaction mixture contains 15.8 or about 15.8 U of sialyase per gram of antibody. In some embodiments, the sialylation reaction mixture contains 18.0 or about 18.0 U of sialyase per gram of antibody.

[0129] In some embodiments, the sialylation reaction mixture is supplemented with about 14.0 to about 20.0 U of sialyase per gram of antibody. In some embodiments, the sialylation reaction mixture is supplemented with about 17.1 to about 18.9 U of sialyase per gram of antibody. In some embodiments, the sialylation reaction mixture is supplemented with 15.8 or about 15.8 U of sialyase per gram of antibody. In some embodiments, the sialylation reaction mixture is supplemented with 18.0 or about 18.0 U of sialyase per gram of antibody.

[0130] In some embodiments, the sialylation reaction mixture contains about 0.1 to about 0.3 mmol of CMP-NANA or a salt thereof per gram of antibody. In some embodiments, the sialylation reaction mixture contains about 0.1425 to about 0.1575 mmol of CMP-NANA or a salt thereof per gram of antibody. In some embodiments, the sialylation reaction mixture contains 0.220 or about 0.220 mmol of CMP-NANA or a salt thereof per gram of antibody. In some embodiments, the sialylation reaction mixture contains 0.150 mmol or about 0.150 mmol of CMP-NANA or a salt thereof per gram of antibody.

[0131] In some embodiments, about 0.01 to about 0.3 mmol of CMP-NANA or a salt thereof per gram of antibody is added to the sialylation reaction mixture at the start of the reaction. In some embodiments, about 0.01425 to about 0.1575 mmol of CMP-NANA or a salt thereof per gram of antibody is added to the sialylation reaction mixture at the start of the reaction.

[0132] In some embodiments, the sialylation reaction mixture is supplemented with about 0.01 to about 0.3 mmol of CMP-NANA or its salt per gram of antibody. In some embodiments, the sialylation reaction mixture is supplemented with about 0.01425 to about 0.1575 mmol of CMP-NANA or its salt per gram of antibody.

[0133] In some embodiments, the total amount of CMP-NANA added to the sialylation reaction mixture is about 0.1 to about 0.3 mmol of CMP-NANA or its salt per gram of antibody. In some embodiments, the total amount of CMP-NANA added to the sialylation reaction mixture is about 0.1425 to about 0.1575 mmol of CMP-NANA or its salt per gram of antibody. In some embodiments, the total amount of CMP-NANA added to the sialylation reaction mixture is 0.220 or about 0.220 mmol of CMP-NANA or its salt per gram of antibody. In some embodiments, the total amount of CMP-NANA added to the sialylation reaction mixture is 0.150 mmol or about 0.150 mmol of CMP-NANA or its salt per gram of antibody.

[0134] In some embodiments, the sialylation reaction mixture is supplemented with CMP-NANA 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the sialylation mixture is supplemented with CMP-NANA less than 7 times.

[0135] In some embodiments, the galactosylation and / or sialylation reaction mixtures each independently contain about 1 to about 20 mM MnCl2. In some embodiments, the galactosylation and / or sialylation reaction mixtures each independently contain about 4.5 to about 5.5 mM MnCl2. In some embodiments, the galactosylation and / or sialylation reaction mixtures each independently contain 7.5 mM or about 7.5 mM MnCl2. In some embodiments, the galactosylation and / or sialylation reaction mixtures each independently contain 5.0 mM or about 5.0 mM MnCl2.

[0136] In some embodiments, the galactosylation and / or sialylation reaction mixture comprises a BIS-TRIS buffer. In some embodiments, the galactosylation and / or sialylation reaction mixture each independently comprises 10 to about 500 mM of BIS-TRIS buffer. In some embodiments, the galactosylation and / or sialylation reaction mixtures each independently contain about 10 to about 400 mM, about 10 to about 300 mM, about 10 to about 300 mM, about 10 to about 200 mM, about 10 to about 100 mM, about 10 to about 50 mM, about 50 to about 500 mM, about 50 to about 400 mM, about 50 to about 300 mM, about 50 to about 200 mM, about 50 to about 100 mM, about 100 to about 500 mM, about 100 to about 400 mM, about 100 to about 300 mM, about 100 to about 200 mM, about 200 to about 500 mM, about 200 to about 400 mM, about 200 to about 300 mM, about 300 to about 500 mM, or about 400 to about 500 mM of BIS-TRIS buffer. In some embodiments, the galactosylation and / or sialylation reaction mixtures are each independently approximately 10 to approximately 100, approximately 10 to approximately 90, approximately 10 to approximately 80, approximately 10 to approximately 70, approximately 10 to approximately 60, approximately 10 to approximately 50, approximately 10 to approximately 40, approximately 10 to approximately 30, approximately 10 to approximately 20, approximately 20 to approximately 100, approximately 20 to approximately 90, approximately 20 to approximately 80, approximately 20 to approximately 70, approximately 20 to approximately 60, approximately 20 to approximately 50, approximately 20 to approximately 40, approximately 20 to approximately 30, approximately 30 to approximately 100, approximately 30 to approximately 90, approximately 30 to approximately 80, approximately 30 to approximately 70, and approximately 30 Includes BIS-TRIS buffer in approximately 60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 mM.

[0137] In some embodiments, the galactosylation and / or sialylation reaction mixtures each independently contain 50 mM or less, 100 mM or less, 150 mM or less, 30 mM or less of sodium chloride, or contain none at all.

[0138] In some embodiments, the buffer for the galactosylation and / or sialation reaction mixture, for example, the Bis-Tris buffer for the galactosylation and / or sialation reaction mixture, each independently contains 50 mM or less, 100 mM or less, 150 mM or less, 30 mM or less of sodium chloride, or contains none at all.

[0139] In some embodiments, the galactosylation and / or sialylation steps are carried out independently at approximately 20 to approximately 50°C. In some embodiments, sialylation is carried out at approximately 20 to approximately 45°C, approximately 20 to approximately 40°C, approximately 20 to approximately 35°C, approximately 20 to approximately 30°C, approximately 20 to approximately 25°C, approximately 25 to approximately 50°C, approximately 25 to approximately 45°C, approximately 25 to approximately 40°C, approximately 25 to approximately 35°C, approximately 25 to approximately 30°C, approximately 30 to approximately 50°C, approximately 30 to approximately 45°C, approximately 30 to approximately 40°C, approximately 30 to approximately 35°C, approximately 35 to approximately 50°C, approximately 35 to approximately 45°C, approximately 35 to approximately 40°C, approximately 40 to approximately 50°C, approximately 40 to approximately 45°C, or approximately 45 to approximately 50°C. In some embodiments, sialylation is carried out at 37°C or approximately 37°C.

[0140] In some embodiments, the galactosylation and / or sialylation steps are carried out independently at approximately pH 5.8 to approximately pH 7.2. In some embodiments, sialylation is carried out at approximately pH 5.8 to approximately pH 7.1, approximately pH 5.8 to approximately pH 7.0, approximately pH 5.8 to approximately pH 6.9, approximately pH 5.8 to approximately pH 6.8, approximately pH 5.8 to approximately pH 6.7, approximately pH 5.8 to approximately pH 6.6, approximately pH 5.8 to approximately pH 6.5, approximately pH 5.8 to approximately pH 6.4, approximately pH 5.8 to approximately pH 6.3, approximately pH 5.8 to approximately pH 6.2, approximately pH 5.8 to approximately pH 6.1, approximately pH 5.8 to approximately pH 6.0, approximately pH 5.8 to approximately pH 5.9, approximately pH 5.9 to approximately pH 7.2, approximately pH 5.9 to approximately pH 7.1, approximately pH 5.9 Approximately pH 7.0, approximately pH 5.9 to approximately pH 6.9, approximately pH 5.9 to approximately pH 6.8, approximately pH 5.9 to approximately pH 6.7, approximately pH 5.9 to approximately pH 6.6, approximately pH 5.9 to approximately pH 6.5, approximately pH 5.9 to approximately pH 6.4, approximately pH 5.9 to approximately pH 6.3, approximately pH 5.9 to approximately pH 6. 2. About pH5.9 to about pH6.0, about pH6.0 to about pH7.2, about pH6.0 to about pH7.1, about pH6.0 to about pH7.0, about pH6.0 to about pH6.9, about pH6.0 to about pH6.8, about pH6.0 to about pH6.7, about pH6.0 to about pH6.6, about pH6 .0 to about pH6.5, about pH6.0 to about pH6.4, about pH6.0 to about pH6.3, about pH6.0 to about pH6.2, about pH5.9 to about pH6.1, about pH6.0 to about pH7.2, about pH6.0 to about pH7.1, about pH6.0 to about pH7.0, about pH6.0 to about p H6.9, approximately pH6.0 to approximately pH6.8, approximately pH6.0 to approximately pH6.7, approximately pH6.0 to approximately pH6.6, approximately pH6.0 to approximately pH6.5, approximately pH6.0 to approximately pH6.4, approximately pH6.0 to approximately pH6.3, approximately pH6.0 to approximately pH6.2, approximately pH6.0 to approximately pH6.1, approximately pH6.1 to about pH7.2, about pH6.1 to about pH7.1, about pH6.1 to about pH7.0, about pH6.1 to about pH6.9, about pH6.1 to about pH6.8, about pH6.1 to about pH6.7, about pH6.1 to about pH6.6, about pH6.1 to about pH6.5, about pH6.1 ~ about pH6.4, about pH6.1 to about pH6.3, about pH6.1 to about pH6.2, about pH6.2 to about pH7.2, about pH6.2 to about pH7.1, about pH6.2 to about pH7.0, about pH6.2 to about pH6.9, about pH6.2 to about pH6.8, about pH6.2 to about pH6.7, about pH6.2 to about pH6.6, about pH6.2 to about pH6.5, about pH6.2 to about pH6.4, about pH6.2 to about pH6.3, about pH6.3 to about pH7.2, about pH6.3 to about pH7.1, about pH6.3 to about pH7.0, about pH6.3 to about pH6.9, about pH6.3 to about pH6.8, about pH6.3 to about pH6.7, about pH6.3 to about pH6.6, about pH6.3 to about pH6.5, about pH6 .3~about pH6.4, about pH6.4~about pH7.2, about pH6.4~about pH7.1, about pH6.4~about pH7.0, about pH6.4~about pH6.9, about pH6.4~about pH6.8, about pH6.4~ About pH6.7, about pH6.4 to about pH6.6, about pH6.4 to about pH6.5, about pH6.5 to about pH7.2, about pH6.5 to about pH7.1, about pH6.5 to about pH7.0, about pH6.5 to about pH 6.9, about pH6.5 to about pH6.8, about pH6.5 to about pH6.7, about pH6.5 to about pH6.6, about pH6.6 to about pH7.2, about pH6.6 to about pH7.1, about pH6.6 to about pH7.0 , about pH6.6 to about pH6.9, about pH6.6 to about pH6.8, about pH6.6 to about pH6.7, about pH6.7 to about pH7.2, about pH6.7 to about pH7.1, about pH6.7 to about pH7.0, about p The experiment will be conducted at pH levels of approximately 6.7 to 6.9, 6.7 to 6.8, 6.8 to 7.2, 6.8 to 7.1, 6.8 to 7.0, 6.8 to 6.9, 6.9 to 7.2, 6.9 to 7.1, 6.9 to 7.0, 7.0 to 7.2, 7.0 to 7.1, or 7.1 to 7.2.

[0141] In some embodiments, the pH of one or more of the reaction mixture is adjusted during galactosylation and / or sialylation to, for example, fall within a preferred range, return to, or nearly return to the starting pH, for example.

[0142] In some embodiments, the galactosylation step is carried out over a period of 60 hours or less, for example, 50 hours or less, 40 hours or less, or preferably 20 hours or less.

[0143] In some embodiments, the galactosylation step is carried out for at least 8, 12, 18, 24, 30, or 40 hours, but not for more than 60 hours.

[0144] In some embodiments, the galactosylation process is carried out over approximately 8, 12, 18, 24, 30, 40, 50, or 60 hours.

[0145] In some embodiments, the sialylation process is carried out over a period of 70 hours or less, for example, 60 hours or less, 50 hours or less, or preferably 40 hours or less.

[0146] In some embodiments, the sialylation process is carried out for at least 8, 12, 18, 24, 30, 40, or 50 hours, but not for more than 70 hours.

[0147] In some embodiments, the sialylation process is carried out over approximately 8, 12, 18, 24, 30, 40, 50, 60, or 70 hours.

[0148] In some embodiments, the total incubation time for galactosylation and sialylation is, for example, 130 hours or less, for example, 120 hours or less, 110 hours or less, 100 hours or less, 90 hours or less, 80 hours or less, preferably 70 hours or less or 60 hours or less, continuously in the same reaction mixture.

[0149] In some embodiments, for example, the total incubation time for galactosylation and sialylation is at least 8, 12, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or 120 hours, but not exceeding 130 hours, for example, in the same reaction mixture consecutively.

[0150] In some embodiments, the total incubation time for galactosylation and sialylation is, for example, about 8, 12, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or 130 hours, consecutively in the same reaction mixture.

[0151] In some embodiments, at least or about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in an antibody, such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0152] In some embodiments, about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans in an antibody, such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0153] In some embodiments, about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycan in the Fab domain of an antibody, such as hsIgG, has sialic acid in both the α1,3 arm and the α1,6 arm, which are connected via NeuAc-α2,6-Gal terminal linkages.

[0154] In some embodiments, about or at least 80% of the branched Fc glycans in IgG have sialic acid in both the α1,3 branching and the α1,6 branching.

[0155] In some embodiments, about or at least 60%, 65%, or 70% of the branched glycans in the Fab domain of an antibody, such as hsIgG, have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages.

[0156] In some embodiments, about or at least 85% of the branched Fc glycans in an antibody, such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0157] In some embodiments, about or at least 60%, 65%, or 70% of the branched glycans in the Fab domain of an antibody, such as hsIgG, have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages.

[0158] In some embodiments, about or at least 90% of the branched Fc glycans in an antibody, such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0159] In some embodiments, about or at least 60%, 65%, or 70% of the branched glycans in the Fab domain of an antibody, such as hsIgG, have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages.

[0160] Exemplary galactosylation and sialylation reactions are shown in the table below.

[0161] [Table 9] [Examples]

[0162] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0163] Example 1: Preparation of hypersialylated IgG IgG in which more than 60% of the entire branched glycan is disialized can be prepared as follows.

[0164] In short, a mixture of IgG antibodies is subjected to sequential enzymatic reactions using β1,4-galactosyltransferase 1 (B4GalT) and α2,6-sialyltransferase (ST6Gal1) enzymes. There is no need to remove B4GalT from the reaction before adding ST6Gal1, nor is there any need to partially or completely purify the product between enzymatic reactions.

[0165] The galactosyltransferase enzyme selectively adds galactose residues to existing asparagine-linked glycans. The resulting galactosylated glycans function as substrates for sialic acid transferase, which selectively adds sialic acid residues to cap the asparagine-linked glycan structure they bind to. Therefore, the entire sialation reaction used two sugar nucleotides (uridine 5'-diphosphogalactose (UDP-Gal)) and cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA)). The latter was periodically replenished to increase the disialylated product relative to the monosialylated product. The reaction included manganese chloride as a cofactor.

[0166] Representative examples of IgG-Fc glycan profiles and reaction products from such reactions starting with IVIg are shown in Figure 4. In Figure 4, the left figure is a schematic diagram of the enzymatic sialylation reaction for converting IgG to hsIgG, and the right figure is the IgG Fc glycan profile of the starting IVIg and hsIgG. In this study, glycan profiles of different IgG subclasses are obtained via glycopeptide mass spectrometry. The peptide sequences used to quantify glycopeptides for different IgG subclasses were IgG1=EEQYNSTYR (SEQ ID NO: 1), IgG2 / 3 EEQFNSTFR (SEQ ID NO: 2), IgG3 / 4 EEQYNSTFR (SEQ ID NO: 3), and EEQFNSTYR (SEQ ID NO: 4).

[0167] Glycan data is shown per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg, the total of all non-sialylated glycans is over 80%, and the total of all sialylated glycans is <20%. For reaction products, the total of all non-sialylated glycans is <20%, and the total of all sialylated glycans is greater than 80%. The nomenclature of the different glycans listed in the glycoprofile uses the Oxford notation for N-linked glycans.

[0168] Example 2: Improvement of sialylation reaction To further improve the disialysis of IgG antibodies, including the disialysis of Fc-domain branched glycans, a wide range of reaction conditions were analyzed. The ST6Ga1-driven sialylation reaction using CMP-NANA as a substrate has properties that make improvement of the reaction difficult, whether evaluated by the overall disialysis level, the time to reach a specific disialysis level, or the amount of enzyme and substrate required to reach a specific overall disialysis level. For example, (a) CMP-NANA is not completely stable and hydrolyzes spontaneously even in the absence of enzymes. (b) ST6Gal1 is thought to catalyze the hydrolysis of CMP-NANA without productive addition to Gal in branched glycans. (c) Cytidine monophosphate (CMP), a byproduct generated by either enzyme addition or CMP-NANA hydrolysis, may act as a competitive inhibitor of ST6Gal1. (d) CMP has been observed to catalyze a reverse enzymatic reaction to remove NeuAc from the newly formed glycan. Therefore, the level of byproducts increases over time, which can lead to a slowdown or even a reversal of the desired sialylation reaction.

[0169] Reaction conditions exist that result in high levels of disialation of IgG antibodies, IVIg, or pooled immunoglobulins on Fc-domain branched glycans. For example, sialylation using ST6Gal1 in MOPS buffer at 37°C and pH 7.4 with relatively high IgG antibody concentrations (e.g., high IVIg concentrations (≧125 mg / mL)) can result in high levels of sialylation of branched glycans, such as branched glycans in the Fc domain, if the sialylation reaction is carried out for a sufficiently long time and CMP-NANA is added during the sialylation process. Nevertheless, it would be desirable to find alternatives that offer other improvements in hsIgG production, such as reducing the substrate or its concentration, shortening the reaction time, or reducing the substrate.

[0170] Adding alkaline phosphatase to remove phosphate from CMP and convert it to non-inhibitory cytidine was considered promising based on the properties of the sialylation reaction. However, this modification yielded only slight benefits under the tested conditions. Variations in the pH of the MOPS buffer did not appear to provide significant benefits under the tested conditions. The addition of various metal ions was also investigated, but did not appear to provide significant benefits under the tested conditions. However, in the process of investigating these variations, it was observed that the addition of TRIS buffer, even as a co-buffer, seemed to provide some benefit. Furthermore, it was observed that a decrease in CMP-NANA concentration may be beneficial under some conditions.

[0171] We searched for various buffers with different pKa values ​​that have some structural similarity to TRIS, as additions to or replacements for MOPS. The buffers tested include those listed in Table 5 below. Sialization was performed on Fc-containing proteins using ST6Gal1 and CMP-NANA.

[0172] [Table 10]

[0173] As shown in Figure 5, the properties of the buffer affected the A2F level (71-85%).

[0174] As shown in Figure 6, the formation of 1,6-A1F varied less across the entire buffer under the tested conditions.

[0175] The sialylation of IVIg was investigated using BIS-TRIS at pH 6.9, TEA at pH 7.5, TEA at pH 8.0, and TRIS at pH 8.0. Furthermore, the effect of various buffers on galactosylation was also investigated, as it may be desirable to use the same buffer for both galactosylation and sialylation, for example, to provide a one-pot reaction. Certain buffers that appeared beneficial for sialylation were observed to be detrimental to galactosylation.

[0176] BIS-TRIS was selected for further testing. Since some of the above tests seemed to suggest that a reduction in CMP-NANA might be beneficial under certain conditions, detailed tests of enzyme and sugar nucleotide excesses and administration regimens were performed using IVIg as a substrate. As part of these tests, the effect of the BIS-TRIS buffer on galactosylation was investigated.

[0177] In the galactosylation of IVIg in BIS-TRIS at pH 6.9, it was found that 50% less B4GalT enzyme could be used compared to when MOPS was used at pH 7.4. When the same amount of UDP-Gal was used, galactosylation was completed within 15 hours. In the subsequent sialylation reaction in BIS-TRIS at pH 6.9, it was found that reducing the total amount of CMP-NANA by 50% dramatically shortened the reaction time (from over 72 hours to 32-33 hours). Tables 6 and 7 below provide examples of improvements observed with BIS-TRIS buffer.

[0178] [Table 11]

[0179] [Table 12]

[0180] Overall, it was found that changing from MOPS pH 7.4 to BIS-TRIS pH 6.9 allowed for a reduction in the amount of enzymes and / or sugar nucleotides used, while simultaneously achieving high levels of sialylation in a significantly shorter time. Therefore, preferred reaction conditions in 50 mM BIS-TRIS (pH 6.9) include galactosylation of an IgG antibody (e.g., pooled IgG antibody, pooled immunoglobulin, or IVIg) as follows: 7.4 mM MnCl2, 38 μmol UDP-Gal / g IgG antibody, and incubation at 37°C for 16–24 hours, followed by 7.5 units of B4GalT / g IgG antibody with sialylation in 7.4 mM MnCl2, 220 μmol CMP-NANA / g IgG antibody (added twice, i.e., half at the start of the reaction and half after 9–10 hours), and 15 units of ST6Gal1 / g IgG antibody with incubation at 37°C for 30–33 hours. The reaction may also be carried out by adding ST6Gal1 and CMP-NANA to the galactosylation reaction. Alternatively, all reactants can be combined at the start, and CMP-NANA can be added as needed.

[0181] Example 3: Enzymatic galactosylation and sialylation for hsIgG production using high-concentration IVIg or IgG antibody Siallylation was performed in two consecutive enzymatic reaction steps using UDP-Gal and CMP-NANA in 50 mM MOPS buffer at pH 7.4. Galactosylation was carried out by reacting IVIg (approximately 135 mg / mL) in 50 mM MOPS buffer at pH 7.4 containing 5-8 mM MnCl2 with 8-15 units of B4GalT / g IVIg and 0.038-0.042 mmol of UDP-Gal / g IVIg. The reaction was allowed to proceed at 37°C for 46-50 hours. Next, 15.8-18 units of ST6Gal / g IVIg and CMP-NANA were added to the reaction, and the concentration of IVIg was adjusted to approximately 120 mg / mL in 50 mM MOPS buffer at pH 7.4. CMP-NANA was added at the start of the sialylation reaction and then added five more times at 8-12 hour intervals over a total reaction time of 70-74 hours at 37°C. The amount of CMP-NANA added was 400 μmol CMP-NANA / g 1VIg. Therefore, each addition was 1 / 6 of the total amount added.

[0182] Next, the reaction mixture was cooled to ambient temperature and diluted with 5x sodium phosphate buffer (PBS) in a 1:1 v / v ratio.

[0183] The total glycans were evaluated for sialylation. Over 97% of the glycans were sialylated, and over 90% were disialylated.

[0184] Example 4: Reaction conditions The galactosylation reaction in the production of hsIgG is relatively simple. However, the sialylation reaction presents several challenges. Firstly, CMP-NANA is not stable and spontaneously hydrolyzes even in the absence of the enzyme. Furthermore, the ST6 enzyme is thought to catalyze the hydrolysis of CMP-NANA without productive addition to the glycan acceptor. Cytidine monophosphate (CMP) is a byproduct generated by either enzymatic addition or CMP-NANA hydrolysis. CMP acts as a competitive inhibitor of ST6. It has also been observed that CMP catalyzes a reverse enzymatic reaction to remove NeuAc from the newly formed glycan. Therefore, over time, as the concentration of CMP-NANA decreases but the byproduct accumulates, the sialylation reaction slows down and reverses. However, this reverse reaction appears far less desirable with BIS-TRIS as a buffer compared to MOPS as a buffer.

[0185] M254 is currently produced using a very high IVIg concentration (approximately 150 mg / mL) in which the high protein concentration improves the reaction rate, by enzymatic sialylation of Fc and Fab glycans with the IVIg formulation in MOPS pH 7.4 buffer at 37°C. The galactosylation step uses an incubation period of 48 hours. To compensate for the above sialylation problem, CMP-NANA is added twice a day (a total of 6 times) over 72 hours, following a single addition of ST6. This is referred to as process 2.0.

[0186] It is desirable to switch to an alternative process, nominally called Process 3.0.0, which is carried out in BIS-TRIS buffer at pH 6.90. Process 3.0.0 uses less B4GalT enzyme and less CMP-NANA, and the total reaction time for both steps is shorter, at 56 hours compared to 120 hours. Therefore, Process 3.0.0 reduces both material costs and manufacturing costs.

[0187] Siallylation of IVIg in BIS-TRIS buffer using process 3.0.0 was performed at two different facilities, both at laboratory scale (≤2g) and at larger scales of 50g and 250g. The degree of disialylation obtained at larger scales using BIS-TRIS buffer (process 3.0.0) was lower than that observed at laboratory scale and lower than that of reactions performed at the same facility using MOPS buffer (process 2.0), but still met the specification of over 80% disialylation (Table 8). Therefore, experiments were conducted to try and understand which reaction conditions had the greatest impact on this difference. While the galactosylation step showed near-ideal results, it is recommended to increase both UDP-Gal and B4GalT by 10% to provide a buffer to ensure the best possible results.

[0188] Furthermore, it was shown that using 30% less CMP-NANA and 10% more ST6 compared to the initially used BIS-TRIS conditions could result in higher sialylation while minimizing the overall increase in material costs.

[0189] [Table 13] 1 Average of 5 GMP runs

[0190] IVIg solution IVIg solutions were prepared using Privigen IVIg formulation buffer that had been replaced with BIS-TRIS buffer. One batch of IVIg was buffer-exchanged using a G25 desalting column equilibrated with BIS-TRIS buffer at pH 6.9, and the IVIg flow-through fraction was concentrated using a 10kDa Vivaspin Turbo15 instrument. Three 5g batches of Privigen IVIg were buffer-exchanged by tangential flow filtration (TFF) to 50mM BIS-TRIS at pH 6.67, 6.93, and 7.11. One batch of IVIg was buffer-exchanged by tangential flow filtration (TFF) to 50mM BIS-TRIS at pH 6.9, and then concentrated using a 10kDa Vivaspin Turbo15 instrument.

[0191] Table 9 shows the IVIg lot used, the buffer exchange method, the pH of the buffer exchange, and the final pH measurement after concentration.

[0192] [Table 14]

[0193] If, after buffer exchange and concentration, the pH of the IVIg solution is outside the preferred range, such as 7.2 to 7.4, preferably about 7.3, the pH should be adjusted.

[0194] General reaction explanation Generally, galactosylation was initiated first thing in the morning. After gently mixing the reagents (IVIg, UDP-Gal, B4GalT enzyme, and MnCl2), the reaction mixture was incubated at 37°C without agitation. The reaction mixture was not sterile filtered. Incubation was continued for various durations. Two 5 μL aliquots were taken at different points in time and then frozen until analysis. At the end of the experiment, the bulk reaction material was left at 4°C.

[0195] The sialylation step was initiated by adding the ST6 enzyme and half of the required CMP-NANA (typically after 24 hours of galactosylation). In some cases, the galactosylated material was initially divided into smaller volumes to perform multiple sialylation reactions. At 9 hours, the second half of CMP-NANA was added. Incubation was continued for varying durations. Two 5 μL aliquots were taken at different points in time and then frozen until analysis. At the end of the experiment, the bulk reaction material was placed at 4°C.

[0196] Degree of glycosylation The degree of glycosylation was quantified by LC-MS against Fc glycopeptides. Table 10 shows the glycans quantified by LC-MS against Fc glycopeptides. Complete galactosylation included all glycans with two galactose residues, regardless of whether they were sialylated or not. Disyalylation was defined as the sum of A2F, A2F + bisected GlcNAc, and A2.

[0197] [Table 15]

[0198] MnCl2 concentration Prior to the start of this experiment, IVIg sialylation experiments were performed with a wide range of MnCl2 amounts (Figure 3). 10 This clearly demonstrates that large amounts of MnCl2 are harmful and suggests that even around 7.5 mM, as used in process 3.0.0, can have different effects.

[0199] Figure 7A shows the increase in the amounts of G1F+NeuAc and G1+NeuAc with increasing MnCl2. These species arise from incomplete galactosylation. Figure 7B shows the increase in the amounts of G0F, G1F, and G2F with increasing MnCl2. This indicates that sialylation is also affected in addition to insufficient galactosylation, i.e., the more MnCl2 there is, the greater the amount of non-sialylated species. Figure 8 repeats these results showing the disialylation levels.

[0200] These results prompted further experiments to examine the lower end of the MnCl2 concentration range from 2.5 mM to 10 mM. 11 This series of reactions used IVIg buffer exchanged using G25 desalting and Vivaspin concentrators.

[0201] Galactosylation (using B4GalT and UDP-Gal, Table 11) was performed, and samples were taken for glycopeptide analysis after 20, 24, 28, and 44 hours. Subsequently, the 44-hour samples were further treated with CMP-NANA and ST6 for 48 hours, and the samples taken for analysis at 28, 32, 36, and 48 hours are referred to as experimental series A. Separately, a set of samples was galactosylated for 24 hours and then sialylated for an additional 48 hours, and aliquots taken at regular intervals are referred to as experimental series B. LCMS glycopeptide data were analyzed using Qual Browser.

[0202]

Table 16

[0203] Figure 9 shows that for all conditions (grouped by MnCl2 concentration), galactosylation of IgG1 increased between 20 hours and 44 hours. Similar results were observed for the other IgG subclasses.

[0204] Figure 10 shows the same data but grouped by time. Here, it can be seen that at any given point, the degree of galactosylation increases between 2.5 mM and 5.0 mM of MnCl2 and then decreases as it progresses to 7.5 mM and then 10 mM. This clearly demonstrates that galactosylation at 5.0 mM of MnCl2 is better than the 7.5 mM of MnCl2 used in process 3.0.0.

[0205] Salt concentration IVIg in a pH 6.9 buffer was subjected to galactosylation and sialylation reactions in the presence of MnCl2 under incubation at 37°C using UDP-Gal / B4GalT and CMP-NANA / ST6, respectively. Various concentrations of sodium chloride in BIS-TRIS buffer were added to obtain final reaction concentrations of 0, 50, 100, 150, and 300 mM sodium chloride. At the end of the galactosylation and sialylation reactions, the samples were isolated for glycan analysis by glycopeptide LC-MS.

[0206] As shown in Figures 11, 12, 13, 14, 15, and 16, the addition of sodium chloride to the galactosylation and sialylation reactions adversely affected the reaction degree in a salt concentration-dependent manner. This effect was observed for all IgG subclasses and was most pronounced in the sialylation step. The presence of 50 mM sodium chloride could reduce the degree of sialylation by 4–9%, depending on the IgG subclass.

[0207] UDP-Gal Stability UDP-Gal was found to be unstable in the presence of MnCl2, producing degradation products (UMP and possibly 1,2-phosphogalactose 1) different from the enzyme-catalyzed transfer product (UDP). This mechanism is thought to follow the pattern shown in Figure 17.

[0208] The amounts of UDP-Gal, UDP, and UMP were evaluated by ion-pairing HPLC on a Supelcosil LC-18-T column using 0.1 M potassium phosphate, 4 mM tetrabutylammonium bisulfate, pH 6.0 mobile phase, and UV detection at 254 nm. Only uridine-supported components were detected by UV; sugars not bound to uridine were not detected. The products were compared to known standards.

[0209] UDP-Gal in BIS-TRIS buffer at pH 6.9 was heated at 37°C for 8 hours in the presence of 0, 5, 10, or 20 mM MnCl2, and then evaluated by ion-pairing HPLC. B4Galt was not included in this experiment. The amount of UDP-Gal loss was MnCl2-dependent and increased with increasing MnCl2 concentration. The only other product observed was UMP. In the absence of MnCl2, UMP was hardly visible.

[0210] As shown in Figures 18 and 19, this nonspecific degradation of UDP-Gal can be detected during the galactosylation of IVIg. IVIg was galactosylated for 24 hours using UDP-Gal, B4GalT, and 5 mM MnCl2 in BIS-TRIS buffer at three different pH levels (6.7, 6.9, and 7.1). High molecular weight IgG proteins were separated from low molecular weight sugar nucleotides using a 500 MWCO spin unit, and the nucleotide-containing fraction was injected into ion-pairing HPLC. Formation of both UMP (peak 1) and UDP (peak 3) was observed, with UMP from nonspecific degradation and UDP from enzyme-catalyzed transfer of IgG to glycans. UMP formation increased with increasing pH from 6.7 to 6.9 and then to 7.1. UDP formation did not appear to be affected by the pH range investigated here.

[0211] Example 5: Preparation of hypersialylated IgG In another example, hsIgG is prepared using BIS-TRIS at pH 7.3. Therefore, preferred reaction conditions in 50 mM BIS-TRIS (pH 7.3) include the following galactosylation of an IgG antibody (e.g., pooled IgG antibody, pooled immunoglobulin, or IVIg): 5.0 mM MnCl2, 42 μmol UDP-Gal / g IgG antibody, and incubation at 37°C for 16–24 hours, followed by 8.0 units of B4GalT / g IgG antibody with sialylation in 5.0 mM MnCl2, 110 μmol CMP-NANA / g IgG antibody (added twice, i.e., half at the start of the reaction and again after 9–10 hours), and 18 units of ST6Gal1 / g IgG antibody with incubation at 37°C for 30–33 hours. The reaction may also be carried out by adding ST6Gal1 and CMP-NANA to the galactosylation reaction.

[0212] This method was performed on a 21g scale and achieved 99% complete IgG1 galactosylation by glycopeptide LC-MS, 96% disiallylated IgG1 by glycopeptide LC-MS, and 94% disiallylation by N-glycan release (InstantPC kit, AdvanceBio Gly-X N-glycan preparation using Agilent). This resulted in the total release of N-glycans, enabling quantitative summation of IgG1, IgG2, IgG3, and IgG4Fc glycans, as well as approximately 15-25% Fab glycosylation present in IVIg.

[0213] array Sequence ID 1 (IgG1) EeqYNSTYR Sequence ID 2 (IgG2 / 3) EeqFNSTFR Sequence ID 3 (IgG3 / 4) EEQYNSTFR Sequence ID 4 (IgG3 / 4) EeqFNSTYR Sequence ID 5 (NP_001488.2 B4GALT1[Biology=Human][GeneID=2683][Isoform=1]) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLH PVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFL TINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS Sequence ID 6 (NP_001365424.1 B4GALT1[Biology=Human][GeneID=2683][Isoform=2]) MPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQ QLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS Sequence ID 7 (NP_001365425.1 B4GALT1[Biology=Human][GeneID=2683][Isoform=3]) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKV AIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS Sequence ID 8 (NP_001365426.1 B4GALT1[Biology=Human][GeneID=2683][Isoform=4]) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPAS NLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQYEKIRRLLW Sequence ID 9 MRLREPLLSGSAAMPGASLQRACR Sequence ID 10 LLVAVCALHLGVTLVYYLAG Sequence ID 11 RDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTI FNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS Sequence ID 12 (B4GalT) GPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIP MNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS Sequence ID 13 (B4GalT) gssplldmGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPM NDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPSprdhhhhhhh Sequence ID 14(NP_001340845.1(NP_003023.1, NP_775323.1)ST6GAL1[Biology=Human][GeneID=6480][Isoform=a]) MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSS SKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREID DHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWEL WDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC Sequence ID 15 (NP_775324.1 ST6GAL1[Biology=Human][GeneID=6480][Isoform=b]) MNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC Sequence ID 16 MIHTNLKKK Sequence ID 17 FSCCVLVFLLFAVICVW Sequence ID 18 KEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPT ANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC Sequence ID 19 (ST6Gal1) AKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKD SLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC Sequence ID 20 (ST6Gal1) gssplldmlehhhhhhhhmAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTILMNSQL VTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC The inventions described in the original claims of this application are listed below. [Invention 1] A method for producing hypersialylated IgG (hsIgG), (a) To provide pooled IgG antibodies, (b) The pooled IgG antibody is transferred to β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion, UDP-Gal or its salt, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl 2 Incubating in a reaction mixture containing the above, thereby generating galactosylated IgG antibodies, (c) The galactosylated IgG antibody is mixed with ST6Gal or its enzymatically active portion, CMP-NANA or its salt, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl 2 Incubating in a reaction mixture containing, A method comprising generating hsIgG. [Invention 2] A method for preparing highly sialylated IgG (hsIgG), (a) To provide pooled IgG antibodies, (b) The pooled IgG antibody is transferred to β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion, UDP-Gal or its salt, ST6Gal or its enzymatically active portion, CMP-NANA or its salt, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl 2 The hsIgG preparation is prepared by incubation in a reaction mixture containing the following: Methods that include... [Invention 3] A method for preparing highly sialylated IgG (hsIgG), (a) To provide pooled IgG antibodies, (b) The pooled IgG antibody is transferred to β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion, UDP-Gal or its salt, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl 2 The process involves incubating in a galactosylation reaction mixture containing the following to generate galactosylated IgG antibodies: (c) Adding ST6Gal or its enzymatically active portion and CMP-NANA or its salt to the galactosylation reaction mixture to produce a sialylation reaction mixture, (d) Incubating the sialylation reaction mixture, A method comprising generating hsIgG. [Invention 4] The method according to any one of inventions 1 to 3, wherein the B4GalT or its enzymatically active portion is at least 85% identical to SEQ ID NO: 13. [Invention 5] The method according to any one of Inventions 1 to 4, wherein the ST6Gal1 or its enzymatically active portion includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 19. [Invention 6] A method according to any one of Inventions 1 to 5, wherein the total incubation time is less than 72 hours. [Invention 7] The method according to any one of inventions 1 to 6, wherein the incubation time of the reaction mixture containing ST6Gal or its enzymatically active portion is less than 40 hours. [Invention 8] The method according to any one of Inventions 1 to 7, wherein each of the reaction mixtures independently comprises BIS-TRIS at about 10 to about 500 mM and about pH 5.5 to about pH 8.5. [Invention 9] The method according to any one of Inventions 1 to 8, wherein each reaction mixture independently comprises a BIS-TRIS buffer of about 50 mM and about pH 7.3. [Invention 10] The method according to any one of Inventions 1 to 9, wherein the pooled IgG antibody is provided as a composition further comprising a BIS-TRIS buffer at approximately pH 7.2. [Invention 11] Each of the reaction mixtures is independently mixed with about 1 to about 20 mM MnCl 2 A method according to any one of inventions 1 to 10, including the method described above. [Invention 12] Each of the reaction mixtures is independently mixed with about 4.5 to about 5.5 mM MnCl 2 A method according to any one of inventions 1 to 11, including the method described above. [Invention 13] The method according to any one of Inventions 1 to 12, wherein the reaction mixture comprises about 0.038 to about 0.046 UDP-Gal or a salt thereof per gram of pooled IgG antibody. [Invention 14] The method according to any one of Inventions 1 to 13, wherein the reaction mixture contains about 0.1425 to about 0.1575 CMP-NANA or a salt thereof per gram of IgG antibody. [Invention 15] The method according to any one of Inventions 1 to 14, wherein the reaction mixture containing CMP-NANA is supplemented with additional CMP-NANA or a salt thereof during incubation. [Invention 16] The method according to Invention 15, wherein the total amount of CMP-NANA or its salt added to the reaction mixture containing CMP-NANA is about 0.1425 to about 0.1575. [Invention 17] The method according to Invention 16, wherein the total amount of CMP-NANA is added to the sialylation reaction mixture in less than seven installments. [Invention 18] The method according to any one of Inventions 1 to 17, wherein the reaction mixture containing B4GalT or its enzymatically active portion contains about 7.2 to about 8.8 U of B4GalT or its enzymatically active portion per gram of pooled IgG. [Invention 19] The method according to any one of Inventions 1 to 18, wherein the reaction mixture containing ST6Gal or its enzymatically active portion contains about 17.1 to about 18.9 U of ST6Gal1 or its enzymatically active portion per gram of pooled IgG. [Invention 20] The incubation is carried out at approximately 20 to 50°C, according to any one of inventions 1 to 19. [Invention 21] The method according to any one of inventions 1 to 20, wherein the incubation is carried out at approximately 37°C. [Invention 22] The method according to any one of Inventions 1 to 21, wherein the IgG antibody comprises IgG antibodies isolated from at least 1,000 donors. [Invention 23] The method according to any one of Inventions 1 to 22, wherein at least 50%, 55%, 60%, 65%, or 70% w / w of the IgG antibody is IgG1 antibody. [Invention 24] The method according to Invention 23, wherein at least 90% of the donor subjects have been exposed to the virus. [Invention 25] The method according to any one of Inventions 1 to 24, wherein approximately 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in the aforementioned hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching. [Invention 26] The method according to any one of Inventions 1 to 25, wherein approximately 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans in the aforementioned hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching. [Discussion 27] The method according to any one of Inventions 1 to 26, wherein at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycan in the Fab domain of the hsIgG has sialic acid in both the α1,3 arm and the α1,6 arm connected via NeuAc-α2,6-Gal terminal linkage. [Invention 28] The method according to any one of Inventions 1 to 24, wherein at least 80% of the branched Fc glycans in the aforementioned hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching. [Invention 29] The method according to Invention 28, wherein at least 60%, 65%, and 70% of the branched glycans in the Fab domain of the hsIgG have sialic acid in both the α1,3 arms and the α1,6 arms connected via NeuAc-α2,6-Gal terminal ligatures. [Invention 30] The method according to any one of Inventions 1 to 24, wherein at least 85% of the branched Fc glycans in the aforementioned hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching. [Invention 31] The method according to Invention 30, wherein at least 60%, 65%, and 70% of the branched glycan in the Fab domain of the hsIgG have sialic acid in both the α1,3 arm and the α1,6 arm connected via NeuAc-α2,6-Gal terminal linkage. [Invention 32] The method according to any one of Inventions 1 to 24, wherein at least 90% of the branched Fc glycans in the aforementioned hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching. [Invention 33] The method according to Invention 32, wherein at least 60%, 65%, and 70% of the branched glycans in the Fab domain of the hsIgG have sialic acid in both the α1,3 arms and the α1,6 arms connected via NeuAc-α2,6-Gal terminal ligatures.

Claims

1. A method for preparing hypersialylated IgG (hsIgG) from pooled IgG antibodies, (a) The pooled IgG antibody is mixed with 7.2 to 8.8 units (U) of β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion per gram of IgG antibody, 38 μmol to 46 μmol of UDP-Gal or its salt per gram of IgG antibody, 50 mM bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and 1 mM to 20 mM MnCl ２ The reaction mixture containing [the specified substance] is incubated at pH 6.8 to 7.3, thereby generating galactosylated IgG antibodies. (b) The galactosylated IgG antibody is mixed with 14 to 20 U of ST6Gal or its enzymatically active moiety per gram of IgG antibody, 100 to 300 μmol of CMP-NANA or its salt per gram of IgG antibody, 50 mM bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and 1 mM to 20 mM MnCl ２ The reaction mixture containing the above is incubated at pH 6.8 to 7.

3. The method comprising preparing the hsIgG by the means described above.

2. A method for preparing hypersialylated IgG (hsIgG) from pooled IgG antibodies, The pooled IgG antibody is mixed with 7.2 to 8.8 units (U) of β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion per gram of IgG antibody, 38 μmol to 46 μmol of UDP-Gal or its salt per gram of IgG antibody, 14 to 20 U of ST6Gal or its enzymatically active portion per gram of IgG antibody, 100 μmol to 300 μmol of CMP-NANA or its salt per gram of IgG antibody, 50 mM bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and 1 mM to 20 mM MnCl ２ The hsIgG is prepared by incubation in a reaction mixture containing at a pH of 6.8 to 7.

3. The method, including the method described above.

3. A method for preparing hypersialylated IgG (hsIgG) from pooled IgG antibodies, (a) The pooled IgG antibody is mixed with 7.2 to 8.8 units (U) of β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion per gram of IgG antibody, 38 μmol to 46 μmol of UDP-Gal or its salt per gram of IgG antibody, 50 mM bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and 1 mM to 20 mM MnCl ２ The galactosylation reaction mixture containing the specified substance is incubated at pH 6.8 to 7.3 to generate galactosylated IgG antibodies. (b) To produce a sialylation reaction mixture, add 14 to 20 U of ST6Gal or its enzymatically active portion per gram of IgG antibody, and 100 μmol to 300 μmol of CMP-NANA or its salt per gram of IgG antibody, (c) The sialylation reaction mixture is incubated at pH 6.8 to 7.

3. The method comprising preparing the hsIgG by the means described above.

4. The method according to any one of claims 1 to 3, wherein the B4GalT or its enzymatically active portion is at least 90% identical to SEQ ID NO:

13.

5. The method according to any one of claims 1 to 4, wherein the ST6Gal or its enzymatically active portion includes an amino acid sequence that is at least 90% identical to SEQ ID NO:

19.

6. The method according to any one of claims 1 to 5, wherein the total incubation time is less than 72 hours.

7. The method according to any one of claims 1 to 6, wherein the incubation time of the reaction mixture containing ST6Gal or its enzymatically active portion is less than 40 hours.

8. The method according to any one of claims 1 to 7, wherein each reaction mixture independently comprises a 50 mM and pH 7.3 BIS-TRIS buffer.

9. The method according to any one of claims 1 to 8, wherein the pooled IgG antibody is in a composition containing a BIS-TRIS buffer at pH 7.

2.

10. Each of the reaction mixtures is independently mixed with 4.5–5.5 mM MnCl ２ The method according to any one of claims 1 to 9, including the method described in any one of claims 1 to 9.

11. The method according to any one of claims 1 to 10, wherein the reaction mixture comprises 142.5 μmol to 157.5 μmol of CMP-NANA or a salt thereof per gram of IgG antibody.

12. The method according to any one of claims 1 to 11, wherein the reaction mixture containing CMP-nana is supplemented with additional CMP-nana or a salt thereof during incubation.

13. The method according to claim 12, wherein the total amount of CMP-NANA is added to the reaction mixture in less than seven installments.

14. The method according to any one of claims 1 to 13, wherein the reaction mixture containing ST6Gal or its enzymatically active moiety contains 17.1 to 18.9 units (U) of ST6Gal or its enzymatically active moiety per gram of pooled IgG.

15. The method according to any one of claims 1 to 14, wherein the reaction mixture is incubated at 20 to 50°C.

16. The method according to any one of claims 1 to 15, wherein the reaction mixture is incubated at 37°C.

17. The method according to any one of claims 1 to 16, wherein the IgG antibody comprises IgG antibodies isolated from at least 1,000 donors.

18. The method according to any one of claims 1 to 17, wherein at least 50% w / w of the IgG antibody is IgG1 antibody.

19. The method according to claim 18, wherein at least 90% of the donors have been exposed to the virus.

20. The method according to any one of claims 1 to 19, wherein 80% of the branched glycans in the hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching.

21. The method according to any one of claims 1 to 20, wherein 80% of the branched Fc glycan in the hsIgG has sialic acid in both the α1,3 branching and the α1,6 branching.

22. The method according to any one of claims 1 to 21, wherein at least 60% of the branched glycan in the Fab domain of the hsIgG has sialic acid in both the α1,3 arm and the α1,6 arm connected via a NeuAc-α2,6-Gal terminal linkage.

23. The method according to any one of claims 1 to 19, wherein at least 80% of the branched Fc glycan in the hsIgG has sialic acid in both the α1,3 branching and the α1,6 branching.

24. The method according to claim 23, wherein at least 60% of the branched glycan in the Fab domain of the hsIgG has sialic acid in both the α1,3 arm and the α1,6 arm connected via a NeuAc-α2,6-Gal terminal linkage.

25. The method according to any one of claims 1 to 19, wherein at least 85% of the branched Fc glycan in the hsIgG has sialic acid in both the α1,3 branching and the α1,6 branching.

26. The method according to claim 25, wherein at least 60% of the branched glycan in the Fab domain of the hsIgG has sialic acid in both the α1,3 arm and the α1,6 arm connected via a NeuAc-α2,6-Gal terminal linkage.

27. The method according to any one of claims 1 to 19, wherein at least 90% of the branched Fc glycan in the hsIgG has sialic acid in both the α1,3 branching and the α1,6 branching.

28. The method according to claim 27, wherein at least 60% of the branched glycan in the Fab domain of the hsIgG has sialic acid in both the α1,3 arm and the α1,6 arm connected via a NeuAc-α2,6-Gal terminal linkage.

29. The method according to any one of claims 1 to 28, comprising (a) below and any one of (b1) to (b3) below. (a) To provide pooled IgG antibodies, and (b1) The pooled IgG antibody is incubated at pH 6.9 in a reaction mixture containing 7.5 units (U) of β1,4-galactosyltransferase (B4GalT) or its enzymatically active moiety per gram of IgG antibody, 38 μmol of UDP-Gal or its salt per gram of IgG antibody, 15 U of ST6Gal or its enzymatically active moiety per gram of IgG antibody, 220 μmol of CMP-NANA or its salt per gram of IgG antibody, 50 mM bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and 7.4 mM MnCl2, thereby generating hypersialylated IgG (hsIgG) antibody. Or, (b2) (i) The pooled IgG antibody is incubated at pH 6.9 and 37°C for 16 to 24 hours in a reaction mixture containing 7.5 units (U) of β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion per gram of IgG antibody, 38 μmol of UDP-Gal or its salt per gram of IgG antibody, 50 mM bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and 7.4 mM MnCl2, thereby generating galactosylated IgG antibody, and (ii) The galactosylated IgG antibody is incubated at pH 6.9 and 37°C for 30 to 33 hours in a reaction mixture containing 15 U of ST6Gal or its enzymatically active moiety per gram of IgG antibody, 220 μmol of CMP-NANA or its salt per gram of IgG antibody, 50 mM bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and 7.4 mM MnCl2, thereby generating hsIgG antibody. Or, (b3) (i) The pooled IgG antibody is incubated at pH 6.9 and 37°C for 16 to 24 hours in a reaction mixture containing 7.5 units (U) of β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion per gram of IgG antibody, 38 μmol of UDP-Gal or its salt per gram of IgG antibody, 50 mM bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer, and 7.4 mM MnCl2, thereby generating galactosylated IgG antibody. (ii) Add 15 U of ST6Gal or its enzymatically active portion per gram of IgG antibody, and 220 μmol of CMP-NANA or its salt per gram of IgG antibody to the galactosylation reaction mixture to produce a sialylation reaction mixture, and (iii) The sialylation reaction mixture is incubated at pH 6.9 at 37°C for 30 to 33 hours to generate hsIgG antibody.

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