Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/005—Glycopeptides, glycoproteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y204/00—Glycosyltransferases (2.4)
  • C12Y204/01—Hexosyltransferases (2.4.1)
  • C12Y204/01038—Beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase (2.4.1.38)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'

Definitions

• IVIg Intravenous immunoglobulin
• IVIg preparations have distinct limitations, such as variable efficacy, clinical risks, high costs, and finite supply. Different IVIg preparations are frequently treated as interchangeable products clinically, but it is well-known that significant differences in product preparations exist that may impact tolerability and activity in selected clinical applications. At the current maximal dosing regimens, only partial and unsustained responses are obtained in many instances. In addition, the long infusion times (4–6 h) associated with the high volume of IVIg treatment consume significant resources at infusion centers and negatively affect patient- reported outcomes, such as convenience and quality of life. The identification of the important anti-inflammatory role of Fc domain sialylation has presented an opportunity to develop more potent immunoglobulin therapies.
• IVIg preparations generally exhibit low levels of sialylation on the Fc domain of the antibodies present. Specifically, they exhibit low levels of di-sialylation of the branched glycans on the Fc region.
• Washburn et al. (Proceedings of the National Academy of Sciences, USA 112: E1297– E1306 (2015)) describes a controlled sialylation process to generate highly tetra-Fc–sialylated IVIg and showed that the process yields a product with consistent enhanced anti-inflammatory activity.
• the sialylation reaction driven by ST6Gal using CMP -NANA as a substrate has characteristics that make improvement of the reaction, whether assessed by overall level of disialylation, time to reach a certain level of disialylation, amount of enzyme and substrate required to reach a certain overall level of disialylation, challenging.
• CMP- NANA is not entirely stable and will spontaneously hydrolyze even in the absence of any enzyme
• ST6Gal1 is thought to catalyze hydrolysis of the CMP -NANA without productive addition to the Gal on a branched glycan
• CMP cytidine monophosphate
• CMP has been observed to catalyze the reverse enzymatic reaction to remove the NeuAc from the newly formed glycan.
• the present disclosure is based, at least in part, on the discovery that the reverse reaction is far less favorable with BIS-TRIS as buffer, e.g., as when compared to MOPS as buffer.
• hsIgG hypersialylated IgG
• methods of producing hypersialylated IgG comprising: (a) providing pooled IgG antibodies; (b) incubating the pooled IgG antibodies in a reaction mixture comprising b l .4-Gal actosyltransferase (B4GalT) or enzymatically active portion thereof, UDP-Gal or salt thereof, Bis (2-hydroxyethyl) aminotris (hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl 2 .
• hsIgG hypersialylated IgG
• methods of preparing hypersialylated IgG comprising: (a) providing pooled IgG antibodies; (b) incubating the pooled IgG antibodies in a reaction mixture comprising ⁇ 1,4-Galactosyltransferase (B4GalT) or enzymatically active portion thereof, UDP-Gal or salt thereof, ST6Gal or enzymatically active portion thereof, CMP -NANA or salt thereof, Bis (2-hydroxyethyl) aminotris (hydroxymethyl)methane (BIS-TRIS) buffer, and MnC'h. thereby creating the hsIgG preparation.
• B4GalT ⁇ 1,4-Galactosyltransferase
• UDP-Gal or salt thereof UDP-Gal or salt thereof
• ST6Gal or enzymatically active portion thereof CMP -NANA or salt thereof
• hsIgG hypersialylated IgG
• methods of preparing hypersialylated IgG comprising:(a) providing pooled IgG antibodies; (b) incubating the pooled IgG antibodies in a galactosylation reaction mixture comprising ⁇ 1,4-Galactosyltransferase (B4GalT) or enzymatically active portion thereof, UDP-Gal or salt thereof, Bis (2-hydroxyethyl) aminotris (hydroxymethyl)methane (BIS-TRIS) buffer, and MnCl 2 , thereby producing galactosylated IgG antibodies; (c) adding ST6Gal or an enzymatically active portion thereof and CMP -NANA or salt thereof to the galactosylation reaction mixture to produce a sialylation reaction mixture; and (d) incubating the sialylation reaction mixture, thereby producing hsIgG
• the B4GalT or enzymatically active portion thereof is at least 85% identical to SEQ ID NO: 13.
• the ST6Gal or enzymatically active portion thereof comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19.
• the total incubation time is less than 72 hours.
• the incubation time of the reaction mixture comprising ST6Gal or enzymatically active portion thereof is less than 40 hours.
• each of the reaction mixture(s) each independently comprise BIS- TRIS at from about 10 to about 500 mM and from about pH 5.5 to about pH 8.5.
• reaction mixture(s) each independently comprise BIS-TRIS buffer at about 50 mM and about pH 7.3.
• the pooled IgG antibodies are provided as a composition further comprising BIS-TRIS buffer at about pH 7.2.
• each of the reaction mixture(s) each independently comprise MnCl 2 at about 1 to about 20 mM.
• each of the reaction mixture(s) each independently comprise MnCl 2 at about 4.5 to about 5.5 mM.
• the reaction mixture comprises from about 0.038 to about 0.046 UDP-Gal or salt thereof per gram of pooled IgG antibodies.
• the reaction mixture comprises about 0.1425 to about 0.1575 CMP -NANA or salt thereof per gram of IgG antibody.
• reaction mixture comprising CMP -NANA is supplemented with additional CMP-NANA or salt thereof during incubation.
• the total amount of CMP-NANA or salt thereof added to the reaction mixture comprising CMP-NANA is from about 0.1425 to about 0.1575.
• the total amount of CMP-NANA is added to the sialylation reaction mixture in less than 7 portions.
• the reaction mixture comprising B4GalT or enzymatically active portion thereof comprises from about 7.2 to or to about 8.8 U B4GalT or enzymatically active portion thereof per gram of pooled IgG. In some embodiments, the reaction mixture comprising ST6Gal1 or enzymatically active portion thereof comprises from about 17.1 to about 18.9 U ST6Gal1 or enzymatically active portion thereof per gram of pooled IgG.
• the incubation takes place at from about 20 to about 50°C.
• the incubation takes place at about 37 °C.
• the IgG antibodies comprise IgG antibodies isolated from at least 1000 donors.
• At least 50%, 55%, 60%, 65% or 70% w/w of the IgG antibodies are IgGl antibodies.
• At least 90% of the donor subject has been exposed to a virus.
• about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• At least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the a 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• At least 80% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• At least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• At least 85% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• At least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• At least 90% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• At least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• immunoglobulin G having a very high level of Fc sialylation, particularly disialylation (sialylation on both the alpha 1,3 branch and the alpha 1,6 branch of the glycan at Asn297 (EU Numbering).
• the methods described herein can provide hypersialylated IgG (hsIgG) in which greater than 70% of the branched glycans on the Fc domain are sialylated on both branches (i.e., on the alpha 1,3 branch and the alpha 1,6 branch).
• HsIgG contains a diverse mixture of IgG antibodies, primarily IgGl antibodies. The diversity of the antibodies is high.
• the immunoglobulins used to prepare hsIgG can be obtained, for example from pooled human plasma (e.g., pooled plasma from at least 1,000 - 30,000 donors).
• the immunoglobulins can be obtained from IVIg, including commercially available IVIg.
• HsIgG has far higher level of sialic acid on the branched glycans on the Fc region than does IVIg. This results in a composition that differs from IVIg in both structure and activity.
• HsIgG can be prepared as described in W02014/179601 or Washburn et al.
• Described herein are improved methods for preparing hsIgG.
• hsIgG hypersialylated
• the method comprising: (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture comprising b 1.4-Gal actosyltransrerase I (B4GalT) and UDP-Gal to produce galactosylated IgG antibodies; (c) incubating the galactosylated IgG antibodies in a reaction mixture comprising ST6Gal1 and CMP -NANA, wherein the galactosylation reaction mixture and the sialylation reaction mixture comprise Bis (2 -hydroxy ethyl) aminotris (hydroxymethyl)methane (BIS-TRIS) buffer, thereby creating the hsIgG preparation.
• B4GalT 1.4-Gal actosyltransrerase I
• UDP-Gal UDP-Gal
• hsIgG hypersialylated
• the method comprising (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture comprising ⁇ 1 ,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, thereby creating the hsIgG preparation.
• the B4GalT is at least 85% identical to SEQ ID NO: 13;
• the ST6Gal1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19;
• step (b) is carried out for at least 8, 12, 18, 24, 30, or 40 hrs;
• step (c) is carried out for at least 8, 12, 18, 24, 30, or 40 hrs;
• step (c) comprises adding ST6Gal1 and CMP-NANA to the reaction mixture of step (a);
• the reactions take place in BIS-TRIS at 10 - 500 mM pH 5.5 - 8.5;
• the reaction mixtures comprise MnCl 2 at 1 - 20 mM;
• the UDP-Gal is present at 5 mM UDP-Gal/g IgG antibody;
• the CMP-NANA is present at 5 pM CMP-NANA/g IgG antibody;
• the incubation takes place at 20-50°C;
• the incubation takes place at 30-45 °C;
• hypersialylated IgG at least 60% (e.g., 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98% up to and including 100%) of branched glycans on the Fc region are di-sialylated (i.e., on both the ⁇ 1,3 branch and the ⁇ 1,6 arm) by way of NeuAc- ⁇ 2,6-Gal terminal linkages.
• less than 50% (e.g., less than 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) of branched glycans on the Fc region are mono-sialylated (i.e., sialylated only on the ⁇ 1,3 branch or only on the ⁇ 1,6 branch) by way of aNeuAc- ⁇ 2,6-Gal terminal linkage.
• the polypeptides are derived from plasma, e.g., human plasma.
• the polypeptides are overwhelmingly IgG polypeptides (e.g., IgGl, IgG2, IgG3 or IgG4 or mixtures thereof), although trace amounts of other contain trace amount of other immunoglobulin subclasses can be present.
• an antibody refers to a polypeptide that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or immunoglobulin variable domain sequence.
• an antibody can include a heavy (H) chain variable region (abbreviated herein as V H ), and a light (L) chain variable region (abbreviated herein as V L ).
• V H heavy chain variable region
• L light chain variable region
• an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
• antibody encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab, F(ab')2, Fd, Fv, and dAb fragments) as well as complete antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).
• the light chains of the immunoglobulin can be of types kappa or lambda.
• constant region refers to a polypeptide that corresponds to, or is derived from, one or more constant region immunoglobulin domains of an antibody.
• a constant region can include any or all of the following immunoglobulin domains: a C H I domain, a hinge region, a C H 2 domain, a CH3 domain (derived from an IgA, IgD, IgG, IgE, or IgM), and a C H 4 domain (derived from an IgE or IgM).
• Fc region refers to a dimer of two “Fc polypeptides,” each “Fc polypeptide” including the constant region of an antibody excluding the first constant region immunoglobulin domain.
• an “Fc region” includes two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers.
• 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 also include part or the entire flexible hinge N-terminal to these domains.
• Fc polypeptide comprises immunoglobulin domains Cgamma2 (C ⁇ 2) and Cgamma3 (C ⁇ 3) and the lower part of the hinge between Cgammal (C ⁇ 1) and C ⁇ 2.
• the human IgG heavy chain Fc polypeptide is usually defined to comprise residues starting P232, to its carboxyl-terminus, wherein the numbering is according to the EU system (Edelman et al, Proc. Natl. Acad. USA, 63, 78-85 (1969)).
• Fc polypeptide comprises immunoglobulin domains Calpha2 (C ⁇ 2) and Calpha3 (C ⁇ 3) and the lower part of the hinge between Calphal (C ⁇ 1) and Ca2.
• An Fc region can be synthetic, recombinant, or generated from natural sources such as IVIg.
• glycocan is a sugar, which can be monomers or polymers of sugar residues, such as at least three sugars, and can be linear or branched.
• a “glycan” can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic 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.).
• natural sugar residues e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.
• glycocan includes homo and heteropolymers of sugar residues.
• glycan also encompasses a glycan component of a glycoconjugate (e.g., of a polypeptide, glycolipid, proteoglycan, etc.).
• a glycoconjugate e.g., of a polypeptide, glycolipid, proteoglycan, etc.
• free glycans including glycans that have been cleaved or otherwise released from a glycoconjugate.
• glycoprotein refers to a protein that contains a peptide backbone covalently linked to one or more sugar moieties (i.e., glycans).
• the sugar moiety(ies) may be in the form of monosaccharides, disaccharides, oligosaccharides, and/or polysaccharides.
• the sugar moiety(ies) may comprise a single unbranched chain of sugar residues or may comprise one or more branched chains.
• Glycoproteins can contain O-linked sugar moieties and/or N-linked sugar moieties.
• IVIg is a preparation of pooled, polyvalent IgG, including all four IgG subgroups, extracted from plasma of at least 1,000 human donors. IVIg is approved as a plasma protein replacement therapy for immune deficient patients. The level of IVIg Fc glycan sialylation varies among IVIg preparations, but is generally less than 20%. The level of disialylation is generally far lower than 20%.
• the term “derived from IVIg” refers to polypeptides which result from manipulation of IVIg. For example, polypeptides purified from IVIg (e.g., enriched for sialylated IgGs or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).
• an “N-glycosylation site of an Fc polypeptide” refers to an amino acid residue within an Fc polypeptide to which a glycan is N-linked.
• an Fc region contains a dimer of Fc polypeptides, and the Fc region comprises two N-glycosylation sites, one on each Fc polypeptide.
• percent (%) of branched glycans refers to the number of moles of glycan X relative to total moles of glycans present, wherein X represents the glycan of interest.
• pharmaceutically effective amount refers to an amount (e.g., dose) effective in treating a patient, having a disorder or condition described herein. It is also to be understood herein that a “pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents.
• kits containing the preparation or product and instructions for use.
• “Pharmaceutical preparations” and “pharmaceutical products” generally refer to compositions in which the final predetermined level of sialylation has been achieved, and which are free of process impurities. To that end, “pharmaceutical preparations” and “pharmaceutical products” are substantially free of ST6Gal1 and/or sialic acid donor (e.g., cytidine 5'- monophospho-N-acetyl neuraminic acid) or the byproducts thereof (e.g., cytidine 5’- monophosphate).
• sialic acid donor e.g., cytidine 5'- monophospho-N-acetyl neuraminic acid
• the byproducts thereof e.g., cytidine 5’- monophosphate
• “Pharmaceutical preparations” and “pharmaceutical products” are generally substantially free of other components of a cell in which the glycoproteins were produced (e.g., the endoplasmic reticulum or cytoplasmic proteins and RNA), if recombinant.
• purified refers to a polynucleotide or a polypeptide that is removed or separated from other components present in its natural environment.
• an isolated polypeptide is one that is separated from other components of a cell in which it was produced (e.g., the endoplasmic reticulum or cytoplasmic proteins and RNA).
• An isolated polynucleotide is one that is separated from other nuclear components (e.g., histones) and/or from upstream or downstream nucleic acids.
• An isolated polynucleotide or polypeptide can be at least 60% free, or at least 75% free, or at least 90% free, or at least 95% free from other components present in natural environment of the indicated polynucleotide or polypeptide.
• sialylated refers to a glycan having a terminal sialic acid.
• mono-sialylated refers to branched glycans having one terminal sialic acid, e.g., on an ⁇ 1,3 branch or an ⁇ 1,6 branch.
• di-sialylated refers to a branched glycan having a terminal sialic acid on two arms, e.g., both an ⁇ 1,3 arm and an ⁇ 1,6 arm.
• FIG. 1 shows a short, branched core oligosaccharide comprising two N- acetylglucos amine and three mannose residues.
• One of the branches is referred to in the art as the “ ⁇ 1,3 arm,” and the second branch is referred to as the “ ⁇ 1,6 arm,”.
• Squares N- acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N- acetylneuraminic acid; triangles: fucose.
• FIG. 2 shows common Fc glycans present in IVIg.
• Squares N-acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N-acetylneuraminic acid; triangles: fucose.
• FIG. 3 shows how immunoglobulins, e.g., IgG antibodies, can be sialylated by carrying out 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.
• FIG. 4 shows the reaction product of a representative example of the IgG-Fc glycan profile for a reaction starting with IVIg.
• the left panel is a schematic representation of enzymatic sialylation reaction to transform IgG to hsIgG; the right panel is the IgG Fc glycan profile for the starting IVIg and hsIgG. Bars, from left to right, correspond to IgG1, IgG2/3, and IgG3/4, respectively.
• FIG. 5 shows the level of A2F formation as a result of sialylation a Fc containing protein with various buffers at various pHs.
• FIG. 6 shows the level of 1,6-A1F formation as a result of sialylation a Fc containing protein with various buffers at various pHs.
• FIG. 7A shows the effect of high MnCl 2 concentration on galactosylation and sialylation of IVIg. Bars, from left to right: GIF+NeuAc; Gl+NeuAc.
• FIG. 7B shows the effect of high MnCl 2 concentration on galactosylation and sialylation of IVIg. Bars, from left to right: 5 mM; 10 mM; 20 mM; 40 mM; 61 mM.
• FIG. 8 shows the effect of high MnCl 2 concentration on disialylation of IVIg.
• FIG. 9 shows the effect of ⁇ 10 mM MnCl 2 concentration on galactosylation of IVIg (grouped by MnCl 2 concentration).
• FIG. 10 shows the effect of ⁇ 10 mM MnCl 2 concentration on galactosylation of IVIg grouped by time.
• FIG. 11 shows the effect of salt on IgGl galactosylation by glycopeptide LCMS.
• FIG. 12 shows the effect of salt on IgGl sialylation by glycopeptide LCMS.
• FIG. 13 shows the effect of salt on IgG2/3 galactosylation by glycopeptide LCMS.
• FIG. 14 shows the effect of salt on IgG2/3 sialylation by glycopeptide LCMS.
• FIG. 15 shows the effect of salt on IgG3/4 galactosylation by glycopeptide LCMS.
• FIG. 16 shows the effect of salt on IgG3/4 sialylation by glycopeptide LCMS.
• FIG. 17 shows a scheme for conversion of UDP-Gal to UMP and UDP.
• FIG. 18 demonstrates non-specific degradation of UDP-Gal could be detected in the galactosylation of IVIg.
• FIG. 19 demonstrates non-specific degradation of UDP-Gal could be detected in the galactosylation of IVIg.
• Antibodies are glycosylated at conserved positions in the constant regions of their heavy chain and on the Fab domain.
• human IgG antibodies have a single N-linked glycosylation site at Asn297 of the CH2 domain.
• Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions.
• the core oligosaccharide normally consists of GlcNAc 2 Man 3 GlcNAc, with differing numbers of outer residues. Variation among individual IgG’s can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).
• the present disclosure encompasses, in part, methods for preparing immunoglobulins (e.g., human IgG) having an Fc region having particular levels of branched glycans that are sialylated on both of the arms of the branched glycan (e.g., with a NeuAc- ⁇ 2,6-Gal terminal linkage).
• immunoglobulins e.g., human IgG
• Fc region having particular levels of branched glycans that are sialylated on both of the arms of the branched glycan (e.g., with a NeuAc- ⁇ 2,6-Gal terminal linkage).
• the levels can be measured on an individual Fc region (e.g., the number of branched glycans that are sialylated on an ⁇ 1,3 arm, an ⁇ 1,6 arm, or both, of the branched glycans in the Fc region), or on the overall composition of a preparation of polypeptides (e.g., the number or percentage of branched glycans that are sialylated on an ⁇ 1,3 arm, an ⁇ 1,6 arm, or both, of the branched glycans in the Fc region in a preparation of polypeptides).
• an individual Fc region e.g., the number of branched glycans that are sialylated on an ⁇ 1,3 arm, an ⁇ 1,6 arm, or both, of the branched glycans in the Fc region
• the overall composition of a preparation of polypeptides e.g., the number or percentage of branched glycans that are sialy
• Naturally derived polypeptides that can be used to prepare hyp ersi alyl ated IgG include, for example, IgG in human serum (particular human serum pooled from more than 1,000 donors), intravenous immunoglobulin (IVIg) and polypeptides derived from IVIg (e.g., polypeptides purified from IVIg (e.g., enriched for sialylated IgGs) or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).
• human serum particular human serum pooled from more than 1,000 donors
• IVIg intravenous immunoglobulin
• polypeptides derived from IVIg e.g., polypeptides purified from IVIg (e.g., enriched for sialylated IgGs) or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).
• N-linked oligosaccharide chains are added to a protein in the lumen of the endoplasmic reticulum.
• an initial oligosaccharide typically 14-sugar
• an asparagine residue contained within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline.
• the structure of this initial oligosaccharide is common to most eukaryotes, and contains three glucose, nine mannose, and two N-acetylglucos amine residues.
• This initial oligosaccharide chain can be trimmed by specific glycosidase enzymes in the endoplasmic reticulum, resulting in a short, branched core oligosaccharide composed of two N-acetylglucosamine and three mannose residues.
• One of the branches is referred to in the art as the “ ⁇ 1,3 arm,” and the second branch is referred to as the “ ⁇ 1,6 arm,” as shown in FIG. 1.
• N-glycans can be subdivided into three distinct groups called “high mannose type,” “hybrid type,” and “complex type,” with a common pentasaccharide core (Man ( ⁇ 1,6)- (Man( ⁇ 1 ,3))-Man( l,4)-GlcpNAc( 1 ,4)-GlcpNAc( 1,N)-Asn) occurring in all three groups.
• one or more monosaccharides units of N-acetylglucosamine may be added to the core mannose subunits to form a “complex glycan.”
• Galactose may be added to the N-acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains that terminate with any of a sialic acid, a galactose or an N-acetylglucosamine residue.
• a fucose residue may be added to an N- acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by specific glycosyl transferases.
• Hybrid glycans comprise characteristics of both high- mannose and complex glycans.
• one branch of a hybrid glycan may comprise primarily or exclusively mannose residues, while another branch may comprise N-acetylglucosamine, sialic acid, galactose, and/or fucose sugars.
• Sialic acids are a family of 9-carbon monosaccharides with heterocyclic ring structures. They bear a negative charge via a carboxylic acid group attached to the ring as well as other chemical decorations 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 usually occur as terminal structures attached to galactose (Gal) residues at the non-reducing termini of both bl and O-linked glycans.
• the glycosidic linkage configurations for these sialic acid groups can be either ⁇ 2,3 or ⁇ 2,6.
• Fc regions are glycosylated at conserved, N-linked glycosylation sites.
• each heavy chain of an IgG antibody has a single N-linked glycosylation site at Asn297 of the CH2 domain.
• IgA antibodies have N-linked glycosylation sites within the CH2 and CH3 domains
• IgE antibodies have N-linked glycosylation sites within the CH3 domain
• IgM antibodies have N-linked glycosylation sites within the CH1, CH2, CH3, and CH4 domains.
• Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions.
• IgG has a single N-linked biantennary carbohydrate at Asn297 of the CH2 domain in each Fc polypeptide of the Fc region, which also contains the binding sites for C1q and Fc ⁇ R.
• the core oligosaccharide normally consists of GlcNAc2Man3GlcNAc, with differing numbers of outer residues. Variation among individual IgG can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).
• Immunoglobulins e.g., IgG antibodies
• Beta-l,4-galactosyltransferase 1 (B4GalT) is a Type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5’- diphosphosegalactose ([[ (2R,3S,4R,5R)-5-(2,4-dioxopyrimidin- 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 ⁇ -1,4 linkage.
• Alpha-2, 6-sialyltransferase 1 is a Type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5’-monophospho-Nacetylneuraminicacid ((2R, 4S,5R, 6R)-5-acetamido-2-[[(2R, 3S,4R, 5R)-5-(4- amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-4- hydroxy-6-(1,2,3-trihydroxypropyl)oxane-2-carboxylic acid; CMP-NANA or CMP-Sialic Acid) to Gal as an a-2,6 linkage.
• the reactions proceed shown in FIG. 3.
• Glycans of polypeptides can be evaluated using any methods known in the art. For example, sialylation of glycan compositions (e.g., level of branched glycans that are sialylated on an ⁇ 1,3 branch and/or an ⁇ 1,6 branch) can be characterized using methods described in WO2014/179601.
• At least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans on the Fc domain have a sialic acid on both the ⁇ 1 ,3 arm and the ⁇ 1 ,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• At least 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain have a sialic acid on both the ⁇ 1 ,3 arm and the ⁇ 1 ,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• the hsIgG compositions prepared by the methods described herein comprises at least 50%, 55%, 60%, 65%, 70% or 75% of the branched glycans on the Fc domain have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm.
• 1 U B4GlaT B4GalT is equal to the formation of 1 nmol Gal- GlcNAc (also referred to as LacNAc) by the transfer of Gal from UDP-Gal and to GlcNAc per minute.
• LacNAc Gal- GlcNAc
• 1 U ST6Gal1 is equal to the formation of 1 nmol NeuAc-Gal- GlcNAc (also referred to as Sa-LacNAc) by the transfer of NeuAc ifomfrom CMP-NANA and to Gal-GlcNAc (LacNAc) per minute.
• Beta-l,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galtl, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of beta- 1, 4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galtl, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same are suitable for use in the methods described herein.
• Beta-1, 4-galactosyltransferase 1 is a Type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5’-diphosphosegalactose (UDP-Gal) to GlcNAc as a ⁇ -1,4 linkage.
• B4Galtl is one of seven beta-1, 4-galactosyltransferase (beta4GalT) genes that each encode type II membrane-bound glycoproteins that appear to have exclusive specificity for the donor substrate UDP -galactose; all transfer galactose in a betal,4 linkage to similar acceptor sugars: GlcNAc, Glc, and Xyl.
• B4Galtl adds galactose to N-acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains.
• B4GalTl is also called GGTB2.
• Four alternative transcripts encoding four isoforms of B4GALT1 (NCBI Gene ID 2683) are described in Table 1. Table 1. Human B4GALT1 isoforms
• the soluble form of B4GalT1 derives from the membrane form by proteolytic processing.
• the cleavage site is at positions 77-78 of B4GALT1 isoform 1 (SEQ ID NO: 5).
• one or more of the amino acids of the B4GalTl corresponding to amino acids 113, 130, 172, 243, 250, 262, 310, 343, or 355 of B4GALT1 isoform 1 (SEQ ID NO: 1
• the enzyme is an enzymatically active portion of, e.g., B4GalTl.
• the enzyme is an enzymatically active portion of B4GALT1 isoform 1
• the enzyme is an enzymatically active portion of B4GALT1 isoform 2 (SEQ ID NO: 6), or an ortholog, mutant, or variant of SEQ ID NO: 6.
• the enzyme is an enzymatically active portion of B4GALT1 isoform 3 (SEQ ID NO: 7), or an ortholog, mutant, or variant of SEQ ID NO: 7.
• the enzyme is an enzymatically active portion of B4GALT1 isoform 4 (SEQ ID NO: 8), or an ortholog, mutant, or variant of SEQ ID NO:
• the enzymatically active portion of B4GalTl does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 9. In some embodiments, the enzymatically active portion of B4GalTl does not comprise a transmembrane domain, e.g., SEQ ID NO: 10. In some embodiments, the enzymatically active portion of B4GalTl does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 9 or a transmembrane domain, e.g., SEQ ID NO: 10.
• the enzymatically active portion of B4GalTl comprises all or a portion of a luminal domain, e.g., SEQ ID NO: 11, or an ortholog, mutants, or variants thereof.
• the enzymatically active portion of B4GalTl comprises amino acids 109-398 of SEQ ID NO: 5, or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of B4GalTl consists of SEQ ID NO: 5, or an ortholog, mutant, or variant of SEQ ID NO: 5.
• a suitable functional portion of an B4GalTl can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 12.
• amino acid sequence that comprises or consists of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 13.
• ST6Gal1 e.g., ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same, are suitable for use in the methods described herein.
• 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 a-2,6 linkage.
• ST6Gal1 is also called as ST6N or SIAT1.
• Four alternative transcripts encoding two isoforms of ST6GAL1 (NCBI Gene ID 6480) are described in Table 1.
• the soluble form of ST6Gal1 derives from the membrane form by proteolytic processing.
• one or more of the amino acids of the 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 is conserved as compared to SEQ ID NO: 14.
• the enzyme is an enzymatically active portion of ST6Gal1 isoform a (SEQ ID NO: 14), or an ortholog, mutant, or variant of SEQ ID NO: 14.
• the enzyme is an enzymatically active portion of ST6Gal1 isoform b (SEQ ID NO: 15), or an ortholog, mutant, or variant of SEQ ID NO: 15.
• the enzymatically active portion of ST6Gal1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 16. In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a transmembrane domain, e.g., SEQ ID NO: 17. In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 16 or a transmembrane domain, e.g., SEQ ID NO: 17.
• the enzymatically active portion of ST6Gal1 comprises all or a portion of a luminal domain, e.g., SEQ ID NO: 18, or an ortholog, mutants, or variants thereof.
• the enzymatically active portion of ST6Gal1 comprises amino acids 87-406 of SEQ ID NO: 14 (SEQ ID NO: 19), or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of ST6Gal1 consists of SEQ ID NO: 19, or an ortholog, mutant, or variant of SEQ ID NO: 19.
• a suitable functional portion of an ST6Gal1 can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 19.
• the ST6Gal1 comprises or consists of SEQ ID NO: 19, the portion of SEQ ID NO: 19 from amino acid 4 to 320, or the portion of SEQ ID NO: 19 from amino acid 5 to 320.
• amino acid sequence that comprises or consists of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 20.
• the methods described herein include galactosylation and sialylation of antibodies.
• Suitable antibodies include, for example, IgG antibodies.
• the antibodies, e.g., IgG antibodies, can be pooled.
• pooled IgG antibodies include IVIg.
• the IgG antibodies comprise IgG antibodies isolated from at least 1000 donors.
• At least 50%, 55%, 60%, 65% or 70% w/w of the IgG antibodies are IgGl antibodies.
• At least 90% of the donor subject has been exposed to a virus.
• the methods described herein include providing a mixture of IgG antibodies.
• 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.
• the mixture of IgG antibodies are isolated from intravenous immunoglobulin.
• the mixture of IgG antibodies are intravenous immunoglobulin.
• the step of isolating a mixture of IgG antibodies from the pooled plasma comprises ethanol precipitation or caprylic acid (also called octanoic acid) precipitation.
• the step of isolating a mixture of IgG antibodies from the pooled plasma comprises biding IgG antibodies to an ion exchange column and eluting the IgG antibodies from an ion exchange column.
• the antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg is provided as part of a solution.
• the concentration of antibod(ies), e.g., antibod(ies) described herein, e.g., pooled IgG, e.g., IVIg is from or from about 100 mg/mL to or to about 200 mg/mL. In some embodiments, the concentration of the antibod(ies) is 150 mg/mL or is about 150 mg/mL.
• the solution consists of or comprises the antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg, and a buffer.
• the buffer is selected from the group consisting of BIS-TRIS, MOPS, MES, PIPES, BES, MOPSO, TEA, POPSO, EPPS, and combinations thereof.
• the solution consists of or comprises the antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg, and BIS-TRIS buffer. In some embodiments, the solution consists of or comprises the antibod(ies) in 50 mM BIS-TRIS buffer.
• the antibod(ies) e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg, and BIS-TRIS buffer.
• the solution consists of or comprises the antibod(ies) in 50 mM BIS-TRIS buffer.
• the solution consists of or comprises the antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg, and BIS-TRIS buffer, e.g., 50 mM BIS-TRIS buffer, at from or from about pH 6.8 to or to about pH 7.4.
• the antibod(ies) described herein e.g., IgG, e.g., pooled IgG, e.g., IVIg
• BIS-TRIS buffer e.g., 50 mM BIS-TRIS buffer
• the solution consists of or comprises the antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg, and BIS-TRIS buffer, e.g., 50 mM BIS- TRIS buffer at from or from about pH 6.8 to or to about pH 7.3, from or from about pH 6.8 to or to about pH 7.2, from or from about pH 6.8 to or to about pH 7.1, from or from about pH 6.8 to or to about pH 7.0, from or from about pH 6.8 to or to about pH 6.9, from or from about pH 6.9 to or to about pH 7.4, from or from about pH 7.3 to or to about pH 7.2, from or from about pH 6.9 to or to about pH 7.1, from or from about pH 6.9 to or to about pH 7.0, from or from about pH 7.0 to or to about pH 7.4, from or from about pH 7.0 to or to about pH 7.3,
• the solution consists of or comprises the antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg, and BIS-TRIS buffer, e.g., 50 mM BIS-TRIS buffer at or at about pH 7.3.
• the antibod(ies) described herein e.g., IgG, e.g., pooled IgG, e.g., IVIg
• BIS-TRIS buffer e.g., 50 mM BIS-TRIS buffer at or at about pH 7.3.
• the methods described herein can comprise a galactosylation step.
• An exemplary galactosylation reaction is depicted in FIG. 3.
• a method for galactosylating antibod(ies), e.g., antibod(ies) described herein by providing a composition (a galactosylation mixture) comprising: antibod(ies), e.g., antibod(ies) described herein; a galatosylating enzyme, e.g., a galatosylating enzyme described herein, e.g., B4GalT or enzymatically active portion of variant thereof; UDP-gal or salt thereof; a buffer, e.g., a buffer described herein, e.g., BIS-TRIS buffer; and optionally MnCl 2 . and incubating the composition under conditions effective for galactosylating the antibody, e.g., as described herein, thereby producing galactosylated antibod
• the methods described herein can comprise a sialylation step.
• An exemplary sialylation reaction is depicted in FIG. 3.
• a method for sialylating, e.g., hyper- sialylating, antibod(ies), e.g., antibod(ies) described herein by providing a composition (a sialylation reaction mixture) comprising: galatosylated antibod(ies), e.g., as described herein; a sialylating enzyme, e.g., a sialylating enzyme described herein, e.g., ST6Gal1 or enzymatically active portion or variant thereof; CMP -NANA or a salt thereof; a buffer, e.g., a buffer described herein, e.g., BIS-TRIS buffer; and optionally MnCl 2 , and incubating the composition under conditions effective for sialylating the antibod(ies), e.g., as described herein.
• the galactosylation step and the sialylation step are carried out sequentially in the same reaction mixture, that is, the galactosylation reaction mixture becomes the sialylation reaction mixture upon addition of the sialylating enzyme and CMP-NANA or salt thereof.
• there galactosylation reaction mixture is not filtered, fractionated, or purified prior to the sialylation step.
• the galactosylation step and the sialylation step are carried out separately, e.g, pre-galactosylated antibod(ies) are provided, though they may have been processed (e.g., filtered, fractionated, or purified) and/or stored prior to the sialylation step.
• the methods described herein can also comprise a sequential galactosylation and sialylation step.
• An exemplary galactosylation and sialylation reaction is depicted in FIG. 3.
• a method for galactosylating and sialylating e.g., hyper-sialylating, antibod(ies), e.g., antibod(ies) described herein, by a) providing a composition (a galactosylation reaction mixture) comprising: antibod(ies), e.g., as described herein; a galactosylating enzyme, e.g., a galactosylating enzyme described herein, e.g., B4GalT or enzymatically active portion or variant thereof; UDP-gal or a salt thereof; a buffer, e.g., a buffer described herein, e.g., BIS- TRIS buffer; and optionally MnCl 2 : and b) in
• one or more component(s) of one or more of the reaction mixture(s) are supplemented during the incubation. That is, the reaction mixture may comprise an amount of the component at the beginning of the reaction (which may change during the course of the reaction), but also be supplemented with additional amounts of the component(s) during the reaction.
• the galactosylation reaction mixture comprises from or from about 50 to or to about 200 mg/mL antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg.
• antibod(ies) e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg.
• the galactosylation reaction mixture comprises from or from about 50 to or to about 200, from or from about 50 to or to about 150, from or from about 50 to or to about 100, from or from about 100 to or to about 200, from or from about 100 to or to about 200, from or from about 100 to or to about 150, or from or from about 150 to or to about 200 mg/mL antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg.
• antibod(ies) described herein e.g., IgG, e.g., pooled IgG, e.g., IVIg.
• the galactosylation reaction mixture comprises 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 antibod(ies), e.g., the antibod(ies) described herein, e.g., IgG, e.g., pooled IgG, e.g., IVIg.
• antibod(ies) described herein e.g., IgG, e.g., pooled IgG, e.g., IVIg.
• the galactosylation reaction mixture comprises from or from about 6.0 to or to about 15.0 U galactosylating enzyme per gram of antibody. In some embodiments, the galactosylation reaction mixture comprises from or from about 7.0 to or to about 9.0 U galactosylating enzyme per gram of antibody. In some embodiments, the galactosylation reaction mixture comprises from or from about 7.2 to or to about 8.8 U of galactosylating enzyme per gram of antibody. In some embodiments, the galactosylation reaction mixture comprises 7.5 or about 7.5 U of galactosylating enzyme per gram of antibody. In some embodiments, the galactosylation reaction mixture comprises 8.0 or about 8.0 U of galactosylating enzyme per gram of antibody.
• the galactosylation reaction mixture is supplemented with from or from about 6.0 to or to about 15.0 U galactosylating enzyme per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with from or from about 7.0 to or to about 9.0 U galactosylating enzyme per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with from or from about 7.2 to or to about 8.8 U of galactosylating enzyme per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with 7.5 or about 7.5 U of galactosylating enzyme per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with 8.0 or about 8.0 U of galactosylating enzyme per gram of antibody.
• the galactosylation reaction mixture comprises from or from about 0.030 to or to about 0.050 mmol UDP-gal or salt thereof per gram of antibody. In some embodiments, the galactosylation mixture comprises from or from about 0.038 to or to about 0.046 mmol UDP-gal or salt thereof per gram of antibody. In some embodiments, the galactosylation reaction mixture comprises 0.038 or about 0.038 mmol UDP-gal or salt thereof per gram of antibody. In some embodiments, the galactosylation reaction mixture comprises 0.042 or about 0.042 mmol UDP-gal or salt thereof per gram of antibody.
• the galactosylation reaction mixture is supplemented with from or from about 0.030 to or to about 0.050 mmol UDP-gal or salt thereof per gram of antibody. In some embodiments, the galactosylation mixture is supplemented with from or from about 0.038 to or to about 0.046 mmol UDP-gal or salt thereof per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with 0.038 or about 0.038 mmol UDP-gal or salt thereof per gram of antibody. In some embodiments, the galactosylation reaction mixture is supplemented with 0.042 or about 0.042 mmol UDP-gal per gram of antibody.
• the sialylation reaction mixture comprises from or from about 14.0 to or to about 20.0 U sialylating enzyme per gram of antibody. In some embodiments, the sialylation reaction mixture comprises from or from about 17.1 to or to about 18.9 U of sialylating enzyme per gram of antibody. In some embodiments, the sialylation reaction mixture comprises 15.8 or about 15.8 U of sialylating enzyme per gram of antibody. In some embodiments, the sialylation reaction mixture comprises 18.0 or about 18.0 U of sialylating enzyme per gram of antibody.
• the sialylation reaction mixture is supplemented with from or from about 14.0 to or to about 20.0 U sialylating enzyme per gram of antibody. In some embodiments, the sialylation reaction mixture is supplemented with from or from about 17.1 to or to about 18.9 U of sialylating enzyme per gram of antibody. In some embodiments, the sialylation reaction mixture is supplemented with 15.8 or about 15.8 U of sialylating enzyme per gram of antibody. In some embodiments, the sialylation reaction mixture is supplemented with 18.0 or about 18.0 U of sialylating enzyme per gram of antibody.
• the sialylation reaction mixture comprises from or from about 0.1 to or to about 0.3 mmol CMP -NANA or salt thereof per gram of antibody. In some embodiments, the sialylation reaction mixture comprises from or from about 0.1425 to or to about 0.1575 mmol CMP -NANA or salt thereof per gram of antibody. In some embodiments, the sialylation reaction mixture comprises 0.220 or about 0.220 mmol CMP-NANA or salt thereof per gram of antibody. In some embodiments, the sialylation reaction mixture comprises 0.150 mmol or about 0.150 mmol CMP-NANA or salt thereof per gram of antibody.
• from or from about 0.01 to or to about 0.3 mmol CMP-NANA or salt thereof is added to the sialylation reaction mixture per gram of antibody at the beginning of the reaction. In some embodiments, from or from about 0.01425 to or to about 0.1575 mmol CMP-NANA or salt thereof is added to the sialylation reaction mixture per gram of antibody at the beginning of the reaction.
• the sialylation reaction mixture is supplemented with from or from about 0.01 to or to about 0.3 mmol CMP-NANA or salt thereof per gram of antibody. In some embodiments, the sialylation reaction mixture is supplemented with from or from about 0.01425 to or to about 0.1575 mmol CMP-NANA or salt thereof per gram of antibody.
• the total amount of CMP-NANA added to the sialylation reaction mixture is from or from about 0.1 to or to about 0.3 mmol CMP-NANA or salt thereof per gram of antibody. In some embodiments, the total amount of CMP-NANA added to the sialylation reaction mixture is from or from about 0.1425 to or to about 0.1575 mmol CMP-NANA or salt thereof 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 CMP-NANA or salt thereof 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 CMP-NANA or salt thereof per gram of antibody.
• the sialylation reaction mixture is supplemented with CMP- NANA one, two, three, four, five, six, seven, eight, nine, or ten times. In some embodiments the sialylation mixture is supplemented with CMP-NANA less than seven times.
• the galactosylation and/or sialylation reaction mixture(s) each independently comprises from or from about 1 to or to about 20 mM nCk In some embodiments, the galactosylation and/or sialylation reaction mixture(s) each independently comprise from or from about 4.5 to or to about 5.5 mM nCk In some embodiments, the galactosylation and/or sialylation reaction mixture(s) each independently comprise 7.5 or about 7.5 mM MnCl 2 In some embodiments, the galactosylation and/or sialylation reaction mixture(s) each independently comprise 5.0 or about 5.0 mM nCk
• the galactosylation and/or sialylation reaction mixture(s) comprise BIS-TRIS buffer. In some embodiments, the galactosylation and/or sialylation reaction mixture(s) each independently comprise from or 10 to or to about 500 mM BIS-TRIS buffer.
• the galactosylation and/or sialylation reaction mixture(s) each independently comprise from or from about 10 to or to about 400, from or from about 10 to or to about 300, from or from about 10 to or to about 300, from or from about 10 to or to about 200, from or from about 10 to or to about 100, from or from about 10 to or to about 50, from or from about 50 to or to about 500, from or from about 50 to or to about 400, from or from about 50 to or to about 300, from or from about 50 to or to about 200, from or from about 50 to or to about 100, from or from about 100 to or to about 500, from or from about 100 to or to about 400, from or from about 100 to or to about 300, from or from about 100 to or to about 200, from or from about 200 to or to about 500, from or from about 200 to or to about 400, from or from about 200 to or to about 300, from or from about 300 to or to about 500, from or from about 300 to or to about 400, or from or from about 400 to or from about 200 to
• the galactosylation and/or sialylation reaction mixture(s) each independently comprise from or from about 10 to or to about 100, from or from about 10 to or to about 90, from or from about 10 to or to about 80, from or from about 10 to or to about 70, from or from about 10 to or to about 60, from or from about 10 to or to about 50, from or from about 10 to or to about 40, from or from about 10 to or to about 30, from or from about 10 to or to about 20, from or from about 20 to or to about 100, from or from about 20 to or to about 90, from or from about 20 to or to about 80, from or from about 20 to or to about 70, from or from about 20 to or to about 60, from or from about 20 to or to about 50, from or from about 20 to or to about 40, from or from about 20 to or to about 30, from or from about 30 to or to about 100, from or from about 30 to or to about 90, from or from about 30 to or to about 80, from or from about 30 to or to about 70,
• the galactosylation and/or sialylation reaction mixture(s) each independently comprise 50 mM or less, 100 mM or less, 150 mM or less, 30 mM or less, or no sodium chloride.
• the galactosyl ati on and/or sialylation steps are each independently carried out from or from about 20 to or to about 50 °C.
• the sialylation is carried out from or from about 20 to or to about 45, from or from about 20 to or to about 40, from or from about 20 to or to about 35, from or from about 20 to or to about 30, from or from about 20 to or to about 25, from or from about 25 to or to about 50, from or from about 25 to or to about 45, from or from about 25 to or to about 40, from or from about 25 to or to about 35, from or from about 25 to or to about 30, from or from about 30 to or to about 50, from or from about 30 to or to about 45, from or from about 30 to or to about 40, from or from about 30 to or to about 35, from or from about 35 to or to about 50, from or from about 35 to or to about 45, from or from about 35 to or to about 40, from or from about 40 to or to about 50, from or from about 40 to or to about 50, from
• the galactosylation and/or sialylation steps are each independently carried out at our about pH 5.8 to or to about pH 7.2.
• the sialylation is carried out at or at about pH 5.8 to or to about pH 7.1, at or at about pH 5.8 to or to about pH 7.0, at or at about pH 5.8 to or to about pH 6.9, at or at about pH 5.8 to or to about pH 6.8, at or at about pH 5.8 to or to about pH 6.7, at or at about pH 5.8 to or to about pH 6.6, at or at about pH 5.8 to or to about pH 6.5, at or at about pH 5.8 to or to about pH 6.4, at or at about pH 5.8 to or to about pH 6.3, at or at about pH 5.8 to or to about pH 6.2, at or at about pH 5.8 to or to about pH 6.1, at or at about pH 5.8 to or to about pH 6.0, at or at about pH 5.8 to or to about pH 5.9, at or at about pH 5.9 to or
• the pH of one or more of the reaction mixtures is adjusted during the galactosylation and/or sialylation, e.g., to fall within the preferred range, e.g., to return to or to about the starting pH.
• the galactosylation step is carried out for 60 hours or less, e.g., 50 hours or less, 40 hours or less, or preferably 20 hours or less or 20 hours or less.
• the galactosylation step is carried out for at least 8, 12, 18, 24, 30, or 40, but no more than 60 hours.
• the galactosylation step is carried out for or for about 8, 12, 18,
• the sialylation step is carried out for 70 hours or less, e.g., 60 hour or less, 50 hours or less, or preferably 40 hours or less.
• the sialylation step is carried out for at least 8, 12, 18, 24, 30, 40, or 50 hours, but no more than 70 hours.
• the sialylation step is carried out for or for about 8, 12, 18, 24, 30, 40, 50, 60, or 70 hours.
• the total incubation time for galactosylation and sialylation is 130 hours or less, e.g., 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.
• 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 no more than 130 hours.
• the total incubation time for galactosylation and sialylation is or is about 8, 12, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or 130 hours.
• At least or about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans on the antibod(ies), e.g., hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1 ,6 branch.
• about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the antibod(ies), e.g., hsIgG have a sialic acid on both the ⁇ 1 ,3 arm and the ⁇ 1 ,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• about or at least 80% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• about or at least 85% of the of the branched Fc glycans on the antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• about or at least 90% of the of the branched Fc glycans on the antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• IgG in which more than 60% of the overall branched glycans are disialylated can be prepared as follows.
• a mixture of IgG antibodies is exposed to a sequential enzymatic reaction using ⁇ 1,4 galactosyltransferase 1 (B4GalT) and ⁇ 2,6-sialyltransferase (ST6Gal1) enzymes.
• B4GalT ⁇ 1,4 galactosyltransferase 1
• ST6Gal1 ⁇ 2,6-sialyltransferase
• the galactosyltransferase enzyme selectively adds galactose residues to pre-existing asparagine-linked glycans.
• the resulting galactosylated glycans serve as substrates to the sialic acid transferase enzyme which selectively adds sialic acid residues to cap the asparagine-linked glycan structures attached to.
• the overall sialylation reaction employed two sugar nucleotides (uridine 5'-diphosphogalactose (UDP-Gal) and cytidine-5'-monophospho-N- acetylneuraminic acid (CMP -NANA)). The latter is replenished periodically to increase disialylated product relative to monosialylated product.
• the reaction includes the co-factor manganese chloride.
• FIG. 4 A representative example of the IgG-Fc glycan profile for such a reaction starting with IVIg and the reaction product is shown in the FIG. 4.
• the left panel is a schematic representation of enzymatic sialylation reaction to transform IgG to hsIgG; the right panel is the IgG Fc glycan profile for the starting IVIg and hsIgG.
• glycan profiles for the different IgG subclasses are derived via glycopeptide mass spectrometry analysis.
• 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).
• the glycan data is shown per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg the sum of all the nonsialylated glycans is more than 80% and the sum of all sialylated glycans is ⁇ 20%. For the reaction product, the sum for all nonsialylated glycans is ⁇ 20% and the sum for all sialylated glycans is more than 80%. Nomenclature for different glycans listed in the glycoprofile use the Oxford notation for N linked glycans. Example 2: Improvement of Sialylation Reaction
• the sialylation reaction driven by ST6Gal using CMP -NANA as a substrate has characteristics that make improvement of the reaction, whether assessed by overall level of disialylation, time to reach a certain level of disialylation, amount of enzyme and substrate required to reach a certain overall level of disialylation, challenging.
• CMP -NANA is not entirely stable and will spontaneously hydrolyze even in the absence of any enzyme
• ST6Gal1 is thought to catalyze hydrolysis of the CMP -NANA without productive addition to the Gal on a branched glycan
• CMP cytidine monophosphate
• CMP has been observed to catalyze the reverse enzymatic reaction to remove the NeuAc from the newly formed glycan.
• reaction conditions that result in IgG antibodies or IVIg or pooled immune globulins with high levels of disialylation on Fc domain branched glycans.
• sialylation with ST6Gal1 in MOPS buffer at pH 7.4 at 37 °C at a relatively high IgG antibodies concentration e.g, a high concentration of IVIg concentration ( ⁇ 125 mg/mL)
• IgG antibodies concentration e.g, a high concentration of IVIg concentration ( ⁇ 125 mg/mL)
• branched glycans e.g., branched glycans on the Fc domain
• CMP-NANA is supplemented over the course of the sialylation reaction.
• 1,6-A1F varies to a lesser extent across the buffers under the conditions examined, as shown in FIG. 6.
• the sialylation of IVIg was investigated in BIS-TRIS pH 6.9, TEA pH 7.5, TEA pH 8.0, and TRIS pH 8.0.
• BIS-TRIS was selected for further study.
• suitable reaction conditions in 50 mM BIS-TRIS pH 6.9 include: galactosylation of IgG antibodies (e.g., pooled IgG antibodies, pooled immunoglobulins or IVIg) are as follows: 7.4 mM MnCl 2 : 38 ⁇ mol UDP-Gal/g IgG antibody; and 7.5 units B4GalT/g IgG antibody with 16-24 hours of incubation at 37°C followed by sialylation in 7.4 mM MnCl 2 ; 220 pmol CMP-NANA/g IgG antibody (added twice: half at the start of the reaction and half after 9- 10 hrs); and 15 units ST6Gal1/g IgG antibody with 30-33 hours of incubation at 37°.
• the reaction can be
• Example 3 Enzymatic Galactosylation and Sialylation for Production of hsIgGs at high concentration IVIg or IgG Antibodies
• Sialylation is accomplished in two sequential enzymatic reaction steps using UDP-Gal and CMP-NANA in 50 mM MOPS buffer pH 7.4.
• Galactosylation occurs by reaction of IVIg (at about 135 mg/ml) with 8 to 15 units B4GalT/g IVIg and 0.038 - 0.042 mmol UDP-Gal /g IVIg in 50 mM MOPS buffer pH 7.4 with 5 to 8 mM MnCl 2 .
• the reaction is allowed to proceed for 46 to 50 hrs and 37°C.
• reaction is then cooled to ambient temperature and diluted with 5X sodium phosphate buffer (PBS) 1:1 v/v.
• PBS sodium phosphate buffer
• Total glycans were assessed for sialylation. Greater than 97% of the glycans were sialylated and greater than 90% of the glycans were disialylated.
• CMP-NANA is not stable and will spontaneously hydrolyze even in the absence of any enzyme.
• ST6 enzyme is thought to also catalyze hydrolysis of the CMP-NANA without productive addition to the glycan acceptor.
• Cytidine monophosphate (CMP) is a side-product, generated either through enzymatic addition or CMP-NANA hydrolysis.
• CMP acts as a competitive inhibitor of ST6.
• CMP has been observed to catalyze the reverse enzymatic reaction to remove the NeuAc from the newly formed glycan.
• M254 is currently produced by the enzymatic sialylation of Fc and Fab glycans of IVIg drug product in MOPS pH 7.4 buffer, all steps at 37 °C, and using a very high IVIg concentration (-150 mg/mL) where high protein concentration improves the reaction kinetics.
• the galactosylation step uses incubation over the course of 48 h.
• a single addition of ST6 is followed by twice daily additions of CMP-NANA (six total) over the course of 72 h. This has been dubbed Process 2.0.
• Process 3.0.0 uses less B4GalT enzyme, less CMP-NANA, and a shorter overall reaction time for both steps 56 h total vs. 120 h. Thus Process 3.0.0 incurs both lower materials cost and lower production costs.
• IVIg solutions were prepared using Privigen IVIg drug product buffer exchanged into BIS-TRIS buffer.
• One batch of IVIg was buffer exchanged using a G25 desalting column equilibrated with BIS-TRIS pH 6.9 buffer followed by concentration of the IVIg flow thru fraction using a 10 kDa Vivaspin Turbo 15 device.
• Three 5 g batches of Privigen IVIg were buffer exchanged by tangential flow filtration (TFF) into 50 mM BIS-TRIS at pH 6.67, 6.93, and 7.11.
• One batch of IVIg was buffer exchanged by tangential flow filtration (TFF) into 50 mM BIS-TRIS at pH 6.9, followed by concentration using a 10 kDa Vivaspin Turbo 15 device.
• the pH of the IVIg solution lies outside of the preferred range, e.g., 7.2 to 7.4, preferably about 7.3, it should be adjusted.
• the sialylation step was initiated (typically after 24 h galactosylation) by addition of ST6 enzyme and 1 ⁇ 2 the required CMP -NANA.
• the galactosylated material was first divide into smaller volumes to run multiple sialylation reactions.
• the second 1 ⁇ 2 CMP- NANA was added.
• Incubation was continued for various times.
• Two 5 uL aliquots were removed at various times and then frozen until analyzed.
• the bulk reaction material was placed at 4 °C.
• the extent of glycosylation was quantified by LCMS on the Fc glycopeptides.
• Table 10 shows the glycans quantified by LCMS on the Fc glycopeptides. Fully galactosylated encompassed all glycans having two galactose residues whether sialylated or not. Disialylation was defined as the sum of A2F, A2F+bisecting GlcNAc, and A2. Table 10. Glycans quantified by LCMS on the Fc glycopeptides.
• FIG. 7A shows the amount of GIF+NeuAc and Gl+NeuAc increase with increasing MnCl 2 . These species result from incomplete galactosylation.
• FIG. 7B shows that the amounts of G0F, G1F, and G2F increase with increasing MnCl 2 . This shows that in addition to poorer galactosylation, sialylation is also affected, i.e. the amount of non-sialylated species is also higher with higher MnCl 2 .
• FIG. 9 shows an increase in galactosylation of IgGl between 20 and 44 h for all conditions (grouped by MnCl 2 concentration). Similar results were seen for the other IgG subclasses.
• FIG. 10 shows the same data but grouped by time.
• galactosylation extent increases from 2.5 mM MnCl 2 to 5.0 mM and then falls going to 7.5 mM and then 10 mM.
• IVIg in pH 6.9 buffer was subjected to the galactosylation and sialylation reaction using UDP-Gal/B4GalT and CMP-NANA/ST6 respectively in the presence of MnCl 2 with 37 °C incubation.
• Various concentrations of sodium chloride in BIS-TRIS buffer were added to obtain a final reaction concentration of 0, 50, 100, 150, and 300 mM sodium chloride.
• Samples were removed for glycan analysis by glycopeptide LCMS at the end of the galactosylation and sialylation reactions.
• UDP-Gal was found to be unstable in the presence of MnCl 2 to give decomposition products (UMP and presumably 1,2-phosphogalactose 1) different from enzyme catalyzed transfer products (UDP). The mechanism is thought to follow the scheme shown in FIG. 17.
• UDP-Gal, UDP, and UMP were assessed by ion pairing HPLC on a Supelcosil LC-18-T column using a 0.1 M potassium phosphate, 4 mM tetrabutylammonium bisulfate, pH 6.0 mobile phase and UV detection at 254 nm. Only components bearing uridine were detected by UV and sugars not bound to uridine could not be detected. Products were compared to known standards. UDP-Gal in BIS-TRIS pH 6.9 buffer was heated at 37 °C in the presence of 0, 5, 10 or 20 mM MnCl 2 for 8 h and then assessed by ion pairing HPLC. No B4Galt was included in this experiment. The amount of UDP-Gal loss was MnCl 2 dependent and increased with increasing MnCl 2 concentration. The only other product observed was UMP. Very little UMP was visible in the absence of MnCl 2 .
• IVIg was galactosylated 24 h using UDP-Gal, B4GalT, and 5 mM MnCl 2 in BIS-TRIS buffer at three different pH (6.7, 6.9, and 7.1).
• the higher molecular weight IgG protein was separated from the low molecular weight sugar nucleotide using a 500 MWCO spin unit and the nucleotide containing fraction was injected onto the ion pairing HPLC.
• hsIgG is prepared using BIS-TRIS pH 7.3.
• suitable reaction conditions in 50 mM BIS-TRIS pH 7.3 include: galactosylation of IgG antibodies (e.g., pooled IgG antibodies, pooled immunoglobulins or IVIg) are as follows: 5.0 mM MnCl 2 ; 42 ⁇ mol UDP- Gal/g IgG antibody; and 8.0 units B4GalT/g IgG antibody with 16-24 hours of incubation at 37°C followed by sialylation in 5.0 mM MnCl 2 ; 110 pmol CMP-NANA/g IgG antibody (added twice: half at the start of the reaction and again after 9-10 hrs); and 18 units ST6Gal1/g IgG antibody with 30-33 hours of incubation at 37°.
• the reaction can be carried out by adding the ST6Gal1 and CMP -NANA to the galactosylation reaction.
• This method carried out at a 21 g scale, achieved 99% full IgGl galactosylation by glycopeptide LCMS, 96% disialylated IgGl by gly copeptide LCMS, and 94% disialylated by N- glycan release (AdvanceBio Gly-X N-glycan prep with InstantPC kit, Agilent). This gives the global release ofN-glycans allowing the quantitative sum of IgGl, IgG2, IgG3, IgG4 Fc glycans as well as the -15-25% Fab glycosylation present in IVIg.

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