Preparation and purification of highly sialylated IgG

Abstract

Methods for preparing and purifying hypersialylated IgG have been described.

Description

[Technical Field]

[0001] (Claiming priority) This application claims the benefits of U.S. Provisional Patent Application No. 63 / 026,875, filed on 19 May 2020. The entire foregoing is incorporated herein by reference.

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

[0003] Intravenous immunoglobulin (IVIg), prepared from pooled plasma from human donors (e.g., pooled plasma 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] Therefore, described herein is a method for preparing immunoglobulin G (IgG) having a very high level of Fc sialylation, in particular disialylation (sialylation of α1,3 branching and α1,6 branching of glycans at Asn297 (EU numbering)). The method described herein can provide highly sialylated IgG (hsIgG) in which more than 70% of the branched glycans in the Fc domain are sialylated at both branches (i.e., α1,3 branching and α1,6 branching). HsIgG contains a diverse mixture of IgG antibodies, mainly IgG1 antibodies. The antibody diversity is high. The immunoglobulin used to prepare hsIgG can be obtained, for example, from pooled human plasma (e.g., pooled plasma 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 glycans in the Fc region than IVIg. This results in a composition that differs 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.

[0007] The methods described herein are improved methods for preparing and purifying hsIgG. These methods produce an unpurified hsIgG composition from which purified hsIgG and, in some embodiments, the enzyme used to prepare hsIgG can be isolated. In some cases, the isolated enzyme can be reused.

[0008] This specification describes a method for producing a purified highly sialylated IgG (hsIgG) composition in which more than 75% of the branched Fc glycans on hsIgG have sialic acid in both α1,3 branching and α1,6 branching, and the method comprises (a) providing an hsIgG composition comprising hsIgG and ST6Gal or its enzymatically active moiety, (b) diluting the composition with citrate buffer of about 50 mM, about pH 4.5 to produce a buffered antibody composition, (c) adding the buffered antibody composition to a chromatography column containing a resin having a sulfonic acid functional group under conditions that bind to hsIgG and ST6Gal or its enzymatically active moiety, and (d) selectively eluting IgG from the column to produce a purified hsIgG composition in which more than 75% of the branched Fc glycans on hsIgG have sialic acid in both α1,3 branching and α1,6 branching.

[0009] In some embodiments, the step of providing an hsIgG composition includes providing a composition containing hsIgG and diluting the composition in 5×PBS at a dilution of about 1:1.

[0010] In some embodiments, the CEX column is SO3 - Includes resins having functional groups.

[0011] In some embodiments, selective elution of hsIgG includes elution in a buffer containing approximately 400 mM or more of NaCl.

[0012] In some embodiments, the buffer is a citrate buffer with a concentration of approximately 50 mM and a pH of approximately 4.5.

[0013] In some embodiments, the method does not include additional filtration, fractionation, or purification steps other than (a) and (b) before performing step (c).

[0014] In some embodiments, in addition to steps (a) and (b), the method includes a single depth filtration step before step (c).

[0015] In some embodiments, the additional depth filtration step is performed between steps (a) and (c).

[0016] In some embodiments, the method does not include additional filtration, fractionation, or purification steps other than a single depth filtration step before performing step (c).

[0017] In some embodiments, the method includes a virus inactivation step before step (c).

[0018] In some embodiments, what is added in step (c) is added at intervals over 1, 2, or 3 times.

[0019] In some embodiments, step (e) is repeated one or more times.

[0020] In some embodiments, step (c) is performed with a residence time (plural available) of 1, 2, 3, 4, 5, or 6 minutes or about 1, 2, 3, 4, 5, or 6 minutes.

[0021] In some embodiments, the method further includes one or more of (f) blue dye chromatography, (g) DE pad filtration step, (h) depth filtration step, or (g) PS20 spike and filtration step after step (e).

[0022] In some embodiments, the purified hsIgG composition contains 80%, 85%, 90%, or 95% or more of the amount of unpurified hsIgG from the composition of step (a).

[0023] In some embodiments, the purified hsIgG composition comprises ST6Gal or an enzymatically active portion thereof at 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, or 30 ppm or less.

[0024] Also provided herein is a method for generating a purified highly sialylated IgG (hsIgG) composition, the method comprising: (a) providing an hsIgG composition comprising hsIgG and ST6Gal or an enzymatically active portion thereof, optionally quenching the reactants; (b) diluting the composition in a buffer suitable for use in a column, thereby generating a buffered antibody composition; (c) adding the buffered antibody composition to a blue dye column, such as the blue column described herein, under conditions that allow 80% or more of the hsIgG to flow through but allow only ST6 or an enzymatically active portion thereof at 100 ppm or less to flow through, thereby generating a purified hsIgG.

[0025] In some embodiments, the blue dye chromatography column is a Trisacryl Blue (TABS) column, and the buffer suitable for use with the column is a citrate buffer of about 100 mM, pH about 4.5, containing about 800 mM NaCl.

[0026] In some embodiments, the blue dye chromatography column is a Capto™ Blue or Capto™ Blue HS column, and the buffer is about 250 mM glycine at pH about 4.5 containing about 800 mM NaCl.

[0027] In some embodiments, the step of providing the hsIgG composition comprises providing a composition comprising hsIgG and diluting the composition in 5×PBS at an approximate 1:1 dilution.

[0028] In some embodiments, the method does not include additional filtration, fractionation, or purification steps other than (a) and (b) prior to performing step (c).

[0029] In some embodiments, the method includes a single deep filtration step prior to step (c), in addition to steps (a) and (b).

[0030] In some embodiments, an additional filtration step is performed between steps (a) and (c).

[0031] In some embodiments, the method does not include any additional filtration, fractionation, or purification steps other than a single deep filtration step before performing step (c).

[0032] In some embodiments, the method includes a virus inactivation step prior to step (c).

[0033] In some embodiments, the addition in step (c) is added one, two, or three times with intervals in between.

[0034] In some embodiments, step (c) is performed for a dwell time of 1, 2, 3, 4, 5, or 6 minutes or approximately 1, 2, 3, 4, 5, or 6 minutes.

[0035] In some embodiments, the method further comprises, after step (e), one or more of the following: (f) blue dye chromatography, (g) DE pad filtration, (h) deep filtration, or (g) PS20 spike and filtration.

[0036] This specification also describes a method for producing a highly sialylated IgG (hsIgG) composition, the method comprising (a) providing hsIgG, (b) precipitating hsIgG by adding a saturated solution of ammonium sulfate to produce a precipitated hsIgG solution, and (c) isolating the precipitated hsIgG composition to produce a purified hsIgG composition.

[0037] In some embodiments, isolating precipitated hsIgG involves filtration and / or centrifugation.

[0038] This specification also provides a method for producing highly sialylated IgG (hsIgG), the method comprising: (a) providing a pooled IgG antibody; (b) incubating the pooled IgG in a reaction mixture comprising β1,4-galactosyltransferase (B4GalT) or its enzymatically active moiety and UDP-Gal or a salt thereof; (c) incubating the galactosylated IgG antibody in a reaction mixture comprising ST6Gal or its enzymatically active moiety and CMP-NANA or a salt thereof; and (d) purifying the hsIgG according to any of the methods described herein.

[0039] This specification also describes a method for preparing highly sialylated IgG (hsIgG), the method comprising (a) providing a pooled IgG antibody, (b) incubating the pooled IgG antibody with a reaction mixture comprising β1,4-galactosyltransferase (B4GalT) or its enzymatic active moiety, UDP-Gal or a salt thereof, ST6Gal or its enzymatic active moiety, and CMP-NANA or a salt thereof, and (c) purifying the hsIgG according to any of the methods described herein, thereby producing an hsIgG preparation.

[0040] This specification also describes a method for preparing highly sialylated IgG (hsIgG), the method 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 enzymatic active moiety and UDP-Gal or a salt thereof to generate a galactosylated IgG antibody; (c) adding ST6Gal or its enzymatic active moiety and CMP-NANA or a salt thereof to the galactosylation reaction mixture to generate a sialylation reaction; (d) incubating the sialylation reaction; and (e) purifying hsIgG according to any of the methods described herein to generate hsIgG.

[0041] This specification also describes a method for preparing a purified highly sialylated IgG (hsIgG) composition, the method comprising: (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture containing β1,4-galactosyltransferase I (β4GalT) or its enzyme active moiety and UDP-Gal to produce galactosylated IgG antibodies; (c) incubating the galactosylated IgG antibodies in a second reaction mixture containing ST6Gal or its enzyme active moiety and CMP-NANA to produce an hsIgG mixture; (d) adding the hsIgG mixture to a protein A column under conditions that bind to the IgG antibody; and (e) eluting hsIgG from the protein A column.

[0042] In some embodiments, the method further includes (f) purifying the hsIgG generated in step (e) using a trisacryl blue column.

[0043] In some embodiments, step (e) includes eluting hsIgG with a buffer containing glycine.

[0044] In some embodiments, the protein A column is washed with acetate buffer between step (d) and step (e).

[0045] In some embodiments, the pH and salt content of the hsIgG generated in step (e) are modified before being added to the trisacryl blue column.

[0046] In some embodiments, step (f) includes eluting hsIgG with a high-salt buffer.

[0047] In some embodiments, the high-salt buffer contains 2 M NaCl.

[0048] In some embodiments, the high-salt buffer contains 2M KCl.

[0049] In some embodiments, the hsIgG produced in step (e) is modified with 0.4 M NaCl and pH 4.5.

[0050] This specification also describes a method for preparing hypersialyzed (hsIgG), the method comprising: (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture containing β1,4-galactosyltransferase (B4GalT) or its enzymatically active portion, UDP-Gal, ST6Gal or its enzymatically active portion, and CMP-NANA to produce an hsIgG preparation; (d) adding the hsIgG preparation to a protein A column under conditions that bind to the IgG antibody; and (e) eluting hsIgG from the protein A column.

[0051] In some embodiments, B4GalT or its enzyme-active portion is at least 90% identical to SEQ ID NO: 12 or SEQ ID NO: 13, and ST6Gal1 or its enzyme-active portion contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO: 20.

[0052] In some embodiments, the IgG antibody comprises IgG antibodies isolated from at least 1000 donors.

[0053] In some embodiments, at least 70% w / w of the IgG antibody is IgG1 antibody.

[0054] In some embodiments, at least 90% of the donor subjects have been exposed to the virus.

[0055] In some embodiments, the step of providing a mixture of IgG antibodies includes (a) providing pooled plasma from at least 1,000 human subjects, and (b) isolating the mixture of IgG antibodies from the pooled plasma.

[0056] In some embodiments, the mixture of IgG antibodies is isolated from intravenous immunoglobulin.

[0057] In some embodiments, the mixture of IgG antibodies is intravenous immunoglobulin.

[0058] In some embodiments, the step of isolating a mixture of IgG antibodies from pooled plasma includes ethanol precipitation or caprylic acid precipitation.

[0059] 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.

[0060] This specification also provides a method for preparing a purified highly sialylated (hsIgG) composition, the method comprising: (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture containing β1,4-galactosyltransferase (B4GalT) or its enzyme active moiety and UDP-Gal to produce galactosylated IgG antibodies; (c) incubating the galactosylated IgG antibodies in a reaction mixture containing ST6Gal or its enzyme active moiety and CMP-NANA to produce an hsIgG composition; and (d) isolating B4GalT or its enzyme active moiety and ST6Gal or its enzyme active moiety from the hsIgG composition.

[0061] In some embodiments, the method further includes isolating B4GalT or its enzymatically active moiety and ST6Gal or its enzymatically active moiety, or both, from the hsIgG composition.

[0062] This specification also provides a method for preparing highly sialylated IgG (hsIgG), the method comprising: (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture containing β1,4-galactosyltransferase (B4GalT) or its enzymatically active moiety, UDP-Gal, ST6Gal or its enzymatically active moiety, and CMP-NANA to produce an hsIgG preparation; and (c) isolating hsIgG from the hsIgG preparation, and isolating B4GalT or its enzymatically active moiety and ST6Gal or its enzymatically active moiety from the hsIgG preparation.

[0063] In some embodiments of the purification methods described herein, about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching before purification.

[0064] In some embodiments of the purification methods described herein, about 80% or 85% of the branched Fc glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching before purification.

[0065] In some embodiments of the purification methods described herein, at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fc domain of hsIgG have sialic acid in both the α1,3 arm and the α1,6 arm, which are connected via NeuAc-α2,6-Gal terminal linkages before purification.

[0066] In some embodiments of the purification methods described herein, at least 80% of the branched Fc glycans on IgG have sialic acid in both the α1,3 branching and the α1,6 branching before purification.

[0067] In some embodiments of the purification methods described herein, at least 60%, 65%, and 70% of the branched glycans on the Fc domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms, which are connected via NeuAc-α2,6-Gal terminal linkages before purification.

[0068] In some embodiments of the purification methods described herein, at least 85% of the branched Fc glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching before purification.

[0069] In some embodiments of the purification methods described herein, at least 60%, 65%, and 70% of the branched glycans on the Fc domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms, which are connected via NeuAc-α2,6-Gal terminal linkages before purification.

[0070] In some embodiments of the purification methods described herein, at least 90% of the branched Fc glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching before purification.

[0071] In some embodiments of the purification methods described herein, at least 60%, 65%, and 70% of the branched glycans on the Fc domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms, which are connected via NeuAc-α2,6-Gal terminal linkages before purification.

[0072] In some embodiments of the purification methods described herein, about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching after purification.

[0073] In some embodiments of the purification methods described herein, about 80% or 85% of the branched Fc glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching after purification.

[0074] In some embodiments of the purification methods described herein, at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fc domain of hsIgG have sialic acid in both the α1,3 arm and the α1,6 arm, which are connected via NeuAc-α2,6-Gal terminal linkages after purification.

[0075] In some embodiments of the purification methods described herein, at least 80% of the branched Fc glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching after purification.

[0076] In some embodiments of the purification methods described herein, at least 60%, 65%, and 70% of the branched glycans on the Fc domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms, which are connected via NeuAc-α2,6-Gal terminal linkages after purification.

[0077] In some embodiments of the purification methods described herein, at least 85% of the branched Fc glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching after purification.

[0078] In some embodiments of the purification methods described herein, at least 60%, 65%, and 70% of the branched glycans on the Fc domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms, which are connected via NeuAc-α2,6-Gal terminal linkages after purification.

[0079] In some embodiments of the purification methods described herein, at least 90% of the branched Fc glycans on hsIgG have sialic acid in both the α1,3 branching and the α1,6 branching after purification.

[0080] In some embodiments of the purification methods described herein, at least 60%, 65%, and 70% of the branched glycans on the Fc domain of hsIgG have sialic acid in both the α1,3 and α1,6 arms, which are connected via NeuAc-α2,6-Gal terminal linkages after purification.

[0081] In some embodiments of the purification methods described herein, the method further includes analyzing the amount of one or more IgG subclasses after the chromatography step.

[0082] This specification also describes a method for preparing a purified highly sialylated IgG (hsIgG) composition, the method 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; (c) incubating the galactosylated IgG antibodies in a second reaction mixture containing ST6Gal1 and CMP-NANA to produce an hsIgG mixture; (d) adding the hsIgG mixture to a protein A column under conditions that bind to the IgG antibodies; and (e) eluting hsIgG from the protein A column.

[0083] In various embodiments, the method further comprises (g) further purification of the hsIgG produced in step (e) using a trisacryl blue column. B4GalT is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2; ST6Gal1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5; step (e) comprises eluting the hsIgG with a glycine-containing buffer; the protein A column is washed with acetate buffer between step (d) and step (e); the pH and salt content of the hsIgG produced in step (e) are changed before being added to the trisacryl blue column; and step (g) comprises eluting the hsIgG with a high-salt buffer; the high-salt buffer comprises 2 M NaCl; the high-salt buffer comprises 2 M KCl; and / or the hsIgG produced in step (e) is changed to 0.4 M NaCl and pH 4.5.

[0084] This specification also describes a method for preparing hypersialyzed (hsIgG), the method 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), UDP-Gal, ST6Gal1, and CMP-NANA to produce an hsIgG preparation; (d) adding the hsIgG composition to a protein A column under conditions that bind to IgG antibodies; and (e) eluting the IgG antibodies from the protein A column.

[0085] This specification also describes a method for preparing a purified highly sialylated IgG (hsIgG) composition, the method 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; (c) incubating the galactosylated IgG antibodies in a reaction mixture containing ST6Gal1 and CMP-NANA to produce an hsIgG composition; and (d) isolating hsIgG from the hsIgG composition and isolating B4GalT and ST6Gal1 from the hsIgG composition.

[0086] A method for preparing highly sialylated (hsIgG) is also described, and this method is The process includes (a) providing a mixture of IgG antibodies, (b) incubating the mixture of IgG antibodies in a reaction mixture containing β1,4-galactosyltransferase I (B4GalT), UDP-Gal, ST6Gal1, and CMP-NANA to produce an hsIgG preparation, and (c) isolating hsIgG from the hsIgG composition and isolating B4GalT and ST6Gal1 from the hsIgG composition.

[0087] In all various embodiments of the method described above, B4GalT is at least 90% identical to SEQ ID NO: 12 or SEQ ID NO: 13, and ST6Gal1 contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO: 20. The IgG antibody contains IgG antibodies isolated from at least 1000 donors. At least 70% w / w of the IgG antibody is IgG1 antibody. At least 90% of the donor subjects have been exposed to the virus. At least about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans in the IgG antibody in the hsIgG preparation have sialic acid in both the α1,3 branching and the α1,6 branching. At least about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the IgG antibody in the hsIgG preparation have sialic acid in both the α1,3 branching and the α1,6 branching. The step of providing a mixture of IgG antibodies includes (a) providing pooled plasma from at least 1,000 human subjects, and (b) isolating the mixture of IgG antibodies from the pooled plasma. The mixture of IgG antibodies is isolated from intravenous immunoglobulin. The mixture of IgG antibodies is intravenous immunoglobulin. The step of isolating the mixture of IgG antibodies from pooled plasma includes ethanol precipitation or caprylic acid precipitation, and the step of isolating the 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.

[0088] In highly sialylated IgG, at least 60% of the branched glycans on the Fc region (e.g., 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98%, including 100%) are disialylated via NeuAc-α2,6-Gal terminal linkages (i.e., on 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 linkages (i.e., sialylated only on the α1,3 branching or only on the α1,6 branching).

[0089] 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 immunoglobulin subclasses may also be present.

[0090] As used herein, the term “antibody” means a polypeptide comprising at least one immunoglobulin variable region, for example, 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 as used herein). 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.

[0091] 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).

[0092] 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 also include some or all of the flexible hinges 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 from P232 to their carboxyl terminus, and their numbering follows 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 Calpha2 (Cα2) and Calpha3 (Cα3), as well as the lower part of the hinge between Calpha1 (Cα1) and Cα2. The Fc region may be synthetic, recombinant, or generated from natural sources such as IVIg.

[0093] 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.

[0094] 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.

[0095] 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 less 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)).

[0096] 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.

[0097] As used herein, “percentage of branched glycans (%)” refers to the number of moles of glycan X relative to the total moles of glycans present, where X represents the glycan in question.

[0098] The terms “medically effective amount” or “therapeutic 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 “medically 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.

[0099] "Pharmaceutical preparations" and "pharmaceutical products" may be included in a kit containing a preparation or product and instructions for use.

[0100] "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'-monophospho-N-acetylneuraminic acid) or their by-products (e.g., cytidine 5'-monophosphate).

[0101] "Pharmaceutical preparations" and "pharmaceutical products," in general, in the case of recombinants, substantially do not contain other components of the cell in which the glycoprotein was produced (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA).

[0102] "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.

[0103] 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.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Methods and materials for use in the invention are described herein, but other preferred methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. In case of any inconsistency, this specification shall prevail, including definitions.

[0105] Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0106] [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 for reactions starting with IVIg are shown. The left figure is a schematic diagram of the enzymatic sialylation reaction for converting 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 an exemplary process for generating hsIgG. [Figure 6] This demonstrates the bonding ability of POROS® resin. [Figure 7] The yield and residual ST6 predictive profiler is shown. [Figure 8] It exhibits an operating range of 90-95% yield and residual ST6 levels below 30 ppm. [Figure 9] This is a flow-through SEC chromatogram. [Figure 10] This is the SEC of the eluate. [Figure 11] This is an overlay of TAB, Capto(trademark)blue, and Capto(trademark)Blue Sephrarose 2FF. [Figure 12] The image shows three runs of recovery demonstrating similar recovery (the bars, from left to right, correspond to TAB, Capto® Blue, and Blue Sepharose® FF). [Figure 13] The chromatogram overlays for runs 1 and 4 are shown. [Figure 14] The results show four runs of residual ST6 demonstrating good clearance between Capto® Blue HS (runs 3 and 4) and Capto® Blue (runs 1 and 2). [Figure 15] This shows a comparison of residual ST6 between Capto® Blue HS and TAB, demonstrating better clearance for Capto® Blue HS. [Figure 16] The IVIg load, FT, and SEC analysis of the eluate are shown. [Figure 17] The mass spectrometry of Capto® Blue HS, IVIg flow-through, and eluate against the starting IVIg is shown. The bars, from left to right, correspond to IgG1, IgG2, IgG3, and IgG4. [Figure 18] The effects of 5×PBS and MOPS in a pH 4.5 buffer on residual ST6 are shown (Execution 1). X axis: mg1VIg / mL resin. [Figure 19] This shows the effect of glycine at pH 4.5 on residual ST6 (execution 2). X axis: mg 1V 6g / mL resin. [Figure 20] This shows the effect of glycine + 100 mM NaCl at pH 4.5 on residual ST6 (execution 2). [Figure 21] This shows the effect of loading (grams / mL column volume for M254) on ST6 clearance by the TAB column. The orange and blue dots represent the residual ST6 levels for the TAB column flow-through fraction in CEX loading and CEX eluate at different loading amounts, respectively. At the current production-scale maximum loading of 1.1 g / mL (1X), similar levels of residual ST6 were achieved for the TAB column flow-through fraction in both loading (CEX loading and CEX elution). [Figure 22]This is an overlay of the SEC analysis for IVIg and precipitated IVIg, showing no change in aggregate levels. [Figure 23] This is an analytical SEC profile of crude reactant versus precipitate, showing a significant reduction in nucleotides. [Modes for carrying out the invention]

[0107] 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 (EU numbering) of 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 via the binding of galactose and / or galactose-sialic acid to one or both of the terminal GlcNAc, or via the binding of a third GlcNAc arm (bisected GlcNAc).

[0108] 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).

[0109] Naturally derived polypeptides that can be used to prepare highly sialylated 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 enriched with sialylated IgG), polypeptides purified from IVIg, or modified IVIg (e.g., enzymatically sialylated IVIg IgG).

[0110] 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."

[0111] N-glycans can be subdivided into three distinct groups called "high-mannose type," "hybrid type," and "complex type," and a common pentasaccharide core (Man(alpha1,6)-(Man(alpha1,3))-Man(beta1,4)-GlcpNAc(beta1,4)-GlcpNAc(beta1,N)-Asn) arises in all three groups.

[0112] Figure 2 shows more common Fc glycans present in IVIg.

[0113] Additionally or alternatively, one or more monosaccharide units of N-acetylglucosamine may be added to the coremannose subunit to form a "complex glycan." Galactose may be added to the N-acetylglucosamine subunit, or a sialic acid subunit may be added to the galactose subunit, resulting in a chain terminated by either sialic acid, galactose, or N-acetylglucosamine residues. Additionally, fucose residues may be added to the N-acetylglucosamine residues of the core oligosaccharide. Each of these additions is catalyzed by a specific glycosyltransferase.

[0114] "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, and / or fucose sugars.

[0115] 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.

[0116] 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.

[0117] Each antibody isotype has different N-linked carbohydrate structures in its constant region. For example, IgG has a single N-linked bibranched 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 (bibranched GlcNAc).

[0118] 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. α-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)oxane-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.

[0119] 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.

[0120] 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.

[0121] In some embodiments of the hsIgG compositions prepared by the method described herein, at least 50%, 55%, 60%, 65%, 70%, or 75% of the branched glycans on the Fc domain have sialic acid in both the α1,3 and α1,6 arms.

[0122] enzyme Galactosyltransferase Beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, and its orthologs, mutants, and variants, including their enzymatically active moieties, are suitable for use in the methods described herein, together with fusion proteins and polypeptides containing them. 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, all of which transfer β1,4-linked galactose to 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.

[0123] [Table 1]

[0124] [Table 2]

[0125] [Table 3]

[0126] [Table 4]

[0127] 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).

[0128] 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).

[0129] 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, variant, 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, variant, 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, variant, 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, variant, or variant of SEQ ID NO: 8.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] Furthermore, amino acid sequences that are at least 80% (85%, 90%, 95%, 98%, or 100%) identical to, or comprise, the amino acid sequence of SEQ ID NO: 13 are preferred for use in the method described herein.

[0135] sialylation enzymes ST6, e.g., ST6Gal1, e.g., human

[0136] [Table 5]

[0137] [Table 6]

[0138] [Table 7]

[0139] [Table 8]

[0140] The soluble form of ST6Gal1 originates from the membrane morphology resulting from proteolytic treatment.

[0141] 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).

[0142] Furthermore, for example, the enzyme active moiety of ST6Gal1 is also provided herein. In some embodiments, the enzyme is the enzyme active moiety 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 enzyme active moiety of ST6Gal1 isoform b (SEQ ID NO: 15), or an ortholog, variant, or variant of SEQ ID NO: 15.

[0143] 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 a cytoplasmic domain, e.g., SEQ ID NO: 16, nor a transmembrane domain, e.g., SEQ ID NO: 17.

[0144] 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 ortholog, variant, or variant.

[0145] 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 ortholog, variant, or variant. In some embodiments, the enzymatically active portion of ST6Gal1 consists of SEQ ID NO: 19, or its ortholog, variant, or variant.

[0146] A preferred functional portion of ST6Gal1 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: 19.

[0147] 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.

[0148] Furthermore, amino acid sequences that are at least 80% (85%, 90%, 95%, 98%, or 100%) identical to, or comprise, the amino acid sequence of SEQ ID NO: 20 are preferred for use in the method described herein.

[0149] antibody The methods described herein may include galactosylation and sialylation of antibodies. Suitable antibodies include, for example, IgG antibodies. Antibodies, for example, IgG antibodies, can be pooled. For example, pooled IgG antibodies may include IVIg.

[0150] In some embodiments, the IgG antibody comprises IgG antibodies isolated from at least 1000 donors.

[0151] In some embodiments, at least 50%, 55%, 60%, 65%, or 70% w / w of the IgG antibody is IgG1 antibody.

[0152] In some embodiments, at least 90% of the donor subjects have been exposed to the virus.

[0153] 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 referred to as 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.

[0154] 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(s), e.g., an antibody described herein, a galactosylating enzyme, e.g., a galactosylating enzyme described herein, such as B4GalT or its enzymatically active moiety or variant, and UDP-gal or a salt thereof; and a method for galactosylating an antibody(s), e.g., an antibody described herein, by incubating the composition under conditions effective for galactosylating the antibody, e.g., as described herein, to produce a galactosylated antibody(s).

[0155] 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(s) as described herein, a sialylation enzyme, such as ST6Gal1 or its enzymatically active moiety or variant, as described herein, and CMP-NANA or a salt thereof; and a method for sialylating a highly sialylated antibody(s), such as the antibody(s) described herein, by incubating the composition under conditions effective for sialylating the antibody(s), as described herein.

[0156] 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, providing pregalactosylated antibodies(s), which may be processed (e.g., filtered, fractionated, or purified) and / or stored before the sialylation step.

[0157] Therefore, the method 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 mixture) comprising: a) an antibody(s), for example, an antibody described herein; a galactosylating enzyme, for example, B4GalT or its enzyme active moiety or variant, as described herein; and UDP-gal or a salt thereof; b) incubating the composition under conditions effective for galactosylating the antibody(s), for example, as described herein; c) adding a sialing enzyme, for example, a sialing enzyme described herein, for example, ST6Gal1 or its enzyme active moiety or variant, and CMP-NANA or a salt thereof to the galactosylation reaction mixture to generate a sialing reaction mixture; and d) incubating the composition under conditions effective for sialing the galactosylating antibody(s), for example, as described herein, to galactosylate and sialylate, for example, hypersialylate the antibody(s), by: a) an antibody(s), for example, an antibody described herein; a galactosylating enzyme, for example, ST6Gal1 or its enzyme active moiety or variant, and CMP-NANA or a salt thereof; and d) incubating the composition under conditions effective for sialylation of the galactosylating antibody(s), for example, as described herein.

[0158] This specification also provides a composition comprising an antibody(s), for example, an antibody as described herein; a galactosylase, for example, a galactosylase as described herein, such as B4GalT or its active portion or variant; UDP-gal or a salt thereof; a sialyase, for example, a sialyase as described herein, such as ST6Gal1 or its active portion or variant; and d) a method for galactosylating and sialylation, for example, hypersialing, an antibody(s), for example, an antibody(s) as described herein, by incubating the composition under conditions effective for galactosylating and sialylation of the antibody(s), for example, as described herein.

[0159] In some embodiments, the galactosylation reaction mixture and / or sialylation reaction mixture comprises bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane (BIS-TRIS) buffer.

[0160] In some embodiments, the galactosylation reaction mixture and / or sialylation reaction mixture contains MnCl2.

[0161] In some embodiments, one or more components of the reaction mixture are replenished during incubation. That is, the reaction mixture may contain the initial amounts of components (which may change during the reaction), but additional amounts of components may be replenished during the reaction.

[0162] In some embodiments, B4GalT comprises or consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 or SEQ ID NO: 13.

[0163] In some embodiments, ST6Gal1 comprises or consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 or SEQ ID NO: 20.

[0164] In some embodiments, for example, at least or about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0165] In some embodiments, for example, about or at least about 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans on a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0166] Furthermore, in some embodiments, for example, about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal linkages.

[0167] In some embodiments, for example, about or at least 80% of the branched Fc glycans on a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0168] In some embodiments, for example, about 60%, 65%, or 70% of the branched glycans on the Fab domain of a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal links.

[0169] In some embodiments, for example, about or at least 85% of the branched Fc glycans on a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0170] In some embodiments, for example, about 60%, 65%, or 70% of the branched glycans on the Fab domain of a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal links.

[0171] In some embodiments, for example, about or at least 90% of the branched Fc glycans on a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 branching and the α1,6 branching.

[0172] In some embodiments, for example, about 60%, 65%, or 70% of the branched glycans on the Fab domain of a sialyzed antibody(s), such as hsIgG, have sialic acid in both the α1,3 and α1,6 arms connected via NeuAc-α2,6-Gal terminal links.

[0173] Isolation and purification methods This specification provides a method for purifying antibodies such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, and hsIgG.

[0174] This specification provides a method for producing purified sialyzed antibodies, such as purified hsIgG, which comprises i) producing sialyzed antibodies, such as hsIgG, as described herein, and ii) purifying sialyzed antibodies, such as described herein.

[0175] An exemplary manufacturing process for the isolation and purification of hsIgG is shown in Figure 5. Variations of the process are described herein.

[0176] In some embodiments, a method for purifying an antibody(s) includes analyzing the amount of one or more IgG subclasses after purification, for example, after a chromatography step.

[0177] Quenching The methods described herein may include, for example, enzymatic reactions such as those described herein, such as quenching of an enzymatic sialylation reaction.

[0178] In some embodiments, quenching includes mixing a composition comprising, for example, a sialyzed antibody(s) as described herein, e.g., a sialyzed reaction mixture, with a buffer, e.g., phosphate-buffered saline (PBS), e.g., 5×PBS, to produce a quenched antibody composition. In some embodiments, quenching includes mixing the composition and the buffer in a 1:1 or approximately 1:1 ratio. In some embodiments, quenching includes mixing the antibody composition and the buffer with one or more volumes of buffer and one volume of the antibody composition.

[0179] deep filtration The methods described herein may include deep filtration. Deep filtration is well known in the art and is described, for example, in Sutherland, "Filtration Overview: A Closer Look at Depth Filtration," Filtration & Separation 45(8):25-28(2008); and Nguyen et al., "Improved HCP Reduction Using a New, All-Synthetic Depth Filtration Media Within an Antibody Purification Process," Biotechnology Journal 14(1):1700771(2019).

[0180] In some embodiments, the method described herein for generating and / or purifying antibodies such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, hsIgG, etc., comprises one or more deep filtration steps. In some embodiments, the method comprises one or more deep filtration steps before the chromatography step. In some embodiments, the method comprises one or more deep filtration steps after the chromatography step. In some embodiments, the method does not include a deep filtration step before chromatography. In some embodiments, the method does not include a deep filtration step after chromatography. In some embodiments, the method does not include a deep filtration step.

[0181] buffer exchange The methods described herein may include buffer exchange. Methods for buffer exchange are well known in the art and are described, for example, in Kurnik, "Buffer Exchange Using Size Exclusion Chromatography, Countercurrent Dialysis, and Tangential Flow Filtration: Models, Development, and Industrial Application," Biotechnology & Bioengineering 45(2):149-58(1995); and Dizon-Maspat et al., "Single Pass Tangential Flow Filtration to Debottleneck Downstream Processing for Therapeutic Antibody Production," Biotechnology & Bioengineering 109(4):962-970(2012).

[0182] In some embodiments, the buffer exchange method is size exclusion chromatography (SEC). In some embodiments, the buffer exchange method is tangential flow filtration (TFF). In some embodiments, the buffer exchange method is countercurrent dialysis (CCD).

[0183] In some embodiments, the buffer exchange method is tangential flow filtration (TFF).

[0184] In some embodiments, the method described herein for generating and / or purifying antibodies such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, hsIgG, etc., comprises one or more buffer exchange steps. In some embodiments, the method comprises one or more buffer exchange steps before the chromatography step. In some embodiments, the method comprises one or more buffer exchange steps after the chromatography step. In some embodiments, the method does not include a buffer exchange step before chromatography. In some embodiments, the method does not include a buffer exchange step after chromatography. In some embodiments, the method does not include a buffer exchange step.

[0185] Virus inactivation / removal Methods described herein may include viral inactivation or viral removal. Methods for viral inactivation and removal are well known in the art and are described, for example, in Horowitz et al., "Strategies for Viral Inactivation," Current Opinion in Hematology 2(6):484-92(1995); and Klutz et al., "Continuous Viral Inactivation at low pH Value in Antibody Manufacturing," Chemical Engineering and Processing: Process Intensification 102:88-11(2016).

[0186] In some embodiments, the virus inactivation method is selected from solvent / detergent inactivation, low pH inactivation, pasteurization, microwave heating, irradiation, high-energy light, and combinations thereof.

[0187] In some embodiments, the virus removal method is selected from affinity chromatography, nanofiltration, and a combination thereof.

[0188] In some embodiments, the method for purifying antibodies such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, and hsIgG comprises one or more virus inactivation or virus removal steps. In some embodiments, the virus inactivation or virus removal step is performed after galactosylation and sialylation. In some embodiments, the virus inactivation or virus removal step is performed after galactosylation and sialylation but before chromatography. In some embodiments, virus inactivation and removal comprises virus inactivation followed by nanofiltration. In some embodiments, the method comprises virus removal (e.g., by nanofiltration) but does not include a separate virus inactivation step.

[0189] Nanofiltration The methods described herein may include nanofiltration. Membranes and methods for nanofiltration are well known in the art and are described, for example, in Mohammad et al., "Nanofiltration Membranes Review: Recent Advances and Future Prospects," Desalination 356:226-54 (2015).

[0190] In some embodiments, the method described herein for generating and / or purifying antibodies such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, hsIgG, etc., comprises one or more nanofiltration steps. In some embodiments, the method comprises one or more nanofiltration steps before the chromatography step. In some embodiments, the method comprises one or more nanofiltration steps after the chromatography step. In some embodiments, the method does not include a nanofiltration step before chromatography. In some embodiments, the method does not include a nanofiltration step after chromatography. In some embodiments, the method does not include a nanofiltration step.

[0191] chromatography Methods for generating and / or purifying antibodies such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, or hsIgG may include chromatography as part of the purification process.

[0192] In some embodiments, the antibody(s) are bound to the chromatography resin. In some embodiments, the antibody(s) flow through the chromatography resin.

[0193] In some embodiments, the impurities (or multiple impurities) (e.g., one or more of the other components of an enzymatic reaction mixture (or multiple), e.g., a galactosylation reaction mixture (or multiple) and / or sialylation reaction mixture (or multiple), including but not limited to enzymes such as B4GalT and / or ST6, nucleotide sugars, and MnCl2) are bound to the chromatography resin. In some embodiments, the impurities flow through the chromatography resin.

[0194] In some embodiments, both the antibody(s) and impurities(s) are bound to the chromatographic resin. In such embodiments, the antibody(s) and impurities(s) can be eluted separately.

[0195] Protein A affinity chromatography Methods for generating and / or purifying antibodies such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, or hsIgG may include an affinity chromatography step in which the antibody binds to a ligand on a chromatography resin, such as a protein A affinity chromatography step.

[0196] The materials and methods for protein A affinity chromatography are well known in the art and are described, for example, in Ramos-de-la-Pena et al., "Protein A chromatography: Challenges and Progress in the Purification of Monoclonal Antibodies," Journal of Separation Science 2019:doi:10.1002 / jssc.201800963.

[0197] In some embodiments, the protein A affinity chromatography resin is MabSelect® PrismA (GE Healthcare).

[0198] In some embodiments, the protein A affinity chromatography resin ligand is selected from the group consisting of protein A, protein A derivatives, protein A mimes, or combinations thereof.

[0199] In some embodiments, the method described herein includes, for example, protein A affinity chromatography followed by blue dye chromatography, as described herein.

[0200] Cation exchange chromatography Methods described herein for generating and / or purifying antibodies such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, hsIgG, etc., may include a cation exchange chromatography (CEX) step, for example, a strong cation exchange chromatography step using a strong cation exchange chromatography resin.

[0201] In some embodiments, the strong cation exchange resin is selected from the group consisting of Poros® XS (e.g., Cytiva 4404336), SP Sepharose High Performance (SP HP) (e.g., Cytiva 28950515), Capto® SP Impres (e.g., Cytiva 17546815), Capto® S (e.g., Cytiva 28926979), Capto® S Impact (e.g., Cytiva 1737174), and combinations thereof.

[0202] Poros (trademark) strong cation exchange (CEX) resin is a 50-μm rigid polymeric ion exchange chromatography resin. Thermoscientific Product Information Sheet Publication No. 100031321 Rev.D. The polyhydroxyl surface coating provides low non-specific binding, and surface functionalization with sulfopropyl results in a strong cation exchanger that can be ionized at pH 1 to 14. The support matrix is cross-linked polystyrene-divinylbenzene, and the surface functional group is sulfopropyl (-CH2CH2CH2SO3 - ). The dynamic binding capacity of Poros (trademark) XS is ≥ 102 mg / mL (5% breakthrough of polyclonal human IgG in 20 mM MES, < 40 mM NaCl, pH 5.0, 300 cm / h, 0.46 cm D × 20 cm L column). The dynamic binding capacity of Poros (trademark) 50HS is 57.0 - 75.3 mg / mL (5% breakthrough of lysozyme in 20 mM MES, pH 6.2, 100 cm / h, 0.46 cm D × 20 cm L column). Ibid

[0203] SP Sepharose High Performance (SPHP) resin is based on a 6% cross-linked agarose matrix with a particle size of 24 - 44 μm (d - ~ 34 μm) using a sulfopropyl ligand (-CH2CH2CH2SO3 50v ). See Product Bulleting “SP Sepharose High Performance”, cytiva.com. It has an operating pH stability of 4 - 13. Ibid It has a dynamic binding capacity of approximately 55 ribonuclease A / mL resin. Ibid

[0204] Capto (trademark) SP Impres is based on a high-flow agarose-based matrix with a bead size of approximately 40 μm. Product literature “Capto (trademark) SP ImpRes, Capto (trademark) Q ImpRes Ion Exchange Chromatography”, cytiva.com. The functional group of Capto (trademark) SP Impres is -CH2CH2CH2SO3 -The pH stability of the same procedure is 4-12. The binding dose (10% breakthrough measured in a Tricorn® 5 / 100 column with a bed height of 10 cm, residence time of 4 minutes (150 cm / h), 20 mM sodium phosphate, pH 7.2 (lysozyme), 50 mM Tris, pH 8.0 (BSA)) is >70 mg lysozyme / mL medium and >95 mg BSA / mL medium.

[0205] Capto(trademark)S is -SO3 as shown below. - This is a powerful cation exchange resin based on a highly crosslinked agarose spherical matrix with a cumulative volume distribution median particle size of approximately 90 μm, utilizing charged groups.

[0206] [ka]

[0207] See Cytiva, "Ion Exchange Resins", 28407452 AG, cytiva.com. This has a dynamic binding dose of >120 mg lysozyme / mL resin (10% breakthrough by front-end analysis at 600 cm / h phase transfer rate on a Tricorn® 5 / 100 column with a 10 cm bed height (1 minute residence time) and >60 mg β-lactoglobulin / mL resin (100 mM citrate, β-lactoglobulin at pH 3 with a 10 cm bed height (1 minute residence time) and 600 cm / h mobile phase transfer rate on a PEEK 7.5 / 100 column) and can be operated at pH 4-12.

[0208] Capto(TM) S Impact is -SO3 -The ligand-based cumulative volume distribution is based on a high-flow agarose-based matrix with a median particle size of approximately 50 μm. See Capto® S ImpAct product sheet, cytiva.com. This has binding capacities of >85 mg BSA / resin mL, >90 mg lysozyme / resin mL, and >100 mg IgG / resin mL. Ibid.

[0209] In some embodiments, the strong cation exchange resin has a binding capacity of 50 mg / mL or more, for example, 75 mg / mL or more, for example, 100 mg / mL or more, as measured as a 5% breakthrough of polyclonal human IgG in a 0.46 cmD × 20 cmL column at 300 cm / hour, 20 mM MES, <40 mM NaCl, pH 5.0.

[0210] In some embodiments, the strong cation exchange resin maintains a binding capacity of 50 mg / mL or higher, e.g., 75 mg / mL or higher, e.g., 100 mg / mL or higher, as measured as the 5% breakthrough of polyclonal human IgG at 300 cm / hour in a 0.46 cmD × 20 cmL column, with 20 mM MES, <40 mM NaCl, and pH 5.0, up to 150 mM NaCl (15 mS / cm).

[0211] In some embodiments, the functional group of the strong cation exchange resin contains SO3-. In some embodiments, the functional group of the strong cation exchange resin is -CH2CH2CH2SO3 - That is the case.

[0212] In some embodiments, cation exchange chromatography is performed in flow-through mode. In some embodiments, cation exchange chromatography is performed in binding and elution mode. In some embodiments, antibody(s), e.g., sialylated antibody(s), e.g., hsIgG, flows through the cation exchange chromatography resin. In some embodiments, antibody(s), e.g., sialylated antibody(s), e.g., hsIgG, binds and is then eluted from the cation exchange chromatography resin.

[0213] In some embodiments, a composition comprising a sialylated antibody(s), such as hsIgG, and one or more impurities is packed into the CEX column. In some embodiments, a sialylation reaction mixture, such as a sialylation mixture comprising a sialylated antibody(s), as described herein, is packed into the CEX column.

[0214] In some embodiments, 10 or about 10-110 or about 110 mg of antibody(s) per mL of resin is packed into the CEX column.In some embodiments, for example, per mL of resin, 10 or about 10 to 100 or about 100, 10 or about 10 to 90 or about 90, 10 or about 10 to 80 or about 80, 10 or about 10 to 70 or about 70, 10 or about 10 to 60 or about 60, 10 or about 10 to 50 or about 50, 10 or about 10 to 40 or about 40, 10 or about 10 to 30 or about 30, 10 or about 10 to 20 or about 20, 20 or about 20 to 110 or about 110, 20 or about 20 to 100 or about 100, 20 or about 20 to 90 or about 90, 20 or about 20 to 80 or about 80, 20 Or approximately 20-70 or approximately 70, 20 or approximately 20-60 or approximately 60, 20 or approximately 20-50 or approximately 50, 20 or approximately 20-40 or approximately 40, 20 or approximately 20-30 or approximately 30, 30 or approximately 30-110 or approximately 110, 30 or approximately 30-100 or approximately 100, 30 or approximately 30-90 or approximately 90, 30 or approximately 30-80 or approximately 80, 30 or approximately 30-70 or approximately 70, 30 or approximately 30-60 or approximately 60, 30 or approximately 30-50 or approximately 50, 30 or approximately 30-40 or approximately 40, 40 or approximately 40-110 or approximately 110, 40 or approximately 40-100 or approximately 1 00, 40 or approximately 40-90 or approximately 90, 40 or approximately 40-80 or approximately 80, 40 or approximately 40-70 or approximately 70, 40 or approximately 40-60 or approximately 60, 40 or approximately 40-50 or approximately 50, 50 or approximately 50-110 or approximately 110, 50 or approximately 50-100 or approximately 100, 50 or approximately 50-90 or approximately 90, 50 or approximately 50-80 or approximately 80, 50 or approximately 50-70 or approximately 70, 50 or approximately 50-60 or approximately 60, 60 or approximately 60-110 or approximately 110, 60 or approximately 60-100 or approximately 100, 60 or approximately 60-90 or approximately 90, 60 or approximately 60 ~80 or approximately 80, 60 or approximately 60~70 or approximately 70, 70 or approximately 70~110 or approximately 110, 70 or approximately 70~100 or approximately 100, 70 or approximately 70~90 or approximately 90, 70 or approximately 70~80 or approximately 80, 80 or approximately 80~110 or approximately 110, 80 or approximately 80~100 or approximately 100, 80 or approximately 80~90 or approximately 90, 90 or approximately 90~110 or approximately 110, 90 or approximately 90~100 or approximately 100, 100 or approximately 100~110 or approximately 110 mg of antibody, such as sialyzed antibodies (multiple antibodies are possible), including hsIgG.

[0215] In some embodiments, the methods described herein include one or more of the following: (a) a quenching step, for example, as described herein; (b) a deep filtration step, for example, as described herein; (c) a buffer exchange step, for example, as described herein; (d) a virus inactivation or removal step, for example, as described herein; and (e) a nanofiltration step, for example, as described herein, prior to CEX chromatography.

[0216] In some embodiments, the methods described herein include one or more of the following: (a) blue dye chromatography as described herein, (b) DE pad filtration as described herein, (c) buffer exchange as described herein, and (d) PS20 spiking and filtration after CEX chromatography, as described herein.

[0217] This specification provides a method for purifying sialylated antibodies(s), in the order of (a) to (d), (a) a composition comprising a sialylated antibody, e.g., hsIgG, and galactosylating and / or sialylation enzymes(s), e.g., B4GalT and / or ST6 or their enzymatic active portion, as described herein, and, if the sialylation reaction mixture is as described herein, optionally, for example, the reaction mixture is diluted 1:1 in PBS, e.g., 5X, as described herein. The process includes (b) quenching in PBS, (c) diluting the composition with citrate buffer (e.g., 6.757 g / L of dehydrated sodium citrate and 5.192 g / L of citrate buffer), for example, 50 mM or about 50 mM, pH 4.5 or pH about 4.5, thereby generating a buffered antibody composition, (d) adding the buffered antibody composition to a CEX column, for example, a CEX column as described herein, under conditions that bind to a sialyzed antibody(s) such as hsIgG and a galactosylating and / or sialylation enzyme(s), and (e.g., a CEX column as described herein, by selectively eluting a sialyzed antibody(s) such as hsIgG from the CEX column, thereby generating a purified sialyzed antibody.

[0218] In some embodiments, the method does not include any additional filtration, fractionation, or purification steps other than (a) and (b) before performing step (c). In some embodiments, in addition to steps (a) and (b), the method includes a single deep filtration step before step (c), for example, between steps (a) and (b), but does not include any additional filtration, fractionation, or purification steps other than the single deep filtration step before performing step (c). In some embodiments, the method includes a virus inactivation step before step (c). In some embodiments, the addition in step (c) is time-interval, for example, a portion of the buffered antibody composition is added to the column two or more times, for example, one, two, or three times. In some embodiments, step (e) is repeated one or more times. In some embodiments, step (c) is performed with a residence time of 1, 2, 3, 4, 5, or 6 minutes or approximately 1, 2, 3, 4, 5, or 6 minutes.

[0219] In some embodiments, selective elution of sialylated antibodies(s) includes eluting a buffer containing 400 mM or more of NaCl, for example, 400 mM or more of NaCl in a citrate buffer at 50 mM or approximately 50 mM, pH 4.5 or approximately pH 4.5.

[0220] In some embodiments, the method further includes, after step (e), one or more of the following: (f) a blue dye chromatography step, such as Trisacyl Blue (TAB) chromatography, for example, as described herein; (g) a DE pad filtration step, such as as described herein; (h) a deep filtration step, such as as described herein; and (g) a PS20 spike and filtration step, such as as described herein.

[0221] In some embodiments, this method uses a sialylation enzyme, such as ST6 or its enzymatically active portion, present at concentrations of 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, or 30 ppm or less, to obtain yields (antibody recovery rates) of 80%, 85%, 90%, or 95% or higher, or approximately 80%, 85%, 90%, or 95% or higher.

[0222] Blue dye affinity chromatography Methods for producing and / or purifying antibodies, such as IgG, pooled IgG, IVIg, sialylated IgG, sialylated pooled IgG, sialylated IVIg, or hsIgG, as described herein, may include a blue dye affinity chromatography step, such as an affinity chromatography step using a blue dye resin, as part of the purification process.

[0223] In some embodiments, the blue dye resin is selected from the group consisting of Blue Trisacryl (trademark) (e.g., Sartorius 25896-010), Capto (trademark) Blue (e.g., Cytiva 17544801), Capto (trademark) Blue HS (e.g., Cytiva 17545202), Blue Sephrarose (trademark) (e.g., Cytiva 17-0948-01), and combinations thereof.

[0224] Blue Trisacryl® resin is based on a 60 μm bead-sized synthetic polymer matrix using blue dye ligands as functional groups. See Product Specifications, Sartorius 25896-010, Sartorius.com. It has an operating pH of 4–10.

[0225] Capto® Blue resins are based on a highly cross-linked agarose matrix with an average particle size of 75 μm. See Product Bulletin, "Capto Blue and Capto Blue (high sub) Affinity Chromatography," cytiva.com. These resins use Cibacron® Blue (1-amino-4-[4-[[4-chloro-6-(2-sulfoanilino)-1,3,5triazine-2-yl]amino]-3-sulfoanilino]-9,10dioxoanthracene-2-sulfonic acid) ligand. Ibid.; PubChem ID 172469. They have an operating pH of 3 to 13. Capto® Blue has a dynamic binding capacity of HSA at a residence time of 4 minutes and a 10% breakthrough of 24 mg / mL, and Capto® Blue HS has a dynamic binding capacity of HSA at a residence time of 4 minutes and a 10% breakthrough of 30 mg / mL. Ibid.

[0226] Blue Sepharose® is based on a highly cross-linked agarose-based matrix (6% spherical) with a median particle size of approximately 90 μm in cumulative volume distribution. See Product Bulletin, "Blue Sepharose 6 Fast Flow Affinity Chromatography," cytiva.com. This uses Cibacron® Blue 3G (1-amino-4-[4-[[4-chloro-6-(3-sulfoanilino)-1,3,5triazine-2-yl]amino]-3-sulfoanilino]-9,10dioxoanthracene-2-sulfonic acid). This has operational pH stability of 4-12 and a total binding capacity of ≥18 mg of human serum albumin / mL resin (excess protein is packed in 0.050 M potassium dihydrogen phosphate pH 7.0 on a PEEK 7.5 / 50 column). The binding capacity is obtained by measuring the amount of bound and eluted proteins in 0.050 M potassium dihydrogen phosphate, 1.5 M potassium chloride, pH 7.0.

[0227] In some embodiments, blue dye affinity chromatography is performed in flow-through mode. In some embodiments, antibodies, such as sialyzed antibodies, such as hsIgG, flow through the blue dye chromatography resin.

[0228] In some embodiments, a composition comprising a sialylated antibody(s), such as hsIgG, and one or more impurities is packed into a blue dye column. In some embodiments, a sialylation reaction mixture, such as a sialylated mixture comprising a sialylated antibody(s) as described herein, is packed into a blue dye column.

[0229] In some embodiments, 10 or about 10-110 or about 110 mg of antibody(s) per mL of resin is packed into the blue dye column.In some embodiments, for example, per mL of resin, 10 or about 10 to 100 or about 100, 10 or about 10 to 90 or about 90, 10 or about 10 to 80 or about 80, 10 or about 10 to 70 or about 70, 10 or about 10 to 60 or about 60, 10 or about 10 to 50 or about 50, 10 or about 10 to 40 or about 40, 10 or about 10 to 30 or about 30, 10 or about 10 to 20 or about 20, 20 or about 20 to 110 or about 110, 20 or about 20 to 100 or about 100, 20 or about 20 to 90 or about 90, 20 or about 20 to 80 or about 80, 20 or Approximately 20-70 or approximately 70, 20 or approximately 20-60 or approximately 60, 20 or approximately 20-50 or approximately 50, 20 or approximately 20-40 or approximately 40, 20 or approximately 20-30 or approximately 30, 30 or approximately 30-110 or approximately 110, 30 or approximately 30-100 or approximately 100, 30 or approximately 30-90 or approximately 90, 30 or approximately 30-80 or approximately 80, 30 or approximately 30-70 or approximately 70, 30 or approximately 30-60 or approximately 60, 30 or approximately 30-50 or approximately 50, 30 or approximately 30-40 or approximately 40, 40 or approximately 40-110 or approximately 110, 40 or approximately 40-100 or approximately 100, 4 0 or approximately 40-90 or approximately 90, 40 or approximately 40-80 or approximately 80, 40 or approximately 40-70 or approximately 70, 40 or approximately 40-60 or approximately 60, 40 or approximately 40-50 or approximately 50, 50 or approximately 50-110 or approximately 110, 50 or approximately 50-100 or approximately 100, 50 or approximately 50-90 or approximately 90, 50 or approximately 50-80 or approximately 80, 50 or approximately 50-70 or approximately 70, 50 or approximately 50-60 or approximately 60, 60 or approximately 60-110 or approximately 110, 60 or approximately 60-100 or approximately 100, 60 or approximately 60-90 or approximately 90, 60 or approximately 60-80 or approximately 80, 60 or approximately 60-70 or approximately 70, 70 or approximately 70-110 or approximately 110, 70 or approximately 70-100 or approximately 100, 70 or approximately 70-90 or approximately 90, 70 or approximately 70-80 or approximately 80, 80 or approximately 80-110 or approximately 110, 80 or approximately 80-100 or approximately 100, 80 or approximately 80-90 or approximately 90, 90 or approximately 90-110 or approximately 110, 90 or approximately 90-100 or approximately 100, 100 or approximately 100-110 or approximately 110 mg of antibody(s), for example, sialyzed antibody(s), for example, hsIgG, is packed into a blue dye column.

[0230] In some embodiments, 10 or about 10 to 110 or about 110 centigrams of antibody per mL of resin is loaded into the blue dye column.In some embodiments, for example, per mL of resin, 10 or about 10 to 100 or about 100, 10 or about 10 to 90 or about 90, 10 or about 10 to 80 or about 80, 10 or about 10 to 70 or about 70, 10 or about 10 to 60 or about 60, 10 or about 10 to 50 or about 50, 10 or about 10 to 40 or about 40, 10 or about 10 to 30 or about 30, 10 or about 10 to 20 or about 20, 20 or about 20 to 110 or about 110, 20 or about 20 to 100 or about 100, 20 or about 20 to 90 or about 90, 20 or about 20 to 80 or about 80, 20 or about 20-70 or approximately 70, 20 or approximately 20-60 or approximately 60, 20 or approximately 20-50 or approximately 50, 20 or approximately 20-40 or approximately 40, 20 or approximately 20-30 or approximately 30, 30 or approximately 30-110 or approximately 110, 30 or approximately 30-100 or approximately 100, 30 or approximately 30-90 or approximately 90, 30 or approximately 30-80 or approximately 80, 30 or approximately 30-70 or approximately 70, 30 or approximately 30-60 or approximately 60, 30 or approximately 30-50 or approximately 50, 30 or approximately 30-40 or approximately 40, 40 or approximately 40-110 or approximately 110, 40 or approximately 40-100 or approximately 100, 40 or Approximately 40-90 or approximately 90, 40 or approximately 40-80 or approximately 80, 40 or approximately 40-70 or approximately 70, 40 or approximately 40-60 or approximately 60, 40 or approximately 40-50 or approximately 50, 50 or approximately 50-110 or approximately 110, 50 or approximately 50-100 or approximately 100, 50 or approximately 50-90 or approximately 90, 50 or approximately 50-80 or approximately 80, 50 or approximately 50-70 or approximately 70, 50 or approximately 50-60 or approximately 60, 60 or approximately 60-110 or approximately 110, 60 or approximately 60-100 or approximately 100, 60 or approximately 60-90 or approximately 90, 60 or approximately 60-80 or approximately 80, 60 or approximately 60-70 or approximately 70, 70 or approximately 70-110 or approximately 110, 70 or approximately 70-100 or approximately 100, 70 or approximately 70-90 or approximately 90, 70 or approximately 70-80 or approximately 80, 80 or approximately 80-110 or approximately 110, 80 or approximately 80-100 or approximately 100, 80 or approximately 80-90 or approximately 90, 90 or approximately 90-110 or approximately 110, 90 or approximately 90-100 or approximately 100, 100 or approximately 100-110 or approximately 110 centigrams of antibody(s), for example, sialyzed antibody(s), for example, hsIgG, are packed into a blue dye column.

[0231] In some embodiments, the methods described herein include one or more of the following: (a) a quenching step as described herein, (b) a deep filtration step as described herein, (c) a buffer exchange step as described herein, (d) a virus inactivation or removal step as described herein, (e) a nanofiltration step as described herein, or (f) a CEX chromatography step as described herein, prior to blue dye chromatography.

[0232] In some embodiments, the method does not include a CEX chromatography step.

[0233] In some embodiments, the methods described herein include one or more of the following: (a) DE pad filtration as described herein, (b) buffer exchange as described herein, for example, and (c) PS20 spiking and filtration after blue dye chromatography, for example, as described herein.

[0234] This specification provides a method for purifying sialylated antibodies, which, in the order of (a) to (c), provide a composition comprising (a) a sialylated antibody, e.g., hsIgG, and galactosylating and / or sialylation enzymes, e.g., B4GalT and / or ST6 or their enzymatic active portion, as described herein, and, for example, if the sialylation reaction mixture is as described herein, optionally, for example, the reaction mixture is diluted 1:1 in PBS, e.g., 5X (b) Quench in PBS, dilute the composition with a buffer suitable for use with the column, for example, in the case of a TAB column, citrate buffer, for example, 100 mM or about 100 mM citrate buffer at pH 4.5 or pH 4.5, or 800 mM or about 800 mM NaCl, or in the case of a CaptBlue column, 250 mM or about 250 mM glycine + 100 mM or about 100 mM sodium chloride at pH 4.5 or pH 4.5, thereby buffering the antibody composition. (c) generating and (c) generating purified sialyzed antibodies by adding the buffered antibody composition to a blue dye column, for example, a blue column as described herein, under conditions that allow 80% or more of the sialyzed antibody, such as hsIgG, e.g., 85% or more, 90% or more, or 95% or more, to flow, but allow only 30, 40, 50, 60, 70, 80, 90, or 100 ppm or less of galactosylating and / or sialyzed enzymes to flow.

[0235] In some embodiments, the method does not include any additional filtration, fractionation, or purification steps other than (a) and (b) before performing step (c). In some embodiments, in addition to steps (a) and (b), the method includes a single deep filtration step before step (c), for example, between steps (a) and (b), but does not include any additional filtration, fractionation, or purification steps other than the single deep filtration step before performing step (c). In some embodiments, the method includes a virus inactivation step before step (c). In some embodiments, the addition in step (c) is time-intervened, for example, a portion of the buffered antibody composition is added to the column two or more times, for example, one, two, or three times. In some embodiments, step (c) is performed with a residence time of 1, 2, 3, 4, 5, or 6 minutes or approximately 1, 2, 3, 4, 5, or 6 minutes.

[0236] In some embodiments, the method further includes, after step (c), one or more of the following: (d) a CEX chromatography step, for example, as described herein; (e) a DE pad filtration step, for example, as described herein; (f) a deep filtration step, for example, as described herein; and (g) a PS20 spike and filtration step, for example, as described herein.

[0237] In some embodiments, the method further includes, after step (c), one or more of the following: (d) a DE pad filtration step, for example, as described herein; (e) a deep filtration step, for example, as described herein; and (f) a PS20 spike and filtration step, for example, as described herein, but without a CEX chromatography step, for example, as described herein.

[0238] In some embodiments, this method uses a sialylation enzyme, such as ST6 or its enzymatically active portion, present at concentrations of 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, or 30 ppm or less, to obtain yields (antibody recovery rates) of 80%, 85%, 90%, or 95% or higher, or approximately 80%, 85%, 90%, or 95% or higher.

[0239] Polysorbate spikes Polysorbate, for example, polysorbate 20, can be added to a composition comprising the antibody(s) described herein, for example, hsIgG, for example, purified hsIgG. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, polysorbate 20 is Super Refined® polysorbate 20 (Croda Health Care).

[0240] Precipitation The methods described herein may include precipitates. In some embodiments, the precipitate is an ammonium sulfate precipitate.

[0241] In some embodiments, the method described herein, for example, for purifying an antibody(s), for example, IgG, for example, pooled IgG, for example, IVIg, for example, for example, sialylated IgG, for example, sialylated pooled IgG, for example, sialylated IVIg, for example, hsIgG, comprises ammonium sulfate precipitation.

[0242] variant In some embodiments, the enzymes described herein are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of an exemplary sequence (e.g., provided herein), and have differences such as up to 1%, 2%, 5%, 10%, 15%, or 20% of residues of the exemplary sequence being replaced by, for example, the mutations described herein or, in addition, by conservative mutations. In preferred embodiments, the variant retains the desired activity of the parent, e.g., β-galactoside α-2,6-sialyltransferase activity or β-1,4-galactosyltransferase activity.

[0243] To determine the percentage of identity between two nucleic acid sequences, these sequences are aligned for optimal comparison purposes (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the reference sequence length, and in some embodiments, at least 90% or 100%. Then, nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, "identity" of nucleic acids is equivalent to "homology" of nucleic acids). The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap.

[0244] The percentage of identity between a target polypeptide or nucleic acid sequence (i.e., the query) and a second polypeptide or nucleic acid sequence (i.e., the target) can be determined in various ways within the scope of the skills in the art using publicly available computer software, for example: Smith Waterman Alignment (Smith, TF and MS Waterman (1981) J Mol Biol 147:195-7), "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher Plus®, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, MO, Ed, pp353-358, BLAST program (Basic Local Alignment Search Tool), (Altschul, SF, W. Gish, et al. (1990) J Mol Biol Software such as 215:403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR). Furthermore, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared. Generally, for a target protein or nucleic acid, the length of comparison may be any length up to the full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of this disclosure, the identity percentage is relative to the full length of the query sequence.

[0245] For the purposes of this disclosure, the comparison of sequences between two sequences and the determination of the identity percentage can be achieved using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0246] Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine, and phenylalanine, tyrosine. [Examples]

[0247] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0248] Example 1: Preparation of highly sialylated IgG IgG in which more than 60% of the entire branched glycan is disialized can be prepared as follows.

[0249] In short, a mixture of IgG antibodies is subjected to sequential enzymatic reactions using β1,4-galactosyltransferase 1 (B4-GalT) 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.

[0250] 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 uses two sugar nucleotides (uridine 5'-diphosphogalactose (UDPGal) and cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA)). The latter is periodically replenished to increase the disialylated product relative to the monosialylated product. The reaction contains manganese chloride as a cofactor.

[0251] A typical example of the IgG-Fc glycan profile of such a reaction starting from IVIg and the reaction product is 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 / 3EEQFNSTFR (SEQ ID NO: 2), IgG3 / 4EEQYNSTFR (SEQ ID NO: 3), and EEQFNSTYR (SEQ ID NO: 4).

[0252] 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.

[0253] Example 2: Alternative sialylation conditions Alternative and suitable reaction conditions for galactosylation and sialylation to produce hsIgG 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 in two parts, i.e., half at the start of the reaction and the other half after 9–10 hours), and 15 units of ST6-Gal1 / g IgG antibody with incubation at 37°C for 30–33 hours. The reaction can be carried out by adding ST6-Gal1 and CMP-NANA to the galactosylation reaction. Alternatively, all reactants can be combined at the start, and CMP-NANA can be added as needed.

[0254] Example 3: Purification of hsIgG For example, it is desirable to have an efficient and economical method for purifying hsIgG prepared by any of the methods described herein. To prepare hsIgG suitable for use in formulations, it is desirable to efficiently remove enzymes, sugar nucleotides, and manganese from the reaction(s) used to prepare the hsIgG. Furthermore, it may be desirable to reduce or eliminate the need for buffer exchange. Finally, since hsIgG can contain various IgG subclasses (e.g., IgG1, IgG2, etc.), it may be desirable to use a method that produces a purified product that does not excessively alter the subclass distribution of the unpurified material.

[0255] Protein A column chromatography was examined. Washing conditions were examined including single salts (NaCl, Na2SO4) and chaotropic salts (arginine, MgCl2, KSCN), buffers (phosphate, TRIS, BIS-TRIS, acetate, formate, glycolate, citrate, MOPS), organic modifiers (ethylene glycol, propylene glycol), additives (imidazole, methyl galactopyranoside, lactose) and pH.

[0256] Using a Mabselect PrismA column (GE Healthcare Life Sciences), the filtered reaction mixture was loaded (preferably in 50 mM, pH 7.4 MOPS), and then washed with eluents containing 50 mM, pH 4.99 sodium acetate, and glycine pH 3.0, so that hsIgG with a subclass profile similar to the initially loaded material (load: IgG1 168%, IgG2 30%; IgG3 1.8% and IgG4 1.4%; eluate: IgG1 70%; IgG2 29%; IgG3 0.9% and IgG4 1.1%) was produced. This column step removed ST6-Gal almost completely. Following the Protein A column step, a polishing step was performed using a Blue Trisacryl M (Pall Corp.) column in flow-through mode. Here, the loading buffer was 0.4 M NaCl, 50 mM sodium citrate pH 4.5, and the washing buffer was 2 M NaCl, 50 mM sodium phosphate pH 7.0. This provided a very clean product. Another type of blue dye column containing a Sepharose-based resin (HiTrap Blue HP, GE Healthcare Life Sciences) was used in binding and elution mode. Here, loading with 50 mM, pH 4.5 sodium citrate and elution in 1.5 M KCl, 32.5 mM sodium citrate, 35% ethylene glycol, pH 4.51 provided a very clean product.

[0257] Example 4: Purification Process and Improvement As shown in Figure 5, various improvements to the hsIgG manufacturing process described below are described in Examples 5-7.

[0258] After sialylation (step 4 in Figure 5), the process involves diluting the reactants 1:1 with 5×PBS and quenching the reaction. The quenched reaction mixture is depth filtered to remove particulates (step 5 in Figure 5), buffer exchanged into a 6 diavolumes of 50 mM Tris, 100 mM NaCl, 10 mM EDTA, pH 8.0 buffer, and subsequently buffer exchanged into 250 mM glycine pH 5.0 (step 6 in Figure 5). This buffer exchange step is to remove reaction components such as nucleotides and manganese chloride. Further, the EDTA buffer is a chelating agent that should also remove manganese chloride from the solution.

[0259] In the glycine buffer, the pH of the process material is lowered to 3.5 for virus inactivation. The buffer is held at low pH for 90 minutes until quenched and the pH is raised to 4.5 (step 7 in Figure 5). The quenched virus inactivation pool is diluted to 4.5 mg / mL at 50 mM citrate, 400 mM NaCl, pH 4.5 before passing through a combination of depth filter / nanofiltration (step 8 in Figure 5). The filtrate is then buffer exchanged into a 6 diavolumes of 50 mM citrate, 400 mM NaCl, pH 4.5 and concentrated to 30 mg / mL (step 9 in Figure 5). The final material is depth filtered one last time before the chromatographic purification step (step 10 in Figure 5).

[0260] The first purification step (step 11 in Figure 5) uses a strong cation exchange resin Poros™ XS in flow-through mode. This means that the material is directly loaded onto the column and impurities (ST6, B4GalT) stick to the resin while the product is collected in the effluent. The flow-through is then loaded onto a trisacryl blue (TAB) column operating also in flow-through mode (step 12 in Figure 5). The TAB resin also has significant capacity to remove the reaction enzymes.

[0261] Example 5: Strong cation exchange resin in binding and elution modes The process involves two TFF steps (buffer exchange / 1 concentration), three deep filtration steps, and two dilutions before chromatography. These steps are expensive (materials / buffers / operating time / product loss). Reducing the number of steps before chromatographic purification reduces cost, operating time, and the amount of material lost while still removing reaction impurities (e.g., nucleotides, enzymes, manganese chloride).

[0262] As described herein, steps 5-10 in Figure 5 can be removed from the process by diluting the quenched (5×PBS) reaction mixture with 50 mM citrate, pH 4.5 and using a strong cation exchange resin Poros® XS in binding and elution mode. The reaction mixture binds to the Poros® XS strong cation exchange resin while the nucleotides and manganese chloride flow into the waste. From here, the bound material can be selectively eluted using the same 50 mM citrate, 400 mM NaCl, pH 4.5 solution used in the current process. Enzymes requiring higher salt concentrations for elution remain bound to the column. Furthermore, the eluted products are now in the same buffer required for TAB resin purification.

[0263] Resin screening Five different strong cation exchange resins, Polps® XS, Capto® S, Capto® SP Impres, SP HP, and Capto® S Impact, were evaluated for their ability to separate IVIg from ST6 spiked in solution. The loading material was prepared by diluting 0.55 mL of IVIg with 100 mg / mL at a 1:10 ratio in 50 mM citrate at pH 4.5, and then spiked into 3.3 mL of ST6 at 16 mg / mL. For each resin, 8 mL of the loading material was added to the column and then eluted by a linear gradient into 50 mM citrate, 1 M NaCl, pH 4.5. The ability of each resin to separate IVIg and ST6 was evaluated by observing chromatograms and calculating the peak resolution using Unicorn, as shown in the following equation (where t is the mean retention time and W is the peak width). Resolution calculations (Table 9) showed that Poros®XS performed best in separating IVIg from ST6.

[0264]

number

[0265] [Table 9]

[0266] Determination of binding capacity Operating Poros® resins in flow-through mode is advantageous because it requires only one cycle and less resin, as the resin only needs to bind impurities. When using Poros® XS resins in binding and elution modes, the total number of cycles required depends on the amount of product that can be bound at once. The loading solution was prepared by diluting IVIg 1:20 with 50 mM citrate pH 4.5. The loading solution was added to the column with residence times of 2, 4, and 6 minutes, and the binding capacity was evaluated at a 5% breakthrough. Figure 6 shows that operating beyond a residence time of 4 minutes, which is shorter than the typical 5-minute residence time for chromatographic operations, does not yield a significantly larger volume. Furthermore, the volume is very high, at approximately 96 mg / resin mL.

[0267] Yield and residual ST6 evaluation The yield and residual ST6 for different loading volumes and elution buffer conductivity were evaluated over several runs to identify the best elution conditions and loading volumes (Table 10). The loading solution was prepared by diluting 2.8 g of IVIg and 500 uL of ST6 to 15.6 mg / mL, and then diluting the solution 1:20 with 50 mM citrate pH 4.5. All steps were performed with a residence time of 5 minutes. The loading material was 2,700 ppm of ST6.

[0268] [Table 10]

[0269] Statistical analysis showed that elution conductivity was the only significant parameter in the regression models for both yield and residual ST6. The predictive profiler in Figure 7 shows that increasing the packing volume did not have a statistically significant effect on the yield.

[0270] Figure 8 shows an operating range with a yield of 90 - 95% and a residual ST6 level of less than 30 ppm. The white space shows an operating range suggesting that an elution conductivity of 39 - 44 mS / cm can be used for a loading capacity of less than 55 mg / mL. As the loading capacity increases, this range becomes narrower. These results essentially confirm that 50 mM citrate, 400 mM NaCl, pH 4.5 (400 mM NaCl corresponding to a conductivity of 40 mS / cm) is the best choice for the elution buffer.

[0271] Purification of the hsIVIg reaction mixture Several runs were performed using hsIVIg generated using a sialylation procedure carried out in MOPS (Table 11). The hsIVIg reaction mixture was quenched with 5×PBS at a 1:1 dilution and then further diluted with 50 mM citrate pH 4.5. For all runs below, 50 mM citrate, 400 mM NaCl, pH 4.5 was used as the elution buffer. A concentration of 7.15 mg / mL corresponds to a 1:20 dilution with 50 mM citrate pH 4.5. A 5 - minute residence time was used for all steps.

[0272]

Table 11

[0273] The flow - through step contains no IgG - only nucleotides and manganese chloride, as seen in the flow - through SEC chromatogram of Figure 9.

[0274] This can be compared to the SEC of the eluate in Figure 10, which shows a dimer peak at a retention time of 6.8 minutes and a monomer peak at a retention time of 7.8 minutes.

[0275] Summary of results As these results show, the current hsIVIg process is significantly improved by directly diluting the reaction mixture to 50 mM citrate at pH 4.5 and loading it into the same Poros® XS resin. Binding hsIVIg to the resin allows for a much simpler pathway for removing nucleotides and manganese chloride (achieved by prior buffer exchange). It is clear that significant ST6 enzyme clearance can be achieved using 50 mM citrate, 400 mM NaCl, and pH 4.5. Furthermore, the Poros® XS eluate pool is in suitable conditions for the subsequent TAB purification step.

[0276] In production-scale processes, a single deep filtration step may still be used. The sialylation reaction mixture is often very turbid and unsuitable for direct loading into chromatographic resin. Therefore, the quenched reaction mixture may be diluted with 50 mM citrate at pH 4.5, and the process material may be deep filtered before loading into Poros® XS resin.

[0277] Example 6: Alternative column The current manufacturing process for M254 uses a trisacryl blue (TAB) column in flow-through mode, which cannot withstand high-concentration sodium hydroxide washing. This results in high levels of bioburden in some batches. Different blue resins were evaluated, and Capto® Blue High Sub (Capto® Blue HS) was found to be a good alternative to the TAB column, offering similar recovery and better enzyme clearance. Furthermore, Capto® Blue High Sub can withstand high-concentration sodium hydroxide washing and has the potential to reduce the number of steps in the current purification process.

[0278] Evaluation of Capto(trademark) Blue and Blue Sephrarose(trademark) Two chromatography resins were evaluated against TAB to determine if recovery was affected. For this purpose, 250 mg of IVIg was diluted 1:1 in 800 mM NaCl + 100 mM citrate, pH 4.5 and directly packed three times. An overlay of one chromatogram from one run is shown in Figure 11, and the recovery of the three runs is reported in Figure 12. As shown, recovery (99%) was unaffected.

[0279] Comparison of Capto(trademark) Blue and Capto(trademark) Blue HS Capto® Blue and Capto® Blue HS were compared to evaluate recovery and enzyme clearance. Multiple runs were performed either by directly loading hsIGIV (post-strong cation exchange column flow-through material) or by loading after dilution. Table 12 summarizes the different loading conditions, and Table 13 shows the mass balance.

[0280] [Table 12]

[0281] As shown in Figure 13, the overlake chromatograms for runs 1 and 4 appear to be very similar.

[0282] [Table 13]

[0283] The recovery of Capto(trademark) Blue HS is very similar to that of Capto(trademark) Blue, and the 96% recovery rate is considered sufficient to replace TAB columns.

[0284] We also evaluated the residual ST6 levels after Capto(trademark)Blue and Capto(trademark)Blue HS, and found that they were low, if not lower, than TAB, as shown in Figures 14 and 15.

[0285] Impact of using Capto® Blue HS on product attributes The impact of IVIg from Capto® Blue HS flow-through and eluate on product quality was evaluated compared to the load. Analysis SEC of the load, flow-through, and eluate was compared. As shown in Figure 16 and Table 14, no differences were observed.

[0286] [Table 14]

[0287] The IgG subclass distribution was evaluated by mass spectrometry (Figure 17). Analysis before and after Capto® Blue High Sub in flow-through mode was compared for two sources of IVIg, but no changes were found.

[0288] Example 7: Elimination of cation exchange column Capto(trademark) Blue HS To reduce the number of steps in the current manufacturing process, we evaluated different conditions and assessed residual ST6. • Experiments using Capto® Blue HS flow-through with a residual ST6 of 651 ppm were used as the load test material, and 5X PBS and MOPS were evaluated with pH 4.5 buffer as the load buffer. The load buffer mimics the load material that is directly packed into the column after the reaction. • Execution 2: Post-virus inactivation material with 1400 ppm residual ST6 was directly filled into Capto® Blue HS resin using 250 mM glycine pH 4.5. • Execution 3: Post-virus inactivation material with 1400 ppm residual ST6 was directly filled into Capto® Blue HS resin using 250 mM glycine + 100 mM sodium chloride pH 4.5.

[0289] As shown in Figures 18 (Run 1), 19 (Run 2), and 20 (Run 3), residual ST6 was evaluated for three runs. Directly filling the reaction mixture with Capto® Blue High Sub significantly reduced the ST6 level. However, increasing the salt content, even with 100 mM NaCl, adversely affected residual ST6.

[0290] Trisacryl Blue (TAB) The current manufacturing process utilizes two chromatography columns, CEX followed by TAB, to reduce the level of ST6 added during the enzymatic reaction. Residual ST6 levels have ranged from 2.5 to 7.2 ppm for previously manufactured drug substance lots. The maximum load in previous manufacturing processes using the TAB column was 1.1 g of 1VI g / ml resin (1X).

[0291] In this study, TAB column execution was performed to understand the potential to increase loading capacity and eliminate CEX columns from the current manufacturing process.

[0292] A pre-loaded 1 mL TAB column was used for the loading test. The TAB column loading test was performed using CEX load and CEX eluate obtained from a 1.5 kg GMP run lot #20006. The TAB column run was performed using an AKTA Avant 150. To achieve residence times similar to those in the manufacturing column procedure, the loading flow rate was maintained at 0.17 mL / min. The load flow-through fraction was collected, the protein concentration was measured by A280, and the residual level of ST6 was determined by ELISA.

[0293] As shown in Figure 21, the residual ST6 levels ranged from 0.1 to 9 ppm for the TAB column flow-through fraction when packed with 0.1 to 1.1 g / mL, with the x-axis representing cumulative protein flow-through. When these flow-through fractions are pooled, it is reasonable to estimate an ST6 level of approximately 5 ppm (1.1 g / mL load), which is similar to that of past manufacturing drug substance lots. The results of this test confirm that the maximum load on the TAB column should be kept below 1.1 g / mL to maintain the ST6 levels observed in DS lots. The ST6 levels were 161 ppm and 62 ppm for the manufacturing-scale CEX load and CEX eluate, respectively, indicating that the CEX column in the current manufacturing process reduces ST6 levels.

[0294] In this study, TAB column execution was performed using CEX loading, in other words, without CEX column purification. As shown in Figure 21, the residual levels of the ST6 value were similar for the TAB column flow-through fractions (0.1-9 ppm) using two loading materials: CEX loading (no CEX column purification, 161 ppm) and CEX eluate (with CEX column, as in the current manufacturing process, 62 ppm).

[0295] The results of this study showed that 1.1 g / ml was the maximum loading of the TAB column that reduced the ST6 level of the active pharmaceutical ingredient to ≤7.2 ppm, similar to previous manufacturing runs (ST6 levels 2.5–7.2 ppm). Furthermore, the results of this study suggest that, in the absence of a CEX column, the TAB column efficiently reduces the ST6 level to meet the current target (API ≤7.2 ppm).

[0296] Example 8: Ammonium sulfate precipitation IVIg is isolated and purified from human plasma by several methods, including precipitation using either ethanol or polyethylene glycol at low temperatures. Here, the precipitation of hsIVIg is evaluated using ammonium sulfate to remove residual ST6.

[0297] For this purpose, IVIg and hsIVIg were precipitated as follows: A saturated solution of ammonium sulfate was prepared by directly adding the powder to 100 mL of water while stirring. Once a significant amount of powder had precipitated, the solution was stirred for 30 minutes, then the precipitate was decanted and added directly to the IVIg solution. Saturated ammonium sulfate solution was added until the IVIg solution turned white and no further color changes were observed. The precipitate was collected by centrifugation or by sterile filtration.

[0298] Figure 22 shows the starting IVIg and the overlay of precipitated IVIg on the precipitated material, and Figure 23 shows hsIVIg before and after precipitation using sterile filtration as a method for recovering the precipitated material. The removal of nucleotides that elute in approximately 11 minutes can be clearly seen. There was also a significant decrease in residual ST6 enzyme, as shown in Table 15.

[0299] A single experiment demonstrates that residual enzymes can be removed by precipitation. The aggregates observed when the material is recovered by filtration are not a problem, indicating that filtration is not a method for collecting IVIg.

[0300] [Table 15]

[0301] 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_001488.2B4GALT1[Biology=Human][GeneID=2683][Isoform=1]) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLH PVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFL TINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS Sequence ID 6 (NP_001365424.1B4GALT1[Biology=Human][GeneID=2683][Isoform=2]) MPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQ QLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS Sequence ID 7 (NP_001365425.1B4GALT1[Biology=Human][GeneID=2683][Isoform=3]) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKV AIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS Sequence ID 8 (NP_001365426.1B4GALT1[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_775324.1ST6GAL1[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

[0302] Other Embodiments Although the present invention has been described in connection with its detailed description, the foregoing description is illustrative of the scope of the invention and is not intended to limit the scope of the invention, and the scope of the invention is understood to be defined by the appended claims. Other aspects, advantages, and modifications are within the following claims.

Claims

1. 1. A method for producing a purified highly sialylated IgG (hsIgG) composition, wherein greater than 75% of branched Fc glycans on said hsIgG have sialic acid at both α1,3 and α1,6 branches, comprising: (a) applying to a chromatography column comprising a resin having sulfonic acid functional groups an hsIgG composition comprising hsIgG and ST6Gal or an enzymatically active portion thereof, and a citrate buffer of about 50 mM and about pH 4.5, under conditions that bind said hsIgG and said ST6Gal or an enzymatically active portion thereof; (b) selectively eluting the IgG from the column, thereby producing the purified hsIgG composition, wherein greater than 75% of the branched Fc glycans on the hsIgG have sialic acid at both the α1,3 branching and the α1,6 branching. The method comprising:

2. The method of claim 1, further comprising: (c) prior to (a), diluting the unpurified hsIgG with 5x PBS at a dilution of approximately 1:

1.

3. The method according to claim 1, wherein the CEX column comprises a resin having an SO 3 − functional group.

4. A method according to any one of claims 1 to 3, wherein selectively eluting the hsIgG comprises eluting in a buffer containing at least about 400 mM NaCl.

5. The method of claim 1, wherein the method does not further comprise an additional filtration, fractionation, or purification step prior to performing step (a).

6. The method of claim 1, wherein the method comprises a single deep filtration step prior to step (a).

7. The method of claim 1, wherein the method comprises a viral inactivation step prior to step (a).

8. The method of claim 1, wherein step (a) is carried out with a residence time(s) of 1, 2, 3, 4, 5, or 6 minutes or about 1, 2, 3, 4, 5, or 6 minutes.

9. The method of claim 1, further comprising, after step (b), subjecting the purified hsIgG composition to one or more of: (i) blue dye chromatography; (ii) a DE pad filtration step; (iii) a depth filtration step; or (iv) a PS20 spiking and filtration step.

10. The method of any one of claims 1 to 9, wherein the purified hsIgG composition comprises no more than 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, or 30 ppm of ST6Gal or an enzymatically active portion thereof.

11. A method for producing a purified highly sialylated IgG (hsIgG) antibody composition, comprising: A buffered antibody composition comprising the hsIgG and the ST6Gal or an enzymatically active portion thereof is applied to a blue dye column under conditions that allow 80% or more of the hsIgG to flow through and allow 100 ppm or less of ST6Gal or an enzymatically active portion thereof to flow through; thereby producing said purified hyper sialylated IgG (hsIgG) antibody composition.

12. The method of claim 11, wherein the blue dye chromatography column is a Trisacryl Blue (TABS) column and the buffer suitable for use with the column is about 100 mM citrate buffer containing about 800 mM NaCl, about pH 4.

5.

13. The method of claim 11, wherein the blue dye chromatography column is a Capto™ Blue or Capto™ Blue HS column and the buffer is about 250 mM glycine with about 800 mM NaCl at a pH of about 4.

5.

14. The method of any one of claims 11 to 13, further comprising: (b) prior to (a), diluting the unpurified hsIgG composition with 5x PBS at a dilution of approximately 1:

1.

15. The method of claim 11, wherein the method does not include an additional filtration, fractionation or purification step prior to (a).

16. The method of claim 11, wherein the method comprises a single deep filtration step prior to (a).

17. The method of claim 11, wherein the method comprises a viral inactivation step prior to step (a).

18. The method of any one of claims 11 to 17, wherein step (a) is carried out with a residence time(s) of 1, 2, 3, 4, 5, or 6 minutes or about 1, 2, 3, 4, 5, or 6 minutes.

19. The method of any one of claims 11 to 18, further comprising, after step (a), one or more of: (i) blue dye chromatography; (ii) a DE pad filtration step; (iii) a depth filtration step; or (iv) a PS20 spiking and filtration step.

20. A method for producing a highly sialylated IgG (hsIgG) composition, comprising: (a) precipitating hsIgG by adding a saturated solution of ammonium sulfate, thereby producing a precipitated hsIgG solution; (b) isolating the precipitated hsIgG; Thereby, purifying the purified hsIgG composition. The method comprising:

21. The method of claim 20, wherein isolating the precipitated hsIgG comprises filtration and / or centrifugation.

22. A method for producing hypersialylated IgG (hsIgG) from pooled IgG antibodies, comprising: (a) incubating the pooled IgG antibodies in a reaction mixture comprising β1,4-galactosyltransferase (B4GalT) or an enzymatically active portion thereof and UDP-Gal or a salt thereof, thereby forming a galactosylated IgG antibody; (b) incubating the galactosylated IgG antibody in a reaction mixture containing ST6Gal or an enzymatically active portion thereof and CMP-NANA or a salt thereof; (c) purifying said hsIgG according to the method of any one of claims 1 to 21, thereby producing said hsIgG. The method comprising:

23. A method for preparing hypersialylated IgG (hsIgG) from a pooled IgG antibody, comprising: (a) incubating the pooled IgG antibodies in a reaction mixture comprising β1,4-galactosyltransferase (B4GalT) or an enzymatically active portion thereof, UDP-Gal or a salt thereof, ST6Gal or an enzymatically active portion thereof, and CMP-NANA or a salt thereof, thereby forming the hsIgG; (b) purifying the hsIgG according to the method of any one of claims 1 to 21; Thereby producing the hsIgG preparation. The method comprising:

24. A method for preparing hypersialylated IgG (hsIgG) from pooled IgG antibodies, comprising: (a) incubating the pooled IgG antibodies in a galactosylation reaction mixture comprising β1,4-galactosyltransferase (B4GalT) or an enzymatically active portion thereof and UDP-Gal or a salt thereof, thereby producing a galactosylated IgG antibody; (b) adding ST6Gal, or an enzymatically active portion thereof, and CMP-NANA, or a salt thereof, to the galactosylation reaction mixture to form a sialylation reaction mixture; (c) incubating the sialylation reaction mixture; (d) purifying the hsIgG according to the method of any one of claims 1 to 21; thereby producing said hsIgG. The method comprising:

25. A method for preparing a purified highly sialylated (hsIgG) composition for pooled IgG antibodies, comprising: (a) incubating the IgG antibody in a first reaction mixture comprising β1,4-galactosyltransferase I (β4GalT) or an enzymatically active portion thereof and UDP-Gal to produce a galactosylated IgG antibody; (b) incubating the galactosylated IgG antibody in a second reaction mixture comprising ST6Gal or an enzymatically active portion thereof and CMP-NANA to generate a hsIgG mixture; (c) applying the hsIgG mixture to the Protein A column under conditions whereby the hsIgG binds to the Protein A column; (d) eluting the purified hsIgG from the Protein A column; The method comprising:

26. The method of claim 25, further comprising: (e) further purifying the hsIgG produced in step (d) using a trisacryl blue column.

27. The method of claim 25 or 26, wherein step (d) comprises eluting the hsIgG from the Protein A column with a buffer containing glycine.

28. The method of claim 25, wherein the protein A column is washed with an acetate buffer between steps (c) and (d).

29. A method according to any one of claims 25 to 28, wherein the pH and salt content of the hsIgG produced in step (d) is altered prior to application to the trisacryl blue column.

30. The method of any one of claims 25 to 29, wherein step (f) comprises eluting the hsIgG with a high salt buffer.

31. The method of claim 30, wherein the high salt buffer comprises 2M NaCl.

32. The method of claim 30, wherein the high salt buffer comprises 2M KCl.

33. The method of claim 29, wherein the pH and salt content of the hsIgG produced in step (d) is changed to 0.4 M NaCl and pH 4.

5.

34. A method for preparing highly sialylated (hsIgG) antibodies from a mixture of IgG antibodies, comprising the steps of: (a) incubating a mixture of said IgG antibodies in a reaction mixture comprising β1,4-galactosyltransferase (B4GalT) or an enzymatically active portion thereof, UDP-Gal, ST6Gal or an enzymatically active portion thereof, and CMP-NANA, thereby producing an hsIgG preparation; (b) applying the hsIgG preparation to a Protein A column under conditions that bind the hsIgG antibodies; (c) eluting the hsIgG from the Protein A column; The method comprising:

35. The method of claim 22, wherein the B4GalT or an enzymatically active portion thereof is at least 90% identical to SEQ ID NO:12 or SEQ ID NO:13, and the ST6Gal or an enzymatically active portion thereof comprises an amino acid sequence at least 90% identical to SEQ ID NO:19 or SEQ ID NO:

20.

36. A method for preparing a purified highly sialylated (hsIgG) composition from a mixture of IgG antibodies, comprising: (a) incubating the mixture of IgG antibodies in a reaction mixture comprising β1,4-galactosyltransferase I (B4GalT) or an enzymatically active portion thereof and UDP-Gal to produce galactosylated IgG antibodies; (b) incubating the galactosylated IgG antibody in a reaction mixture comprising ST6Gal or an enzymatically active portion thereof and CMP-NANA to produce the hsIgG composition; and (c) isolating the B4GalT or an enzymatically active portion thereof, and the ST6Gal or an enzymatically active portion thereof from the hsIgG composition. The method comprising:

37. (a) incubating a mixture of said IgG antibodies in a reaction mixture comprising β1,4-galactosyltransferase (B4GalT) or an enzymatically active portion thereof, UDP-Gal, ST6Gal or an enzymatically active portion thereof, and CMP-NANA, thereby producing an hsIgG preparation; (b) isolating the hsIgG from the hsIgG preparation, and isolating the B4GalT or the enzymatically active portion thereof and the ST6Gal or the enzymatically active portion thereof from the hsIgG preparation. The method comprising:

38. The method of any one of the preceding claims, wherein approximately 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the hsIgG have sialic acid on both the α1,3 branching and the α1,6 branching prior to purification.

39. The method of any one of the preceding claims, wherein approximately 80% or 85% of the branched Fc glycans on the hsIgG have sialic acid at both the α1,3 branching and the α1,6 branching before purification.

40. The method of any one of the preceding claims, wherein at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the hsIgG have sialic acid on both the α1,3 arm and the α1,6 arm, and the sialic acid is attached via a NeuAc-α2,6-Gal terminal linkage prior to purification.

41. A method according to any one of claims 1 to 24, wherein at least 80% of the branched Fc glycans on the hsIgG have sialic acid at both the α1,3 branch and the α1,6 branch before purification.

42. The method of any one of the preceding claims, wherein approximately 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the hsIgG have sialic acid on both the α1,3 branches and the α1,6 branches after purification.

43. The method of any one of the preceding claims, wherein approximately 80% or 85% of the branched Fc glycans on the hsIgG have sialic acid at both the α1,3 branching and the α1,6 branching after purification.

44. The method of any one of the preceding claims, wherein at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the hsIgG have sialic acid on both the α1,3 arm and the α1,6 arm connected via NeuAc-α2,6-Gal terminal linkages after purification.

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