Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/005—Glycopeptides, glycoproteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation

Definitions

• the present disclosure relates to methods for preparing and purifying hypersialylated
• Intravenous immunoglobulin which is prepared from the pooled plasma of human donors (e.g., pooled plasma from at least 1,000 donors), is used to treat a variety of inflammatory disorders.
• IVIg preparations have distinct limitations, such as variable efficacy, clinical risks, high costs, and finite supply. Different IVIg preparations are frequently treated as interchangeable products clinically, but it is well-known that significant differences in product preparations exist that may impact tolerability and activity in selected clinical applications. At the current maximal dosing regimens, only partial and unsustained responses are obtained in many instances.
• IVIg preparations generally exhibit low levels of sialylation on the Fc domain of the antibodies present. Specifically, they exhibit low levels of di-sialylation of the branched glycans on the Fc region. Washburn et al. ( Proceedings of the National Academy of Sciences, USA 112: E1297-
• immunoglobulin G having a very high level of Fc sialylation, particularly disialylation (sialylation on both the alpha 1,3 branch and the alpha 1,6 branch of the glycan at Asn297 (EU Numbering).
• the methods described herein can provide hypersialylated IgG (hsIgG) in which greater than 70% of the branched glycans on the Fc domain are sialylated on both branches (i.e., on the alpha 1,3 branch and the alpha 1,6 branch).
• HsIgG contains a diverse mixture of IgG antibodies, primarily IgGl antibodies. The diversity of the antibodies is high.
• the immunoglobulins used to prepare hsIgG can be obtained, for example from pooled human plasma (e.g., pooled plasma from at least 1,000 - 30,000 donors).
• the immunoglobulins can be obtained from IVIg, including commercially available IVIg.
• HsIgG has far higher level of sialic acid on the branched glycans on the Fc region than does IVIg. This results in a composition that differs from IVIg in both structure and activity.
• HsIgG can be prepared as described in W02014/179601 or Washburn et al. (Proceedings of the National Academy of Sciences, USA 112: E1297-E1306 (2015)), both of which are hereby incorporated by reference.
• Described herein are improved methods for preparing and purifying hsIgG.
• the methods generate an unpurified hsIgG composition from which both purified hsIgG and, in some embodiments, the enzymes used to generate to prepare the hsIgG can be isolated. In some cases, the isolated enzymes can be reused.
• hsIgG hypersialylated IgG
• hsIgG hypersialylated IgG
• a purified hypersialylated IgG composition with greater than 75% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch comprising: (a) providing a hsIgG composition comprising hsIgG and ST6Gal or an enzymatically active portion thereof; (b) diluting the composition in a citrate buffer at about 50 mM, about pH 4.5, thereby producing a buffered antibody composition; (c) applying the buffered antibody composition to a chromatography column comprising a resin with a sulfonic acid functional group under conditions that bind the hsIgG as well as the ST6Gal or enzymatically active portion thereof; and (d) selectively eluting the IgG from the
• the step of providing a hsIgG composition comprises providing a composition comprising hsIgG and diluting the composition in 5X PBS at about a 1 : 1 dilution.
• the CEX column comprises a resin having a SO 3 - functional group.
• selectively eluting the hsIgG comprises eluting in a buffer comprising about 400 mM or more NaCl.
• the buffer is citrate buffer at about 50 mM and about pH 4.5.
• the method does not contain any additional filtering, fractionation, or purification steps other than (a) and (b) before carrying out step (c).
• the method comprises a single depth filtering step, in addition to steps (a) and (b) before step (c).
• the additional depth filtering step is carried out between steps (a) and (c).
• the method does not comprise any additional filtering, fractionation, or purification steps beyond the single depth filtration step before carrying out step
• the method comprises a viral inactivation step before step (c).
• step (c) is spaced out over 1, 2, or 3 applications. In some embodiments, step (e) is repeated one or more times.
• step (c) is carried out at resident time(s) of from or from about 1, 2, 3, 4, 5, or 6 minutes.
• the method further comprises, following step (e), one or more of: (f) blue dye chromatography (g) DE pad filtration step, (h) depth filtration step or (g) PS20 spiking & filtration step.
• the purified hsIgG composition comprises 80%, 85%, 90%, or 95% or more of the amount of unpurified hsIgG from the composition of step (a).
• the purified hsIgG composition comprises 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, or 30 ppm or less ST6Gal or enzymatically active portion thereof.
• a method of producing a purified hypersialylated IgG (hsIgG) composition comprising: (a) providing a hsIgG composition comprising hsIgG and ST6Gal or an enzymatically active portion thereof and optionally quenching the reaction; (b) diluting the composition in a buffer suitable for use with the column, thereby producing a buffered antibody composition; (c) applying the buffered antibody composition to a blue dye column, e.g., a blue column described herein, under conditions that allow 80% or more of the hsIgG to flow through, but allows only 100 ppm or less of the ST6 or enzymatically active portion thereof to flow through, thereby producing purified hsIgG.
• a blue dye column e.g., a blue column described herein
• the blue dye chromatography column is a Trisacryl Blue (TABS) column and the buffer suitable for use with the column is citrate buffer, about 100 mM, about pH 4.5, with about 800 mM NaCl.
• TABS Trisacryl Blue
• the blue dye chromatography column is a CaptoTM Blue or CaptoTM Blue HS column and the buffer is about 250 mM glycine with about 800 mM NaCl at about pH 4.5.
• the step of providing a hsIgG composition comprises providing a composition comprising hsIgG and diluting the composition in 5X PBS at about a 1 : 1 dilution.
• the method does not contain any additional filtering, fractionation, or purification steps other than (a) and (b) before carrying out step (c).
• the method comprises a single depth filtering step, in addition to steps (a) and (b) before step (c).
• the additional filtering step is carried out between steps (a) and (c).
• the method does not comprise any additional filtering, fractionation, or purification steps beyond the single depth filtration step before carrying out step
• the method comprises a viral inactivation step before step (c).
• the applying in step (c) is spaced out over 1, 2, or 3 applications.
• step (c) is carried out at resident time(s) of from or from about 1,
• the method further comprises, following step (e), one or more of: (f) blue dye chromatography (g) DE pad filtration step, (h) depth filtration step or (g) PS20 spiking & filtration step.
• hsIgG hypersialylated IgG composition
• methods of producing a hypersialylated IgG comprising: (a) providing hsIgG; (b) precipitating the hsIgG by adding saturated solution of ammonium sulfate, thereby producing a precipitated hsIgG solution; and (c) isolating the precipitated hsIgG, thereby producing a purified hsIgG composition
• isolating the precipitated hsIgG comprises filtration and/or centrifugation.
• hsIgG hypersialylated IgG
• methods of producing hypersialylated IgG comprising: (a) providing pooled IgG antibodies; (b) incubating the pooled IgG antibodies in a reaction mixture comprising ⁇ 1,4-Galactosyltransferase (B4GalT) or enzymatically active portion thereof, and UDP-Gal or salt thereof; and(c) incubating the galactosylated IgG antibodies in a reaction mixture comprising ST6Gal, or enzymatically active portion thereof, and CMP -NANA or salt thereof, (d) purifying the hsIgG according to any of the methods described herein.
• B4GalT ⁇ 1,4-Galactosyltransferase
• hsIgG hypersialylated IgG
• methods of preparing hypersialylated IgG comprising: (a) providing pooled IgG antibodies; (b) incubating the pooled IgG antibodies in a reaction mixture comprising ⁇ 1,4-Galactosyltransferase (B4GalT) or enzymatically active portion thereof, UDP-Gal or salt thereof, ST6Gal or enzymatically active portion thereof, and CMP- NANA or salt thereof; and (c) purifying the hsIgG according to any of the methods described herein, thereby creating the hsIgG preparation.
• B4GalT ⁇ 1,4-Galactosyltransferase
• hsIgG hypersialylated IgG
• methods of preparing hypersialylated IgG comprising: (a) providing pooled IgG antibodies; (b) incubating the pooled IgG antibodies in a galactosylation reaction mixture comprising ⁇ 1,4-Galactosyltransferase (B4GalT) or enzymatically active portion thereof, and UDP-Gal or salt thereof, thereby producing galactosylated IgG antibodies; (c) adding ST6Gal or an enzymatically active portion thereof and CMP -NANA or salt thereof to the galactosylation reaction mixture to produce a sialylation reaction mixture; (d) incubating the sialylation reaction mixture; and (e) purifying the hsIgG according to any of the methods described herein, thereby producing hsIgG
• Also described herein are methods of preparing purified hypersialylated (hsIgG) composition comprising: (a) providing a mixture of IgG antibodies, (b) incubating the mixture of IgG antibodies in a reaction mixture comprising ⁇ 1,4-Galactosyltransferase I ( ⁇ 4GalT) or enzymatically active portion thereof and UDP-Gal to produce galactosylated IgG antibodies; (c) incubating the galactosylated IgG antibodies in a second reaction mixture comprising ST6Gal or enzymatically active portion thereof and CMP -NANA to produce a hsIgG mixture; (d) applying the hsIgG mixture to a protein A column under conditions that bind IgG antibodies; and (e) eluting hsIgG from the protein A column.
• the method further comprises (f) further purifying the hsIgG produced in step (e) using a trisacryl blue column.
• step (e) comprises eluting the hsIgG with a buffer comprising glycine.
• the protein A column is washed with an acetate buffer between step (d) and step (e).
• step (e) the pH and salt content of hsIgG produced in step (e) is altered before applying it to the trisacryl blue column.
• step (f) comprises eluting the hsIgG with a high salt buffer.
• the high salt buffer comprises 2 M NaCl.
• the high salt buffer comprises 2 M KC1
• the hsIgG produced in step (e) is altered to 0.4 M NaCl and pH 4.5.
• Also described herein are methods of preparing hypersialylated (hsIgG) comprising: (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture comprising ⁇ 1,4-Galactosyltransferase (B4GalT) or enzymatically active portion thereof, UDP-Gal, ST6Gal or enzymatically active portion thereof and CMP -NANA, thereby creating an hsIgG preparation; (d) applying the hsIgG preparation to a protein A column under conditions that bind IgG antibodies; and (e) eluting the hsIgG from the protein A column.
• B4GalT ⁇ 1,4-Galactosyltransferase
• the B4GalT or enzymatically active portion thereof is at least 90% identical SEQ ID NO: 12 or SEQ ID NO: 13 and the ST6Gal or enzymatically active portion thereof comprises an amino acid sequence that is at least 90% identical SEQ ID NO: 19 or SEQ ID NO: 20
• the IgG antibodies comprise IgG antibodies isolated from at least
• At least 70% w/w of the IgG antibodies are IgGl antibodies.
• At least 90% of the donor subjects have been exposed to a virus.
• the step of providing a mixture of IgG antibodies comprises: (a) providing pooled plasma from at least 1000 human subjects; and (b) isolating a mixture of IgG antibodies from the pooled plasma.
• the mixture of IgG antibodies are isolated from intravenous immunoglobulin.
• the mixture of IgG antibodies are intravenous immunoglobulin.
• the step of isolating a mixture of IgG antibodies from the pooled plasma comprises ethanol precipitation or caprylic acid precipitation.
• the step of isolating a mixture of IgG antibodies from the pooled plasma comprises biding IgG antibodies to an ion exchange column and eluting the IgG antibodies from an ion exchange column.
• Also provided herein are methods of preparing purified hypersialylated (hsIgG) composition comprising: (a) providing a mixture of IgG antibodies, (b) incubating the mixture of IgG antibodies in a reaction mixture comprising ⁇ 1,4-Gal actosyUransferase (B4GalT) or enzymatically active portion thereof and UDP-Gal to produce galactosylated IgG antibodies; (c) incubating the galactosylated IgG antibodies in a reaction mixture comprising ST6Gal or enzymatically active portion thereof and CMP -NANA to produce an hsIgG composition; and (d) isolating B4GalT or enzymatically active portion thereof and ST6Gal or enzymatically active portion thereof from the hsIgG composition.
• B4GalT ⁇ 1,4-Gal actosyUransferase
• the method further comprises isolating one or both of B4GalT or enzymatically active portion thereof and ST6Gal or enzymatically active portion thereof from the hsIgG composition.
• Also provided herein are methods of preparing hypersialylated (hsIgG) comprising: (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture comprising ⁇ 1,4-Galactosyltransferase (B4GalT) or enzymatically active portion thereof, UDP-Gal, ST6Gal or enzymatically active portion thereof and CMP -NANA, thereby creating an hsIgG preparation; and (c) isolating hsIgG from the hsIgG prepartion and isolating B4GalT or enzymatically active portion thereof and ST6Gal or enzymatically active portion thereof from the hsIgG preparation.
• B4GalT ⁇ 1,4-Galactosyltransferase
• about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch before purification.
• about 80%, or 85% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch before purification.
• At least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through aNeuAc- ⁇ 2,6-Gal terminal linkage before purification.
• At least 80% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch before purification.
• At least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage before purification.
• At least 85% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch before purification.
• at least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage before purification.
• At least 90% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch before purification.
• At least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage before purification.
• about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch after purification.
• about 80%, or 85% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch after purification.
• At least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage after purification.
• At least 80% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch after purification.
• At least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage after purification.
• At least 85% of the branched Fc glycans on the hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch after purification.
• At least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through aNeuAc- ⁇ 2,6-Gal terminal linkage after purification.
• At least 90% of the branched Fc glycans on the hsIgG have a sialic acid on both the al,3 branch and the al,6 branch after purification.
• At least 60%, 65%, 70% of the branched glycans on the Fab domain of the hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through aNeuAc- ⁇ 2,6-Gal terminal linkage after purification.
• the method further comprises analyzing the amount of one or more IgG subclasses after a chromatography step.
• Also described herein is a method of preparing purified hypersialylated (hsIgG) composition, the method comprising: (a) providing a mixture of IgG antibodies, (b) incubating the mixture of IgG antibodies in a reaction mixture comprising ⁇ 1, 4-Galactosyltransferase I (B4GalT) and UDP-Gal to produce galactosylated IgG antibodies; (c) incubating the galactosylated IgG antibodies in a second reaction mixture comprising ST6Gall and CMP- NANA to produce a hsIgG mixture; (d) applying the hsIgG mixture to a protein A column under conditions that bind IgG antibodies; and (e) eluting hsIgG from the protein A column.
• hsIgG hypersialylated
• the method further comprises: (g) further purifying the hsIgG produced in step (e) using a trisacryl blue column;
• the B4GalT is at least 90% identical SEQ ID NO:l or SEQ ID NO:2;
• the ST6Gall comprises an amino acid sequence that is at least 90% identical SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5;
• step (e) comprises eluting the hsIgG with a buffer comprising glycine;
• the protein A column is washed with an acetate buffer between step (d) and step (e); the pH and salt content of hsIgG produced in step (e) is altered before applying it to the trisacryl blue column;
• 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 KC1; and/or the hsIgG produced in step (e) is altered
• hsIgG hypersialylated
• the method comprising (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture comprising b 1.4-Gal actosyltransrerase I (B4GalT), UDP-Gal, ST6Gall and CMP -NANA, thereby creating an hsIgG preparation; (d) applying the hsIgG composition to a protein A column under conditions that bind IgG antibodies; and (e) eluting the IgG antibodies from the protein A column.
• hsIgG hypersialylated composition
• the method comprising (a) providing a mixture of IgG antibodies, (b) incubating the mixture of IgG antibodies in a reaction mixture comprising ⁇ 1,4-Galactosyltransferase I (B4GalT) and UDP-Gal to produce galactosylated IgG antibodies; (c) incubating the galactosylated IgG antibodies in a reaction mixture comprising ST6Gal1 and CMP -NANA to produce an hsIgG composition; and (d) isolating hsIgG from the hsIgG composition and isolating B4GalT and ST6Gall from the hsIgG composition.
• hsIgG hypersialylated
• the method comprising (a) providing a mixture of IgG antibodies; (b) incubating the mixture of IgG antibodies in a reaction mixture comprising ⁇ 1,4-Galactosyltransferase I (B4GalT), UDP-Gal, ST6Gall and CMP -NANA, thereby creating an hsIgG preparation; and (c) isolating hsIgG from the hsIgG composition and isolating B4GalT and ST6Gall from the hsIgG composition.
• B4GalT ⁇ 1,4-Galactosyltransferase I
• the B4GalT is at least 90% identical SEQ ID NO: 12 or SEQ ID NO: 13 and the ST6Gall comprises an amino acid sequence that is at least 90% identical SEQ ID NO: 19 or SEQ ID NO: 20;
• the IgG antibodies comprise IgG antibodies isolated from at least 1000 donors; at least 70% w/w of the IgG antibodies are IgGl antibodies; at least 90% of the donor subjects have been exposed to a virus; at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the IgG antibodies in the hsIgG preparation have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch; at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the IgG antibodies in the hsIgG preparation have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6
• hypersialylated IgG at least 60% (e.g., 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98% up to and including 100%) of branched glycans on the Fc region are di-sialylated (i.e., on both the ⁇ 1,3 branch and the ⁇ 1,6 arm) by way of NeuAc- ⁇ 2,6-Gal terminal linkages.
• less than 50% (e.g., less than 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) of branched glycans on the Fc region are mono-sialylated (i.e., sialylated only on the ⁇ 1,3 branch or only on the ⁇ 1,6 branch) by way of a NeuAc- ⁇ 2,6-Gal terminal linkage.
• the polypeptides are derived from plasma, e.g., human plasma.
• the polypeptides are overwhelmingly IgG polypeptides (e.g., IgG1, IgG2, IgG3 or IgG4 or mixtures thereol), although trace amounts of other of other immunoglobulin subclasses can be present.
• an antibody refers to a polypeptide that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or immunoglobulin variable domain sequence.
• an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as V L ).
• VH heavy chain variable region
• L light chain variable region
• an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
• antibody encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab, F(ab') 2 , Fd, Fv, and dAb fragments) as well as complete antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereol).
• the light chains of the immunoglobulin can be of types kappa or lambda.
• constant region refers to a polypeptide that corresponds to, or is derived from, one or more constant region immunoglobulin domains of an antibody.
• a constant region can include any or all of the following immunoglobulin domains: a CHI domain, a hinge region, a CH 2 domain, a CH 3 domain (derived from an IgA, IgD, IgG, IgE, or IgM), and a CH4 domain (derived from an IgE or IgM).
• Fc region refers to a dimer of two “Fc polypeptides,” each “Fc polypeptide” including the constant region of an antibody excluding the first constant region immunoglobulin domain.
• an “Fc region” includes two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers.
• Fc polypeptide refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include part or the entire flexible hinge N-terminal to these domains.
• Fc polypeptide comprises immunoglobulin domains Cgamma2 (C ⁇ 2) and Cgamma3 (C ⁇ 3) and the lower part of the hinge between Cgammal (C ⁇ 1) and C ⁇ 2.
• the human IgG heavy chain Fc polypeptide is usually defined to comprise residues starting P232, to its carboxyl-terminus, wherein the numbering is according to the EU system (Edelman et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)).
• Fc polypeptide comprises immunoglobulin domains Calpha2 (C ⁇ 2) and Calpha3 (C ⁇ 3) and the lower part of the hinge between Calphal (C ⁇ 1) and C ⁇ 2.
• An Fc region can be synthetic, recombinant, or generated from natural sources such as IVIg.
• glycocan is a sugar, which can be monomers or polymers of sugar residues, such as at least three sugars, and can be linear or branched.
• a “glycan” can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and/or modified sugars (e.g., 2'- fluororibose, 2'-deoxyribose, phosphomannose, 6'sulfo N-acetylglucosamine, etc.).
• natural sugar residues e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.
• glycan includes homo and heteropolymers of sugar residues.
• glycan also encompasses a glycan component of a gly coconjugate (e.g., of a polypeptide, gly colipid, proteoglycan, etc.).
• glycan component of a gly coconjugate e.g., of a polypeptide, gly colipid, proteoglycan, etc.
• free gly cans including gly cans that have been cleaved or otherwise released from a gly coconjugate.
• glycoprotein refers to a protein that contains a peptide backbone covalently linked to one or more sugar moieties (i.e., gly cans).
• the sugar moiety(ies) may be in the form of monosaccharides, disaccharides, oligosaccharides, and/or polysaccharides.
• the sugar moiety(ies) may comprise a single unbranched chain of sugar residues or may comprise one or more branched chains.
• Glycoproteins can contain O-linked sugar moieties and/or N-linked sugar moieties.
• IVIg is a preparation of pooled, polyvalent IgG, including all four IgG subgroups, extracted from plasma of at least 1,000 human donors. IVIg is approved as a plasma protein replacement therapy for immune deficient patients. The level of IVIg Fc glycan sialylation varies among IVIg preparations, but is generally less than 20%. The level of disialylation is generally far lower than 20%.
• the term “derived from IVIg” refers to polypeptides which result from manipulation of IVIg. For example, polypeptides purified from IVIg (e.g., enriched for sialylated IgGs or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).
• an “N-glycosylation site of an Fc polypeptide” refers to an amino acid residue within an Fc polypeptide to which a glycan is N-linked.
• an Fc region contains a dimer of Fc polypeptides, and the Fc region comprises two N-glycosylation sites, one on each Fc polypeptide.
• percent (%) of branched gly cans refers to the number of moles of glycan X relative to total moles of gly cans present, wherein X represents the glycan of interest.
• pharmaceutically effective amount refers to an amount (e.g., dose) effective in treating a patient, having a disorder or condition described herein. It is also to be understood herein that a “pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents.
• kits containing the preparation or product and instructions for use.
• “Pharmaceutical preparations” and “pharmaceutical products” generally refer to compositions in which the final predetermined level of sialylation has been achieved, and which are free of process impurities. To that end, “pharmaceutical preparations” and “pharmaceutical products” are substantially free of ST6Gall and/or sialic acid donor (e.g., cytidine 5'- monophospho-N-acetyl neuraminic acid) or the byproducts thereof (e.g., cytidine 5’- monophosphate).
• sialic acid donor e.g., cytidine 5'- monophospho-N-acetyl neuraminic acid
• the byproducts thereof e.g., cytidine 5’- monophosphate
• “Pharmaceutical preparations” and “pharmaceutical products” are generally substantially free of other components of a cell in which the glycoproteins were produced (e.g., the endoplasmic reticulum or cytoplasmic proteins and RNA), if recombinant.
• purified refers to a polynucleotide or a polypeptide that is removed or separated from other components present in its natural environment.
• an isolated polypeptide is one that is separated from other components of a cell in which it was produced (e.g., the endoplasmic reticulum or cytoplasmic proteins and RNA).
• An isolated polynucleotide is one that is separated from other nuclear components (e.g., histones) and/or from upstream or downstream nucleic acids.
• An isolated polynucleotide or polypeptide can be at least 60% free, or at least 75% free, or at least 90% free, or at least 95% free from other components present in natural environment of the indicated polynucleotide or polypeptide.
• sialylated refers to a glycan having a terminal sialic acid.
• mono-sialylated refers to branched glycans having one terminal sialic acid, e.g., on an ⁇ 1,3 branch or an ⁇ 1,6 branch.
• di-sialylated refers to a branched glycan having a terminal sialic acid on two arms, e.g., both an ⁇ 1,3 arm and an ⁇ 1,6 arm.
• FIG. 1 shows a short, branched core oligosaccharide comprising two N- acetylglucosamine and three mannose residues.
• One of the branches is referred to in the art as the “ ⁇ 1,3 arm,” and the second branch is referred to as the “ ⁇ 1,6 arm,”.
• Squares N- acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N- acetylneuraminic acid; triangles: fucose.
• FIG. 2 shows common Fc glycans present in IVIg.
• Squares N-acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N-acetylneuraminic acid; triangles: fucose.
• FIG. 3 shows how immunoglobulins, e.g., IgG antibodies, can be sialylated by carrying out a galactosylation step followed by a sialylation step.
• Squares N-acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N-acetylneuraminic acid; triangles: fucose.
• FIG. 4 shows the reaction product of a representative example of the IgG-Fc glycan profile for a reaction starting with IVIg.
• the left panel is a schematic representation of enzymatic sialylation reaction to transform IgG to hsIgG; the right panel is the IgG Fc glycan profile for the starting IVIg and hsIgG. Bars, from left to right, correspond to IgGl, IgG2/3, and IgG3/4, respectively.
• FIG. 5 shows an exemplary production process for hsIgG.
• FIG. 6 shows binding capacity of the PorosTM resin.
• FIG. 7 shows a prediction profiler for yield and residual ST6.
• FIG. 8 shows the operating range for a yield between 90- 95% and residual ST6 levels less than 30ppm.
• FIG. 9 is an SEC chromatogram of flow through.
• FIG. 10 is an SEC of eluate.
• FIG. 11 is an overlay of TAB, CaptoTM blue and CaptoTM Blue Sepharose 2 FF.
• FIG. 12 shows recovery of the three runs (bars, from left to right:TAB, CaptoTM Blue and Blue SepharoseTM FF) showing similar recovery.
• FIG. 13 shows chromatograms overlay of run 1 and 4.
• FIG. 14 shows residual ST6 of the 4 runs that demonstrates a better clearance by CaptoTM Blue HS (runs 3 and 4) versus CaptoTM Blue (Runs 1 and 2).
• FIG. 15 shows a comparison of residual ST6 of CaptoTM Blue HS and TAB showing a better clearance of the CaptoTM Blue HS.
• FIG. 16 shows analytical SEC of the IVIg load, FT, and eluate.
• FIG. 17 shows mass Spectrometry of CaptoTM Blue HS IVIg flow through and eluate against the starting IVIg. Bars, from left to right: IgGl, IgG2, IgG3, IgG4.
• FIG. 18 shows the impact of 5X PBS and MOPS at pH 4.5 buffer on residual ST6 (run 1).
• X axis mg IVIg/mL resin.
• FIG. 19 shows the impact of Glycine pH 4.5 on residual ST6 (Run2).
• X axis mg IVIg/mL resin.
• FIG. 20 shows the impact of Glycine +100mM NaCl at pH 4.5 on residual ST6 (Run2).
• FIG. 21 shows the amount of loading (grams of M254/ml column volume) impact on the clearance of ST6 by TAB Column.
• Orange and blue dots represent residual level of ST6 for the TAB column flow-through fractions at different loading amounts of CEX load and CEX eluate respectively. Achieved similar level of residual ST6 for the TAB column flow-through fractions with both the loads (CEX load and CEX eluate) at current manufacturing scale maximum loading 1.1g/ml (1X).
• FIG. 22 is an overlay of the analytical SEC of IVIg and precipitated IVIg showing no change on the aggregate level.
• FIG. 23 is an analytical SEC profile of the crude reaction versus the precipitated material showing a significant reduction of nucleotides.
• Antibodies are glycosylated at conserved positions in the constant regions of their heavy chain and on the Fab domain.
• human IgG antibodies have a single N-linked glycosylation site at Asn297 (EU Numbering) of the CH2 domain.
• Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions.
• the core oligosaccharide normally consists of GlcNAc 2 Man 3 GlcNAc, with differing numbers of outer residues.
• Variation among individual IgG’s can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).
• the present disclosure encompasses, in part, methods for preparing immunoglobulins (e.g., human IgG) having an Fc region having particular levels of branched glycans that are sialylated on both of the arms of the branched glycan (e.g., with aNeuAc- ⁇ 2,6-Gal terminal linkage).
• the levels can be measured on an individual Fc region (e.g., the number of branched glycans that are sialylated on an ⁇ 1,3 arm, an ⁇ 1,6 arm, or both, of the branched glycans in the Fc region), or on the overall composition of a preparation of polypeptides (e.g., the number or percentage of branched glycans that are sialylated on an ⁇ 1,3 arm, an ⁇ 1,6 arm, or both, of the branched glycans in the Fc region in a preparation of polypeptides).
• an individual Fc region e.g., the number of branched glycans that are sialylated on an ⁇ 1,3 arm, an ⁇ 1,6 arm, or both, of the branched glycans in the Fc region
• the overall composition of a preparation of polypeptides e.g., the number or percentage of branched glycans that are sialy
• Naturally derived polypeptides that can be used to prepare hypersialylated IgG include, for example, IgG in human serum (particular human serum pooled from more than 1,000 donors), intravenous immunoglobulin (IVIg) and polypeptides derived from IVIg (e.g., polypeptides purified from IVIg (e.g., enriched for sialylated IgGs) or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).
• human serum particular human serum pooled from more than 1,000 donors
• IVIg intravenous immunoglobulin
• polypeptides derived from IVIg e.g., polypeptides purified from IVIg (e.g., enriched for sialylated IgGs) or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).
• N-linked oligosaccharide chains are added to a protein in the lumen of the endoplasmic reticulum.
• an initial oligosaccharide typically 14-sugar
• an asparagine residue contained within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline.
• the structure of this initial oligosaccharide is common to most eukaryotes, and contains three glucose, nine mannose, and two N-acetylglucosamine residues.
• This initial oligosaccharide chain can be trimmed by specific glycosidase enzymes in the endoplasmic reticulum, resulting in a short, branched core oligosaccharide composed of two N-acetylglucosamine and three mannose residues.
• One of the branches is referred to in the art as the “ ⁇ 1,3 arm,” and the second branch is referred to as the “ ⁇ 1,6 arm,” as shown in FIG. 1.
• N-glycans can be subdivided into three distinct groups called “high mannose type,” “hybrid type,” and “complex type,” with a common pentasaccharide core (Man ( ⁇ 1,6)- (Man( ⁇ 1.3))-Mhh( ⁇ 1,4)-GlcpNAc( ⁇ 1,4)-GlcpNAc( ⁇ 1,N)-Asn) occurring in all three groups.
• one or more monosaccharides units of N-acetylglucosamine may be added to the core mannose subunits to form a “complex glycan.”
• Galactose may be added to the N-acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains that terminate with any of a sialic acid, a galactose or an N-acetylglucosamine residue.
• a fucose residue may be added to an N- acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by specific glycosyl transferases.
• Hybrid glycans comprise characteristics of both high-mannose and complex glycans.
• one branch of a hybrid glycan may comprise primarily or exclusively mannose residues, while another branch may comprise N-acetylglucosamine, sialic acid, galactose, and/or fucose sugars.
• Sialic acids are a family of 9-carbon monosaccharides with heterocyclic ring structures. They bear a negative charge via a carboxylic acid group attached to the ring as well as other chemical decorations including N-acetyl and N-glycolyl groups.
• the two main types of sialic acid residues found in polypeptides produced in mammalian expression systems are N-acetyl- neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). These usually occur as terminal structures attached to galactose (Gal) residues at the non-reducing termini of both N- and O-linked glycans.
• the glycosidic linkage configurations for these sialic acid groups can be either ⁇ 2,3 or ⁇ 2,6.
• Fc regions are glycosylated at conserved, N-linked glycosylation sites.
• each heavy chain of an IgG antibody has a single N-linked glycosylation site at Asn297 of the CH2 domain.
• IgA antibodies have N-linked glycosylation sites within the CH2 and CH3 domains
• IgE antibodies have N-linked glycosylation sites within the CH3 domain
• IgM antibodies have N-linked glycosylation sites within the CH1, CH2, CH3, and CH4 domains.
• Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions.
• IgG has a single N-linked biantennary carbohydrate at Asn297 of the CH2 domain in each Fc polypeptide of the Fc region, which also contains the binding sites for Clq and Fc ⁇ R.
• the core oligosaccharide normally consists of GlcNAc2Man3GlcNAc, with differing numbers of outer residues. Variation among individual IgG can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).
• Immunoglobulins e.g., IgG antibodies
• Beta-l,4-galactosyltransferase 1 (B4GalT) is a Type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5’- diphosphosegalactose ([[ (2R,3S,4R,5R)-5-(2.4-dioxopyrimidin- 1 -yl)-3.4-dihydroxyoxolan-2- yl]methoxy-hydroxyphosphoryl] [(2R,3R,4S,5R.6R)-3.4.5-trihydroxy-6-(hydroxymethyl)oxan-2- yl] hydrogen phosphate; UDP-Gal) to GlcNAc as a ⁇ -1,4 linkage.
• Alpha-2, 6-sialyltransferase 1 is a Type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5’-monophospho-N-acetylneuraminicacid ((2R,4S,5R,6R)-5-acetamido-2-[[(2R,3S,4R,5R)-5-(4- amino-2-oxopyrimidin-l-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-4- hydroxy-6-(l,2,3-trihydroxypropyl)oxane-2-carboxylic acid; CMP-NANA or CMP-Sialic Acid) to Gal as an a-2,6 linkage.
• the reactions proceed shown in FIG. 3.
• Glycans of polypeptides can be evaluated using any methods known in the art. For example, sialylation of glycan compositions (e.g., level of branched glycans that are sialylated on an ⁇ 1,3 branch and/or an ⁇ 1,6 branch) can be characterized using methods described in WO2014/179601.
• At least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans on the Fc domain have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• At least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• the hsIgG compositions prepared by the methods described herein comprises at least 50%, 55%, 60%, 65%, 70% or 75% of the branched glycans on the Fc domain have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm.
• Beta-l,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galtl, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of beta- 1, 4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galtl, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same are suitable for use in the methods described herein.
• B4Galtl is one of seven beta- 1,4- galactosyltransferase (beta4GalT) genes that each encode type II membrane-bound glycoproteins that appear to have exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a beta 1,4 linkage to similar acceptor sugars: GlcNAc, Glc, and Xyl.
• B4Galtl adds galactose to N-acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains.
• B4GalTl is also called GGTB2.
• Four alternative transcripts encoding four isoforms of B4GALT1 (NCBI Gene ID 2683) are described in Table 1. Table 1. Human B4GALT1 isoforms
• the soluble form of B4GalTl derives from the membrane form by proteolytic processing.
• the cleavage site is at positions 77-78 of B4GALT1 isoform 1 (SEQ ID NO: 5).
• one or more of the amino acids of the B4GalTl corresponding to amino acids 113, 130, 172, 243, 250, 262, 310, 343, or 355 of B4GALT1 isoform 1 (SEQ ID NO: 5) is conserved as compared to (SEQ ID NO: 5).
• the enzyme is an enzymatically active portion of, e.g., B4GalTl. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 1 (SEQ ID NO: 5), or an ortholog, mutant, or variant of SEQ ID NO: 5. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 2 (SEQ ID NO: 6), or an ortholog, mutant, or variant of SEQ ID NO: 6. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 3 (SEQ ID NO: 7), or an ortholog, mutant, or variant of SEQ ID NO: 7. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 4 (SEQ ID NO: 8), or an ortholog, mutant, or variant of SEQ ID NO: 8
• the enzymatically active portion of B4GalTl does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 9. In some embodiments, the enzymatically active portion of B4GalTl does not comprise a transmembrane domain, e.g., SEQ ID NO: 10. In some embodiments, the enzymatically active portion of B4GalTl does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 9 or a transmembrane domain, e.g., SEQ ID NO: 10.
• the enzymatically active portion of B4GalTl comprises all or a portion of a luminal domain, e.g., SEQ ID NO: 11, or an ortholog, mutants, or variants thereof.
• the enzymatically active portion of B4GalTl comprises amino acids 109-398 of SEQ ID NO: 5, or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of B4GalTl consists of SEQ ID NO: 5, or an ortholog, mutant, or variant of SEQ ID NO: 5.
• a suitable functional portion of an B4GalTl can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 12.
• amino acid sequence that comprises or consists of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 13.
• ST6Gall e.g., ST6Gall, e.g., human ST6Gall, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of ST6Gall, e.g., human ST6Gall, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same, are suitable for use in the methods described herein.
• Alpha-2, 6-sialyltransferase 1 (ST6) is a Type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5’-monophospho-N-acetylneuraminic acid (CMP-NANA) to Gal as an a-2,6 linkage.
• ST6Gall is also called as ST6N or SIAT1.
• Four alternative transcripts encoding two isoforms of ST6GAL1 (NCBI Gene ID 6480) are described in Table 5. Table 5.
• the soluble form of ST6Gall derives from the membrane form by proteolytic processing.
• one or more of the amino acids of the ST6Gall corresponding to amino acids 142, 149, 161, 184, 189, 212, 233, 335, 353, 354, 364, 365, 369, 370, 376, or 406 of ST6Gall isoform a is conserved as compared to SEQ ID NO: 14.
• an enzymatically active portion of, e.g., ST6Gall is an enzymatically active portion of STG6Gall isoform a (SEQ ID NO: 14), or an ortholog, mutant, or variant of SEQ ID NO: 14. In some embodiments, the enzyme is an enzymatically active portion of STG6Gall isoform b (SEQ ID NO: 15), or an ortholog, mutant, or variant of SEQ ID NO: 15.
• the enzymatically active portion of ST6Gall does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 16. In some embodiments, the enzymatically active portion of ST6Gall does not comprise a transmembrane domain, e.g., SEQ ID NO: 17. In some embodiments, the enzymatically active portion of ST6Gall does not comprise a cytoplasmic domain, e.g., SEQ ID NO: 16 or a transmembrane domain, e.g., SEQ ID NO: 17.
• the enzymatically active portion of ST6Gall comprises all or a portion of a luminal domain, e.g., SEQ ID NO: 18, or an ortholog, mutants, or variants thereof.
• the enzymatically active portion of ST6Gall comprises amino acids 87-406 of SEQ ID NO: 14 (SEQ ID NO: 19), or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of ST6Gall consists of SEQ ID NO: 19, or an ortholog, mutant, or variant of SEQ ID NO: 19.
• a suitable functional portion of an ST6Gall can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 19.
• the ST6Gall comprises or consists of SEQ ID NO: 19, the portion of SEQ ID NO: 19 from amino acid 4 to 320, or the portion of SEQ ID NO: 19 from amino acid 5 to 320.
• amino acid sequence that comprises or consists of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO: 20.
• the methods described herein can include galactosylation and sialylation of antibodies.
• Suitable antibodies include, for example, IgG antibodies.
• the antibodies, e.g., IgG antibodies, can be pooled.
• pooled IgG antibodies include IVIg.
• the IgG antibodies comprise IgG antibodies isolated from at least 1000 donors.
• At least 50%, 55%, 60%, 65% or 70% w/w of the IgG antibodies are IgGl antibodies.
• At least 90% of the donor subject has been exposed to a virus.
• the methods described herein include providing a mixture of IgG antibodies.
• providing a mixture of IgG antibodies includes (a) providing pooled plasma from at least 1000 human subjects; and (b) isolating a mixture of IgG antibodies from the pooled plasma.
• the mixture of IgG antibodies are isolated from intravenous immunoglobulin.
• the mixture of IgG antibodies are intravenous immunoglobulin.
• the step of isolating a mixture of IgG antibodies from the pooled plasma comprises ethanol precipitation or caprylic acid (also called octanoic acid) precipitation.
• the step of isolating a mixture of IgG antibodies from the pooled plasma comprises biding IgG antibodies to an ion exchange column and eluting the IgG antibodies from an ion exchange column.
• the methods described herein can comprise a galactosylation step.
• An exemplary galactosylation reaction is depicted in LIG. 3.
• a method for galactosylating antibod(ies), e.g., antibod(ies) described herein by providing a composition (a galactosylation mixture) comprising: antibod(ies), e.g., antibod(ies) described herein; a galactosylating enzyme, e.g., a galactosylating enzyme described herein, e.g., B4GalT or enzymatically active portion of variant thereof; UDP-gal or salt thereof; and incubating the composition under conditions effective for galactosylating the antibody, e.g., as described herein, thereby producing galactosylated antibod(ies).
• a galactosylation mixture comprising: antibod(ies), e.g., antibod(ies) described herein; a galact
• the methods described herein can comprise a sialylation step.
• An exemplary sialylation reaction is depicted in LIG. 3.
• a method for sialylating e.g., hyper- sialylating, antibod(ies), e.g., antibod(ies) described herein, by providing a composition (a sialylation reaction mixture) comprising: galactosylated antibod(ies), e.g., as described herein; a sialylating enzyme, e.g., a sialylating enzyme described herein, e.g., ST6Gall or enzymatically active portion or variant thereof; CMP-NANA or a salt thereof; and incubating the composition under conditions effective for sialylating the antibod(ies), e.g., as described herein.
• a sialylation reaction mixture comprising: galactosylated antibod(ies), e.g., as described herein; a sialylating
• the galactosylation step and the sialylation step are carried out sequentially in the same reaction mixture, that is, the galactosylation reaction mixture becomes the sialylation reaction mixture upon addition of the sialylating enzyme and CMP-NANA or salt thereof.
• there galactosylation reaction mixture is not filtered, fractionated, or purified prior to the sialylation step.
• the galactosylation step and the sialylation step are carried out separately, e.g, pre-galactosylated antibod(ies) are provided, though they may have been processed (e.g., filtered, fractionated, or purified) and/or stored prior to the sialylation step.
• the methods described herein can also comprise a sequential galactosylation and sialylation step.
• An exemplary galactosylation and sialylation reaction is depicted in FIG. 3.
• a method for galactosylating and sialylating e.g., hyper-sialylating, antibod(ies), e.g., antibod(ies) described herein, by a) providing a composition (a galactosylation reaction mixture) comprising: antibod(ies), e.g., as described herein; a galactosylating enzyme, e.g., a galactosylating enzyme described herein, e.g., B4GalT or enzymatically active portion or variant thereof; UDP-gal or a salt thereof; and b) incubating the composition under conditions effective for galactosylating the antibod(ies), e.g., as described herein; c) adding a sia
• the galactosylation reaction mixture and/or the sialylation reaction mixture comprises Bis (2 -hydroxy ethyl) aminotris (hydroxymethyl)methane (BIS-TRIS) buffer. In some embodiments, the galactosylation reaction mixture and/or the sialylation reaction mixture comprises MnCl 2 .
• one or more component(s) of one or more of the reaction mixture(s) are supplemented during the incubation. That is, the reaction mixture may comprise an amount of the component at the beginning of the reaction (which may change during the course of the reaction), but also be supplemented with additional amounts of the component(s) during the reaction.
• the B4GalT comprises or consists of an amino acid sequence is at least 90% identical SEQ ID NO: 12 or SEQ ID NO: 13.
• the ST6Gall comprises or consists of an amino acid sequence that is at least 90% identical SEQ ID NO: 19 or SEQ ID NO: 20.
• At least or about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched Fc glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• about or at least 80% of the branched Fc glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• about or at least 85% of the of the branched Fc glycans on the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the sialylated antibod(ies), e.g., hsIgG have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through a NeuAc- ⁇ 2,6-Gal terminal linkage.
• about or at least 90% of the of the branched Fc glycans on the sialylated antibod(ies), e.g., hsIgG have a sialic acid on both the ⁇ 1,3 branch and the ⁇ 1,6 branch.
• about or at least 60%, 65%, 70% of the branched glycans on the Fab domain of the sialylated antibod(ies), e.g., hsIgG, have a sialic acid on both the ⁇ 1,3 arm and the ⁇ 1,6 arm that is connected through aNeuAc- ⁇ 2,6-Gal terminal linkage.
• IgG e.g., pooled IgG, e.g., IVIg
• sialylated IgG e.g., sialylated pooled IgG, e.g., sialylated IVIg, e.g., hsIgG
• FIG. 5 An exemplary manufacturing process for isolation and purification of hsIgG is shown in FIG. 5. Variations of that process are described herein.
• the methods for purifying antibod(ies) comprises analyzing the amount of one or more IgG subclasses after purification, e.g., after a chromatography step.
• the methods described herein can include quenching of an enzymatic reaction, e.g., an enzymatic sialylation reaction, e.g., as described herein.
• quenching comprises mixing the composition comprising sialylated antibod(ies), e.g., sialylation reaction mixture, e.g., as described herein, with a buffer, e.g., phosphate buffered saline (PBS), e.g., 5X PBS, thereby producing a quenched antibody composition.
• a buffer e.g., phosphate buffered saline (PBS), e.g., 5X PBS
• quenching comprises mixing the composition and the buffer at or at about 1:1.
• quenching comprises mixing the antibody composition and the buffer at 1 volume buffer or more to one volume antibody composition.
• Depth filtration is well known in the art and described, e.g., in Sutherland, “Filtration Overview: A Closer Look at Depth Filtration,” Filtration & Separation 45(8):25-28 (2008); 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).
• the methods described herein include one or more depth filtration steps.
• the method includes one or more depth filtration steps before a chromatography step.
• the method includes one or more depth filtration steps after a chromatography step.
• the method does not include a depth filtration step prior to chromatography.
• the method does not include a depth filtration step after chromatography.
• the method does not include a depth filtration step.
• the methods described herein can include buffer exchange.
• Methods for buffer exchange are well known in the art and described, e.g., in Kumik, “Buffer Exchange Using Size Exclusion Chromatography, Countercurrent Dialysis, and Tangential Flow Filtration: Models, Development, and Industrial Application,” Biotechnology & Bioengineering 45(2): 149-58 (1995); Dizon-Maspat et al., “Single Pass Tangential Flow Filtration to Debottleneck Downstream Processing for Therapeutic Antibody Production,” Biotechnology &
• the buffer exchange method is size exclusion chromatography (SEC). In some embodiments, the buffer exchange method is tangential flow filtration (TFF).
• the buffer exchange method is countercurrent dialysis (CCD).
• the buffer exchange method is tangential flow filtration (TFF).
• the methods described herein include one or more buffer exchange steps.
• the method includes one or more buffer exchange steps before a chromatography step.
• the method includes one or more buffer exchange steps after a chromatography step.
• the method does not include a buffer exchange step prior to chromatography.
• the method does not include a buffer exchange step after chromatography.
• the method does not include a buffer exchange step.
• the methods described herein can include viral inactivation or viral removal.
• Methods for viral inactivation and removal are well known in the art and described, e.g., in Horowitz et al., “Strategies for Viral Inactivation,” Current Opinion in Hematology 2(6):484-92 (1995); Klutz et al., “Continuous Viral Inactivation at low pH Value in Antibody Manufacturing,” Chemical Engineering and Processing: Process Intensification 102:88-11 (2016).
• the viral inactivation method is selected from solvent/detergent inactivation, low pH inactivation, pasteurization, microwave heating, irradiation, high-energy light, and combinations thereof.
• the viral removal method is selected from affinity chromatography, nanofiltration, and combinations thereof.
• the methods described herein include one or more viral inactivation or viral removal steps.
• the viral inactivation or viral removal step is carried out after galactosylation and sialylation.
• the viral inactivation or viral removal step is carried out after galactosylation and sialylation but before chromatography.
• the viral inactivation and removal includes viral inactivation followed by nanofiltration.
• the method comprises viral removal (e.g., by nanofiltration), but does not include a separate viral inactivation step.
• the methods described herein can include nanofiltration.
• Membranes and methods for nanofiltration are well known in the art and described, e.g., in Mohammad et al., “Nanofiltration Membranes Review: Recent Advances and Future Prospects,” Desalination 356:226-54 (2015).
• the methods described herein include one or more nanofiltration steps.
• the method includes one or more nanofiltration steps before a chromatography step.
• the method includes one or more nanofiltration steps after a chromatography step.
• the method does not include a nanofiltration step prior to chromatography.
• the method does not include a nanofiltration step after chromatography.
• the method does not include a nanofiltration step.
• the methods described herein e.g., methods for producing and/or purifying antibod(ies), e.g., IgG, e.g., pooled IgG, e.g., IVIg, e.g., e.g., sialylated IgG, e.g., sialylated pooled IgG, e.g., sialylated IVIg, e.g., hsIgG, can include chromatography as part of the purification process.
• antibod(ies) e.g., IgG, e.g., pooled IgG, e.g., IVIg, e.g., sialylated IgG, e.g., sialylated pooled IgG, e.g., sialylated IVIg, e.g., hsIgG
• the antibod(ies) bind to the chromatography resin. In some embodiments, the antibod(ies) flow through the chromatography resin.
• the impurity(ies) e.g., one or more of the other components of the enzymatic reaction mixture(s), e.g., galactosylation reaction mixture(s) and/or sialylation reaction mixture(s), including, but not limited to, enzymes, e.g., B4GalT and/or ST6, nucleotide sugars, and MnCl 2
• the impurities flow through the chromatography resin.
• both the antibod(ies) and the impurity(ies) bind to the chromatography resin.
• the antibod(ies) and impurity(ies) can be separately eluted.
• the methods described herein e.g., methods for producing and/or purifying antibod(ies), e.g., IgG, e.g., pooled IgG, e.g., IVIg, e.g., e.g., sialylated IgG, e.g., sialylated pooled IgG, e.g., sialylated IVIg, e.g., hsIgG, can include an affinity chromatography step in which the antibod(ies) bind to the ligand of the chromatography resin, e.g., a Protein A affinity chromatography step.
• an affinity chromatography step in which the antibod(ies) bind to the ligand of the chromatography resin, e.g., a Protein A affinity chromatography step.
• Protein A affinity chromatography Materials and methods for Protein A affinity chromatography are well known in the art and described, e.g., 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.
• the Protein A affinity chromatography resin is MabSelectTM PrismA (GE Healthcare).
• the Protein A affinity chromatography resin ligand is selected from the group consisting of Protein A, a Protein A derivative, a Protein A mimetic, or a combination thereof.
• the methods describe herein comprise Protein A Affinity Chromatography followed by Blue Dye Chromatography, e.g., as described herein. Cation Exchange Chromatography
• the methods described herein e.g., methods for producing and/or purifying antibod(ies), e.g., IgG, e.g., pooled IgG, e.g., IVIg, e.g., e.g., sialylated IgG, e.g., sialylated pooled IgG, e.g., sialylated IVIg, e.g., hsIgG, can include a cation exchange chromatography (CEX) step, e.g., a strong cation exchange chromatography step using a strong cation exchange chromatography resin.
• CEX cation exchange chromatography
• the strong cation exchange resin is selected from the group consisting of PorosTM XS (e.g., Cytiva 4404336), SP Sepharose High Performance (SP HP) (e.g., Cytiva 28950515), CaptoTM SP Impres (e.g., Cytiva 17546815), CaptoTM S (e.g., Cytiva 28926979), CaptoTM S Impact (e.g., Cytiva 1737174), and combinations thereof.
• PorosTM XS e.g., Cytiva 4404336
• SP HP SP Sepharose High Performance
• CaptoTM SP Impres e.g., Cytiva 17546815
• CaptoTM S e.g., Cytiva 28926979
• CaptoTM S Impact e.g., Cytiva 1737174
• the PorosTM strong cation exchange (CEX) resins are 50- ⁇ m, rigid, polymeric, ion- exchange chromatography resins.
• a polyhydroxyl surface coating provides low non-specific binding and surface functionalization with sulphopropyl yields a strong cation-exchanger ionizable with pH 1-14.
• the support matrix is cross-linked polystyrene-divinylbenzene and the surface functionality is sulfopropyl (-CH 2 CH 2 CH 2 SO 3 -) ⁇ Id.
• the dynamic binding capacity of PorosTM XS is > 102 mg/mL (5% breakthrough of Polyclonal Human IgG in 20 mM MES ⁇ 40 mM NaCl, pH 5.0 at 300 cm/hour in 0.46 cmD x 20 cmL column). Id. The dynamic binding capacity of PorosTM 50 HS 57.0-75.3 mg/mL (5% breakthrough with Lysozyme in 20 mM MES, pH 6.2 at 100 cm/hour in 0.46 cmD x 20 cmL column). Id.
• SP Sepharose High Performance (SP HP) resin is based on a 6% cross-linked agarose matrix with a particle size of 24-44 ⁇ m (d 50v ⁇ 34 ⁇ m) that uses a sulphopropyl ligand (-CH 2 CH 2 CH 2 SO 3 -) ⁇ See Product Bulleting “SP Sepharose High Performance”, cytiva.com. It has an operational pH stability of 4-13. Id. It has a dynamic binding capacity of ⁇ 55 ribonuclease A/mL resin. Id.
• CaptoTM SP ImpRes is based on a high-flow agarose base matrix with a bead size of about 40 ⁇ m.
• the functional group of CaptoTM SP Impres is -CH 2 CH 2 CH 2 SO 3 -. Id.
• the working operating pH stability is 4 to 12.
• the binding capacity (10% breakthrough measured at a residence time of 4 min (150 cm/h) in a TricomTM 5/100 column with 10 cm bed height; 20 mM sodium phosphate, pH 7.2 (lysozyme) and 50 mM Tris, pH 8.0 (BSA)) is > 70 mg lysozyme/mL medium and > 95 mg BSA/mL medium.
• CaptoTM S is a strong cation exchange resin based on a highly cross-linked agarose, spherical matrix with a median particle size of the cumulative volume distribution of about 90 ⁇ m that uses the -SO3- charged group shown below
• CaptoTM S Impact is based on a high-flow agarose base matrix with a median particle size of the cumulative volume distribution of about 50 ⁇ m that uses a -SO3- ligand. See CaptoTM S ImpAct product sheet, cytiva.com. It has a binding capacity of >85 mg BSA/mL resin, >90 mg lysozyme/mL resin, >100 mg IgG/ mL resin. Id.
• the strong cation exchange resin has a binding capacity of 50 mg/mL or more, e.g., 75 mg/mL or more, e.g., 100 mg/mL or more, measured as 5% breakthrough of Polyclonal Human IgG in 20 mM MES ⁇ 40 mM NaCl, pH 5.0 at 300 cm/hour in 0.46 cmD x 20 cmL column.
• the strong cation exchange resin maintains binding capacity of 50 mg/mL or more, e.g., 75 mg/mL or more, e.g., 100 mg/mL or more, measured as 5% breakthrough of Polyclonal Human IgG in 20 mM MES ⁇ 40 mM NaCl, pH 5.0 at 300 cm/hour in 0.46 cmD x 20 cmL column, at up to 150 mM NaCl (15 mS/cm).
• the functional group of the strong cation exchange resin comprises SO3-. In some embodiments, the functional group of the strong cation exchange resin is -CH 2 CH 2 CH 2 SO 3 -.
• the cation exchange chromatography is carried out in flow through mode. In some embodiments, the cation exchange chromatography is carried out in bind and elute mode.
• the antibod(ies), e.g., sialylated antibod(ies), e.g., hsIgG flow through the cation exchange chromatography resin. In some embodiments, the antibod(ies), e.g., sialylated antibod(ies), e.g., hsIgG, are bound and then eluted from the cation exchange chromatography resin.
• a composition comprising sialylated antibod(ies), e.g., hsIgG, and one or more impurities is loaded on to the CEX column.
• the composition is a sialylation reaction mixture, e.g., a sialylation mixture comprising sialylated antibod(ies), e.g., as described herein, is loaded on to the CEX column.
• from or from about 10 to or to about 110 mg antibod(ies), per mL resin are loaded on to the CEX column.
• the methods described herein include one or more of: (a) a quenching step, e.g., as described herein, (b) a depth filtration step, e.g., as described herein (c) a buffer exchange step, e.g., as described herein, (d) a virus inactivation or removal step, e.g., as described herein, and (e) a nanofiltration step, e.g., as described herein, before CEX chromatography.
• a quenching step e.g., as described herein
• a depth filtration step e.g., as described herein
• a buffer exchange step e.g., as described herein
• a virus inactivation or removal step e.g., as described herein
• a nanofiltration step e.g., as described herein, before CEX chromatography.
• the methods described herein comprise one or more of: (a) blue dye chromatography, e.g., as described herein, (b) DE pad filtration, e.g., as described herein, (c) buffer exchange, e.g., as described herein, and (d) PS20 Spiking & Filtration, e.g., as described herein, after CEX chromatography.
• a method for purifying sialylated antibod(ies) which includes, in order from (a) to (d): (a) providing a composition comprising sialylated antibod(ies), e.g., hsIgG and galactosylation and/or sialylation enzyme(s), e.g., B4GalT and/or ST6 or enzymatically active portion thereof, e.g., as described herein, and, for example, if it is a sialylation reaction mixture, e.g., as described herein, optionally quenching the reaction, e.g., as described herein, e.g., in PBS, e.g., in 5X PBS at a 1:1 dilution; (b) diluting the composition, e.g., with citrate buffer (e.g., 6.757 g/L sodium citrate dehydrate and 5.192 g/L citric acid buffer), e.g., citrate buffer (e
• the method does not contain any additional filtering, fractionation, or purification steps other than (a) and (b) before carrying out step (c).
• the method comprises a single depth filtering step, in addition to steps (a) and (b) before step (c), e.g., between steps (a) and (b), but does not comprise any additional filtering, fractionation, or purification steps beyond the single depth filtration step before carrying out step (c).
• the method comprises a viral inactivation step before step (c).
• step (c) is spaced out in time, e.g., a portion of the buffered antibody composition is applied to the column over more than one application, e.g., over 1, 2, or 3 applications.
• step (e) is repeated one or more times.
• step (c) is carried out at resident time(s) of from or from about 1, 2, 3, 4, 5, or 6 minutes.
• selectively eluting the sialylated antibod(ies) comprises eluting in a buffer comprising 400 mM or more NaCl, e.g., a citrate buffer at or at about 50 mM, 400 mM or more NaCl, at or at about pH 4.5.
• the method further comprises, following step (e), one or more of: (f) a blue dye chromatography step, e.g., a Trisacyl Blue (TAB) chromatography, e.g., as described herein, (g) a DE Pad filtration step, e.g., as described herein, (h) a depth filtration step, e.g., as described herein; and (g) a PS20 spiking & filtration step, e.g., as described herein.
• a blue dye chromatography step e.g., a Trisacyl Blue (TAB) chromatography
• TAB Trisacyl Blue
• this method produces yields (antibody recovery rate) of or of about 80%, 85%, 90%, or 95% or more with sialylating enzyme, e.g., ST6 or enzymatically active portion thereof, present at 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, or 30 ppm or less.
• sialylating enzyme e.g., ST6 or enzymatically active portion thereof
• the methods described herein e.g., methods for producing and/or purifying antibod(ies), e.g., IgG, e.g., pooled IgG, e.g., IVIg, e.g., e.g., sialylated IgG, e.g., sialylated pooled IgG, e.g., sialylated IVIg, e.g., hsIgG, can include blue dye affinity chromatography as part of the purification process, e.g., an affinity chromatography step using a blue dye resin.
• the blue dye resin is selected from the group consisting of Blue TrisacrylTM (e.g., Sartorius 25896-010), CaptoTM Blue (e.g., Cytiva 17544801), CaptoTM Blue HS, (e.g., Cytiva 17545202), Blue SepharoseTM (e.g., Cytiva 17-0948-01), and combinations thereof.
• Blue TrisacrylTM e.g., Sartorius 25896-010
• CaptoTM Blue e.g., Cytiva 17544801
• CaptoTM Blue HS e.g., Cytiva 17545202
• Blue SepharoseTM e.g., Cytiva 17-0948-01
• the Blue TrisacrylTM resin is based on a synthetic polymer matrix with a bead size of 60 ⁇ m that uses a blue dye ligand as the functional group. See Product Specifications, Sartorius 25896-010, Sartorius.com. It has an operational pH of 4-10. Id.
• CaptoTM Blue resins are based on a highly cross-linked agarose matrix with an average particle size of 75 ⁇ m. See Product Bulletin, “Capto Blue and Capto Blue (high sub) Affinity Chromatography,” cytiva.com. They use a CibacronTM Blue (1-amino-4-[4-[[4-chloro- 6-(2-sulfoanilino)-1,3,5-triazin-2-yl]amino]-3-sulfoanilino]-9,10-dioxoanthracene-2-sulfonic acid) ligand. Id; PubChem ID 172469. They have an operation pH of 3-13. CaptoTM Blue has a dynamic binding capacity of HSA at 10% breakthrough of 24 mg/mL at 4 min residence time and CaptoTM Blue HS has a dynamic binding capacity of HSA at 10% breakthrough of 30 mg/mL at 4 min residence time. Id.
• Blue SepharoseTM is based on a highly cross-linked agarose base matrix (6% spherical) with a median particle size of the cumulative volume distribution of ⁇ 90 ⁇ m. See Product Bulletin, “Blue Sepharose 6 Fast Flow Affinity Chromatography,” cytiva.com. It uses a CibacronTM Blue 3G (l-amino-4-[4-[[4-chloro-6-(3-sulfoanilino)-1,3,5-triazin-2-yl]amino]-3- sulfoanilino]-9,10-dioxoanthracene-2-sulfonic acid).
• the blue dye affinity chromatography is carried out in flow through mode.
• the antibod(ies) e.g., sialylated antibod(ies), e.g., hsIgG, flow through the blue dye chromatography resin.
• a composition comprising sialylated antibod(ies), e.g., hsIgG, and one or more impurities is loaded on to the blue dye column.
• the composition is a sialylation reaction mixture, e.g., a sialylation mixture comprising sialylated antibod(ies), e.g., as described herein, is loaded on to the blue dye column.
• from or from about 10 to or to about 110 mg antibod(ies), per mL resin are loaded on to the blue dye column.
• from or from about 10 to or to about 110 centigrams antibod(ies), per mL resin are loaded on to the blue dye column.
• the methods described herein include one or more of: (a) a quenching step, e.g., as described herein, (b) a depth filtration step, e.g., as described herein (c) a buffer exchange step, e.g., as described herein, (d) a virus inactivation or removal step, e.g., as described herein, and (e) a nanofiltration step, e.g., as described herein, or (f) a CEX chromatography step, e.g., as described herein, before blue dye chromatography.
• the method does not comprise a CEX chromatography step.
• the methods described herein comprise one or more of: (a) DE pad filtration, e.g., as described herein, (b) buffer exchange, e.g., as described herein, and (c) PS20 spiking & filtration, e.g., as described herein, after blue dye chromatography.
• a method for purifying sialylated antibod(ies) which includes, in order from (a) to (c): (a) providing a composition comprising sialylated antibod(ies), e.g., hsIgG and galactosylation and/or sialylation enzyme(s), e.g., B4GalT and/or ST6 or enzymatically active portion thereof, e.g., as described herein, and, for example, if it is a sialylation reaction mixture, e.g., as described herein, optionally quenching the reaction, e.g., as described herein, e.g., in PBS, e.g., in 5X PBS at a 1:1 dilution; (b) diluting the composition in a buffer suitable for use with the column, e.g., for a TAB column with citrate buffer, e.g., citrate buffer at or at about 100 mM, at or
• the method does not contain any additional filtering, fractionation, or purification steps other than (a) and (b) before carrying out step (c).
• the method comprises a single depth filtering step, in addition to steps (a) and (b) before step (c), e.g., between steps (a) and (b), but does not comprise any additional filtering, fractionation, or purification steps beyond the single depth filtration step before carrying out step (c).
• the method comprises a virus inactivation step before step (c).
• step (c) is spaced out in time, e.g., a portion of the buffered antibody composition is applied to the column over more than one application, e.g., over 1, 2, or 3 applications. In some embodiments, step (c) is carried out at resident time(s) of from or from about 1, 2, 3, 4, 5, or 6 minutes.
• the method further comprises, following step (c), one or more of: (d) a CEX chromatography step, e.g., as described herein, (e) a DE pad filtration step, e.g., as described herein, (f) a depth filtration step, e.g., as described herein; and (g) a PS20 spiking & filtration step, e.g., as described herein.
• step (c) one or more of: (d) a CEX chromatography step, e.g., as described herein, (e) a DE pad filtration step, e.g., as described herein, (f) a depth filtration step, e.g., as described herein; and (g) a PS20 spiking & filtration step, e.g., as described herein.
• the method further comprises, following step (c), one or more of: (d) a DE pad filtration step, e.g., as described herein, (e) a depth filtration step, e.g., as described herein; and (f) a PS20 spiking & filtration step, e.g., as described herein, but does not comprise a CEX chromatography step, e.g., as described herein.
• this method produces yields (antibody recovery rate) of or of about 80%, 85%, 90%, or 95% or more with sialylating enzyme, e.g., ST6 or enzymatically active portion thereof, present at 100 ppm, 90 ppm, 80 ppm, 70 ppm, 60 ppm, 50 ppm, 40 ppm, or 30 ppm or less.
• sialylating enzyme e.g., ST6 or enzymatically active portion thereof
• Polysorbate e.g., polysorbate 20
• the polysorbate is polysorbate 20.
• the polysorbate 20 is Super RefinedTM polysorbate 20 (Croda Health Care).
• the methods described herein can include precipitation.
• the precipitation is ammonium sulfate precipitation.
• the methods described herein include ammonium sulfate precipitation.
• the enzyme(s) 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., as provided herein), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein.
• the variant retains desired activity of the parent, e.g., ⁇ -galactoside a-2,6-sialyltransferase activity or ⁇ -1,4- galactosyltransferase activity.
• desired activity of the parent e.g., ⁇ -galactoside a-2,6-sialyltransferase activity or ⁇ -1,4- galactosyltransferase activity.
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%.
• nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
• nucleic acid “identity” is equivalent to nucleic acid “homology”.
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• Percent identity between a subject polypeptide or nucleic acid sequence (i.e. a query) and a second polypeptide or nucleic acid sequence (i.e. target) is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7);
• the length of comparison can be any length, up to and including full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%).
• percent identity is relative to the full length of the query sequence.
• the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
• 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.
• IgG in which more than 60% of the overall branched glycans are disialylated can be prepared as follows.
• a mixture of IgG antibodies is exposed to a sequential enzymatic reaction using ⁇ 1,4 galactosyltransferase 1 (B4GalT) and ⁇ 2,6-sialyltransferase (ST6Gall) enzymes.
• B4GalT ⁇ 1,4 galactosyltransferase 1
• ST6Gall ⁇ 2,6-sialyltransferase
• the galactosyltransferase enzyme selectively adds galactose residues to pre-existing asparagine-linked glycans.
• the resulting galactosylated glycans serve as substrates to the sialic acid transferase enzyme which selectively adds sialic acid residues to cap the asparagine-linked glycan structures attached to.
• the overall sialylation reaction employed two sugar nucleotides (uridine 5'-diphosphogalactose (UDP-Gal) and cytidine-5'-monophospho-N- acetylneuraminic acid (CMP -NANA)). The latter is replenished periodically to increase disialylated product relative to monosialylated product.
• the reaction includes the co-factor manganese chloride.
• FIG. 4 A representative example of the IgG-Fc glycan profile for such a reaction starting with IVIg and the reaction product is shown in the FIG. 4.
• the left panel is a schematic representation of enzymatic sialylation reaction to transform IgG to hsIgG; the right panel is the IgG Fc glycan profile for the starting IVIg and hsIgG.
• glycan profiles for the different IgG subclasses are derived via gly copeptide mass spectrometry analysis.
• IgG1 EEQYNSTYR (SEQ ID NO: 1), IgG2/3 EEQFNSTFR (SEQ ID NO: 2), IgG3/4 EEQYNSTFR (SEQ ID NO: 3) and EEQFNSTYR (SEQ ID NO: 4).
• the glycan data is shown per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg the sum of all the nonsialylated glycans is more than 80% and the sum of all sialylated glycans is ⁇ 20%. For the reaction product, the sum for all nonsialylated glycans is ⁇ 20% and the sum for all sialylated glycans is more than 80%. Nomenclature for different glycans listed in the gly coprofile use the Oxford notation for N linked glycans.
• An alternative suitable reaction conditions for galactosylation and sialylation to create hsIgG in 50 mM BIS-TRIS pH 6.9 include: galactosylation of IgG antibodies (e.g., pooled IgG antibodies, pooled immunoglobulins or IVIg) are as follows: 7.4 mM MnCl 2 ; 38 ⁇ mol UDP- Gal/g IgG antibody; and 7.5 units B4GalT/g IgG antibody with 16-24 hours of incubation at 37°C followed by sialylation in 7.4 mM MnCl 2 : 220 ⁇ mol CMP-NANA/g IgG antibody (added twice: half at the start of the reaction and half after 9-10 hrs); and 15 units ST6-Gall/g IgG antibody with 30-33 hours of incubation at 37°C.
• the reaction can be carried out by adding the ST6-Gall and CMP -NANA to the galactosylation reaction. Alternatively,
• hsIgG can include a variety of IgG subclasses (e.g., IgG1, IgG2, etc.) it can be desirable to employ methods that produce a purified produce that does not unduly alter the subclass distribution of the unpurified material.
• Protein A column chromatography was examined. Wash conditions including simple (NaCl, Na 2 SO 4 ) and chaotropic (arginine, MgCl 2 . KSCN) salts, buffer (phosphate, TRIS, BIS- TRIS, acetate, formate, glycolate, citrate, MOPS), organic modifier (ethylene glycol, propylene glycol), additives (imidazole, methyl galactopyranoside, lactose), and pH were examined.
• the Protein A column step was followed by a polishing step employing a Blue Trisacryl M (Pall Corp.) column in flow-thru mode.
• the loading buffer was 0.4 M NaCl, 50 mM Na citrate pH 4.5 and the wash buffer was 2 M NaCl, 50 mM sodium phosphate pH 7.0. This provided a very clean product.
• a different type of blue dye column with a sepharose base resin HiTrap Blue HP; GE Healthcare Life Sciences
• loading in 50 mM sodium citrate pH 4.5 and elution in 1.5 M KC1, 32.5 mM sodium citrate, 35% ethylene glycol, pH 4.51 provided a very clean product.
• the process comprises dilution of the reaction material 1 : 1 with 5x PBS to quench the reaction.
• the quenched reaction mixture is depth filtered to remove particulates (step 5 in FIG. 5) and is buffer exchanged into 50 mM tris, 100 mM NaCl, 10 mM EDTA pH 8.0 buffer for six diavolumes followed by buffer exchange into 250 mM glycine pH 5.0 (step 6 in FIG. 5).
• This buffer exchange step is to remove reaction components like nucleotides and manganese chloride.
• the EDTA buffer is a chelating agent that should also remove manganese chloride from solution.
• the pH of the process material is lowered to 3.5 for viral inactivation. It is held at the low pH for 90 minutes before being quenched and the pH raised to 4.5 (step 7 in FIG. 5).
• the quenched viral inactivation pool is diluted down to 4.5 mg/mL with 50 mM citrate, 400 mM NaCl pH 4.5 before being passed through a depth filter/nano filter combination (step 8 in FIG. 5).
• the filtrate is then buffer exchanged into 50 mM citrate, 400 mM NaCl pH 4.5 for 6 diavolumes and concentrated to 30 mg/mL (step 9 in FIG. 5).
• the final material is depth filtered one last time before the chromatography purification steps (step 10 in FIG. 5)
• the first purification step uses the strong cation exchange resin, PorosTM XS in the flow through mode. That means the material is directly loaded onto the column and the product is collected in the effluent while the impurities (ST6, B4GalT) stick to the resin.
• the flow through is then loaded onto a trisacryl blue (TAB) column that is also operated in flow through mode (step 12 in FIG. 5).
• TAB resin also has significant capacity to remove reaction enzymes.
• the process comprises two TFF steps (buffer exchanges/one concentration), three depth filtration steps, and two dilutions before chromatography. These steps are expensive (material / buffer / operating time / lost product). Reducing the number of steps before the chromatography purification reduces cost, operation time, and the amount of material lost while still removing reaction impurities (e.g., nucleotides, enzymes, manganese chloride). As described herein, steps 5-10 in FIG. 5 can be eliminated from the process by diluting the quenched (5x PBS) reaction mixture with 50 mM citrate pH 4.5 and using the strong cation exchange resin, PorosTM XS, in the bind and elute mode.
• reaction impurities e.g., nucleotides, enzymes, manganese chloride
• the reaction mixture will bind to the PorosTM XS strong cation exchange resin while the nucleotides and manganese chloride flow through 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. The enzymes, requiring a higher salt concentration for elution, will remain bound to the column. In addition, the eluted product is now in the same buffer that is required for the TAB resin purification.
• the load material was prepared by diluting 0.55 mL IVIg at 100 mg/mL 1:10 with 50mM citrate pH 4.5 and then spiking in 3.3 mL of ST6 at 16 mg/mL. For each resin, 8 mL of the load material was applied to the column and then eluted by linear gradient into 50mM citrate, 1 M NaCl pH 4.5.
• PorosTM resin in flow through mode is advantageous because it allows for only one cycle and a smaller resin requirement since the resin only needs to bind the impurities.
• the number of cycles required for complete processing depends on how much product it can bind at one time.
• a load solution was made by diluting IVIg 1:20 with 50 mM citrate pH 4.5. The load solution was applied to the column at 2-, 4- and 6-minute residence times and the binding capacity evaluated at a 5% breakthrough.
• FIG. 6 shows that not much capacity is gained by operating above a 4 minute residence time, lower than the 5-minute residence time typical for chromatography operations.
• the capacity is very high at ⁇ 96 mg/mL resin.
• the yield and residual ST6 for differing load capacity and elution buffer conductivity was evaluated over a number of runs to identify the best elution conditions and loading capacity (Table 10).
• the loading solution was prepared by diluting 2.8 g of IVIg, and 500 uL of ST6 at 15.6 mg/mL and diluting the solution 1:20 with 50mM citrate pH 4.5. All steps were operated at a 5-minute residence time. Load material is 2,700 ppm in ST6.
• FIG. 8 shows the operating range for a yield between 90- 95% and residual ST6 levels less than 30 ppm.
• the white space shows the operating range which suggest that elution conductivities between 39-44 mS/cm can be used for loading capacities less than 55 mg/mL- resin. As the loading capacity increases, this range becomes narrower.
• the flow through step contains no IgG only nucleotides and manganese chloride as seen by the SEC chromatogram of the flow through in FIG. 9.
• the current manufacturing process of M254 uses a trisacryl blue (TAB) column in flow- through mode which does not withstand high concentration of sodium hydroxide wash. This results in a higher level of bioburden in some batches.
• TAB trisacryl blue
• CaptoTM Blue High Sub CaptoTM Blue HS
• CaptoTM Blue High Sub can withstand a high sodium hydroxide wash and has the potential to reduce the number of steps of the current purification process.
• CaptoTM Blue and CaptoTM Blue HS have been compared to evaluate the recovery and enzyme clearance. Multiple runs were executed where either hsIGIV (post strong cation exchange column flow-through material) was loaded directly or loaded after dilution. Table 12 summarizes different load conditions while Table 13 is showing the mass balance:
• Residual ST6 after CaptoTM Blue and CaptoTM Blue HS was also evaluated and found to be as low if not lower than TAB as shown in FIG. 14 and FIG. 15. Impact of using CaptoTM Blue HS on product attributes
• IVIg from CaptoTM Blue HS flow through and eluate were compared to the load to evaluate impact on the product quality.
• Analytical SEC of the load, flow-through and eluate were compared; no differences were found, as demonstrated in FIG. 16 and Table 14.
• CaptoTM Blue HS In order to reduce the number of current manufacturing process steps, different conditions were evaluated and residual ST6 was evaluated:
• Run1 CaptoTM Blue HS flow through of an experiment with residual ST6 of 651ppm was used as loading test material to evaluate 5X PBS and MOPS at pH 4.5 buffer as loading buffer which will mimic loading material directly into the column after the reaction.
• TAB Trisacryl Blue
• the current manufacturing process has two chromatography columns, CEX followed by TAB to reduce the level of added ST6 during enzymatic reaction. Residual level of ST6 was 2.5-7.2 ppm for the historical manufactured drug substance lots. Maximum loading for the historical manufacturing process using the TAB column was 1.1g IVIg/ml resin (1X).
• TAB column loading study runs were performed with CEX load and CEX eluate obtained from 1.5Kg GMP run lot # 20006.
• TAB column runs were performed by using AKTA Avant 150. Load flow rate was maintained at 0.17ml/min to achieve residence time similarly to manufacturing column operation. Load flow-through fractions were collected, protein concentration measured by A280, and residual level of ST6 determined by ELISA.
• level of residual ST6 was 0.1 - 9 ppm for the TAB column flow- through fractions when loading 0.1 - 1.1 g/ml where the x-axis is the cumulative protein flow- through. If these flow-through fractions were pooled then it is reasonable to estimate the level of ST6 around 5ppm (with 1.1 g/ml loading), in other words similar to the historical manufacturing drug substance lots. Results from the present study confirm that maximum loading should be kept ⁇ 1.1 g/ml for the TAB column to maintain the ST6 levels seen in DS lots. Level of ST6 values were 161ppm and 62ppm for the manufacturing scale CEX load and CEX eluate indicated CEX column in the current manufacturing process reduce the level of ST6.
• TAB column run was performed with CEX load, in other words without CEX column purification.
• residual level of ST6 values were similar for the TAB column flow-through fractions (0.1-9 ppm) with two load materials, CEX load (without CEX column purification, 161 ppm) and CEX eluate (with CEX column as in the current manufacturing process, 62ppm).
• results in the present study indicated 1.1 g/ml as maximum loading for the TAB column to decrease the level of ST6 ⁇ 7.2ppm for drug substance similarly to historical manufacturing runs (ST6 level 2.5-7.2ppm). Additionally, results in the present study suggest without CEX column, TAB column efficiently decreases the level of ST6 to meet the current target (drug substance ⁇ 7.2ppm).
• IVIg is isolated and purified from human plasma in some methods by precipitation using either ethanol or polyethylene glycol at low temperature.
• precipitation of hsIVIg using ammonium sulfate to remove residual ST6.
• IVIg and hsIVIg were precipitated as follows.
• a saturated solution of ammonium sulfate was prepared by adding the powder directly into a 100 mL of water while stirring. Once a significant amount of the powder was precipitated the solution was left stirring for 30 min then decanted from the precipitate and added as is to the IVIG solution. The saturated ammonium sulfate solution was added until the solution of the IVIG turned white and no change in the color was seen. The precipitate was collected by centrifugation or by sterile filtration.
• FIG. 22 shows an overlay of the precipitated IVIg versus the starting IVIg and the precipitated material and FIG. 23 shows hsIVIg before and after precipitation using sterile filtration as a way of collecting the precipitated material.
• FIG. 23 shows hsIVIg before and after precipitation using sterile filtration as a way of collecting the precipitated material.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Medicinal Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Engineering & Computer Science (AREA)
• Analytical Chemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Immunology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Microbiology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Water Supply & Treatment (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Priority Applications (10)

Applications Claiming Priority (2)

Publications (1)

Family

ID=78708028

Family Applications (1)

Country Status (11)

Families Citing this family (1)

Citations (2)

Family Cites Families (5)

• 2021
  • 2021-05-19 KR KR1020227044052A patent/KR20230012560A/ko active Pending
  • 2021-05-19 CA CA3182023A patent/CA3182023A1/en active Pending
  • 2021-05-19 US US17/926,102 patent/US20230193339A1/en active Pending
  • 2021-05-19 WO PCT/US2021/033156 patent/WO2021236769A1/en not_active Ceased
  • 2021-05-19 MX MX2022014520A patent/MX2022014520A/es unknown
  • 2021-05-19 JP JP2022571265A patent/JP2023526527A/ja active Pending
  • 2021-05-19 AU AU2021275117A patent/AU2021275117A1/en active Pending
  • 2021-05-19 CN CN202180036183.0A patent/CN115715296A/zh active Pending
  • 2021-05-19 BR BR112022023377A patent/BR112022023377A2/pt unknown
  • 2021-05-19 EP EP21809739.2A patent/EP4153617A4/en active Pending
• 2022
  • 2022-12-17 CO CONC2022/0018364A patent/CO2022018364A2/es unknown

Patent Citations (2)

Also Published As

Similar Documents

Legal Events