TREATMENT WITH HIGHLY SILYLATED IGG COMPOSITIONS

Abstract

Compositions comprising a highly sialylated IgG preparation and methods for treating a patient using such preparations are described. This application is based, in part, on the surprising discovery that a dose of a highly sialylated IgG (hsIgG) preparation that is about 1%-10% of the effective dose for IVIG can be effective for treating disorders that are treated with IVIG.

Claims

1. A method for treating a disorder, the method comprising administering a hsIgG preparation to a subject at a dose that is 1%-10% of the effective dose of IVIG for treating the disorder.

2. The method of claim 1, wherein the hsIgG preparation is administered at a dose of 5 mg/kg to 100 mg/kg.

3. The method of claim 1, wherein the disorder is an inflammatory disorder.

4. The method of claim 1, wherein the subject is suffering from antibody deficiency.

5. The method of claim 4, wherein the subject is suffering from primary antibody deficiency.

6. The method of claim 1, wherein the disorder is associated with the presence of autoantibodies.

7. The method of claim 1 wherein the dose of hsIVIG is as effective as the effective dose of IVIG.

8. The method of claim 1, wherein the hsIgG preparation is administered at the same frequency as the effective dose of IVIG.

9. The method of claim 1, wherein the disorder is a neurological disorder.

10. The method of claim 9, wherein the neurological disorder is selected from the group consisting of: dermatomyositis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), myasthenia gravis and stiff person syndrome.

11. The method of claim 1, wherein the disorder is selected from the group consisting of: immune cytopenias, parvovirus B19 associated red cell aplasia, hypogammaglobulinaemia secondary to myeloma and chronic lymphatic leukaemia and post-bone marrow transplantation.

12. The method of claim 1, wherein the disorder is selected from the group consisting of: sculitis, systemic lupus erythematosis (SLE), mucous membrane pemphigoid and uveitis and in dermatology it is used most commonly to treat Kawasaki syndrome, dermatomyositis, toxic epidermal necrolysis and the blistering diseases.

13. The method of claim 1, wherein the disorder is FDA-approved for treatment with IVIG or IVIG is indicated for treatment of the disorder.

14. The method of claim 13, wherein the hsIgG preparation is 1%-10% of the FDA approved IVIG dose for the disorder.

15. The method of claim 1, wherein the disorder is selected from the group consisting of: Myocarditis Acute motor axonal neuropathy Adiposis dolorosa Anti-Glomerular Basement Membrane nephritis; Goodpasture syndrome Antiphospholipid syndrome (APS, APLS) Antisynthetase syndrome; Myositis, ILD ataxic neuropathy (acute & chronic) Autoimmune enteropathy (AIE) Autoimmune neutropenia Autoimmune retinopathy Autoimmune thyroiditis Autoimmune urticaria Dermatitis herpetiformis Epidermolysis bullosa acquisita Essential mixed cryoglobulinemia Granulomatosis with polyangiitis (GPA) Mixed connective tissue disease (MCTD) Neuromyotonia Optic neuritis Paraneoplastic cerebellar degeneration Anti-N-Methyl-D-Aspartate (Anti-NMDA) Receptor Encephalitis Autoimmune hemolytic anemia Autoimmune thrombocytopenic purpura Chronic inflammatory demyelinating polyneuropathy Dermatomyositis Gestational pemphigoid Graves' disease Guillain-Barré syndrome IgG4-related disease Lambert-Eaton myasthenic syndrome Lupus nephritis Myositis Multifocal motor neuropathy Myasthenia gravis Neuromyelitis optica Pemphigus vulgaris Polymyositis and Systemic Lupus Erythematosus (SLE).

16. The method of claim 1, wherein the disorder is selected from the group consisting of: Acute disseminated encephalomyelitis (ADEM) Autoimmune Angioedema (Acquired angioedema type II) Autoimmune hepatitis (Type I & Type II) Autoimmune hypophysitis; Lymphocytic hypophysitis Autoimmune inner ear disease (AIED) Evans syndrome Graves ophthalmopathy Hashimoto's encephalopathy IgA vasculitis (IgAV) Latent autoimmune hepatitis Linear IgA disease (LAD) Lupus vasculitis Membranous glomerulonephritis Microscopic polyangiitis (MPA) Mooren's ulcer Morphea Opsoclonus myoclonus syndrome Ord's thyroiditis Palindromic rheumatism Paraneoplastic opsoclonus—myoclonus-ataxia with neuroblastoma Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS) Postpericardiotomy syndrome Primary biliary cirrhosis (PBC) Rasmussen Encephalitis Rheumatoid vasculitis Schnitzler syndrome Sydenham chorea Undifferentiated connective tissue disease (UCTD), and Miller Fisher Syndrome.

17. The method of claim 1, wherein 60% of the glycans on the IgG in the hsIgG preparation have a sialic acid on both the α1,3 branch and the α1,6 branch.

18. The method of claim 17, wherein at least 60% of the branched glycans on the Fab domain have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage; and at least 60% of the branched glycan on the Fc domain have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.

19. A method of treating CIDP in a subject having CIDP comprising administering a hsIgG preparation at an effective dose than is 10% or less than 10% of the effective dose for IVIG.

20. The method of claim 19, wherein the effective dose for IVIG is 200-2000 mg/kg.

21. The method of claim 19, wherein the hsIgG preparation is administered at an effective dose that is 10% or less than 10% of the effective dose for IVIG.

22. The method of claim 19, wherein the hsIgG preparation is administered at a dose of 1% of the effective dose for IVIG.

23. The method of claim 19, wherein the hsIgG preparation is administered at a dose of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/kg.

24. A method of treating ITP in a subject having ITP comprising administering a hsIgG preparation at an effective dose that is 10% or less than 10% of the of the effective dose for IVIG.

25. The method of claim 24, wherein the effective dose for IVIG is 1000-2000 mg/kg mg/kg.

26. The method of claim 24, wherein the hsIgG preparation is administered at an effective dose that is 10% or less than 10% of the effective dose for IVIG.

27. The method of claim 24, wherein the hsIgG preparation is administered at a dose of 1%-5% of the effective dose for IVIG.

28. The method of claim 24, wherein the hsIgG preparation is administered at a dose of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/kg.

29. A method of treating wAIHA in a subject having wAIHA comprising administering a hsIgG preparation at an effective dose that is 10% or less than 10% of the of the effective dose for IVIG.

30. The method of claim 29, wherein the effective dose for IVIG is 1000 mg/kg.

31.-53. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0110] FIG. 1 is a schematic depiction of an example of a branched glycan that is sialylated on both the α 1,3 arm and the α 1,6 arm by way of NeuAc-α 2,6-Gal terminal linkage.

[0111] FIG. 2 is a schematic illustration of a method for preparing hsIgG The beginning substrate can be IVIG or a fraction thereof.

[0112] FIG. 3 is a schematic illustration of the sialylation of a branched glycan by ST6Gal1.

[0113] FIG. 4 is a graph showing the time course of IVIg Fc glycoform proportions in the presence of CMP-NANA and ST6Gal1. Galactosylated IVIg was incubated with 20 mM CMP-NANA and 0.3 U/mg ST6Gal1 at 37° C. Aliquots were removed at different time points, and the relative proportions of IVIg glycoforms were determined by glycopeptide LC-MS/MS analyses.

[0114] FIGS. 5A and B depicts the results of an analysis of IVIG dose-response in a murine ITP model (ns, Not significant. *P<0.05; **P<0.01; ***P<0.001)

[0115] FIG. 6 depicts the results of a study comparing of therapeutic dosing of 0.1 g/kg hsIgG with IVIg at 0.1 and 1 g/kg in a murine model of ITP.

[0116] FIG. 7 depicts the results of a comparison of hsIgG and IVIG in a human patient with ITP.

DETAILED DESCRIPTION

[0117] The present disclosure concerns methods of treatment using a hsIgG preparation. A hsIgG preparation is a preparation comprising a mixture of IgG antibodies (e.g., prepared from IVIG) in which at least 60% of the branched glycans are disialylated, i.e., have a sialic acid on the α1,3 arm (e.g., with a NeuAc-α2,6-Gal terminal linkage) and on the α1,6 arm (e.g., with a NeuAc-α2,6-Gal terminal linkage).

[0118] An hsIgG preparation disclosed herein can be used to treat disorders that are treated with IVIG Importantly, the hsIgG preparations are far more potent than IVIG permitting effective treatment at a far lower dose and/or less frequent treatment. For example, as shown herein An hsIgG preparation can be 10 to 100-times more potent than IVIG allowing treatment at a dose that is 1%-10% of the effective or approved IVIG dose.

[0119] Current treatments utilizing IVIG have distinct limitations, which include, but are not limited to, variable efficacy, clinical risks, high costs, and finite supply. At the current maximal dosing regimens, only partial and poorly sustained responses are obtained in many instances. In addition, the long infusion times (4-6 h) associated with the high volume of IVIg treatment (IVIG is commonly dosed at 1,000-2,000 mg/kg) consume significant resources at infusion centers and negatively affect patient-reported outcomes, such as convenience and quality of life.

[0120] A hsIgG preparation, because it is so much more potent than IVIG, can provide shorter infusion times, a far lower amount of protein administered, and, in some cases, less frequent administration. For this reason, a hsIgG preparation can provide a greatly improved patient experience and higher quality of life compared to treatment with IVIG In some cases, conditions which must be treated with intravenous administration of IVIG can be treated with subcutaneous administration (injection or infusion) of hsIgG e.g., by means of a subcutaneous pump, allowing administration by the patient at home. In addition, an effective dose of hsIgG can be prepared from a far lower amount of IVIG than would be required for an effective dose of IVIG This is an important advantage because IVIG is costly in limited supply.

[0121] The level of sialylation can be measured on an individual Fc region (e.g., the number of branched glycans that have a sialic acid an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region), or on the overall composition of a preparation of glycoproteins (e.g., the number or percentage of branched glycans that are have a sialic acid on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region in a preparation of glycoproteins).

[0122] Methods of Sialylating IgG Preparation

[0123] As described in Washburn et al. (Proc Natl Acad Sci 112(11):E1297-306), ST6 GalI sialyltransferase catalyzes the transfer of sialic acid from a sialic acid donor (e.g., cytidine 5′-monophospho-N-acetyl neuraminic acid) to a terminal galactose residue of glycans through an α2,6 linkage in an ordered fashion. ST6 sialyltransferase transfers a sialic acid to an α1,3 arm of a branched glycan, which can be followed by transfer of a second sialic acid to an α1,6 arm (yielding a disialylated branched glycan), and can further be followed by removal of sialic acid from an α1,3 arm (yielding a branched glycan having a sialic acid on an α1,6 arm). Accordingly, by controlling and/or modulating activity (e.g., kinetics) of ST6 sialyltransferase, glycoproteins having particular sialylation patterns can be produced.

[0124] Methods for producing hsIgG are described, for example in Washburn et al. and in US 2016/0108450, hereby incorporated by reference. Deletion mutants of ST6Gal-1 sialyltransferase (Engel et al., BMC Proceedings 7(Suppl 6):P110, 2013) can also be useful for preparing hsIgG

[0125] A different sialylation method, which fails to meaningfully increase Fab sialylation, is described in Huang et al. (J Am Chem Soc 134(29):12308-12318, 2012). Despite the lack of Fab sialylation, IVIG sialylated by this method may be useful.

[0126] A hsIgG preparation can be manufactured from commercially available IVIG as follows. Bulk IVIG 10% solution (0.1 g/mL), is pooled and diluted with 3-(N-morpholino) propanesulfonic acid (MOPS) pH 7.4 buffer. The solution is diafiltered (DF) with five diavolumes (DVs) of MOPS pH 7.4 buffer using 30 kDa tangential flow filtration (TFF) membranes (membranes composed of polyethersulfone). The buffer exchanged IVIG solution is then concentrated, followed by depth and 0.2 μm filtrations resulting in a final IVIG concentration of >150 mg/mL. Sialylation is accomplished in two enzymatic reaction steps using two sugar nucleotides dissolved in MOPS pH 7.4 buffer. First, galactosylation occurs by reaction with beta-1,4 galactosyltransferase enzyme (B4GalT), uridine diphosphate galactose (UDP-Gal) and manganese chloride in MOPS pH 7.4 buffer. The reaction solution is adjusted to approximately 135 mg/mL by addition of MOPS buffer and is maintained at approximately 37° C. for approximately 48 hours. Following galactosylation, the material is further incubated for approximately 72 hours with the addition of human α 2,6-sialyltransferase enzyme (ST6-GalI) and cytidine 5′monophospho-N-acetyl neuraminic acid (CMP-NANA), and adjusted to approximately 120 mg/mL with MOPS buffer pH 7.4. CMP-NANA is charged portion wise over the course of reaction at approximately 12 hour intervals.

Glycan Evaluation

[0127] Glycans of glycoproteins can be evaluated using any methods known in the art. For example, sialylation of glycan compositions (e.g., level of branched glycans that are have a sialic acid on an α1,3 arm and/or an α1,6 arm) can be characterized using methods described in, e.g., Barb, Biochemistry 48:9705-9707 (2009); Anumula, J. Immunol. Methods 382:167-176 (2012); Gilar et al., Analytical Biochem. 417:80-88 (2011); Wuhrer et al., J. Chromatogr. B. 849:115-128 (2007). In some embodiments, in addition to evaluation of sialylation of glycans, one or more parameters described in Table 1 are evaluated.

[0128] In some instances, glycan structure and composition as described herein are analyzed, for example, by one or more, enzymatic, chromatographic, mass spectrometry (MS), chromatographic followed by MS, electrophoretic methods, electrophoretic methods followed by MS, nuclear magnetic resonance (NMR) methods, and combinations thereof. Exemplary enzymatic methods include contacting a glycoprotein preparation with one or more enzymes under conditions and for a time sufficient to release one or more glycan(s) (e.g., one or more exposed glycan(s)). In some instances, the one or more enzymes include(s) PNGase F. Exemplary chromatographic methods include, but are not limited to, Strong Anion Exchange chromatography using Pulsed Amperometric Detection (SAX-PAD), liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof. Exemplary mass spectrometry (MS) include, but are not limited to, tandem MS, LC-MS, LC-MS/MS, matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), and combinations thereof. Exemplary electrophoretic methods include, but are not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using antibodies that recognize specific glycan structures, and combinations thereof. Exemplary nuclear magnetic resonance (NMR) include, but are not limited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR), and combinations thereof.

[0129] In some instances, techniques described herein may be combined with one or more other technologies for the detection, analysis, and or isolation of glycans or glycoproteins. For example, in certain instances, glycans are analyzed in accordance with the present disclosure using one or more available methods (to give but a few examples, see Anumula, Anal. Biochem., 350(1):1, 2006; Klein et al., Anal. Biochem., 179:162, 1989; and/or Townsend, R. R. Carbohydrate Analysis” High Performance Liquid Chromatography and Capillary Electrophoresis., Ed. Z. El Rassi, pp 181-209, 1995; WO2008/128216; WO2008/128220; WO2008/128218; WO2008/130926; WO2008/128225; WO2008/130924; WO2008/128221; WO2008/128228; WO2008/128227; WO2008/128230; WO2008/128219; WO2008/128222; WO2010/071817; WO2010/071824; WO2010/085251; WO2011/069056; and WO2011/127322, each of which is incorporated herein by reference in its entirety). For example, in some instances, glycans are characterized using one or more of chromatographic methods, electrophoretic methods, nuclear magnetic resonance methods, and combinations thereof. In some instances, methods for evaluating one or more target protein specific parameters, e.g., in a glycoprotein preparation, e.g., one or more of the parameters disclosed herein, can be performed by one or more of following methods.

[0130] In some instances, methods for evaluating one or more target protein specific parameters, e.g., in a glycoprotein preparation, e.g., one or more of the parameters disclosed herein, can be performed by one or more of following methods.

TABLE-US-00001 TABLE 1 Exemplary methods of evaluating parameters: Method(s) Relevant Literature Parameter C18 UPLC Mass Spec.* Chen and Flynn, Anal. Glycan(s) Biochem., 370: 147-161 (e.g., N-linked glycan, (2007) exposed N-linked glycan, Chen and Flynn, J. Am. glycan detection, glycan Soc. Mass Spectrom., identification, and 20: 1821-1833 (2009) characterization; site specific glycation; glycoform detection (e.g., parameters 1-7); percent glycosylation; and/or aglycosyl) Peptide LC-MS Dick et al., Biotechnol. C-terminal lysine (reducing/non- Bioeng., 100: 1132-1143 reducing) (2008) Yan et al., J. Chrom. A., 1164: 153-161 (2007) Chelius et al., Anal. Chem., 78: 2370-2376 (2006) Miller et al., J. Pharm. Sci., 100: 2543-2550 (2011) LC-MS (reducing/non- Dick et al., Biotechnol. C-terminal lysine reducing/alkylated) Bioeng., 100: 1132-1143 (2008) Goetze et al., Glycobiol., 21: 949-959 (2011) Weak cation exchange Dick et al., Biotechnol. C-terminal lysine (WCX) chromatography Bioeng., 100: 1132-1143 (2008) LC-MS (reducing/non- Dick et al., Biotechnol. N-terminal pyroglu reducing/alkylated) Bioeng., 100: 1132-1143 (2008) Goetze et al., Glycobiol., 21: 949-959 (2011) PeptideLC-MS Yan et al., J. Chrom. A., N-terminal pyroglu (reducing/non- 1164: 153-161 (2007) reducing) Chelius et al., Anal. Chem., 78: 2370-2376 (2006) Miller et al., J. Pharm. Sci., 100: 2543-2550 (2011) Peptide LC-MS Yan et al., J. Chrom. A., Methionine oxidation (reducing/non- 1164: 153-161 (2007); reducing) Xie et al., mAbs, 2: 379-394 (2010) Peptide LC-MS Miller et al., J. Pharm. Sci., Site specific glycation (reducing/non- 100: 2543-2550 (2011) reducing) Peptide LC-MS Wang et al., Anal. Chem., Free cysteine (reducing/non- 83: 3133-3140 (2011); reducing) Chumsae et al., Anal. Chem., 81: 6449-6457 (2009) Bioanalyzer (reducing/ Forrer et al., Anal. Glycan (e.g., N-linked non-reducing)* Biochem., 334: 81-88 (2004) glycan, exposed N-linked glycan) (including, for example, glycan detection, identification, and characterization; site specific glycation; glycoform detection; percent glycosylation; and/or aglycosyl) LC-MS (reducing/non- Dick et al., Biotechnol. Glycan (e.g., N-linked reducing/alkylated)* Bioeng., 100: 1132-1143 glycan, exposed N-linked * Methods include removal (2008) glycan) (e.g., enzymatic, Goetze et al., Glycobiol., (including, for example, chemical, and physical) 21: 949-959 (2011) glycan detection, of glycans Xie et al., mAbs, 2: identification, and 379-394 (2010) characterization; site specific glycation; glycoform detection; percent glycosylation; and/or aglycosyl) Bioanalyzer (reducing/ Forrer et al., Anal. Light chain: Heavy chain non-reducing) Biochem., 334: 81-88 (2004) Peptide LC-MS Yan et al., J. Chrom. A., Non-glycosylation-related (reducing/non- 1164: 153-161 (2007) peptide modifications reducing) Chelius et al., Anal. Chem., (including, for example, 78: 2370-2376 (2006) sequence analysis and Miller et al., J. Pharm. identification of sequence Sci., 100: 2543-2550 (2011) variants; oxidation; succinimide; aspartic acid; and/or site-specific aspartic acid) Weak cation exchange Dick et al., Biotechnol. Isoforms (including, for (WCX) chromatography Bioeng., 100: 1132-1143 example, charge variants (2008) (acidic variants and basic variants); and/or deamidated variants) Anion-exchange Ahn et al., J. Chrom. B, Sialylated glycan chromatography 878: 403-408 (2010) Anion-exchange Ahn et al., J. Chrom. B, Sulfated glycan chromatography 878: 403-408 (2010) 1,2-diamino-4,5- Hokke et al., FEBS Lett., Sialic acid methylenedioxybenzene 275: 9-14 (1990) (DMB) labeling method LC-MS Johnson et al., Anal. C-terminal amidation Biochem., 360: 75-83 (2007) LC-MS Johnson et al., Anal. N-terminal fragmentation Biochem., 360: 75-83 (2007) Circular dichroism Harn et al., Current Trends Secondary structure spectroscopy in Monoclonal Antibody (including, for example, Development and alpha helix content and/or Manufacturing, S. J. Shire beta sheet content) et al., eds, 229-246 (2010) Intrinsic and/or ANS Harn et al., Current Trends Tertiary structure dye fluorescence in Monoclonal Antibody (including, for example, Development and extent of protein folding) Manufacturing, S. J. Shire et al., eds, 229-246 (2010) Hydrogen-deuterium Houde et al., Anal. Chem., Tertiary structure and exchange-MS 81: 2644-2651 (2009) dynamics (including, for example, accessibility f amide protons to solvent water) Size-exclusion Carpenter et al., J. Pharm. Extent of aggregation chromatography Sci., 99: 2200-2208 (2010) Analytical Pekar and Sukumar, Anal. ultracentrifugation Biochem., 367: 225-237 (2007)

[0131] The literature recited above are hereby incorporated by reference in their entirety or, in the alternative, to the extent that they pertain to one or more of the methods for determining a parameter described herein.

[0132] Pharmaceutical Compositions and Administration

[0133] A hsIgG can be incorporated into a pharmaceutical composition. Pharmaceutical compositions for intravenous administration of a hsIgG preparation can be formulated by methods known to those skilled in the art. For example, the pharmaceutical composition can be formulated by suitably combining the hsIgG preparation with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. The amount of active ingredient included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided.

[0134] The sterile composition for injection can be formulated in accordance with conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80™, HCO-50 and the like.

[0135] Non-limiting examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampule.

[0136] In some embodiments, the dose is 1%-10% of the FDA approved (or other national or international regulatory agency) IVIG dose or the effective IVIG dose for a disorder. In some embodiments, the FDA (or other national or international regulatory agency) approved dose or effective dose of IVIG is 200 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 1000 mg/kg, or 2000 mg/kg. In some embodiments, a composition comprising a hsIgG preparation is administered at a dose of about 4, 5, 6, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 975, or 1000 mg/kg. In some embodiments, a composition comprising a hsIgG preparation is administered daily, weekly, semiweekly, biweekly, monthly, semimonthly, bimonthly, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, once every 14 days, once every 21 days, once every 28 days, once daily for two consecutive days in a 28-day cycle, or with the same administration frequency as the FDA approved IVIG dose. In some embodiments, a composition is administered in a single dose. In some embodiments, a composition is administered in multiple doses. The dose and method of administration varies depending on the weight, age, condition, and the like of the patient, and can be suitably selected as needed by those skilled in the art.

[0137] As described in Washburn et al., analysis of sialylation by ST6Gal1 revealed that ST6Gal1 can not only catalyze the transfer of the sialic acid from CMP-NANA sugar nucleotide to the Fc glycan but also, facilitate the removal of the sialic acid from the sialylated product. The reactions are shown schematically in FIG. 3.

[0138] As Washburn et al. reported, under certain reaction conditions, sialylation of the α1,3 branch of the biantennary glycan (to form A1F-1,3) was rapid and essentially complete by 30 min, whereas the doubly sialylated species (A2F) formed at an ˜10× slower rate and stopped accumulating by 24 h. After 24 h, a monosialylated species with the sialic acid on the α1,6 branch (A1F-1,6) started to form and continued accumulating steadily, reaching ˜35% of glycosylated species by 64 h. Concomitantly, A2F exhibited a steady decline from 71% at 20 h to 44% at 64 h, suggesting that the A1F-1,6 glycoform was generated from the removal of sialic acid residues on the more exposed α1,3 branch of disialylated A2F. Additional incubation led to the cleavage of the 1,6 sialic acid in the A1F-1,6 glycoform, generating the asialylated but fully galactosylated and fucosylated species G2F. G2F, which was present in trace amounts at the beginning of the reaction, appeared at measurable amounts at 40 h and continued increasing, reaching 15% by 64 h as the levels of A2F and A1F-1,6 declined.

[0139] Washburn et al. further reported that these observations were critical in optimizing the parameters to maximize the yield of the A2F species while minimizing the A1F-1,3, A1F-1,6, and G2F glycoforms. By evaluating a matrix of parameters affecting the transient state of the G2F⇄A1F-1,3⇄A2F⇄A1F-1,6⇄G2F glycoform distribution, they found that the desialylation component of the reaction (A2F⇄A1F-1,6⇄G2F) was facilitated by spontaneous decomposition of CMP-NANA in the reaction. Wasburn et al. concluded that replenishing the CMP-NANA in the sialylation reaction assists in maximize the A2F yield and found that periodic dosing of fresh CMP-NANA maximized the A2F glycan levels after 24 h without the formation of substantial A1F-1,6 or G2F species.

Galactosylation and Sialylation of IVIG

[0140] The sialylation of IVIG by the sialyltransferase ST6 was analyzed. IVIG was first galactosylated and then sialylated. The reactions were performed sequentially. There was no purification between galactosylation and sialylation reactions. The relative abundance of glycoforms was analyzed following the sialylation reactions.

[0141] A. Galactosylation

[0142] A reaction was set up that contained the following components at the concentrations indicated:

TABLE-US-00002 Final Constituent concentration MOPS (pH 7.4) 25 mM MnCl.sub.2 10 mM IVIG 12.5 mg/ml B4GalT1 (90 u/ml) 400 mu/ml UDP-Galactose 50 mM

[0143] The reaction was incubated for 72 hours at 37° C.

[0144] B. Sialylation

[0145] To an aliquot of the galactosylation reaction were added CMP-NANA, MOPS buffer and ST6Gal1. The final volume was adjusted so that the final concentration of components in the reaction was as indicated.

TABLE-US-00003 Final Constituent concentration MOPS (pH 7.4) 50 mM MnCl.sub.2 8 mM IVIG 10 mg/ml CMP-NANA 20 mM ST6Gal1 (SEQ ID NO: 1) 0.6 mg ST6/mg

[0146] The reaction was incubated at 37° C. Aliquots were extracted at various times frozen at −20° C. for later analyses.

[0147] C. Results

[0148] FIG. 4 depicts the time course of IVIG Fc domain glycoform proportions over the course of a sialylation reaction performed essentially as described above in the presence of CMP-NANA and ST6Gal1. Briefly, galactosylated IVIG was incubated with 20 mM CMP-NANA and 0.3 U/mg ST6Gal1 at 37° C. Aliquots were removed at different time points, and the relative proportions of IVIG glycoforms were determined by glycopeptide LC-MS/MS analyses. As shown in FIG. 4, the predominant glycoform changed over time from G2F to A1F (1,3) to A2F to A1F (1,6) during the course of a reaction due to competing addition (forward reaction) and removal (back reaction) steps.

Analysis of a hsIgG Preparation

[0149] A hsIgG preparation manufactured from commercially available IVIG was prepared essentially as described above with periodic addition of CMP-NANA during the sialylation reaction. The starting IVIG material and the resulting hsIgG preparation were extensively analyzed before and after sialylation. With regard to Fc domain sialylation, the hsIgG preparation was substantially tetrasialylated (i.e., the branched glycan on each Fc chain was sialylated on both branches. More detailed analyses of glycosylation were performed in an isotype- and site-specific manner to discriminate the different Fc isotypes and Fab glycosylation. Fc glycosylation was changed from predominantly asialylated species (e.g., G0F and G1F) in the starting IVIG material to more than 90% disialylated species (e.g., A2F, A2, and A2F+BG1cNAc) in each IgG isotype in the hsIgG preparation. Analysis of the Fab glycans further revealed that, although the IVIG starting material contained a significant distribution of monosialylated (˜35%) and disialylated (˜35%) glycans in the Fab, the distribution shifted to a higher level of disialylated glycans in the hsIgG preparation (˜75%).

hsIgG is about 10 Times Potent than IVIG in Prevent Platelet Destruction in a Murine Model of ITP

[0150] Washburn et al. compared a hsIgG preparation to IVIG in a murine anti-CD41 antibody-induced thrombocytopeania model. Briefly, 6A6-IgG2a antibodies were produced by transient transfection of 293T cells followed by purification of recombinant antibodies from serum-free cell culture supernatants with protein G beads (GE Healthcare) as suggested by the manufacturer. IVIG preparations were diluted in glycine buffer (saline) for experiments. Chronic ITP was induced by daily injections of 0.1 μg/g 6A6-IgG2a antiplatelet antibody. Mice were rendered thrombocytopenic until the end of the experiment (day 3). Platelet counts were determined before and 4 h after daily antibody injection of α 1:4 dilution in PBS in a hematology system (Advia 120; Bayer HealthCare). Platelet counts before antibody injection were set to 100%. A dose-response profile for IVIG in the ITP model (FIG. 5). After maximum platelet depletion with the antiplatelet antibody, mice were treated with IVIG from 0.1 to 1 g/kg, and platelet levels were measured on the day of the IVIg treatment (day 1) and the two subsequent days after treatment (days 2 and 3 of the experiment). A clear dose-response was observed in this model within this range (FIG. 5). Complete activity of IVIg was lost at 0.1 g/kg. Comparing the hsIgG preparation and IVIG in the ITP model, a clear enhancedefficacy was observed. As shown in FIG. 6, therapeutic treatment with hsIgG at 0.1 g/kg returned platelet levels at days 2 and 3 to levels similar to those obtained with IVIg at 1 g/kg.

Studies in Human Patients

[0151] A hsIgG preparation (at least 80% of the branched glycans were sialylated on both the α 1,3 arm and the α 1,6 arm by way of NeuAc-α 2,6-Gal terminal linkages) was compared to IVIG in human patients with ITP. In one study, a single ascending dose was given to normal healthy volunteers over α 3.5 month period. In this double-blind, placebo controlled study, patients were given a single dose of hsIgG every 14 days in the following order: 3 mg/kg, 10 mg/kg, 30 mg/kg, 60 mg/kg, 120 mg/kg, and 250 mg/kg. End point measurements were safety and tolerability.

[0152] In a separate study, a single ascending dose was given to ITP patients over α 4 month period. ITP patients were given a single dose of hsIVIG in the following fixed order: 60 mg/kg, 120 mg/kg, 250 mg/kg, 500 mg/kg, and 1000 mg/kg. The hsIgG dose was administered, and after 28 days, 1000 mg/kg IVIG was administered to the same patient. Platelet levels were measured throughout the study. End point measurements were safety and tolerability.

[0153] In a randomized study, ITP patients were started on either hsIgG or IVIG and switched mid-study. In one subsection, 5 patients were started on a high hsIVIG dose and another 5 patients were started on 1,000 mg/kg IVIG. Midway through to 2 month study, the patients that started on a high hsIVIG dose were given 1,000 mg/kg IVIG for the remainder of the study, and the patients that started on 1,000 mg/kg IVIG were given a high hsIgG dose for the remainder of the study. Platelet response was measured throughout. In another subsection, 5 patients were started on a low hsIgG dose and another 5 patients were started on 1,000 mg/kg IVIG. Midway through to 2 month study, the patients that started on a low hsIgG dose were given 1,000 mg/kg IVIG for the remainder of the study, and the patients that started on 1,000 mg/kg IVIG were given a low hsIG dose for the remainder of the study. Platelet response was measured throughout. The potency of the hsIVIG was determined.

A hsIgG Preparation is More Potent than IVIG in Human ITP Patient

[0154] A hsIgG preparation was compared to IVIG in a human patient with ITP. In this hsIgG preparation at least 80% of the branched glycans were sialylated on both the α 1,3 arm and the α 1,6 arm by way of NeuAc-α 2,6-Gal terminal linkages. The patient was dosed with 43 mg/kg hsIVIG. As shown in FIG. 7, this patient had similar reticulocyte count when administered a hsIgG preparation at 43 mg/kg as at the 1000 mg/kg IVIG dose. Thus, the hsIgG preparation was about 25-fold more potent than IVIG in preventing platelet destruction in this patient.

OTHER EMBODIMENTS

[0155] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.