Siarylated glycoprotein

Abstract

Described herein are liquid pharmaceutical compositions comprising immunoglobulins.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 068,098, filed on 20 August 2020. The entire foregoing is incorporated herein by reference.

[0002] (Field of Invention) Liquid pharmaceutical compositions containing immunoglobulins are described herein. [Background technology]

[0003] The identification of the crucial role of sialylation of the Fc domain has presented an opportunity to develop potent immunoglobulin therapies. One commercially available immunoglobulin source is intravenous immunoglobulin (IVIg), which is prepared from pooled plasma from human donors (e.g., pooled plasma from at least 1,000 donors) and is used to treat various inflammatory diseases. Commercial IVIg preparations generally exhibit low levels of sialylation of the Fc domain of the present antibody. Specifically, they exhibit low levels of dicialylation of branched glycans in the Fc region. Furthermore, IVIg preparations have clear limitations, including variability in efficacy, high cost, and limited supply. [Overview of the project] [Means for solving the problem]

[0004] Pharmaceutical compositions comprising hypersialylated immunoglobulin (hsIgG) are described herein. HsIgG has very high levels of sialic acid in the branched glycans on the Fc region of the immunoglobulin, for example, at least 50% (60%, 70%, 80%, 90% or more) of the branched glycans on the Fc region of the immunoglobulin are sialylated via NeuAc-α2,6-Gal terminal bindings in both the α1,3 and α1,6 arms of the branched glycans. HsIgG comprises a diverse mixture of IgG antibodies, mainly IgG1 antibodies. Antibody diversity is high. The immunoglobulin used to prepare hsIgG can be obtained, for example, from pooled human plasma (e.g., pooled plasma from at least 1,000 to 30,000 donors).

[0005] The pharmaceutical compositions described herein provide pharmaceutically acceptable hsIgG compositions that are stable against many stressors associated with transport (e.g., temperature, agitation, freeze-thaw cycles, and / or photosensitivity). The pharmaceutical compositions described herein provide pharmaceutically acceptable hsIgG compositions that can be transported and handled in liquid form. The formulations are also stable when diluted, for example, with 5% dextrose for intravenous administration. The formulations are stable for, for example, at least 7 months at 5°C, at least 1 month at 25°C, 2 years at 2–8°C, and / or 2 weeks at 15–30°C.

[0006] A liquid pharmaceutical composition comprising immunoglobulin in approximately 10 mM sodium acetate, approximately 0.02% (w / v) polysorbate 20, and at least one of approximately 250 mM glycine or approximately 5% (w / v) sorbitol, wherein at least 50% of the branched glycans on the Fc region of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds, and the pH of the composition is 4 to 7 is described herein.

[0007] In some embodiments, the liquid pharmaceutical composition contains 250 mM glycine. In some embodiments, the liquid pharmaceutical composition contains 5% (w / v) sorbitol.

[0008] In some embodiments, the immunoglobulin concentration is 50-275 mg / mL. In some embodiments, the immunoglobulin concentration is 50-250 mg / mL.

[0009] In some embodiments, the liquid pharmaceutical composition contains 5% (w / v) sorbitol and the immunoglobulin concentration is 100-275 mg / mL. In some embodiments, the liquid pharmaceutical composition contains 5% (w / v) sorbitol and the immunoglobulin concentration is 70-130 mg / mL, 90-110 mg / mL, or 80-120 mg / mL.

[0010] In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding.

[0011] In some embodiments, at least 90% of the immunoglobulins are IgG immunoglobulins. In some embodiments, at least 95% of the immunoglobulins are IgG immunoglobulins.

[0012] In some embodiments, 5-20% of the immunoglobulin is a dimer. In some embodiments, 5-10% of the immunoglobulin is a dimer. In some embodiments, at least 80% of the immunoglobulin is a monomer or a dimer. In some embodiments, at least 85% of the immunoglobulin is a monomer or a dimer. In some embodiments, at least 90% of the immunoglobulin is a monomer or a dimer. In some embodiments, 5-20% of the IgG immunoglobulin is a dimer. In some embodiments, 5-10% of the IgG immunoglobulin is a dimer. In some embodiments, at least 80% of the IgG immunoglobulin is a monomer or a dimer. In some embodiments, at least 85% of the IgG immunoglobulin is a monomer or a dimer. In some embodiments, at least 90% of the IgG immunoglobulin is a monomer or a dimer.

[0013] In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding.

[0014] In some embodiments, the pH is 4.0–5.5, 4.0–4.5, 4.5–5.0, 5.0–5.5, 4.2–4.7, 4.7–5.3, or 5.1–5.3. In some embodiments, the pH is 4.0–5.5, 4.0–4.5, 4.5–5.0, 5.0–5.5, 4.2–4.7, 4.7–5.3, or 5.1–5.3. In some embodiments, the liquid pharmaceutical composition contains 5% (w / v) sorbitol and has a pH of 5.2–5.5 or 5.3–5.4. In some embodiments, the pH is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0015] In some embodiments, the composition has fewer than 1,000 particles having a diameter of 10 to 100 micrometers after being stirred at 1,000 RPM for 8 hours at 2 to 8°C. In some embodiments, the composition has fewer than 500 particles having a diameter of 10 to 100 micrometers after being stirred at 1,000 RPM for 8 hours at 2 to 8°C. In some embodiments, the composition has fewer than 200 particles having a diameter of 10 to 100 micrometers after being stirred at 1,000 RPM for 8 hours at 2 to 8°C.

[0016] In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of immunoglobulins are dicialized by NeuAc-α2,6-Gal terminal binding after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. At least 60%, 70%, 80%, 90%, or 95% of the branched glycans on immunoglobulins are dicialized by NeuAc-α2,6-Gal terminal binding after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of immunoglobulins are dicialized by NeuAc-α2,6-Gal terminal binding after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. In some embodiments, 5–10% of the immunoglobulins are dimers after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. In some embodiments, at least 85% of the immunoglobulins are monomers or dimers after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.In some embodiments, at least 90% of the immunoglobulin is a monomer or dimer after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0017] In some embodiments, the formulation is stable for at least 7 months at 5°C, at least 1 month at 25°C, for 2 years at 2 to 8°C, and / or for 2 weeks at 15 to 30°C.

[0018] In some embodiments, the storage is in a sealed United States Pharmacopeia Type 1 glass vial. In some embodiments, the storage is in a sealed 2R Type 1 glass injection vial.

[0019] Prefilled syringes comprising the liquid pharmaceutical composition are also described herein.

[0020] In some embodiments, the liquid pharmaceutical composition is frozen.

[0021] Also described herein is a liquid pharmaceutical composition comprising immunoglobulin in about 10 mM sodium acetate, about 0.02% (w / v) polysorbate 20, and at least one of about 250 mM glycine or about 5% (w / v) sorbitol, wherein at least 50% of the branched glycans on the immunoglobulin are disialylated via NeuAc-α2,6-Gal terminal linkages, and the pH of the composition is 4 to 7.

[0022] In some embodiments, the liquid pharmaceutical composition comprises 250 mM glycine. In some embodiments, the liquid pharmaceutical composition comprises 5% (w / v) sorbitol. In some embodiments, the concentration of immunoglobulin is 50 to 275 mg / mL. In some embodiments, the concentration of immunoglobulin is 50 to 250 mg / mL. In some embodiments, the liquid pharmaceutical composition comprises 5% (w / v) sorbitol, and the concentration of immunoglobulin is 100 to 275 mg / mL. In some embodiments, the liquid pharmaceutical composition comprises 5% (w / v) sorbitol, and the concentration of immunoglobulin is 70 to 130 mg / mL, 90 to 110 mg / mL, or 80 to 120 mg / mL.

[0023] In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the immunoglobulin are disialylated via NeuAc-α2,6-Gal terminal linkages. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of the immunoglobulin are disialylated via NeuAc-α2,6-Gal terminal linkages. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of the immunoglobulin are disialylated via NeuAc-α2,6-Gal terminal linkages.

[0024] In some embodiments, at least 90% of the immunoglobulin is IgG immunoglobulin. In some embodiments, at least 95% of the immunoglobulin is IgG immunoglobulin.

[0025] In some embodiments, 5-20% of the immunoglobulin is a dimer. In some embodiments, 5-10% of the immunoglobulin is a dimer. In some embodiments, at least 80% of the immunoglobulin is a monomer or a dimer. In some embodiments, at least 85% of the immunoglobulin is a monomer or a dimer. In some embodiments, at least 90% of the immunoglobulin is a monomer or a dimer. In some embodiments, 5-20% of the IgG immunoglobulin is a dimer. In some embodiments, 5-10% of the IgG immunoglobulin is a dimer. In some embodiments, at least 80% of the IgG immunoglobulin is a monomer or a dimer. In some embodiments, at least 85% of the IgG immunoglobulin is a monomer or a dimer. In some embodiments, at least 90% of the IgG immunoglobulin is a monomer or a dimer.

[0026] At least 60%, 70%, 80%, 90%, or 95% of the branched glycans on IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding.

[0027] In some embodiments, the pH is 4.0–5.5, 4.0–4.5, 4.5–5.0, 5.0–5.5, 4.2–4.7, 4.7–5.3, or 5.1–5.3. In some embodiments, the pH is 4.0–5.5, 4.0–4.5, 4.5–5.0, 5.0–5.5, 4.2–4.7, 4.7–5.3, or 5.1–5.3. In some embodiments, the liquid pharmaceutical composition contains 5% (w / v) sorbitol and has a pH of 5.2–5.5 or 5.3–5.4. In some embodiments, the pH is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0028] In some embodiments, the composition has fewer than 1,000 particles having a diameter of 10 to 100 micrometers after being stirred at 1,000 RPM for 8 hours at 2 to 8°C. In some embodiments, the composition has fewer than 500 particles having a diameter of 10 to 100 micrometers after being stirred at 1,000 RPM for 8 hours at 2 to 8°C. In some embodiments, the composition has fewer than 200 particles having a diameter of 10 to 100 micrometers after being stirred at 1,000 RPM for 8 hours at 2 to 8°C.

[0029] In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on immunoglobulins are dicialized by NeuAc-α2,6-Gal terminal binding after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc region of immunoglobulins are dicialized by NeuAc-α2,6-Gal terminal binding after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. In some embodiments, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of immunoglobulins are dicialized by NeuAc-α2,6-Gal terminal binding after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0030] In some embodiments, 5–10% of the immunoglobulin is a dimer after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0031] In some embodiments, at least 85% of the immunoglobulin is monomer or dimer after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. In some embodiments, at least 90% of the immunoglobulin is monomer or dimer after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. In some embodiments, the formulation is stable for at least 7 months at 5°C, at least 1 month at 25°C, 2 years at 2–8°C, and / or 2 weeks at 15–30°C.

[0032] In some embodiments, storage is in a sealed United States Pharmacopeia Type 1 glass vial. In some embodiments, storage is in a sealed 2R Type 1 glass injection vial.

[0033] Pre-filled syringes containing liquid pharmaceutical compositions are also described herein.

[0034] In some embodiments, the liquid pharmaceutical composition is frozen.

[0035] Methods for treating a disease, comprising administering the liquid pharmaceutical composition described in any of the preceding claims in a dose that is 1% to 10% of the effective dose of IVIG for treating the disease, are also described herein.

[0036] In some embodiments, the hsIgG preparation is administered in doses of 5 mg / kg to 100 mg / kg.

[0037] In some embodiments, the disease is an inflammatory disease. In some embodiments, the subject suffers from an antibody deficiency. In some embodiments, the subject suffers from a primary antibody deficiency. In some embodiments, the disease is associated with the presence of autoantibodies.

[0038] In some embodiments, the dose of hsIVIG is as effective as the effective dose of IVIG. In some embodiments, the hsIgG preparation is administered at the same frequency as the effective dose of IVIG.

[0039] In some embodiments, the disease is a neurological disorder. In some embodiments, the neurological disorder is selected from the group consisting of dermatomyositis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), myasthenia gravis, and generalized rigidity syndrome.

[0040] In some embodiments, the disease is selected from the group consisting of immune cytopenia, parvovirus B19-associated erythropoiesis, secondary hypogammaglobulinemia in myeloma and chronic lymphocytic leukemia, and post-bone marrow transplantation.

[0041] In some embodiments, the disease is selected from the group consisting of vasculitis, systemic lupus erythematosus (SLE), mucosal pemphigoid, and uveitis, and is most commonly used in dermatology to treat Kawasaki syndrome, dermatomyositis, toxic epidermal necrolysis, and bullous diseases.

[0042] In some embodiments, the disease is approved by the FDA for treatment with IVIG, or IVIG is indicated for the treatment of the disease.

[0043] In some embodiments, the hsIgG formulation is 1% to 10% of the FDA-approved dose of IVIG for the disease.

[0044] In some embodiments, the disease is myocarditis, acute motor axonal neuropathy, painful steatosis, anti-glomerular basement nephritis, Goodpasture syndrome, antiphospholipid syndrome (APS, APLS), anti-synthetase syndrome, myositis, ILD, ataxic neuropathy (acute and chronic), autoimmune enteropathy (AIE), autoimmune neutropenia, autoimmune retinopathy, autoimmune thyroiditis, autoimmune urticaria, herpetiform dermatitis, acquired epidermolysis bullosa, essential mixed cryoglobulinemia, granulomatosis with polyangiitis (GPA), mixed connective tissue disease The following conditions are selected from: 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, bullous pemphigoid of pregnancy, Graves' disease, Guillain-Barré syndrome, IgG4-related disease, Lambert-Eaton myasthenia gravis, lupus nephritis, myositis, multifocal motor neuropathy, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, polymyositis, systemic lupus erythematosus (SLE), and combinations thereof.

[0045] In some embodiments, the disease is acute disseminated encephalomyelitis (ADEM), autoimmune angioedema (acquired angioedema type II), autoimmune hepatitis (types I and 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), Mollen's ulcer, focal scleroderma, opsoclonus-myoclonus syndrome, Ord's thyroiditis The following conditions are selected from the group consisting of thyroiditis, recurrent rheumatoid arthritis, paraneoplastic opsoclonus-myoclonus ataxia with neuroblastoma, pediatric autoimmune neuropsychiatric disorder associated with Streptococcus (PANDAS), post-pericardiotomy syndrome, primary biliary cirrhosis (PBC), Rasmussen's encephalitis, rheumatoid vasculitis, Schnitzler syndrome, Sydenham's chorea, undifferentiated connective tissue disease (UCTD), and Miller-Fischer syndrome, as well as combinations thereof.

[0046] Methods for treating CIDP in subjects having CIDP, comprising administering an hsIgG preparation in an effective dose of 10% or less than 10% of the effective dose of IVIG, are also described herein. In some embodiments, the effective dose for treating CIDP with IVIG is 200 to 2000 mg / kg. In some embodiments, the hsIgG preparation is administered in an effective dose of 10% or less of the effective dose for treating CIDP IVIG. In some embodiments, the hsIgG preparation is administered in a dose of 1% of the effective dose for treating CIDP IVIG. In some embodiments, the hsIgG preparation is administered in doses of approximately 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.

[0047] Also described herein are methods for treating ITP in subjects having ITP, comprising administering an hsIgG preparation in an effective dose that is 10% or less than 10% of the effective dose of IVIG. In some embodiments, the effective dose for treating ITP with IVIG is 1000 to 2000 mg / kg. In some embodiments, the hsIgG preparation is administered in an effective dose that is 10% or less than the effective dose for treating ITP with IVIG. In some embodiments, the hsIgG preparation is administered in a dose of 1% to 5% of the effective dose for treating ITP with IVIG. In some embodiments, the hsIgG preparation is administered in 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.

[0048] Also described herein are methods for treating wAIHA in subjects having wAIHA, comprising administering an hsIgG preparation in an effective dose that is 10% or less than 10% of the effective dose of IVIG.

[0049] In some embodiments, the effective dose for treating wAIHA with IVIG is 1000 mg / kg. In some embodiments, the hsIgG preparation is administered at a dose of less than 10% of the effective dose for treating wAIHA with IVIG. In some embodiments, the hsIgG preparation is administered at a dose of 1% to 5% of the effective dose for treating wAIHA with IVIG. In some embodiments, the hsIgG preparation is administered at a dose of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg.

[0050] Also described herein are methods for treating Guillain-Barré syndrome in subjects having the syndrome, comprising administering an hsIgG preparation in an effective dose that is 10% or less than 10% of the effective dose of IVIG. In some embodiments, the effective dose for treating Guillain-Barré syndrome with IVIG is 1000 to 2000 mg / kg. In some embodiments, the hsIgG preparation is administered in a dose of less than 10% of the effective dose for treating Guillain-Barré syndrome with IVIG. In some embodiments, the hsIgG preparation is administered in a dose of 1% to 5% of the effective dose for treating Guillain-Barré syndrome with IVIG. In some embodiments, the hsIgG preparation is administered in a dose of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / kg.

[0051] Also described herein are methods for treating primary humoral immunodeficiency disease (PID) in subjects having PID, comprising administering an hsIgG preparation in an effective dose of 10% or less than 10% of the effective dose of IVIG. In some embodiments, the effective dose for treating PID with IVIG is 200 to 800 mg / kg. In some embodiments, the hsIgG preparation is administered in a dose of less than 10% of the effective dose for treating PID with IVIG. In some embodiments, the hsIgG preparation is administered in a dose of 1% to 5% of the effective dose for treating PID with IVIG. In some embodiments, the hsIgG preparation is administered in a dose of approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / kg.

[0052] A method for treating Kawasaki disease in a subject with Kawasaki disease, comprising administering an hsIgG preparation at an effective dose of 10% or less than 10% of the effective dose of IVIG, is also described herein.

[0053] In some embodiments, the effective dose for treating Kawasaki disease with IVIG is 1000–2000 mg / kg. In some embodiments, the hsIgG preparation is administered at a dose of less than 10% of the effective dose for treating Kawasaki disease with IVIG. In some embodiments, the hsIgG preparation is administered at a dose of 1%–5% of the effective dose for treating Kawasaki disease with IVIG. In some embodiments, the hsIgG preparation is administered at a dose of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / kg.

[0054] In some embodiments of the methods described herein, the dose of the pharmaceutical composition has an efficacy similar to that of an effective dose of IVIG.

[0055] In some embodiments of any of the methods described herein, at least one side effect resulting from an effective dose of IVIG is mitigated by administering the pharmaceutical composition.

[0056] In some embodiments of any of the methods described herein, the pharmaceutical composition is administered subcutaneously.

[0057] A syringe suitable for subcutaneous injection containing a pharmaceutical composition according to any one of the above claims in a quantity of 2 mL or less is also described herein.

[0058] Also described herein are liquid pharmaceutical compositions comprising immunoglobulin in 10 mM sodium acetate and at least one of about 250 mM glycine or about 5% (w / v) sorbitol, wherein at least 50% of the branched glycans on the Fc region of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds, and the pH of the composition is 4 to 7. In some embodiments, the liquid pharmaceutical composition comprises immunoglobulin in 10 mM sodium acetate. In some embodiments, the liquid pharmaceutical composition further comprises 0.02% (w / v) polysorbate 20. In some embodiments, the liquid pharmaceutical composition comprises 250 mM glycine. In some embodiments, the liquid pharmaceutical composition further comprises 5% (w / v) sorbitol. In some embodiments, the pH is 4.0–5.5, 4.0–4.5, 4.5–5.0, 5.0–5.5, 4.2–4.8, 4.7–5.3, or 5.1–5.3. In some embodiments, the pH is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0059] In various embodiments, the immunoglobulin concentration is 50-250 mg / mL, the immunoglobulin concentration is 70-130 mg / mL, the immunoglobulin concentration is 80-120 mg / mL, the immunoglobulin concentration is 90-110 mg / mL, and at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc region of the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal binding, and at least 60%, 70%, 80%, or 90% of the branched glycans on the immunoglobulin Alternatively, 95% are diciallylated by NeuAc-α2,6-Gal terminal binding, and at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab region of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding, and at least 90% of the immunoglobulin is IgG immunoglobulin, and at least 95% of the immunoglobulin is IgG immunoglobulin, and 5-20% of the immunoglobulin is dimer, and 5-10% of the immunoglobulin is dimer, and immunoglobulin At least 80% of phosphorus is monomers or dimers, at least 85% of immunoglobulins are monomers or dimers, at least 90% of immunoglobulins are monomers or dimers, 5-20% of IgG immunoglobulins are dimers, 5-10% of IgG immunoglobulins are dimers, at least 80% of IgG immunoglobulins are monomers or dimers, at least 85% of IgG immunoglobulins are monomers or dimers, and at least 90% of IgG immunoglobulins are monomers or dimers. It is a nomer or dimer, and at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc region of IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding, and at least 60%, 70%, 80%, 90%, or 95% of the branched chain glycans on IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal binding, and at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab region of IgG immunoglobulin are NeuAc-α2,Disialylated by 6-Gal terminal bonding, the composition has fewer than 1000 particles with a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C, the composition has fewer than 500 particles with a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C, the composition has fewer than 200 particles with a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C, and branched glycans on the Fc region of immunoglobulins. At least 60%, 70%, 80%, 90%, or 95% of the can is dicialized by NeuAc-α2,6-Gal terminal binding after being stored at -70°C to 40°C, -70°C to 25°C, 0°C to 5°C, 0°C to 25°C, or 0°C to 40°C, or approximately -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months, and immunoglobulin At least 60%, 70%, 80%, 90%, or 95% of the branched glycans on robulin are dicial by NeuAc-α2,6-Gal terminal linkage after storage at -70°C to 40°C, -70°C to 25°C, 0°C to 5°C, 0°C to 25°C, or 0°C to 40°C, or approximately -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. It is ionized, and at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab region of the immunoglobulin are ionized, and after storage at -70°C to 40°C, -70°C to 25°C, 0°C to 5°C, 0°C to 25°C, or 0°C to 40°C, or approximately -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months, NeuAc-α2,It is disialylated by a 6-Gal terminal bond, and 5-10% of the immunoglobulin is vaccinated at -70°C to 40°C, -70°C to 25°C, 0°C to 5°C, 0°C to 25°C, or 0°C to 40°C, or approximately -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or 1, 2, 3, 4, 6, After storage for 8, 10, 12, 14, 16, 18, 20, 22, or 24 months, the dimer was present, and at least 85% of the immunoglobulin was present at -70°C to 40°C, -70°C to 25°C, 0°C to 5°C, 0°C to 25°C, or 0°C to 40°C, or approximately -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 2 After storage for 2 or 24 weeks, or 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months, the immunoglobulin is monomer or dimer, and at least 90% of the immunoglobulin is monomer or dimer after storage at -70°C to 40°C, -70°C to 25°C, 0°C to 5°C, 0°C to 25°C, or 0°C to 40°C, or approximately -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months, and the storage is in sealed United States Pharmacopeia Type 1 glass vials, and the storage is in sealed 2R Type 1 glass injection vials. ,

[0060] In hsIgG, at least 50% (e.g., 60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98%, and up to 100%) of the branched glycans on the Fc region of the immunoglobulin have sialic acid residues on both the α1,3 and α1,6 arms (i.e., they are diciallylated by NeuAc-α2,6-Gal terminal linkages). In some embodiments, in addition to Fc sialylation, at least 50% (e.g., 60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98%, or up to 100%) of the branched glycans on the Fab region are diciallylated by NeuAc-α2,6-Gal terminal linkages. In some cases, at least 85% (including 87%, 90%, 92%, 94%, 95%, 97%, 98%, or 100%) of the entire branched glycan (total glycans on the Fc domain and Fab domain) is dicialized by NeuAc-α2,6-Gal terminal binding. In some embodiments, less than 50% (e.g., less than 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%) of the branched glycan on the Fc region is monosialized via NeuAc-α2,6-Gal terminal binding (e.g., sialized only on the α1,3 arm or α1,6 arm). The HsIgG formulation is primarily an IgG antibody (e.g., at least 80%, 85%, 90%, or 95% by weight of the immunoglobulin is an IgG antibody of various isotypes).

[0061] As used herein, the term “Fc region” refers to a dimer of two “Fc polypeptides,” each “Fc polypeptide” containing the constant region of the antibody excluding the CH1 domain. In some embodiments, the “Fc region” contains two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. The “Fc polypeptide” refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include some or all of the flexible hinges present at the N-terminus of these domains.

[0062] As used herein, “glycan” is a sugar and may be a monomer or polymer of sugar residues, such as at least three sugars, and may be linear or branched. “Glycan” may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo N-acetylglucosamine, etc.). The term “glycan” includes homopolymers and heteropolymers of sugar residues. The term “glycan” also includes the glycan component of complex carbohydrates (e.g., polypeptides, glycolipids, proteoglycans, etc.). The term also includes free glycans, which include glycans that have been cleaved or otherwise released from complex carbohydrates.

[0063] As used herein, the term “glycoprotein” refers to a protein containing a peptide backbone covalently linked to one or more sugar moieties (i.e., glycans). Sugar moieties may be in the form of monosaccharides, disaccharides, oligosaccharides, and / or polysaccharides. Sugar moieties may contain a single unbranched sugar residue or one or more branched chains. Glycoproteins may contain O-linked sugar moieties and / or N-linked sugar moieties.

[0064] IVIg is a pooled polyvalent immunoglobulin preparation containing all four IgG isotypes, extracted from the plasma of at least 1,000 human donors. Forms of IVIg approved for use in the United States include Gammagard (Baxter Healthcare Corporation), Gammaplex (Bio Products Laboratory), Bivigam (Biotest Pharmaceuticals Corporation), Carimmune NF (CSL Behring AG), Gamunes-C (Grifols Therapeutics, Inc.), Glebogamma DID (Instituto Grifols, SA), and Octagam (Octapharma Pharmazeutika Produktionsges Mbh). IVIg is approved as a plasma protein replacement therapy for immunocompromised patients and other uses. The level of IVIg Fc glycan sialylation varies among IVIg preparations but is generally less than 20%. The level of dicialylation is generally much lower.

[0065] As used herein, “N-glycosylation site of Fc polypeptide” refers to an amino acid residue within the Fc polypeptide to which a glycan is N-linked. In some embodiments, the Fc region contains a dimer of the Fc polypeptide, and the Fc region contains one or two N-glycosylation sites on each Fc polypeptide.

[0066] As used herein, “percentage of branched glycans (%)” refers to the number of moles of glycan X relative to the total moles of glycans present, where X represents the glycan in question.

[0067] The terms “medically effective amount” or “therapeutic amount” refer to an amount (e.g., dose) that is effective in treating a patient with any of the diseases or conditions described herein. It should also be understood that “medically effective amount” may be interpreted herein as an amount that, when administered alone or in combination with other therapeutic agents, or when taken in any dose or route, produces the desired therapeutic effect.

[0068] "Pharmaceutical preparations" and "pharmaceutical products" may be included in a kit containing a preparation or product and instructions for use.

[0069] "Pharmaceutical formulations" and "pharmaceutical products" generally refer to compositions in which a final predetermined level of sialylation is achieved and which are free of process impurities. Therefore, "pharmaceutical formulations" and "pharmaceutical products" are substantially free of ST6Gal sialyltransferase and / or sialic acid donors (e.g., cytidine 5'-monophosphone-N-acetylneuraminic acid) or their by-products (e.g., cytidine 5'-monophosphate).

[0070] "Pharmaceutical preparations" and "pharmaceutical products," in general, in the case of recombinants, substantially do not contain other components of the cell in which the glycoprotein was produced (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA).

[0071] "Purified" (or "isolated") refers to a polynucleotide or polypeptide that is removed or separated from other components present in the natural environment. For example, an isolated polypeptide is one that has been isolated from other components of the cell in which it was produced (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA). An isolated polynucleotide is one that has been isolated from other nuclear components (e.g., histones) and / or upstream or downstream nucleic acids. An isolated polynucleotide or polypeptide does not have to contain 60%, or at least 75%, or at least 90%, or at least 95% of the other components present in the natural environment of the indicated polynucleotide or polypeptide.

[0072] As used herein, the term “sialylated” refers to a glycan having a terminal sialic acid. The term “monosiallylated” refers to a branched glycan having one terminal sialic acid, e.g., an α1,3 arm or an α1,6 arm. The term “diciallylated” refers to a branched glycan having terminal sialic acids in both arms, e.g., an α1,3 arm and an α1,6 arm.

[0073] Throughout this application, various embodiments may be presented in scope form. It should be understood that the scope form description is merely for convenience and brevity and should not be interpreted as a firm limitation on the scope of this disclosure. Therefore, the scope description should be considered to specifically disclose all possible sub-scopes and the individual numbers within those scopes. For example, a scope description such as 1-6 should be considered to have specifically disclosed sub-scopes such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within those scopes, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the scope.

[0074] As used herein and in the claims, the singular forms "a," "an," and "the" also include the plural forms unless the context clearly indicates otherwise. For example, the term "sample" includes multiple samples, including a mixture thereof.

[0075] The terms “determining,” “measuring,” “evaluating,” “assessing,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detection). These terms may include quantitative, qualitative, or quantitative and qualitative determinations. Evaluations may be relative or absolute. “Detecting the presence” may include determining the quantity of something that is present, in addition to determining whether it is present or not, depending on the context.

[0076] As used herein, the term “approximately” refers to the number obtained by adding or subtracting 10% from that number. The term “approximately” refers to the range obtained by subtracting 10% of its lowest value and adding 10% of its highest value from that range.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Methods and materials for use in the invention are described herein, but other preferred methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. In case of any inconsistency, this specification shall prevail, including definitions.

[0078] Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0079] [Figure 1] A schematic example of a branched glycan is shown. The light circle is Gal, the dark circle is Man, the triangle is Fuc, the rhombus is NANA, and the square is GlcNAc. [Figure 2] This diagram shows the enzymatic sialylation reaction for converting pooled immunoglobulins to hsIgG (left), and schematic diagrams of IgG Fc glycan profiles for initial IVIg and hsIgG enzymatically prepared from IVIg (right). Glycan profiles for different IgG subclasses are derived by glycopeptide mass spectrometry. The peptide sequences used for quantification of glycopeptides of different IgG subclasses were IgG1=EEQYNSTYR (SEQ ID NO: 1), IgG2 / 3 EEQFNSTFR (SEQ ID NO: 2), IgG3 / 4 EEQYNSTFR (SEQ ID NO: 3), and EEQFNSTYR (SEQ ID NO: 4). [Figure 3] The aggregate curves are superimposed for solutions of M254 at various concentrations from 100 mg / mL to 250 mg / mL, measured using scattered light intensity at 473 nm over a temperature gradient from 20°C to 90°C. [Modes for carrying out the invention]

[0080] Immunoglobulins are glycosylated at conserved sites within the constant region of their heavy chain. For example, human IgG has a single N-linked glycosylation site at Asn297 in the CH2 domain. Each immunoglobulin type has a different variety of N-linked carbohydrate structures within the constant region. In the case of human IgG, the core oligosaccharide typically consists of GlcNAc2Man3GlcNAc with a different number of outer residues. Variations between individual IgGs can arise through the binding of galactose and / or galactose-sialic acid in one or both of the terminal GlcNAc, or through the binding of a third GlcNAc arm (bisected GlcNAc).

[0081] This disclosure partially encompasses pharmaceutical formulations comprising pooled human immunoglobulin having an Fc region, wherein both branched glycans within the Fc region have a specific level of sialylated branched glycans (e.g., with NeuAc-α2,6-Gal terminal binding).

[0082] Immunoglobulins Preparations of pooled polyvalent human immunoglobulins, including IVIg preparations, are highly complex due to their significant heterogeneity in several respects. These include immunoglobulins pooled from hundreds or even over 1,000 individuals. While at least approximately 90% or 95% of the immunoglobulins are IgG isotypes (all subclasses), other isotypes, including IgA and IgM, are present. Immunoglobulin preparations in IVIg and pooled polyvalent human immunoglobulin preparations vary in both specificity and glycosylation patterns.

[0083] Hypersialylation of pooled polyvalent immunoglobulins alters the glycans present on the immunoglobulins. For some glycans, the alteration involves the addition of one or more galactose molecules and one or more sialic acid molecules. For other glycans, the alteration involves the addition of only one or more sialic acid molecules. Furthermore, although essentially all IgG antibodies, the dominant immunoglobulin in a pooled polyvalent immunoglobulin formulation has glycosylation sites on each polypeptide forming the Fc region, and not all IgG antibodies have glycosylation sites on the Fab domain. Altering the glycosylation of immunoglobulin formulations alters the structure and activity of individual immunoglobulins in the formulation, and importantly, alters the interactions between individual immunoglobulins, as well as the bulk behavior of the immunoglobulin formulation.

[0084] The widely used formulations for IVIg preparations are completely unsuitable for hypersialylated immunoglobulin (hsIgG) formulations, at least when used for hsIgG, because these formulations are not stable against the shear stress that occurs during the normal transport of pharmaceutical formulations. When subjected to this type of shear stress, quasi-visible particles are formed in the hsIgG formulation. Such quasi-visible particles in antibody formulations are known to cause serious adverse events at the injection site and off-target immune responses. Quasi-visible particles in antibody formulations may also activate the complement system, causing embolism and other negative immunogenic reactions. It has been found that the addition of nonionic surfactants makes hsIgG formulations more stable against shear stress and significantly reduces the formation of quasi-visible particles.

[0085] Naturally occurring polypeptides that can be used to prepare hsIgG include, for example, immunoglobulins isolated from pooled human serum. HsIgG can also be prepared from IVIg and IVIg-derived polypeptides. HsIgG can be prepared as described in International Publication 2014 / 179601. The preparation of hsIgG is also described in Washburn et al (Proc Natl Acad Sci USA. 2015 Mar 17;112(11):E1297-306). The level of sialylation in hsIgG formulations can be measured on the Fc domain (e.g., the number of sialylated branched glycans in the α1,3 arms, α1,6 arms, or both of the branched glycans within the Fc domain), or by overall sialylation (e.g., the number or percentage of sialylated branched glycans in the α1,3 arms, α1,6 arms, or both of the branched glycans in the polypeptide formulation, regardless of whether it is in the Fc domain or the Fab domain).

[0086] In some cases, pooled serum used as a source of immunoglobulins for preparing hsIgG is isolated from a specific population of individuals that produces antibodies against one or more viruses, such as COVID-19, SARS, parainfluenza, and influenza, but does not have an active infection. In some cases, immunoglobulins are isolated from populations of individuals in which more than 50%, 55%, 60%, or 75% produce antibodies against selected viruses.

[0087] N-linked oligosaccharide chains are attached to proteins within the lumen of the endoplasmic reticulum. Specifically, an initial oligosaccharide (typically 14-saccharide) is attached to the amino group on the side chain of an asparagine residue contained within the Asn-X-Ser / Thr target consensus sequence, where X can be any amino acid other than proline. The structure of this initial oligosaccharide is common to most eukaryotes and contains three glucose, nine mannose, and two N-acetylglucosamine residues. This initial oligosaccharide chain can be trimmed by a specific glycosidase enzyme in the endoplasmic reticulum to obtain a short, branched core oligosaccharide consisting of two N-acetylglucosamine and three mannose residues. One of the branches is referred to in the art as the "α1,3 arm," as shown in Figure 1, and the other branch is referred to as the "α1,6 arm."

[0088] N-glycans can be subdivided into three distinct groups called "high-mannose type," "hybrid type," and "complex type," and a common pentasaccharide core (Man(alpha1,6)-(Man(alpha1,3))-Man(beta1,4)-GlcpNAc(beta1,4)-GlcpNAc(beta1,N)-Asn) arises in all three groups.

[0089] After initial processing in the endoplasmic reticulum, polypeptides are transferred to the Golgi apparatus, where further processing can occur. If glycans are transferred to the Golgi before being completely trimmed into their core pentasaccharide structure, "high-mannose glycans" are obtained.

[0090] Additionally or alternatively, one or more monosaccharide units of N-acetylglucosamine may be added to the coremannose subunit to form a "complex glycan." Galactose may be added to the N-acetylglucosamine subunit, or a sialic acid subunit may be added to the galactose subunit, resulting in a chain with one of the sialic acid, galactose, or N-acetylglucosamine residues at the end. Additionally, a fucose residue may be added to the N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by a specific glycosyltransferase.

[0091] "Hybrid glycans" possess characteristics of both high-mannose and complex glycans. For example, one branch of a hybrid glycan may contain primarily or exclusively mannose residues, while another branch may contain N-acetylglucosamine, sialic acid, galactose, and / or fucose sugars.

[0092] Sialic acids are a family of nine-carbon monosaccharides with heterocyclic structures. They possess a negative charge via a carboxylic acid group attached to the ring, as well as other chemical embellishments including N-acetyl and N-glycolyl groups. The two main types of sialyl residues found in polypeptides produced in mammalian expression systems are N-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). These typically arise as terminal structures attached to galactose (Gal) residues at the non-reducing ends of both N- and O-linked glycans. The glycosidic bond configuration for these sialyl groups can be either α2,3 or α2,6.

[0093] The Fc region is glycosylated at a conserved N-linked glycosylation site. For example, each heavy chain of an IgG antibody is C H The two-domain Asn297 has a single N-linked glycosylation site. The IgA antibody is C H 2 and C Hhas an N-linked glycosylation site in the 3 domain, and the IgE antibody has a C H has an N-linked glycosylation site in the 3 domain, and the IgM antibody has a C H 1, C H 2, C H 3, and C H 4 domain has an N-linked glycosylation site.

[0094] Each antibody isotype has various different N-linked carbohydrate structures in the constant region. For example, IgG has C in each Fc polypeptide of the Fc region, which also contains binding sites for C1q and FcγR H 2 domain has a single N-linked biantennary carbohydrate at Asn297. For human IgG, the core oligosaccharide usually consists of GlcNAc2Man3GlcNAc with different numbers of outer residues. Variation between individual IgGs can arise via the attachment of galactose and / or galactose-sialic acid at one or both terminal GlcNAcs, or the attachment of a third GlcNAc arm (bisecting GlcNAc). Glycans of a polypeptide can be assessed using any method known in the art. For example, sialylation of a glycan composition (e.g., the level of branched glycans sialylated on the α1,3 arm and / or α1,6 arm) can be characterized using the method described in International Publication No. WO2014 / 179601.

[0095] Immunoglobulin composition A composition containing hsIgG can include antibody monomers, dimers, and antibody aggregates, in addition to said components. In some cases, pH can be used to adjust the proportion of monomers, dimers, and aggregates in the composition when measured as percent purity by weight via size exclusion chromatography.

[0096] In some cases, lowering the pH increases the weight percent of monomer + dimer in the solution. In some cases, lowering the pH increases the weight percent of monomer in the solution. In some cases, raising the pH reduces the percent monomer in the solution.

[0097] In some cases, the weight percent aggregate is 3.0% by weight / weight or less (e.g., 2.7, 2.5, 2.3, 2.0, 1.7, 1.5, 1.3, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1% by weight / weight or less).

[0098] In some cases, the monomer + dimer weight percentage is 97.0% or more by weight (e.g., 98% or more by weight, or 99% or more by weight). In some cases, the monomer weight percentage is 80% or more by weight, 83% or more by weight, 85% or more by weight, or 87% or more by weight.

[0099] In some cases, the pH is between 4.0 and 7.0. In some cases, the pH is approximately between 4.0 and 7.0, for example, 4.0 to 6.0, 4.0 to 5.0, 5.0 to 7.0, 5.0 to 6.0, or 6.0 to 7.0.

[0100] In some cases, the pH is 5.5 or less (e.g., 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, or 4.0). In some cases, the pH is 5.2 to 5.5, for example, 5.2 to 5.4, 5.2 to 5.3, 5.3 to 5.5, 5.3 to 5.4, or 5.4 to 5.5. In some cases, the pH is approximately 5.2 to 5.5, for example, approximately 5.2 to 5.4, approximately 5.2 to 5.3, approximately 5.3 to 5.5, approximately 5.3 to 5.4, or approximately 5.4 to 5.5. In some cases, the pH is 5.3 or approximately 5.3. In some cases, the pH is 5.4 or approximately 5.4.

[0101] In some cases, pH is measured before filling. In some cases, pH is measured after filtration. In some cases, pH is measured within one week or approximately one week after filtration.

[0102] In some cases, pH is measured after filtration and storage at temperatures below 40°C, for example, 35°C, 30°C, 25°C, 20°C, 15°C, 10°C, and 5°C. In some cases, pH is measured after filtration and storage at temperatures below approximately 40°C, for example, approximately 35°C, 30°C, 25°C, 20°C, 15°C, 10°C, 5°C, and 0°C. In some cases, pH is measured after filtration and storage at -70°C or approximately -70°C.

[0103] In some cases, the pH after filtration and at -70°C to 40°C, for example, -70 to 40, -70 to 35, -70 to 30, -70 to 25, -70 to 20, -70 to 15, -70 to 10, -70 to 5, -70 to 0, 0 to 40, 0 to 35, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, 5 to 35, 5 to 30, 5 to 25, 5 to 20. Measurements are taken after storage at 5-15, 5-10, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35, or 35-40°C. In some cases, the pH after filtration and at approximately -70°C to approximately 40°C, for example, approximately -70 to approximately 40, approximately -70 to approximately 35, approximately -70 to approximately 30, approximately -70 to approximately 25, approximately -70 to approximately 20, approximately -70 to approximately 15, approximately -70 to approximately 10, approximately -70 to approximately 5, approximately -70 to approximately 0, approximately 0 to approximately 40, approximately 0 to approximately 35, approximately 0 to approximately 30, approximately 0 to approximately 25, approximately 0 to approximately 20, approximately 0 to approximately 15, approximately 0 to approximately 10, approximately 0 to approximately 5, approximately 5 to approximately 35, approximately 5 to approximately 30, approximately 5 to approximately 25, approximately 5 to approximately 20, approximately Measurements are taken after storage at approximately 5-15°C, 5-10°C, 10-40°C, 10-35°C, 10-30°C, 10-25°C, 10-20°C, 10-15°C, 15-40°C, 15-35°C, 15-30°C, 15-25°C, 15-20°C, 20-40°C, 20-35°C, 20-30°C, 20-25°C, 25-40°C, 25-35°C, 25-30°C, 30-40°C, or 30-35°C.

[0104] In some cases, the pH of the pharmaceutical composition is adjusted so that the weight percentage of the monomer is modified. In other cases, the pH of the pharmaceutical composition is decreased so that the weight percentage of the monomer increases.

[0105] In some cases, the pH of the pharmaceutical composition increases as the weight percent of the dimer increases.

[0106] In some cases, the pH is such that the monomer weight percentage is 85% by weight or higher (e.g., 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% by weight or higher).

[0107] In some cases, the composition is a high-concentration hsIgG composition. In some cases, the concentration of hsIgG in the composition is 100 mg / mL to 275 mg / mL, for example, 100 to 250, 100 to 200, 100 to 175, 100 to 125, 125 to 275, 125 to 250, 125 to 200, 125 to 175, 175 to 275, 175 to 250, 175 to 200, 200 to 275, 200 to 250, or 250 to 275 mg / mL. In some cases, the concentration of hsIgG in the composition is approximately 100 mg / mL to approximately 275 mg / mL, for example, approximately 100 to approximately 250, approximately 100 to approximately 200, approximately 100 to approximately 175, approximately 100 to approximately 125, approximately 125 to approximately 275, approximately 125 to approximately 250, approximately 125 to approximately 200, approximately 125 to approximately 175, approximately 175 to approximately 275, approximately 175 to approximately 250, approximately 175 to approximately 200, approximately 200 to approximately 275, approximately 200 to approximately 250, or approximately 250 to approximately 275 mg / mL. In some cases, the concentration of hsIgG in the concentration is 100, 125, 175, 200, 250, or 275 mg / mL, or approximately 100, 125, 175, 200, 250, or 275 mg / mL.

[0108] In some cases, the composition includes one or more buffering agents. In some cases, the buffering agents are selected from the group consisting of sodium acetate, histidine, sodium phosphate, and combinations thereof.

[0109] In some cases, the composition includes a tension modifier. In some cases, the tension modifier is selected from the group consisting of glycine, sorbitol, and combinations thereof.

[0110] In some cases, the composition contains a surfactant. In some cases, the surfactant is polysorbate. In some cases, the polysorbate is polysorbate 20.

[0111] In some cases, the composition includes both a buffer and a tension modifier. In some cases, the composition includes a buffer, a tension modifier, and a polysorbate (e.g., polysorbate 20).

[0112] In some cases, the composition comprises a sodium acetate buffer and a sorbitol tonicity modifier. In some cases, the composition comprises a sodium acetate buffer, a sorbitol tonicity modifier, and a polysorbate (e.g., polysorbate 20).

[0113] In some cases, the buffering agent is present in the composition at a concentration of 5 to 20 mM, for example, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 mM. In some cases, the buffering agent is present in the composition at a concentration of about 5 to about 20 mM, for example, about 5 to about 15, about 5 to about 10, about 10 to about 20, about 10 to about 15, or about 15 to about 20 mM. In some cases, the buffering agent is present in the composition at a concentration of 10 mM or about 10 mM.

[0114] In some cases, polysorbate 20 is present in the composition at a concentration of 0.01% to 0.03%, for example, 0.01% to 0.02% or 0.02% to 0.03%. In some cases, polysorbate 20 is present in the composition at a concentration of approximately 0.01% to approximately 0.03%, for example, approximately 0.01% to approximately 0.02% or approximately 0.02%. In some cases, polysorbate 20 is present in the composition at a concentration of 0.02% or approximately 0.02%.

[0115] In some cases, glycine is present in the composition at concentrations of 200–300 mM, for example, 225–300, 225–275, 225–250, 250–300, 250–275, or 275–300 mM. In some cases, glycine is present in the composition at concentrations of about 200–about 300 mM, for example, about 225–about 300, about 225–about 275, about 225–about 250, about 250–about 300, about 250–about 275, or about 275–about 300 mM. In some embodiments, glycine is present in the composition at concentrations of 250 mM or about 250 mM.

[0116] In some cases, sorbitol may be present in the composition at 1% to 10% (w / v), for example, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10% (w / v). In some cases, sorbitol is present in concentrations of approximately 1% to 10% (w / v), for example, approximately 1 to 9, approximately 1 to 8, approximately 1 to 7, approximately 1 to 6, approximately 1 to 5, approximately 1 to 4, approximately 1 to 3, approximately 1 to 2, approximately 2 to 10, approximately 2 to 9, approximately 2 to 8, approximately 2 to 7, approximately 2 to 6, approximately 2 to 5, approximately 2 to 4, approximately 2 to 3, approximately 3 to 10, approximately 3 to 9, approximately 3 to 8, approximately 3 to 7, approximately 3 to 6. It is present in the composition at approximately 3-5%, 3-4%, 4-10%, 4-9%, 4-8%, 4-7%, 4-6%, 4-5%, 5-10%, 5-9%, 5-8%, 5-7%, 5-6%, 6-10%, 6-9%, 6-8%, 6-7%, 7-10%, 7-9%, 7-8%, 8-10%, 8-9%, or 9-10% (w / v). In some cases, sorbitol is present in the composition at 5% or approximately 5%.

[0117] Storage of immunoglobulin compositions In some cases, the hsIgG compositions described herein are stored under various conditions before administration.

[0118] In some cases, the hsIgG compositions described herein retain dicialylation during storage.

[0119] Accordingly, in some cases, in the hsIgG compositions described herein, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal binding after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0120] In some cases, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans of immunoglobulins are dicialized by NeuAc-α2,6-Gal terminal binding after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0121] In some cases, at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of immunoglobulins are dicialized by NeuAc-α2,6-Gal terminal binding after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0122] In some cases, 5-10% of immunoglobulins are dimers after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0123] In some cases, at least 85% of immunoglobulins are monomers or dimers after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0124] In some cases, at least 90% of immunoglobulins are monomers or dimers after storage at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[0125] In some cases, the composition is stable for at least 7 months at 5°C, at least 1 month at 25°C, for 2 years at 2–8°C, and / or for 2 weeks at 15–30°C.

[0126] In some cases, the compositions described herein are stored in sealed United States Pharmacopeia Type 1 glass vials. In some cases, the hsIgG compositions described herein are stored in sealed 2R Type 1 glass injection vials.

[0127] In some cases, the compositions described herein are provided in a pre-filled syringe.

[0128] Pharmaceutical composition and administration Persialylated IgG can be incorporated into pharmaceutical compositions. For example, a pharmaceutical composition may be formulated by suitably combining hsIgG with pharmaceutically acceptable vehicles or media such as sterile water and saline, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, diluents, vehicles, preservatives, and binders, and then mixing them in unit dose forms required by generally accepted pharmaceutical practice. The amount of active ingredient in the pharmaceutical formulation is such that a suitable dose within a specified range is provided.

[0129] hsIgG can be formulated for intravenous administration. hsIgG can also be formulated for subcutaneous administration.

[0130] Pharmaceutical compositions can be formulated according to conventional pharmaceutical practices using distilled water for injection as a vehicle. For example, a saline or 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 solubilizer, such as an alcohol such as ethanol, a polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as Polysorbate 80 (trademark) or HCO-50.

[0131] Non-limiting examples of oily liquids include sesame oil and soybean oil, which may be combined with benzyl benzoate or benzyl alcohol as a solubilizer. Other items that may be included are buffers such as phosphate buffers or sodium acetate buffers, analgesics such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The prepared injection solution may be packaged in containers such as those described herein.

[0132] The preferred method of administration can be selected based on the patient's age and condition. The preferred dose of hsIgG prepared by the method described herein may be approximately the same as or less than the preferred or approved dose of commercially available IVIg preparations (e.g., less than 20%, 35%, 40%, 50%, 60%, 70%, or 80%). The dose and method of administration may vary depending on the patient's weight, age, condition, etc., and can be appropriately selected by those skilled in the art as needed.

[0133] In some embodiments, the dose is 1% to 10% of the FDA-approved (or other national or international regulatory body) IVIG dose or the effective IVIG dose for the disease. In some embodiments, the FDA-approved (or other national or international regulatory body) dose or the effective IVIG dose is 200 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 1000 mg / kg, or 2000 mg / kg. In some embodiments, the composition containing the hsIgG preparation is administered in doses of approximately 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, the composition containing the hsIgG preparation is administered daily, once a week, twice a week, every other week, monthly, every two weeks, every three days, every four days, every five days, every six days, every seven days, once every 14 days, once every 21 days, once every 28 days, once every two consecutive days in a 28-day cycle, or at the same frequency as the FDA-approved IVIG dose. In some embodiments, the composition is administered as a single dose. In some embodiments, the composition is administered in multiple doses. The dose and method of administration may vary depending on the patient's weight, age, condition, etc., and can be appropriately selected by those skilled in the art as needed.

[0134] As described by Washburn et al., analysis of sialylation by ST6Gal1 revealed that It was revealed that ST6Gal1 not only catalyzes the transfer of sialic acid from CMP-NANA sugar nucleotides to Fc glycans, but can also promote the removal of sialic acid from sialylated products. These reactions are schematically shown in Figure 3.

[0135] As reported by Washburn et al., under specific reaction conditions, sialylation of the α1,3 branch of dibranched glycans (to form A1F-1,3) was rapid and essentially completed in 30 minutes, while the disiallylated species (A2F) formed at approximately 10 times slower rates, with accumulation ceasing after 24 hours. After 24 hours, the monosiallylated species (A1F-1,6) with sialic acid on the α1,6 branch began to form, and accumulation steadily continued, reaching approximately 35% of the glycosylated species by 64 hours. Concurrently, A2F showed a steady decrease from 71% at 20 hours to 44% at 64 hours, suggesting that the A1F-1,6 glycoform was generated from the removal of sialic acid residues on more exposed α1,3 branchings of the disiallylated A2F. Further incubation led to the cleavage of 1,6-sialic acid in the A1F-1,6 glycoform, generating the species G2F, which was not sialylated but fully galactosylated and fucosylated. G2F, present in trace amounts at the start of the reaction, appeared in measurable amounts at 40 hours and continued to increase, reaching 15% at 64 hours as the levels of A2F and A1F-1,6 decreased.

[0136] Washburn et al. further report that these observations highlight the importance of optimizing parameters to maximize A2F species yield while minimizing A1F-1,3, A1F-1,6, and G2F glycoforms. By evaluating a matrix of parameters influencing the transient state of the G2F⇔A1F-1,3⇔A2F⇔A1F-1,6⇔G2F glycoform distribution, it was found that the desialylation component of the reaction (A2F⇔A1F-1,6⇔G2F) was promoted by the spontaneous degradation of CMP-NANA in the reaction. Wasburn et al. concluded that supplementing with CMP-NANA in the sialylation reaction helped maximize the A2F yield, and that periodic administration of fresh CMP-NANA after 24 hours without the formation of significant A1F-1,6 or G2F species maximized the level of A2F glycans. [Examples]

[0137] The present invention is further described in the following embodiments, but these do not limit the scope of the present invention as described in the claims.

[0138] Example 1: Persialylated IgG formulated in different buffers Over 60% of the branched Fc region glycan was disialylated into hypersialylated IgG, which was prepared as generally described in International Publication No. 2014 / 179601.

[0139] In short, IVIg is subjected to a one-pot sequential enzymatic reaction using β1,4-galactosyltransferase 1 (B4-GalT) and α2,6-sialyltransferase (ST6-Gal1) enzymes. The galactosyltransferase enzyme selectively adds galactose residues to the existing asparagine-binding glycan in IVIg. The resulting galactosylated glycan functions as a substrate for sialylatesase, which selectively adds sialic acid residues to cap the asparagine-binding glycan structure bound to IVIg. Therefore, the total sialylation reaction was carried out using two sugar nucleotides (UDPGal and CMP-NANA). The latter was periodically replenished to increase the disialylated product relative to the monosialylated product. The reaction contains manganese chloride as a cofactor.

[0140] Representative examples of the corresponding IgG-Fc glycan profiles for the starting IVIg and reaction products are shown in the right panel of Figure 2. Glycan data is shown per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg, the total of all non-siallylated glycans is over 80%, and the total of all siallylated glycans is <20%. For the reaction products, the total of all non-siallylated glycans is <20%, and the total of all siallylated glycans is greater than 80%. The nomenclature of the different glycans listed in the glycoprofile uses the Oxford notation for N-linked glycans. Figure 2 is a schematic diagram (left) of the enzymatic sialization reaction for converting IVIg to hsIgG; it shows the IgG Fc glycan profiles for starting IVIg and hsIgG. Glycan profiles for different IgG subclasses are derived by glycopeptide mass spectrometry. The peptide sequences used to quantify glycopeptides of different IgG subclasses were IgG1=EEQYNSTYR (SEQ ID NO: 1), IgG2 / 3 EEQFNSTFR (SEQ ID NO: 2), IgG3 / 4 EEQYNSTFR (SEQ ID NO: 3), and EEQFNSTYR (SEQ ID NO: 4) (right).

[0141] Commercially available, non-persialylated IVIg is generally stable in glycine and generally does not form quasi-visible particles when stirred, for example, during transport.

[0142] Example 2: Various hsIgG formulations Stability assays were performed to evaluate the range of liquid formulations and determine the optimal conditions for providing maximum stability of M254 (hypersialized immunoglobulin) at high concentrations. Formulation differences included buffer, pH, and tonicity modifiers, but the concentration and surfactant percentages were kept constant throughout all samples. Various stresses associated with pharmaceutical processing, storage, and transportation were then induced.

[0143] Formulations composed of low-pH sodium acetate buffer were consistently stable over time at various temperatures. In particular, the combination of sodium acetate buffer and sorbitol tonicity modifier showed increased stability compared to the combination of sodium acetate buffer and glycine. Histidine buffer and sodium phosphate buffer had higher pH values, were less stable, and exhibited variability compared to sodium acetate buffer.

[0144] As shown in Table 1, 14 different formulations (F1 to F14) with pH levels ranging from 4.2 to 7.5 at 100 mg / mL were prepared for the tests described herein.

[0145] [Table 1]

[0146] Example 3: Stabilized glycine preparation of hsIgG using different buffers The filtered sample formulations were transferred to glass vials and exposed to four stress temperatures (-70°C, 5°C, 25°C, or 40°C) for 12 weeks. After exposure to stress temperatures, comparable concentrations and pH levels were observed for each formulation compared to their corresponding non-stress conditions. The results are shown in Table 2.

[0147] The sodium acetate buffer formulations (F1, F3), like the controls (F13, F14), contained no visible particles. All other buffer formulations (F5, F7, F9, and F11) showed an increase in milky white color with increasing pH at all temperatures. At 40°C, all formulations showed a yellow color with increasing pH, but contained no visible particles.

[0148] [Table 2]

[0149] Example 4: Stabilized glycine preparations of hsIgG using different buffers at various temperatures The filtered sample formulations were transferred to glass vials and stored at stress temperatures (-70°C, 5°C, 25°C, or 40°C) for 12 weeks. After storage at stress temperatures, the purity of the formulations was tested by SE-HPLC. The results are shown in Tables 3-5.

[0150] The sodium acetate buffer formulation showed less aggregate formation than all other formulations, including the control. F3 showed the least amount of aggregate formation across the entire stress temperature range. F1 showed minimal aggregate formation at all stress temperatures, similar to the control, except at 40°C where aggregate formation increased.

[0151] At time 0, formulations with a pH of 6 or higher showed lower monomer peak percentages compared to formulations with lower pH levels. Formulations with a pH of 5 or lower showed the highest monomer percentages compared to other formulations, consistent with previous studies.

[0152] All formulations showed an increase in dimer peak percentage compared to time 0. As seen in previous studies, a greater increase in dimer formation correlated with an increase in pH. Acetate buffer retained monomer and dimer percentages better under temperature stress than all other formulations, including the control, and exhibited less dimer conversion.

[0153] [Table 3]

[0154] [Table 4]

[0155] [Table 5]

[0156] Example 5: Stability of N-glycans, sialylation, and dicialylation in hsIgG glycine formulations using different buffers. The filtered sample formulations were transferred to glass vials and stored at stress temperatures (-70°C, 5°C, 25°C, or 40°C) for 12 weeks. After storage at stress temperatures, the formulations were tested for purity by HILIC-HPLC. At time 0, all formulations showed comparable A2F peak percentages (68.4%–68.8%). The results are shown in Tables 6–9.

[0157] Storage at -70°C and 5°C yielded equivalent A2F peak percentages (70.0% to 70.9% for the former and 69.9% to 70.4% for the latter), total sialylation (99.5% to 99.6%), monosialylation percentage (6.7% to 7.7%), and dicialylation percentage (91.4% to 92.8%).

[0158] At 25°C, F1 showed a decrease in A2F. All other formulations with a pH of 5.0 or higher showed a slight increase in peak A2F.

[0159] At 40°C, F1 showed a significant change in A2F, most likely due to the lower pH, while formulations with higher pH (>pH 6.5, F9, F11) showed the smallest change in N-glycan peak percentage.

[0160] F3 (sodium acetate buffer) showed similar A2F peaks, total sialylation percentage, monosialylation percentage, and dicialylation percentage to the control at all stress temperatures. F1 (also sodium acetate buffer) showed the greatest variability compared to all other formulations.

[0161] [Table 6]

[0162] [Table 7]

[0163] [Table 8]

[0164] [Table 9]

[0165] Example 6: Charge variants in hsIgG stabilized using various buffers at various stress temperatures. The filtered sample formulations were transferred to glass vials and stored at stress temperatures (-70°C, 5°C, 25°C, or 40°C) for 12 weeks. After storage at stress temperatures, the formulations were tested for changes in charge variants and isoelectric point peaks by imaged capillary isoelectric focusing (icIEF) to understand the formulation stability at different stress temperatures. At time 0, all formulations showed similar combined peak percentages for acidic, neutral, and basic conditions, as well as a major peak isoelectric point around 7.4. All major peak isoelectric points remained around 7.4 regardless of temperature stress. The results are shown in Tables 10-13.

[0166] The formulations showed only slight changes in isoform percentages, and no significant trends were observed at -70°C, 5°C, and 5°C. At 25°C, all formulations (except F14) showed a slight decrease in the basic peak percentage (-0.15 to 3.41%), while corresponding increases were observed in the neutral and acidic percentages.

[0167] At 40°C, F3 (sodium acetate buffer formulation) showed acidic, neutral, and basic peak percentages comparable to the control formulation, with only slight changes from time 0. All formulations showed a decrease in basic peak percentage (except F1), as well as an increase in acidic peak percentage (except F1) and an increase in neutral peak percentage (except F11). F11 (sodium phosphate buffer) showed a large shift in peak percentage from time 0.

[0168] [Table 10]

[0169] [Table 11]

[0170] [Table 12]

[0171] [Table 13]

[0172] Example 7: Stabilized sorbitol preparation of hsIgG The filtered sample formulations were transferred to glass vials and exposed to stress temperatures (-70°C, 5°C, 25°C, or 40°C) for 12 weeks. After exposure to stress temperatures, comparable concentrations and pH levels were observed for each formulation compared to their corresponding non-stress conditions. The results are shown in Table 14.

[0173] The sodium acetate buffer formulations (F2, F4), like the controls (F13, F14), contained no visible particles. All other buffer formulations (F6, F8, F10, and F12) showed an increase in milky white color with increasing pH at all temperatures. At 40°C, all formulations showed a yellow color with increasing pH, but contained no visible particles.

[0174] [Table 14]

[0175] Example 8: Stabilization of hsIgG using various buffers at various temperatures and formulations of sorbitol The filtered sample formulations were transferred to glass vials and stored at stress temperatures (-70°C, 5°C, 25°C, or 40°C) for 12 weeks. After storage at stress temperatures, the purity of the formulations was tested by SE-HPLC. The results are shown in Tables 15-17.

[0176] The sodium acetate buffer formulation showed less aggregate formation than all other formulations, including the control. All sorbitol formulations showed reduced aggregate formation compared to previous studies using 250 mM glycine, and the sodium acetate buffer with 5% sorbitol showed the least amount of aggregate formation.

[0177] After 12 weeks, the lower pH formulation using sorbitol showed minimal aggregation and dimerization (F2, F4).

[0178] At -70°C, 5°C, and 25°C, formulations with a pH of 6 or higher began to show a slight increase in aggregate percentage compared to the result at time 0. At 40°C, all formulations except F2 showed an increase in dimer percentage compared to time 0.

[0179] After 12 weeks, F2 and F4 showed the smallest change in dimer percentage, while the control formulation showed an increase of 3.1%–3.2%. Furthermore, F2 and F4 showed a low flocculation percentage (+1.2%–1.7%), while the control showed more flocculation (3.9%–4.2%). At each pH, ​​formulations containing sorbitol showed less flocculation than formulations containing glycine. Overall, the lower pH formulations containing sorbitol (F2 and F4) showed the smallest change in monomer percentage compared to time 0 (-1.3% and -1.8%, respectively), while all other formulations showed a monomer decrease of more than 3.0%.

[0180] [Table 15]

[0181] [Table 16]

[0182] [Table 17]

[0183] Example 9: Stability of N-glycans, sialylation, and dicialylation % in sorbitol formulations of hsIgG using various buffers. The filtered sample formulations were transferred to glass vials and stored at stress temperatures (-70°C, 5°C, 25°C, or 40°C) for 12 weeks. After storage at stress temperatures, the formulations were tested for purity by HILIC-HPLC. At time 0, all formulations showed comparable A2F peak percentages (68.4%–68.8%). The results are shown in Tables 18–21.

[0184] Storage at -70°C and 5°C yielded equivalent A2F peak percentages (70% to 70.9% for the former and 69.9% to 70.4% for the latter), total sialylation (99.5% to 99.7%), monosialylation percentage (6.8% to 8.1%), and dicialylation percentage (91.5% to 92.9%).

[0185] At 25°C, F2 showed a decrease in A2F. However, this was not as pronounced as in its glycine counterpart formulation. All other formulations with a pH of 5.0 or higher showed a slight increase in peak A2F.

[0186] At 40°C, F2 showed no significant changes in the A2F, total sialylation percentage, monosialylation percentage, and dicialylation percentage peaks, similar to those observed in its glycine counterpart at similar pH levels. F4 (sodium acetate formulation) was comparable to the control, showing only a very minimal decrease in the A2F peak and total sialylation percentage, an increase in monosialylation percentage, and a similar decrease in dicialylation percentage.

[0187] Formulations with higher pH levels showed the smallest change in sialylation, while those with the lowest pH levels showed the largest change.

[0188] [Table 18]

[0189] [Table 19]

[0190] [Table 20]

[0191] [Table 21]

[0192] Example 10: Charge variants in sorbitol formulations that stabilize hsIgG using various buffers at various stress temperatures. The filtered sample formulations were transferred to glass vials and stored at stress temperatures (-70°C, 5°C, 25°C, or 40°C) for 12 weeks. After storage at stress temperatures, the formulations were tested for changes in charge variants and isoelectric focus peaks by imaging capillary isoelectric focusing (icIEF) to understand the formulation stability at different stress temperatures.

[0193] At time 0, all formulations exhibited similar combined peak percentages for acidic, neutral, and basic conditions, as well as a main peak isoelectric point around 7.4. All main peak isoelectric points remained around 7.4 regardless of temperature stress. The results are shown in Tables 22-25.

[0194] At -70°C and 5°C, no significant trends were observed in the formulations, and only slight changes in isoform percentages were observed.

[0195] At 25°C, formulations F2 and F4 showed acidic, neutral, and basic peak percentages comparable to the control formulation, with only slight changes from time 0 (<1%). At 25°C, all formulations (except F14) showed a slight decrease in basic peak percentage, with corresponding increases in neutral and acidic percentages. The decrease was in the basic peak percentage, which was lower than that of their glycine counterparts.

[0196] At 40°C, formulations F2 and F4 showed the smallest change in peak percentage, comparable to the control formulation. All formulations showed a decrease in basic peak percentage. All sorbitol formulations, unlike their glycine counterparts, showed an increase in acidic and neutral peak percentage. Formulations at higher pH levels showed the most significant change compared to time 0, and the sorbitol formulations showed less change than their glycine counterparts.

[0197] [Table 22]

[0198] [Table 23]

[0199] [Table 24]

[0200] [Table 25]

[0201] Example 11: High-concentration formulation Six formulations of M254 hsIgG at pH 5.3, containing 10 mM sodium acetate, 5% (w / v) sorbitol, and 0.02% (w / v) polysorbate 20, were prepared at various IgG concentrations (100 mg / mL, 125 mg / mL, 175 mg / mL, 200 mg / mL, 250 mg / mL, and 275 mg / mL). A control was also prepared using 100 mg / mL IgG, 250 mM glycine, and 0.02% (w / v) polysorbate 20 (pH 5.2).

[0202] The formulation was stored at 5°C, 25°C, and 40°C. Appearance, pH, and concentration (A 280 ), turbidity (A 650 Size exclusion chromatography (SEC) and UNCLE (Unchained Labs) were used to characterize the samples before packing and at 1 week. Using UNCLE, the aggregation curve was evaluated, and the aggregation temperature was determined using the scattered light intensity at 473 nm over a temperature gradient from 20°C to 90°C. Osmotic pressure was tested on the pre-packing samples. Viscosity was tested at time 0 (after filtration) and at 1 week. After storage at different temperatures for 1 week, there were no changes in visual appearance (all formulations remained clear and colorless under different storage conditions and did not contain visible particles), pH (Table 26), or turbidity (Table 27). Concentrations decreased for all formulations at 1 week (Table 28). Osmotic pressure increased with increasing hsIgG concentration (Table 29), and viscosity also increased with increasing hsIgG concentration (Table 29; Table 30). No significant increase in soluble aggregates was observed even with increasing concentration. Across all storage temperatures, the 100 mg / mL and 125 mg / mL hsIgG samples maintained slightly higher monomer percentages compared to samples with concentrations of 175 mg / mL or higher (Table 31). UNCLE data showed that increasing concentration only slightly reduced product stability, resulting in low Tag levels (Figure 3).

[0203] [Table 26] * Sample names are based on pre-filling concentrations.

[0204] [Table 27] * Sample names are based on pre-filling concentrations.

[0205] [Table 28] * Sample names are based on pre-filling concentrations.

[0206] [Table 29] * Sample names are based on pre-filling concentrations.

[0207] [Table 30] * Sample names are based on pre-filling concentrations.

[0208] [Table 31] * Sample names are based on pre-filling concentrations.

[0209] Other Embodiments Although the present invention has been described in connection with its detailed description, the foregoing description is illustrative of the scope of the invention and is not intended to limit the scope of the invention, and the scope of the invention is understood to be defined by the appended claims. Other aspects, advantages, and modifications are within the following claims. The inventions described in the original claims of this application are listed below. [Invention 1] A liquid pharmaceutical composition comprising immunoglobulin in approximately 10 mM sodium acetate, approximately 0.02% (w / v) polysorbate 20, and at least one of approximately 250 mM glycine or approximately 5% (w / v) sorbitol, wherein at least 50% of the branched glycans on the Fc region of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds, and the pH of the composition is 4 to 7. [Invention 2] A liquid pharmaceutical composition according to Invention 1, comprising 250 mM glycine. [Invention 3] A liquid pharmaceutical composition according to Invention 1, comprising 5% (w / v) sorbitol. [Invention 4] The liquid pharmaceutical composition according to Invention 1, wherein the concentration of the immunoglobulin is 50 to 275 mg / mL. [Invention 5] The liquid pharmaceutical composition according to Invention 1, wherein the concentration of the immunoglobulin is 50 to 250 mg / mL. [Invention 6] The liquid pharmaceutical composition according to Invention 3, wherein the concentration of the immunoglobulin is 100 to 275 mg / mL. [Invention 7] The liquid pharmaceutical composition according to Invention 3, wherein the concentration of the immunoglobulin is 70-130 mg / mL, 90-110 mg / mL, or 80-120 mg / mL. [Invention 8] A liquid pharmaceutical composition according to any one of Inventions 1 to 7, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages. [Invention 9] A liquid pharmaceutical composition according to any one of Inventions 1 to 8, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds. [Invention 10] A liquid pharmaceutical composition according to any one of Inventions 1 to 9, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages. [Invention 11] A liquid pharmaceutical composition according to any one of Inventions 1 to 10, wherein at least 90% of the immunoglobulin is IgG immunoglobulin. [Invention 12] The liquid pharmaceutical composition according to Invention 11, wherein at least 95% of the immunoglobulin is IgG immunoglobulin. [Invention 13] A liquid pharmaceutical composition according to any one of Inventions 1 to 12, wherein 5 to 20% of the immunoglobulin is a dimer. [Invention 14] The liquid pharmaceutical composition according to Invention 13, wherein 5-10% of the immunoglobulin is a dimer. [Invention 15] A liquid pharmaceutical composition according to any one of Inventions 1 to 14, wherein at least 80% of the immunoglobulin is a monomer or dimer. [Invention 16] The liquid pharmaceutical composition according to Invention 15, wherein at least 85% of the immunoglobulin is a monomer or dimer. [Invention 17] The liquid pharmaceutical composition according to Invention 16, wherein at least 90% of the immunoglobulin is a monomer or dimer. [Invention 18] A liquid pharmaceutical composition according to any one of Inventions 1 to 17, wherein 5 to 20% of the IgG immunoglobulin is a dimer. [Invention 19] The liquid pharmaceutical composition according to Invention 18, wherein 5-10% of the IgG immunoglobulin is a dimer. [Invention 20] A liquid pharmaceutical composition according to any one of Inventions 1 to 19, wherein at least 80% of the IgG immunoglobulin is a monomer or dimer. [Invention 21] The liquid pharmaceutical composition according to Invention 20, wherein at least 85% of the IgG immunoglobulin is a monomer or dimer. [Invention 22] The liquid pharmaceutical composition according to Invention 21, wherein at least 90% of the IgG immunoglobulin is a monomer or dimer. [Invention 23] A liquid pharmaceutical composition according to any one of Inventions 1 to 22, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycan on the Fc domain of the IgG immunoglobulin is diciallylated by a NeuAc-α2,6-Gal terminal bond. [Invention 24] A liquid pharmaceutical composition according to any one of Inventions 1 to 23, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds. [Invention 25] A liquid pharmaceutical composition according to any one of Inventions 1 to 24, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of the IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages. [Invention 26] A liquid pharmaceutical composition according to any one of Inventions 1 to 25, wherein the pH is 4.0 to 5.5, 4.0 to 4.5, 4.5 to 5.0, 5.0 to 5.5, 4.2 to 4.7, 4.7 to 5.3, or 5.1 to 5.3. [Discussion 27] The liquid pharmaceutical composition according to Invention 1, wherein the pH is 4.0-5.5, 4.0-4.5, 4.5-5.0, 5.0-5.5, 4.2-4.7, 4.7-5.3, or 5.1-5.3. [Invention 28] The liquid pharmaceutical composition according to Invention 6, wherein the pH is 5.2 to 5.5 or 5.3 to 5.4. [Invention 29] A liquid pharmaceutical composition according to any one of Inventions 1 to 25, wherein the pH is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. [Invention 30] The liquid pharmaceutical composition according to any one of Inventions 1 to 29, wherein the composition, after being stirred at 1000 RPM for 8 hours at 2 to 8°C, has fewer than 1000 particles having a diameter of 10 to 100 micrometers. [Invention 31] The liquid pharmaceutical composition according to Invention 30, wherein the composition, after being stirred at 1000 RPM for 8 hours at 2 to 8°C, has fewer than 500 particles having a diameter of 10 to 100 micrometers. [Invention 32] The liquid pharmaceutical composition according to Invention 31, wherein the composition, after being stirred at 1000 RPM for 8 hours at 2 to 8°C, has fewer than 200 particles having a diameter of 10 to 100 micrometers. [Invention 33] A liquid pharmaceutical composition according to any one of Inventions 1 to 32, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal linkage after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 34] A liquid pharmaceutical composition according to any one of Inventions 1 to 33, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal bonds after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 35] A liquid pharmaceutical composition according to any one of Inventions 1 to 34, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal linkage after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 36] A liquid pharmaceutical composition according to any one of Inventions 1 to 35, wherein 5-10% of the immunoglobulin is a dimer after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 37] A liquid pharmaceutical composition according to any one of Inventions 1 to 36, wherein at least 85% of the immunoglobulin is a monomer or dimer after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 38] The liquid pharmaceutical composition according to Invention 37, wherein at least 90% of the immunoglobulin is a monomer or dimer after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 39] A liquid pharmaceutical composition according to any one of Inventions 1 to 38, wherein the formulation is stable for at least 7 months at 5°C, at least 1 month at 25°C, for 2 years at 2 to 8°C, and / or for 2 weeks at 15 to 30°C. [Invention 40] The liquid pharmaceutical composition according to any one of Inventions 1 to 39, wherein the storage is in a sealed United States Pharmacopeia Type 1 glass vial. [Invention 41] The liquid pharmaceutical composition according to any one of Inventions 1 to 39, wherein the storage is in a sealed 2R Type 1 glass injection vial. [Invention 42] A pre-filled syringe containing a liquid pharmaceutical composition described in any one of Inventions 1 to 39. [Invention 43] The liquid pharmaceutical composition according to any one of Inventions 1 to 39, wherein the liquid pharmaceutical composition is frozen. [Invention 44] A liquid pharmaceutical composition comprising immunoglobulin in approximately 10 mM sodium acetate, approximately 0.02% (w / v) polysorbate 20, and at least one of approximately 250 mM glycine or approximately 5% (w / v) sorbitol, wherein at least 50% of the branched glycans on the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds, and the pH of the composition is 4 to 7. [Invention 45] A liquid pharmaceutical composition according to Invention 44, comprising 250 mM glycine. [Invention 46] A liquid pharmaceutical composition according to Invention 44, comprising 5% (w / v) sorbitol. [Invention 47] The liquid pharmaceutical composition according to Invention 44, wherein the concentration of the immunoglobulin is 50 to 275 mg / mL. [Invention 48] The liquid pharmaceutical composition according to Invention 44, wherein the concentration of the immunoglobulin is 50 to 250 mg / mL. [Invention 49] The liquid pharmaceutical composition according to Invention 46, wherein the concentration of the immunoglobulin is 100 to 275 mg / mL. [Invention 50] The liquid pharmaceutical composition according to Invention 46, wherein the concentration of the immunoglobulin is 70-130 mg / mL, 90-110 mg / mL, or 80-120 mg / mL. [Invention 51] A liquid pharmaceutical composition according to any one of Inventions 44 to 50, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds. [Invention 52] A liquid pharmaceutical composition according to any one of Inventions 44 to 51, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages. [Invention 53] A liquid pharmaceutical composition according to any one of Inventions 44 to 52, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages. [Invention 54] A liquid pharmaceutical composition according to any one of Inventions 44 to 53, wherein at least 90% of the immunoglobulin is IgG immunoglobulin. [Invention 55] The liquid pharmaceutical composition according to Invention 54, wherein at least 95% of the immunoglobulin is IgG immunoglobulin. [Invention 56] A liquid pharmaceutical composition according to any one of Inventions 44 to 55, wherein 5 to 20% of the immunoglobulin is a dimer. [Invention 57] A liquid pharmaceutical composition according to any one of Inventions 44 to 56, wherein 5 to 10% of the immunoglobulin is a dimer. [Invention 58] A liquid pharmaceutical composition according to any one of Inventions 44 to 57, wherein at least 80% of the immunoglobulin is a monomer or dimer. [Invention 59] The liquid pharmaceutical composition according to Invention 58, wherein at least 85% of the immunoglobulin is a monomer or dimer. [Invention 60] The liquid pharmaceutical composition according to Invention 59, wherein at least 90% of the immunoglobulin is a monomer or dimer. [Invention 61] A liquid pharmaceutical composition according to any one of Inventions 44 to 60, wherein 5 to 20% of the IgG immunoglobulin is a dimer. [Invention 62] The liquid pharmaceutical composition according to Invention 61, wherein 5-10% of the IgG immunoglobulin is a dimer. [Invention 63] A liquid pharmaceutical composition according to any one of Inventions 44 to 62, wherein at least 80% of the IgG immunoglobulin is a monomer or a dimer. [Invention 64] The liquid pharmaceutical composition according to Invention 63, wherein at least 85% of the IgG immunoglobulin is a monomer or dimer. [Invention 65] The liquid pharmaceutical composition according to Invention 64, wherein at least 90% of the IgG immunoglobulin is a monomer or dimer. [Invention 66] A liquid pharmaceutical composition according to any one of Inventions 44 to 65, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds. [Invention 67] A liquid pharmaceutical composition according to any one of Inventions 44 to 66, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc domain of the IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages. [Invention 68] A liquid pharmaceutical composition according to any one of Inventions 44 to 67, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of the IgG immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages. [Invention 69] A liquid pharmaceutical composition according to any one of Inventions 44 to 68, wherein the pH is 4.0 to 5.5, 4.0 to 4.5, 4.5 to 5.0, 5.0 to 5.5, 4.2 to 4.7, 4.7 to 5.3, or 5.1 to 5.3. [Invention 70] The liquid pharmaceutical composition according to Invention 44, wherein the pH is 4.0-5.5, 4.0-4.5, 4.5-5.0, 5.0-5.5, 4.2-4.7, 4.7-5.3, or 5.1-5.3. [Invention 71] The liquid pharmaceutical composition according to Invention 46, wherein the pH is 5.2 to 5.5 or 5.3 to 5.4. [Discussion 72] A liquid pharmaceutical composition according to any one of inventions 44 to 68, wherein the pH is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. [Invention 73] The liquid pharmaceutical composition according to any one of Inventions 44 to 72, wherein the composition, after being stirred at 1000 RPM for 8 hours at 2 to 8°C, has fewer than 1000 particles having a diameter of 10 to 100 micrometers. [Invention 74] The liquid pharmaceutical composition according to Invention 73, wherein the composition, after being stirred at 1000 RPM for 8 hours at 2 to 8°C, has fewer than 500 particles having a diameter of 10 to 100 micrometers. [Invention 75] The liquid pharmaceutical composition according to Invention 73, wherein the composition, after being stirred at 1000 RPM for 8 hours at 2 to 8°C, has fewer than 200 particles having a diameter of 10 to 100 micrometers. [Invention 76] A liquid pharmaceutical composition according to any one of Inventions 44 to 75, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal bonds after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 77] A liquid pharmaceutical composition according to any one of Inventions 44 to 75, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fc region of the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal bonds after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 78] A liquid pharmaceutical composition according to any one of Inventions 44 to 77, wherein at least 60%, 70%, 80%, 90%, or 95% of the branched glycans on the Fab domain of the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal linkage after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Discussion 79] A liquid pharmaceutical composition according to any one of Inventions 44 to 78, wherein 5-10% of the immunoglobulin is a dimer after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 80] A liquid pharmaceutical composition according to any one of Inventions 44 to 79, wherein at least 85% of the immunoglobulin is a monomer or dimer after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 81] The liquid pharmaceutical composition according to Invention 80, wherein at least 90% of the immunoglobulin is a monomer or dimer after being stored at -70°C, 4°C, 5°C, 25°C, or 40°C for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 weeks, or for 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months. [Invention 82] A liquid pharmaceutical composition according to any one of Inventions 44 to 82, wherein the formulation is stable for at least 7 months at 5°C, at least 1 month at 25°C, for 2 years at 2 to 8°C, and / or for 2 weeks at 15 to 30°C. [Invention 83] The liquid pharmaceutical composition according to any one of Inventions 44 to 82, wherein the storage is in a sealed United States Pharmacopeia Type 1 glass vial. [Invention 84] The liquid pharmaceutical composition according to any one of Inventions 44 to 82, wherein the storage is in a sealed 2R Type 1 glass injection vial. [Invention 85] A pre-filled syringe containing a liquid pharmaceutical composition described in any one of inventions 44 to 82. [Invention 86] The liquid pharmaceutical composition according to any one of inventions 44 to 82, wherein the liquid pharmaceutical composition is frozen. [Invention 87] A method for treating a disease, comprising administering a liquid pharmaceutical composition according to any one of Inventions 1 to 86 in a dose that is 1% to 10% of an effective dose of IVIG for treating the disease. [Invention 88] The method according to Invention 87, wherein the hsIgG preparation is administered in a dose of 5 mg / kg to 100 mg / kg. [Invention 89] The method according to Invention 87, wherein the disease is an inflammatory disease. [Invention 90] The method according to Invention 87, wherein the subject is suffering from an antibody deficiency. [Invention 91] The method according to Invention 90, wherein the subject is suffering from a primary antibody deficiency. [Invention 92] The method according to Invention 87, wherein the disease is related to the presence of autoantibodies. [Invention 93] The method according to Invention 87, wherein the dose of hsIVIG is as effective as the effective dose of IVIG. [Invention 94] The method according to Invention 87 or 93, wherein the hsIgG preparation is administered at the same frequency as the effective dose of IVIG. [Invention 95] The method according to Invention 87, wherein the disease is a neurological disease. [Invention 96] The method according to Invention 95, wherein the neurological disorder is selected from the group consisting of dermatomyositis, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), myasthenia gravis, and generalized rigidity syndrome. [Identification 97] The method according to Invention 87, wherein the disease is selected from the group consisting of immune cytopenia, parvovirus B19-associated erythropoiesis, secondary hypogammaglobulinemia in myeloma and chronic lymphocytic leukemia, and post-bone marrow transplantation. [Invention 98] The method according to Invention 87, wherein the disease is selected from the group consisting of vasculitis, systemic lupus erythematosus (SLE), mucosal pemphigoid, and uveitis, and is most commonly used in dermatology to treat Kawasaki syndrome, dermatomyositis, toxic epidermal necrolysis, and bullous diseases. [Distribution 99] The method of Invention 87, wherein the disease is approved by the FDA for treatment with IVIG, or IVIG is indicated for the treatment of the disease. [Invention 100] The method according to Invention 99, wherein the hsIgG preparation is 1% to 10% of the FDA-approved dose of IVIG for the disease. [Invention 101] The aforementioned disorders include myocarditis, acute motor axonal neuropathy, painful steatosis, anti-glomerular basement nephritis, Goodpasture syndrome, antiphospholipid antibody syndrome (APS, APLS), anti-synthetase syndrome, myositis, interstitial lung disease (ILD), ataxic neuropathy (acute and chronic), autoimmune enteropathy (AIE), autoimmune neutropenia, autoimmune retinopathy, autoimmune thyroiditis, autoimmune urticaria, dermatitis herpetiformis, acquired epidermolysis bullosa, essential mixed cryoglobulinemia, granulomatosis with polyangiitis (GPA), mixed connective tissue disease (MCTD), neuromyotonia, and optic neuritis. The method according to Invention 87, selected from the group consisting of paraneoplastic cerebellar degeneration, anti-N-methyl-D-aspartate (anti-NMDA) receptor encephalitis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, dermatomyositis, pemphigoid of pregnancy, Graves' disease, Guillain-Barré syndrome, IgG4-related disease, Lambert-Eaton myasthenia gravis, lupus nephritis, myositis, multifocal motor neuropathy, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, polymyositis, and systemic lupus erythematosus (SLE), and combinations thereof. [Invention 102] The aforementioned diseases include acute disseminated encephalomyelitis (ADEM), autoimmune angioedema (acquired angioedema type II), autoimmune hepatitis (types I and 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), Mollen's ulcer, focal scleroderma, and opsoclonus myoclosis. The method according to Invention 87, selected from the group consisting of Streptococcal syndrome, Oud thyroiditis, recurrent rheumatoid arthritis, paraneoplastic opsoclonus-myoclonus ataxia with neuroblastoma, pediatric autoimmune neuropsychiatric disorders 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-Fischer syndrome, and combinations thereof. [Invention 103] A method for treating CIDP in a subject having CIDP, comprising administering an hsIgG preparation at an effective dose of 10% or less than 10% of the effective dose of IVIG. [Invention 104] The method according to Invention 103, wherein the effective dose of IVIG is 200 to 2000 mg / kg. [Invention 105] The method according to invention 103 or 104, wherein the hsIgG preparation is administered in an effective dose that is 10% or less of the effective dose of IVIG. [Invention 106] The method according to Invention 105, wherein the hsIgG preparation is administered at a dose of 1% of the effective dose of IVIG. [Invention 107] The method according to Invention 103, wherein the hsIgG preparation is administered in a dose of approximately 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. [Invention 108] A method for treating ITP in a subject having ITP, comprising administering an hsIgG preparation in an effective dose that is 10% or less than 10% of the effective dose of IVIG. [Invention 109] The method according to Invention 108, wherein the effective dose of IVIG is 1000 to 2000 mg / kg. [Invention 110] The method according to invention 108 or 109, wherein the hsIgG preparation is administered in an effective dose that is 10% or less of the effective dose of IVIG. [Invention 111] The method according to Invention 110, wherein the hsIgG preparation is administered in a dose of 1% to 5% of the effective dose of IVIG. [Invention 112] The method according to Invention 108, wherein the hsIgG preparation is administered in a dose of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / kg. [Invention 113] A method for treating wAIHA in a subject having wAIHA, comprising administering an hsIgG preparation in an effective dose that is 10% or less than 10% of the effective dose of IVIG. [Invention 114] The method according to Invention 113, wherein the effective dose of IVIG is 1000 mg / kg. [Invention 115] The method according to invention 113 or 114, wherein the hsIgG preparation is administered in a dose of less than 10% of the effective dose of IVIG. [Invention 116] The method according to Invention 115, wherein the hsIgG preparation is administered in a dose of 1% to 5% of the effective dose of IVIG. [Invention 117] The method according to Invention 113, wherein the hsIgG preparation is administered in a dose of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg. [Invention 118] A method for treating Guillain-Barré syndrome in a subject having Guillain-Barré syndrome, comprising administering an hsIgG preparation in an effective dose that is 10% or less than 10% of the effective dose of IVIG. [Invention 119] The method according to Invention 118, wherein the effective dose of IVIG is 1000 to 2000 mg / kg. [Invention 120] The method according to invention 118 or 119, wherein the hsIgG preparation is administered in a dose of less than 10% of the effective dose of IVIG. [Invention 121] The method according to invention 120, wherein the hsIgG preparation is administered in a dose of 1% to 5% of the effective dose of IVIG. [Invention 122] The method according to Invention 118, wherein the hsIgG preparation is administered in a dose of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / kg. [Invention 123] A method for treating PID (Primary Humoral Immunodeficiency Disease) in a subject having PID, comprising administering an hsIgG preparation in an effective dose that is 10% or less than 10% of the effective dose of IVIG. [Invention 124] The method according to Invention 123, wherein the effective dose of IVIG is 200 to 800 mg / kg. [Invention 125] The method according to invention 123 or 124, wherein the hsIgG preparation is administered in a dose of less than 10% of the effective dose of IVIG. [Invention 126] The method according to Invention 125, wherein the hsIgG preparation is administered in a dose of 1% to 5% of the effective dose of IVIG. [Inquiry 127] The method according to Invention 123, wherein the hsIgG preparation is administered in a dose of approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / kg. [Invention 128] A method for treating Kawasaki disease in a subject having Kawasaki disease, comprising administering an hsIgG preparation in an effective dose that is 10% or less than 10% of the effective dose of IVIG. [Invention 129] The method according to Invention 128, wherein the effective dose of IVIG is 1000 to 2000 mg / kg. [Invention 130] The method according to Invention 129, wherein the hsIgG preparation is administered in a dose of less than 10% of the effective dose of IVIG. [Invention 131] The method according to Invention 130, wherein the hsIgG preparation is administered in a dose of 1% to 5% of the effective dose of IVIG. [Invention 132] The method according to Invention 128, wherein the hsIgG preparation is administered in a dose of approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / kg. [Invention 133] The method according to any one of Inventions 87 to 132, wherein the dose of the pharmaceutical composition has the same efficacy as the effective dose of IVIG. [Invention 134] The method according to any one of inventions 87 to 133, wherein at least one side effect caused by the effective dose of the IVIG is mitigated by administering the pharmaceutical composition. [Invention 135] The method according to any one of Inventions 87 to 134, wherein the pharmaceutical composition is administered subcutaneously. [Invention 136] A syringe suitable for subcutaneous injection, containing a pharmaceutical composition described in any one of inventions 1 to 86 in an amount of 2 mL or less.

Claims

1. A liquid pharmaceutical composition comprising persialylated immunoglobulin (hsIgG) in 10 mM sodium acetate, 0.02% (w / v) polysorbate 20, and at least one of 250 mM glycine or 5% (w / v) sorbitol, wherein at least 60% of the branched glycans on the Fc region and / or Fab region of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal bonds, and the pH of the composition is 4 to 7.

2. The liquid pharmaceutical composition according to claim 1, comprising 250 mM glycine.

3. The liquid pharmaceutical composition according to claim 1, comprising 5% (w / v) sorbitol.

4. The liquid pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of the immunoglobulin is 50 to 275 mg / mL.

5. The liquid pharmaceutical composition according to any one of claims 1 to 4, wherein at least 60% of the branched glycans on the Fc region and / or Fab region of the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages.

6. The liquid pharmaceutical composition according to any one of claims 1 to 5, wherein at least 90% of the immunoglobulin is IgG immunoglobulin.

7. (a) 5-20% of the immunoglobulin is a dimer, or (b) At least 80% of the immunoglobulin is a monomer or dimer, (c) 5-20% of the IgG immunoglobulin is a dimer. A liquid pharmaceutical composition according to any one of claims 1 to 6.

8. The liquid pharmaceutical composition according to any one of claims 1 to 7, wherein the composition, after being stirred at 1000 RPM for 8 hours at 2 to 8°C, has fewer than 1000 particles having a diameter of 10 to 100 micrometers.

9. After storing at -70°C to 40°C for 1 week to 24 months, (a) At least 60% of the branched glycans on the immunoglobulin are diciallylated by NeuAc-α2,6-Gal terminal linkages, (b) The Fc domain and / or Fab domain of the immunoglobulin are dicialized by NeuAc-α2,6-Gal terminal binding. or, (c) 5-10% of the immunoglobulin is a dimer, or (d) At least 85% of the immunoglobulin is monomer, A liquid pharmaceutical composition according to any one of claims 1 to 8.

10. A pre-filled syringe containing the liquid pharmaceutical composition according to any one of claims 1 to 9.

11. The liquid pharmaceutical composition according to any one of claims 1 to 9, wherein the liquid pharmaceutical composition is frozen.

12. A liquid pharmaceutical composition according to any one of claims 1 to 9 for use in a method for treating a disease, wherein the method comprises administering the liquid pharmaceutical composition in a dose that is 1% to 10% of an effective dose of IVIG.

13. The liquid pharmaceutical composition according to claim 12, wherein the liquid pharmaceutical composition is used to be administered in a dose of 5 mg / kg to 250 mg / kg.

14. The liquid pharmaceutical composition according to claim 12 or 13, wherein the disease is selected from inflammatory diseases, antibody deficiencies, and neurological diseases.

15. The liquid pharmaceutical composition according to claim 12, wherein the dose of hsIVIG is effective to the same extent as the effective dose of IVIG.

16. The liquid pharmaceutical composition according to claim 12 or 15, wherein the hsIVIG is used to be administered at the same frequency as the effective dose of the IVIG.

17. A syringe suitable for subcutaneous injection, comprising a pharmaceutical composition according to any one of claims 1 to 9 in an amount of 2 mL or less.

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