Sialylated glycoproteins

Abstract

Pharmaceutical formulations containing supersialylated immunoglobulins are described. The formulations are stable to shear stress. The pharmaceutical compositions described herein provide pharmaceutically acceptable hsIgG compositions that are stable to shear stress (e.g., when the formulation is subjected to shear stress, e.g., agitation, such as sustained transport, no significant numbers of sub-visible particles are formed, and therefore can be transported and handled in liquid form).

Description

[Technical field]

[0001] (Priority) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 836,016, filed April 18, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Intravenous immunoglobulin (IVIg) is prepared from pooled plasma of human donors (e.g., pooled plasma from at least 1,000 donors) and is composed predominantly of IgG antibodies, primarily IgG1 antibodies, although IVIg may also contain trace amounts of other antibody subclasses. Commercially available IVIg preparations generally exhibit low levels of sialylation on the Fc domain of the antibodies present. Specifically, the antibodies in commercial IVIg preparations exhibit low levels of disialylation of branched glycans on the Fc region. Summary of the Invention [Means for solving the problem]

[0003] Described herein are pharmaceutical compositions comprising hypersialylated immunoglobulins (hsIgGs). HsIgGs have very high levels of sialic acid on branched glycans on the Fc region of the immunoglobulin, e.g., at least 50% (60%, 70%, 80%, 90% or more) of the branched glycans on the Fc region of the immunoglobulin are sialylated via NeuAc-α 2,6-Gal terminal linkages on both the α1,3 and α1,6 arms of the branched glycans.

[0004] The pharmaceutical compositions described herein provide pharma- ceutically acceptable hsIgG compositions that are stable to shear stress (e.g., the formulation does not form appreciable numbers of subvisible particles when subjected to shear stress, e.g., agitation during transport), and thus can be transported and handled in liquid form. The formulations are also stable upon dilution, e.g., dilution with 5% dextrose for intravenous administration. The formulations are stable, e.g., 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.

[0005] A liquid pharmaceutical composition comprising an immunoglobulin in 250 mM glycine 0.02% (w / v) polysorbate 20, pH 4-7, wherein at least 50% of the branched glycans on the Fc region of the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

[0006] 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. At least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages, at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages, at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages, at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab region of the immunoglobulin are disialylated by NeuAc-α disialylated by 2,6-Gal terminal linkages, at least 90% of the immunoglobulins are IgG immunoglobulins, at least 95% of the immunoglobulins are IgG immunoglobulins, 5-20% of the immunoglobulins are dimers, 5-10% of the immunoglobulins are dimers, at least 80% of the immunoglobulins are monomers or dimers, at least 85% of the immunoglobulins are monomers or dimers, and at least 90% of the immunoglobulins are monomers. or 5-20% of the IgG immunoglobulins are dimers, 5-10% of the IgG immunoglobulins are dimers, at least 80% of the IgG immunoglobulins are monomers or dimers, at least 85% of the IgG immunoglobulins are monomers or dimers, at least 90% of the IgG immunoglobulins are monomers or dimers, and at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of the IgG immunoglobulins are NeuAc-α at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the IgG immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages, at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab region of the IgG immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages, the pH is 4.7 to 5.5, the pH is 5.1 to 5.3.The composition has less than 1000 particles having a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C, the composition has less than 500 particles having a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C, the composition has less than 200 particles having a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C, and at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of the immunoglobulin are NeuAc-α after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months. disialylated by 2,6-Gal terminal linkages and at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the immunoglobulin are NeuAc-α after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months Disialylated by 2,6-Gal terminal linkages and at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab region of the immunoglobulin are NeuAc-α after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months disialylated by 2,6-Gal terminal linkages, 5-10% of the immunoglobulins are dimers after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months, at least 85% of the immunoglobulins are monomers or dimers after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months, at least 90% of the immunoglobulins are monomers or dimers after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months, storage is in sealed U.S. Pharmacopeia Type 1 glass vials, storage is in sealed 2R Type 1 glass injection vials.

[0007] 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 α 1,3 and α 1,6 arms (i.e., are disialylated by a NeuAc-α 2,6-Gal terminal linkage). In some embodiments, in addition to the 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 disialylated by a NeuAc-α 2,6-Gal terminal linkage. In some cases, at least 85% (including 87%, 90%, 92%, 94%, 95%, 97%, 98%, or 100%) of all branched glycans (the sum of the glycans on the Fc domain and the Fab domain) are disialylated by a NeuAc-α 2,6-Gal terminal linkage. In some embodiments, less than 50% (e.g., less than 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) of the branched glycans on the Fc region are monosialylated (e.g., sialylated only on the α 1,3 arm or the α 1,6 arm) via a NeuAc-α 2,6-Gal terminal linkage. The immunoglobulin is an HsIgG preparation, predominantly an IgG antibody (e.g., at least 80%, 85%, 90%, 95% weight / weight of the immunoglobulin, and is an IgG antibody of various isotypes).

[0008] As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides," each of which comprises the constant region of an antibody, excluding the CH1 domain. In some embodiments, an "Fc region" comprises two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may include some or all of the flexible hinge N-terminal to these domains.

[0009] As used herein, a "glycan" is a sugar, which may be a monomer or polymer of at least three sugar residues, such as sugars, and may be linear or branched. A "glycan" may contain 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 homo- and heteropolymers of sugar residues. The term "glycan" also includes glycan components of glycoconjugates (e.g., polypeptides, glycolipids, proteoglycans, etc.). The term also includes free glycans, including glycans that have been cleaved or otherwise released from glycoconjugates.

[0010] As used herein, the term "glycoprotein" refers to a protein that contains a peptide backbone covalently attached to one or more sugar moieties (i.e., glycans). The sugar moieties may be in the form of monosaccharides, disaccharides, oligosaccharides, and / or polysaccharides. The sugar moieties may comprise a single unbranched sugar residue or may comprise one or more branched chains. Glycoproteins may contain O-linked and / or N-linked sugar moieties.

[0011] IVIg is a preparation of pooled polyvalent immunoglobulins containing all four IgG isotypes extracted from the plasma of at least 1,000 human donors. Among the forms of IVIg approved for use in the United States are 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 between IVIg preparations but is generally less than 20%. The level of disialylation is generally much lower.

[0012] As used herein, "N-glycosylation site of an Fc polypeptide" refers to an amino acid residue in an Fc polypeptide to which a glycan is N-linked. In some embodiments, an Fc region contains a dimer of Fc polypeptides, and the Fc region comprises two N-glycosylation sites, one on each Fc polypeptide.

[0013] As used herein, "percent (%) branched glycan" refers to the number of moles of glycan X relative to the total moles of glycans present, where X represents the glycan of interest.

[0014] The term "pharmacologically effective amount" or "therapeutically effective amount" refers to an amount (e.g., dosage) effective to treat a patient having a disorder or condition described herein. It is also understood that, as used herein, a "pharmacologically effective amount" may be interpreted as an amount that provides the desired therapeutic effect, either taken alone or in combination with other therapeutic agents, once, or taken in any dosage or route.

[0015] The "pharmaceutical formulations" and "pharmaceutical products" may be included in kits which contain the formulation or product and instructions for use.

[0016] A "pharmaceutical formulation" and a "pharmaceutical product" generally refer to a composition in which a final, predetermined level of sialylation has been achieved and which is free of process impurities. As such, a "pharmaceutical formulation" and a "pharmaceutical product" are substantially free of ST6Gal sialyltransferase and / or sialic acid donor (e.g., cytidine 5'-monophosphono-N-acetylneuraminic acid) or its by-products (e.g., cytidine 5'-monophosphate).

[0017] A "pharmaceutical preparation" and a "pharmaceutical product" are generally substantially free of the cell in which the glycoprotein, if recombinant, is produced (eg, endoplasmic or cytoplasmic proteins and RNA).

[0018] "Purified" (or "isolated") refers to a polynucleotide or polypeptide that is removed or separated from other components present in its natural environment. For example, an isolated polypeptide is one that is separated from other components of the cell in which it is produced (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA). An isolated polynucleotide is one that is separated from other nuclear components (e.g., histones) and / or upstream or downstream nucleic acids. An isolated polynucleotide or polypeptide may be 60%, or at least 75%, or at least 90%, or at least 95% free from other components present in the natural environment of the designated polynucleotide or polypeptide.

[0019] As used herein, the term "sialylated" refers to a glycan having a terminal sialic acid. The term "mono-sialylated" refers to a branched glycan having one terminal sialic acid, e.g., the α1,3 arm or the α1,6 arm. The term "disialylated" refers to a branched glycan having terminal sialic acids on both arms, e.g., the α1,3 arm and the α1,6 arm. [Brief description of the drawings]

[0020] [Figure 1] Examples of branched glycans are shown diagrammatically: light circle is Gal, dark circle is Man, triangle is Fuc, diamond is NANA, and square is GlcNAc. [Diagram 2] Left panel: Schematic of the enzymatic sialylation reaction to convert pooled immunoglobulins to hsIgG. Right panel: IgG Fc glycan profile of starting IVIg and of hsIgG enzymatically prepared from IVIg. Glycan profiles of different IgG subclasses are derived by glycopeptide mass spectrometry. The peptide sequences used to quantify glycopeptides of different IgG subclasses are IgG1=EEQYNSTYR, IgG2 / 3 EEQFNSTFR, IgG3 / 4 EEQYNSTFR, and EEQFNSTYR. [Diagram 3] 1 shows a vial of hsIgG in a conventional formulation used for IVIg subjected to shear stress. [Figure 4] 1 shows a vial of hsIgG in a formulation of the present disclosure subjected to shear stress. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Immunoglobulins are glycosylated at conserved positions in the constant region of the heavy chain. For example, human IgG has a single N-linked glycosylation site at Asn297 in the CH2 domain. Each immunoglobulin type has a variety of different N-linked carbohydrate structures in the constant region. In the case of human IgG, the core oligosaccharide usually consists of GlcNAc2Man3GlcNAc with different numbers of outer residues. Variation between individual IgGs can occur through the attachment of galactose and / or galactose-sialic acid at one or both of the terminal GlcNAcs, or the attachment of a third GlcNAc arm (bisected GlcNAc).

[0022] The disclosure encompasses, in part, pharmaceutical preparations that include pooled human immunoglobulins having Fc regions with specific levels of branched glycans that are sialylated on both branched glycans within the Fc region (e.g., with NeuAc-α 2,6-Gal terminal linkages).

[0023] Pooled polyvalent human immunoglobulin preparations, including IVIg preparations, are highly complex because they are highly heterogeneous in several respects. They contain pooled immunoglobulins from hundreds or even more than 1000 individuals. At least about 90% or 95% of the immunoglobulins are of the IgG isotype (of all subclasses), although other isotypes are present, including IgA and IgM. The immunoglobulins in IVIg and pooled polyvalent human immunoglobulin preparations vary in both specificity and glycosylation patterns.

[0024] Supersialylation of pooled polyvalent immunoglobulins modifies the glycans present on the immunoglobulins. For some glycans, the modification involves the addition of one or more galactose molecules and one or more sialic acid molecules. For other glycans, the modification involves only the addition of one or more sialic acid molecules. Furthermore, essentially all IgG antibodies, the predominant immunoglobulins in a pooled polyvalent immunoglobulin preparation, have glycosylation sites on each polypeptide that forms the Fc region, and all IgG antibodies have no glycosylation sites on the Fab domain. Modifying the glycosylation of an immunoglobulin preparation modifies the structure and activity of the individual immunoglobulins in the preparation and, importantly, modifies the interactions between the individual immunoglobulins and the bulk behavior of the immunoglobulin preparation.

[0025] The widely used formulations used for IVIg preparations are not entirely suitable for pharmaceutical preparations of supersialylated immunoglobulins (hsIgG) because, at least when used for hsIgG, the formulations are not stable to the shear stresses that occur in the normal transport of pharmaceutical preparations. When subjected to this type of shear stress, there are subvisible particles formed in hsIgG formulations. It is known that such subvisible particles in antibody preparations can cause serious adverse events at the injection site and turn off the target immune response. Subvisible particles in antibody preparations can also activate the complement system, causing embolization and other negative immunogenic reactions. It has been found that the addition of a non-ionic surfactant makes the hsIgG formulation more stable to shear stress and significantly reduces the formation of subvisible particles.

[0026] Naturally derived 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 WO 2014 / 179601. Preparation of hsIgG is also described in Washbur et al (Proc Natl Acad Sci USA 2015 Mar. 17;112(11):E1297-306). The sialylation level in a hsIgG preparation can be measured on the Fc domain (e.g., the number of branched glycans sialylated on the α1,3 arm, α1,6 arm, or both of the branched glycans in the Fc domain) or global sialylation (e.g., the number or percentage of branched glycans sialylated on the α1,3 arm, α1,6 arm, or both of the branched glycans in a preparation of the polypeptide, regardless of whether it is the Fc domain or the Fab domain).

[0027] In some cases, the pooled serum used as a source of immunoglobulins for preparing hsIgG is isolated from a particular population of individuals producing antibodies against one or more viruses, e.g., COVID-19, SARS, parainfluenza, influenza, etc. In some cases, the immunoglobulins are isolated from a population of individuals where greater than 50%, 55%, 60%, 75% produce antibodies against the selected virus.

[0028] N-linked oligosaccharide chains are added to proteins in the lumen of the endoplasmic reticulum. Specifically, an initial oligosaccharide (typically 14 sugars) is added to the amino group on the side chain of an asparagine residue contained within the target consensus sequence of Asn-X-Ser / Thr, where X can be any amino acid except proline. This initial oligosaccharide structure is common to most eukaryotes and contains three glucose, nine mannose, and two N-acetylglucosamine residues. This initial oligosaccharide chain can be trimmed by specific glycosidase enzymes in the endoplasmic reticulum 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" and the second branch as the "α1,6 arm", as shown in FIG. 1.

[0029] N-glycans can be subdivided into three distinct groups, termed "high mannose type", "hybrid type", and "complex type", with a common pentasaccharide core (Man(alpha1,6)-(Man(alpha1,3))-Man(beta1,4)-GlcpNAc(beta1,4)-GlcpNAc(beta1,N)-Asn) occurring in all three groups.

[0030] After initial processing in the endoplasmic reticulum, the polypeptide is transferred to the Golgi where further processing can occur. If the glycan is transferred to the Golgi before being completely trimmed to the core pentasaccharide structure, then "high mannose glycans" are obtained.

[0031] Additionally or alternatively, one or more monosaccharide units of N-acetylglucosamine may be added to the core mannose subunit to form a "complex glycan." Galactose may be added to the N-acetylglucosamine subunit and sialic acid subunits to the galactose subunit, resulting in chains terminating in either sialic acid, galactose, or N-acetylglucosamine residues. Additionally, fucose residues may be added to the N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by a specific glycosyltransferase.

[0032] A "hybrid glycan" contains 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.

[0033] Sialic acids are a family of 9-carbon monosaccharides with a heterocyclic ring structure. They have a negative charge via the carboxylic acid group attached to the ring, as well as other chemical decorations including N-acetyl and N-glycolyl groups. The two main types of sialic residues found in polypeptides produced in mammalian expression systems are N-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). They usually occur as terminal structures attached to galactose (Gal) residues at the non-reducing ends of both N- and O-linked glycans. The glycosidic bond configuration for these sialic groups can be either α2,3 or α2,6.

[0034] The Fc region is glycosylated at conserved N-linked glycosylation sites. For example, each heavy chain of an IgG antibody contains H The IgA antibody has a single N-linked glycosylation site at Asn297 in the C2 domain. H 2 domain and C HThe IgE antibody has an N-linked glycosylation site within the C3 domain. H The IgM antibody has an N-linked glycosylation site within the C3 domain. H 1. C H 2. C H 3, and C H There are N-linked glycosylation sites within 4 domains.

[0035] Each antibody isotype has a variety of different N-linked carbohydrate structures in the constant region. For example, IgG has C H It has a single N-linked biantennary carbohydrate at Asn297 of the 2 domain and also contains the binding sites for C1q and FcγR. For human IgG, the core oligosaccharide is usually composed of GlcNAc2Man3GlcNAc with different numbers of outer residues. Variation between individual IgGs can occur through the attachment of galactose and / or galactose-sialic acid at one or both of the terminal GlcNAcs, or the attachment of a third GlcNAc arm (bisect GlcNAc). The glycans of a polypeptide can be evaluated using any method known in the art. For example, the sialylation of the glycan composition (e.g., the level of branched glycans sialylated on the α1,3 arm and / or the α1,6 arm) can be characterized using the methods described in WO2014 / 179601.

[0036] A composition containing hsIgG can include in addition to antibody monomers, dimers, and aggregates of antibodies. In some cases, pH can be used to adjust the proportion of monomers, dimers, and aggregates in the composition, as measured by size exclusion chromatography in terms of purity by weight percent. In some cases, lowering the pH increases the weight percent of monomers+dimers in the solution. In some cases, lowering the pH increases the weight percent of monomers in the solution. In some cases, raising the pH decreases the monomer% in the solution. In some cases, the weight percent aggregates is 3.0% 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% weight / weight or less). In some cases, the weight percent of monomers+dimers is 97.0% weight / weight or more (e.g., 98% weight / weight or more, or 99% weight / weight or more). In some cases, the monomer weight % is 80% w / w or more, 83% w / w or more, 85% w / w or more, or 87% w / w or more. In some cases, the pH is 5.3 or less (e.g., 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 less, or 4.0 or less). In some cases, the pH of the pharmaceutical composition is adjusted to modify the monomer weight %. In some cases, the pH of the pharmaceutical composition is decreased to increase the monomer weight %. In some cases, the pH of the pharmaceutical composition is increased to increase the dimer weight %. In some cases, the pH is such that the monomer weight % is 90% w / w or more (e.g., 91, 92, 93, 94, 95, 96, 97, 98, or 99% w / w or more).

[0037] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0038] Example 1: Supersialylated IgG formulated in 250 mM glycine A hypersialylated IgG in which more than 60% of the branched Fc region glycans were disialylated was prepared generally as described in WO 2014 / 179601.

[0039] Briefly, IVIg is subjected to a one-pot sequential enzymatic reaction using the β1,4 galactosyltransferase 1 (B4-GalT) and α2,6-sialyltransferase (ST6-Gal1) enzymes. The galactosyltransferase enzyme selectively adds galactose residues to existing asparagine-linked glycans in IVIg. The resulting galactosylated glycans serve as substrates for sialyltransferases, which selectively add sialic acid residues to cap the asparagine-linked glycan structures attached to IVIg. Thus, all sialylation reactions used two sugar nucleotides (UDPGal and CMP-NANA). The latter was periodically replenished to increase the disialylated product relative to the monosialylated product. The reaction includes the cofactor manganese chloride.

[0040] Representative examples of the corresponding IgG-Fc glycan profiles of the starting IVIg and the reaction product are shown in the right panel of FIG. 1. Glycan data are shown per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg, the sum of all non-sialylated glycans is >80% and the sum of all sialylated glycans is <20%. For the reaction product, the sum of all non-sialylated glycans is <20% and the sum of all sialylated glycans is >80%. The nomenclature of the different glycans listed in the glycoprofile uses the Oxford notation for N-linked glycans.

[0041] Non-supersialized IVIg, including commercially available IVIg, is generally stable in glycine and generally does not form subvisible particles when agitated, e.g., during shipping. Thus, an initial supersialylated IgG (hsIVIg) formulation was prepared at 109 mg / mL in 250 mM glycine. The pH ranged from 4.7 to 5.5. The formulation was an opaque, colorless, clear to pale yellow solution. This formulation was also tested after filtration through a 0.2 micrometer PES filter membrane in a PETG container. Table 1 provides both pre- and post-filtration characteristics of this supersialylated IgG formulation. The glycine-only formulation appeared to have acceptable product properties both pre- and post-filtration.

[0042] [Table 1]

[0043] Further study of the 250 mM glycine (pH 4.7-5.5) formulation revealed that the hsIgG was subjected to agitation stress. The filtered samples were transferred to glass vials, which were shaken at 1000 RPM for up to 8 hours under refrigerated temperature (2-8°C) conditions. Samples were tested before agitation and after 4 and 8 hours of agitation. As can be seen from the photograph in Figure 3, agitation resulted in the formation of sub-visible particles that made the solution turbid. The 250 mM (pH 4.7-5.5) formulation was found to be turbid even when transported in the passenger compartment of the aircraft. This suggested that the formulation formed sub-visible particles during distribution to healthcare providers and patients. Sub-visible particles can cause serious adverse events at the injection site and turn off the target immune response. Sub-visible particles can also activate the complement system, cause embolism, and other negative immunogenic responses. It is therefore important to design a formulation that is stable and does not form sub-visible particles upon agitation.

[0044] Example 2: Stabilized formulation of hsIgG Studies were conducted to develop a formulation of hsIVIG that is stable to agitation stress, yet still maintains the desired product attributes present in the 250 mM glycine formulation.

[0045] Low concentrations of polysorbate 20 (2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl dodecanoate; polyoxyethylene(20)sorbitan monolaurate) improved the ability of hsIgG formulations to withstand agitation stress while retaining desirable product properties. Notably, the presence of this nonionic surfactant did not significantly alter the relative amounts of antibody monomers, dimers, and higher aggregates.

[0046] [Table 2]

[0047] Samples of the filtered 250 mM glycine / 0.02% polysorbate 20 (pH 4.7-5.5) formulation were transferred to glass vials that were shaken at 1000 RPM for up to 8 hours under refrigerated temperature (2-8 °C) conditions. Samples were tested before agitation and after 4 and 8 hours of agitation. As can be seen from the photographs in Figure 4, the formulation was stable to agitation stress.

[0048] Table 3 below provides information on the product attributes of the formulations both before and after agitation stress. As can be seen, the addition of 0.02% polysorbate significantly improved agitation stability but did not alter the monomer / dimer ratio. The addition of 0.02% polysorbate 20 did not significantly affect protein concentration or the percentage of monomer, dimer, aggregates, and low molecular weight species before or after exposure to agitation stress.

[0049] Significantly lower subvisible particle concentrations were observed in the polysorbate 20-containing formulations compared to the original formulation. After exposure to agitation stress (4 and 8 h), comparable particle concentrations were observed for both formulations compared to the corresponding non-stressed conditions.

[0050] [Table 3]

[0051] Example 3: Effect of dilution on 5% dextrose injection To prepare the hsIgG for administration to patients, the concentrated hsIgG formulation is sterile filtered through two 0.2 micrometer PES filter membranes into sterile USP Type 1 glass vials to create the drug product. The vials are transported to the clinical site where they are administered within 72 hours of manufacture (starting from the time of filtration). At the clinical site, the drug product (100 mg / mL) is diluted to 60 mg / mL prior to administration using 5% Dextrose Injection, USP in an IV bag. The diluted product is administered to patients using a system with a standard infusion line and optional 0.2 micrometer in-line filter.

[0052] To be successful, the formulation must be stable throughout these steps, including dilution with 5% Dextrose Injection, USP.

[0053] In this study, 100 mg / mL hsIgG was prepared in 250 mM glycine / 0.02% polysorbate 20. The resulting solution was filtered through two 0.2 micrometer PES filtration membranes into a PETG container. The transferred sample aliquot was transferred to a glass vial and diluted to 60 mg / mL hsIgG with 5% Dextrose Injection USP. Samples were tested under the following conditions: a) diluted to 60 mg / mL hsIgG in 5% Dextrose Injection, USP; b) hsIgG was diluted to 60 mg / mL in 5% Dextrose Injection, USP and stored at 5° C. for 72 hours. c) Dilution to 60 mg / mL hsIgG in 5% Dextrose Injection, USP, storage at 5° C. for 72 hours, filtered using a 0.2 micron PES filter.

[0054] Under all conditions, the initial formulation, 100 mg / mL hsIgG in 250 mM glycine, was used as a control. The results of this study are shown in Table 4, and it can be seen that the 250 mM glycine / 0.02% polysorbate 20 formulation exhibited good stability upon dilution.

[0055] [Table 4]

[0056] In this study, 100 mg / mL (hsIgG) was prepared in 250 mM glycine / 0.02% (w / v) polysorbate 20. The resulting solution was filtered through two 0.2 micrometer PES filtration membranes into a PETG container. Samples from the PETG container were tested under the following conditions: a) T zero (stability start); b) Store at 5°C for 1 month. c) Store at 5℃ and 25℃ for 3 months. d) Store at 5°C for 7 months.

[0057] The results of this study are shown in Table 5, where it can be seen that the 250 mM glycine / 0.02% polysorbate 20 formulation exhibited good stability upon storage.

[0058] [Table 5]

[0059] Example 4: Effect of pH on Purity In this study, three formulations were evaluated: H0: 100 mg / mL hsIgG, 250 mM glycine, pH 5.2; H1: 100 mg / mL hsIgG, 250 mM glycine, pH 5.2, 0.02% PS20; H2: 100 mg / mL hsIgG, 250 mM glycine, pH 4.2; and H3: 100 mg / mL hsIgG, 250 mM glycine, pH 4.2, 0.02% PS20. The resulting formulations were filtered through a 0.2 μM PES filter membrane into a particle-free PETG container. The formulations were then transferred to 2R-type 1 glass vials and tested.

[0060] [Table 6]

[0061] Samples with lower pH correlated with higher purity by SEC-HPLC of monomer. Samples with lower pH correlated with higher purity by SEC-HPLC of monomer+dimer and lower aggregates.

[0062] Example 5: Effect of Polysorbate 20 on Subvisible Particle Formation In this study, the effect of polysorbate 20 on resistance to shear stress was investigated. Three formulations were prepared: H0: 100 mg / mL M254, 250 mM glycine, pH 5.2; H1: 100 mg / mL M254, 250 mM glycine, pH 5.2, 0.02% PS20; H2: 100 mg / mL M254, 250 mM glycine, pH 4.2; and H3: 100 mg / mL M254, 250 mM glycine, pH 4.2, 0.02% PS20. The formulations were filtered through a 0.2 μM PES filter membrane into a particle-free PETG container. The formulations were then transferred to 2R type 1 glass vials and tested.

[0063] [Table 7]

[0064] Example 6: Effect of non-ionic surfactants on shear stress The ability of other surfactants to protect a 100 mg / mL hsIgG formulation from the adverse effects of shear stress was investigated. PS20 at 0.02%, 0.06% or 0.10% was found to be effective, as was polysorbate 80 (2-hydroxyethyl 2-deoxy-3,5-bis-O-(2-hydroxyethyl)-6-O-{2-[(9E)-octadec-9-enoyloxy]ethyl}hexofuranoside, polyoxyethylene 20 sorbitan monooleate, PS80) or F68 (polyoxyethylene polyoxypropylene block copolymer CAS number 9003-11-6. PubChem SID 24898182) at the same concentrations. In each case, vials containing 2.4 mL of the formulation and a control without surfactant were stirred at 1,000 rpm for 4 hours at ambient temperature and analyzed visually by size exclusion HPLC and particle imaging analysis. After stirring, the non-surfactant samples showed slight haze compared to their static counterparts. The surfactant-containing samples were clear and free of visible particles. By SE_HPLC analysis, all surfactant-containing samples showed comparable monomer percentages (88.2%-89.3%). After stirring, the non-surfactant samples showed higher particle concentrations compared to their static counterparts. Due to their significantly higher particle concentrations, the stirred non-surfactant samples did not need to be digitally filtered. The surfactant containing samples had very low levels of sub-visible particles compared to the no surfactant samples.

[0065] Although the present invention has been described in connection with its detailed description, it is understood that the foregoing description is illustrative of the scope of the invention and is not intended to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The present invention may include the following aspects. [1] 1. A liquid pharmaceutical composition comprising an immunoglobulin in 250 mM glycine, 0.02% (w / v) polysorbate 20, pH 4-7, wherein at least 50% of the branched glycans on the Fc region of said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages. [2] The liquid pharmaceutical composition according to [1], wherein the immunoglobulin concentration is 50 to 250 mg / mL. [3] The liquid pharmaceutical composition according to [2], wherein the immunoglobulin concentration is 70 to 130 mg / mL. [4] The liquid pharmaceutical composition according to [2], wherein the immunoglobulin concentration is 80 to 120 mg / mL. [5] The liquid pharmaceutical composition according to [2], wherein the immunoglobulin concentration is 90 to 110 mg / mL. [6] The liquid pharmaceutical composition according to any one of [1] to [5], wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc domain of the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages. [7] The liquid pharmaceutical composition according to any one of [1] to [6], wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages. [8] The liquid pharmaceutical composition according to any one of [1] to [7], wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab domain of the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages. [9] The liquid pharmaceutical composition according to any one of [1] to [8], wherein at least 90% of the immunoglobulins are IgG immunoglobulins.

[10] The liquid pharmaceutical composition according to [9], wherein at least 95% of the immunoglobulins are IgG immunoglobulins.

[11] The liquid pharmaceutical composition according to any one of [1] to

[10] , wherein 5 to 20% of the immunoglobulin is a dimer.

[12] The liquid pharmaceutical composition according to any one of [1] to

[11] , wherein 5 to 10% of the immunoglobulin is a dimer.

[13] The liquid pharmaceutical composition according to any one of [1] to

[12] , wherein at least 80% of the immunoglobulin is a monomer or a dimer.

[14] The liquid pharmaceutical composition according to

[13] , wherein at least 85% of the immunoglobulin is a monomer or a dimer.

[15] The liquid pharmaceutical composition according to

[13] , wherein at least 90% of the immunoglobulin is a monomer or a dimer.

[16] The liquid pharmaceutical composition according to any one of [1] to

[15] , wherein 5 to 20% of the IgG immunoglobulins are dimers.

[17] The liquid pharmaceutical composition according to

[16] , wherein 5 to 10% of the IgG immunoglobulin is a dimer.

[18] The liquid pharmaceutical composition according to any one of [1] to

[17] , wherein at least 80% of the IgG immunoglobulins are monomers or dimers.

[19] The liquid pharmaceutical composition according to

[18] , wherein at least 85% of the IgG immunoglobulins are monomers or dimers.

[20] The liquid pharmaceutical composition according to

[18] , wherein at least 90% of the IgG immunoglobulins are monomers or dimers.

[21] The liquid pharmaceutical composition according to any one of [1] to

[20] , wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc domain of the IgG immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

[22] The liquid pharmaceutical composition according to any one of [1] to

[21] , wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the IgG immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

[23] The liquid pharmaceutical composition according to any one of [1] to

[22] , wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab domain of the IgG immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

[24] The liquid pharmaceutical composition according to [1], wherein the pH is 4.7 to 5.5.

[25] The liquid pharmaceutical composition according to [1], wherein the pH is 5.1 to 5.3.

[26] The liquid pharmaceutical composition according to any one of [1] to

[25] , wherein the composition has less than 1000 particles having a diameter of 10 to 100 micrometers after stirring at 1000 RPM for 8 hours at 2 to 8°C.

[27] The liquid pharmaceutical composition according to any one of [1] to

[26] , wherein the composition has less than 500 particles having a diameter of 10 to 100 micrometers after stirring at 1000 RPM for 8 hours at 2 to 8°C.

[28] The liquid pharmaceutical composition according to any one of [1] to

[27] , wherein the composition has less than 200 particles having a diameter of 10 to 100 micrometers after stirring at 1000 RPM for 8 hours at 2 to 8°C.

[29] The liquid pharmaceutical composition according to any one of [1] to

[28] , wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc domain of the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages after storage at 4° C. for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

[30] The liquid pharmaceutical composition according to any one of [1] to

[29] , wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages after storage at 4° C. for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

[31] The liquid pharmaceutical composition according to any one of [1] to

[30] , wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab domain of the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages after storage at 4° C. for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

[32] The liquid pharmaceutical composition according to any one of [1] to

[31] , wherein 5 to 10% of the immunoglobulin is a dimer after storage at 4° C. for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[33] The liquid pharmaceutical composition according to any one of [1] to

[32] , wherein at least 85% of the immunoglobulins are monomers or dimers after storage at 4° C. for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

[34] The liquid pharmaceutical composition according to any one of [1] to

[33] , wherein at least 90% of the immunoglobulin is a monomer or a dimer after storage at 4° C. for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months.

[35] The liquid pharmaceutical composition according to any one of [1] to

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

[36] The liquid pharmaceutical composition described in any one of

[26] to

[35] , wherein the storage item is in a sealed United States Pharmacopoeia Type 1 glass vial.

[37] The liquid pharmaceutical composition according to any one of

[26] to

[35] , wherein the stored item is in a sealed 2R type 1 glass injection vial.

Claims

1. 1. A liquid pharmaceutical composition comprising 50-250 mg / mL of an immunoglobulin in 250 mM glycine 0.02% (w / v) polysorbate 20 at pH 4-7, wherein at least 50% of the branched glycans on the Fc region of said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages, and said liquid pharmaceutical composition has agitation stability against particle formation.

2. 2. The liquid pharmaceutical composition of claim 1, wherein the concentration of the immunoglobulin is 70 to 130 mg / mL.

3. 2. The liquid pharmaceutical composition of claim 1, wherein the concentration of the immunoglobulin is 80 to 120 mg / mL.

4. 2. The liquid pharmaceutical composition of claim 1, wherein the concentration of the immunoglobulin is 90 to 110 mg / mL.

5. The liquid pharmaceutical composition according to any one of claims 1 to 4, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

6. The liquid pharmaceutical composition according to any one of claims 1 to 5, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

7. The liquid pharmaceutical composition according to any one of claims 1 to 6, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab domain of said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

8. The liquid pharmaceutical composition according to any one of claims 1 to 7, wherein at least 90% of said immunoglobulins are IgG immunoglobulins.

9. 9. The liquid pharmaceutical composition of claim 8, wherein at least 95% of said immunoglobulins are IgG immunoglobulins.

10. The liquid pharmaceutical composition according to any one of claims 1 to 9, wherein 5 to 20% of said immunoglobulins are dimers.

11. The liquid pharmaceutical composition according to any one of claims 1 to 10, wherein 5 to 10% of said immunoglobulins are dimers.

12. The liquid pharmaceutical composition according to any one of claims 1 to 11, wherein at least 80% of said immunoglobulins are monomers or dimers.

13. 13. The liquid pharmaceutical composition of claim 12, wherein at least 85% of the immunoglobulins are monomers or dimers.

14. 13. The liquid pharmaceutical composition of claim 12, wherein at least 90% of the immunoglobulins are monomers or dimers.

15. The liquid pharmaceutical composition according to any one of claims 8 and 9, wherein 5-20% of said IgG immunoglobulins are dimers.

16. 16. The liquid pharmaceutical composition of claim 15, wherein 5-10% of said IgG immunoglobulins are dimers.

17. 10. The liquid pharmaceutical composition according to claim 8, wherein at least 80% of said IgG immunoglobulins are monomers or dimers.

18. 18. The liquid pharmaceutical composition of claim 17, wherein at least 85% of said IgG immunoglobulins are monomers or dimers.

19. 18. The liquid pharmaceutical composition of claim 17, wherein at least 90% of the IgG immunoglobulins are monomers or dimers.

20. The liquid pharmaceutical composition of any one of claims 1 to 19, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of the immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

21. The liquid pharmaceutical composition according to any one of claims 1 to 20, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

22. The liquid pharmaceutical composition according to any one of claims 1 to 21, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab domain of said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages.

23. The liquid pharmaceutical composition according to claim 1, wherein the pH is from 4.7 to 5.

5.

24. The liquid pharmaceutical composition according to claim 1, wherein the pH is from 5.1 to 5.

3.

25. 25. The liquid pharmaceutical composition of any one of claims 1-24, wherein each mL of said composition has less than 1000 particles having a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C.

26. 26. The liquid pharmaceutical composition of any one of claims 1-25, wherein each mL of said composition has less than 500 particles having a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C.

27. 27. The liquid pharmaceutical composition of any one of claims 1-26, wherein each mL of said composition has less than 200 particles having a diameter of 10-100 micrometers after stirring at 1000 RPM for 8 hours at 2-8°C.

28. 28. The liquid pharmaceutical composition of any one of claims 1 to 27, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

29. 29. The liquid pharmaceutical composition of any one of claims 1 to 28, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

30. 30. The liquid pharmaceutical composition of any one of claims 1 to 29, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab domain of said immunoglobulin are disialylated by NeuAc-α 2,6-Gal terminal linkages after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

31. 31. The liquid pharmaceutical composition of any one of claims 1 to 30, wherein 5-10% of said immunoglobulins are dimers after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

32. 32. The liquid pharmaceutical composition of any one of claims 1 to 31, wherein at least 85% of said immunoglobulins are monomers or dimers after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

33. 33. The liquid pharmaceutical composition of any one of claims 1 to 32, wherein at least 90% of said immunoglobulins are monomers or dimers after storage at 4°C for 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months.

34. A liquid pharmaceutical composition according to any one of claims 1 to 33, which is stable for at least 7 months at 5°C, for at least 1 month at 25°C, for 2 years at 2-8°C, and / or for 2 weeks at 15-30°C.

35. 35. The liquid pharmaceutical composition of claim 34, wherein said liquid pharmaceutical composition storage is in a sealed United States Pharmacopeia Type 1 glass vial.

36. 35. The liquid pharmaceutical composition of claim 34, wherein said liquid pharmaceutical composition storage is in a sealed 2R type 1 glass injection vial.

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