阅读说明：本技术 唾液酸化糖蛋白 (Sialylated glycoproteins ) 是由 S·D·帕蒂尔 于 2020-04-17 设计创作，主要内容包括：本发明描述了包含高度唾液酸化免疫球蛋白的药物制剂。所述制剂对剪切应力稳定。本文所述的药物组合物提供药学上可接受的hsIgG组合物,该组合物对剪切应力是稳定的(例如,当制剂经受剪切应力(诸如搅拌)时,例如在运输期间不形成大量的亚可见颗粒),因此可以液体形式运输和处理。(The present invention describes pharmaceutical formulations comprising highly sialylated immunoglobulins. The formulation is stable to shear stress. The pharmaceutical compositions described herein provide pharmaceutically acceptable hsIgG compositions that are stable to shear stress (e.g., do not form substantial amounts of sub-visible particles when the formulation is subjected to shear stress (such as stirring), e.g., during transport), and thus can be transported and handled in liquid form.)

1. A liquid pharmaceutical composition comprising an immunoglobulin in 250mM 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 via the NeuAc- α 2,6-Gal terminal bond.

2. The liquid pharmaceutical composition of claim 1, wherein the concentration of the immunoglobulin is 50mg/mL to 250 mg/mL.

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

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

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

6. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc domain of the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal bond.

7. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the immunoglobulins are disialylated via the NeuAc-a 2,6-Gal terminal linkage.

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

9. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 90% of the immunoglobulins are IgG immunoglobulins.

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

11. The liquid pharmaceutical composition according to any one of the preceding claims, wherein 5-20% of the immunoglobulins are dimers.

12. The liquid pharmaceutical composition according to any one of the preceding claims, wherein 5-10% of the immunoglobulins are dimers.

13. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 80% of the immunoglobulins are monomers or dimers.

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

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

16. The liquid pharmaceutical composition according to any one of the preceding claims, wherein 5-20% of the IgG immunoglobulins are dimers.

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

18. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 80% of the IgG immunoglobulins are monomers or dimers.

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

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

21. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc domain of the IgG immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal bond.

22. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the IgG immunoglobulins are disialylated via the NeuAc-a 2,6-Gal terminal linkage.

23. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab domain of the IgG immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal bond.

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

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

26. The liquid pharmaceutical composition according to any one of the preceding claims, wherein the composition has less than 1000 particles with a diameter between 10 and 100 microns after stirring at 1000RPM for 8 hours at 2-8 ℃.

27. The liquid pharmaceutical composition according to any one of the preceding claims, wherein the composition has less than 500 particles with a diameter between 10 and 100 microns after stirring at 1000RPM for 8 hours at 2-8 ℃.

28. The liquid pharmaceutical composition according to any one of the preceding claims, wherein the composition has less than 200 particles with a diameter between 10 and 100 microns after stirring at 1000RPM for 8 hours at 2-8 ℃.

29. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc domain of the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal bond after storage at 4 ℃ 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 the preceding claims, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal linkage after storage at 4 ℃ 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 the preceding claims, wherein at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab domain of the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal bond after 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months of storage at 4 ℃.

32. The liquid pharmaceutical composition according to any one of the preceding claims, wherein 5-10% of the immunoglobulins are dimers after 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months of storage at 4 ℃.

33. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 85% of the immunoglobulins are monomers or dimers after 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months of storage at 4 ℃.

34. The liquid pharmaceutical composition according to any one of the preceding claims, wherein at least 90% of the immunoglobulins are monomers or dimers after 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months of storage at 4 ℃.

35. The liquid pharmaceutical composition of any one of the claims, wherein the formulation is stable for at least 7 months at 5 ℃, at least one month at 25 ℃, two years at 2-8 ℃ and/or two weeks at 15-30 ℃.

36. The liquid pharmaceutical composition of any one of claims 26-35, wherein the storage is in a sealed usp type 1 glass vial.

37. The liquid pharmaceutical composition of any one of claims 26-35, wherein the storage is in a sealed glass injection vial type 2R 1.

Background

Intravenous immunoglobulin (IVIg) is prepared from pooled plasma of human donors (e.g., pooled plasma from at least 1,000 donors), and although mostly composed of IgG antibodies (primarily IgG1 antibodies), IVIg may also contain trace amounts of other antibody subclasses. Commercially available IVIg preparations typically exhibit low levels of sialylation on the Fc domain of the antibodies present. In particular, antibodies in commercial IVIg preparations exhibit low levels of bi-sialylation of branched glycans on the Fc region.

Disclosure of Invention

Described herein are pharmaceutical compositions comprising highly sialylated immunoglobulins (hsIgG). hsIgG has very high levels of sialic acid on the branched glycans on the Fc region of an immunoglobulin, e.g., at least 50% (60%, 70%, 80%, 90% or more) of the branched glycans on the Fc region of an immunoglobulin are sialylated via the NeuAc- α 2,6-Gal terminal bonds on both the α 1,3 and α 1,6 arms of the branched glycans.

The pharmaceutical compositions described herein provide pharmaceutically acceptable hsIgG compositions that are stable to shear stress (e.g., do not form large numbers of sub-visible particles when the formulation is subjected to shear stress, e.g., stirring during transport), and thus can be transported and handled in liquid form. The formulations are also stable upon dilution, e.g., dilution in 5% dextrose for intravenous administration. The formulation is stable, for example, at 5 ℃ for at least 7 months, and at 25 ℃ for at least one month, at 2 ℃ to 8 ℃ for two years, and/or at 15 ℃ to 30 ℃ for two weeks.

Described herein are liquid pharmaceutical compositions comprising an immunoglobulin in 250mM 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 via the NeuAc- α 2,6-Gal terminal bond.

In various embodiments: the concentration of the immunoglobulin is 50mg/mL-250 mg/mL; the concentration of the immunoglobulin is 70mg/mL-130 mg/mL; the concentration of the immunoglobulin is 80mg/mL-120 mg/mL; the concentration of the immunoglobulin is 90mg/mL-110 mg/mL; at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal linkage; at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal linkage; at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab region of the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal linkage; at least 90% of the immunoglobulins are IgG immunoglobulins; at least 95% of the immunoglobulins are IgG immunoglobulins; 5% -20% of the immunoglobulin is a dimer; 5% -10% of the immunoglobulin is a dimer; at least 80% of the immunoglobulins are monomeric or dimeric; at least 85% of the immunoglobulins are monomeric or dimeric; at least 90% of the immunoglobulins are monomers or dimers, and 5% -20% of the IgG immunoglobulins are dimers; 5% -10% of IgG immunoglobulins are dimers; at least 80% of the IgG immunoglobulins are monomeric or dimeric; at least 85% of the IgG immunoglobulins are monomeric or dimeric; at least 90% of the IgG immunoglobulins are monomeric or dimeric; at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of an IgG immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal linkage; at least 60%, 70%, 80%, 90% or 95% of the branched glycans on IgG immunoglobulins are disialylated via the NeuAc- α 2,6-Gal terminal linkage; at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab region of the IgG immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal linkage; the pH value is 4.7-5.5; the pH value is 5.1-5.3; after stirring at 1000RPM for 8 hours at 2 ℃ to 8 ℃, the composition has less than 1000 particles with a diameter between 10 microns and 100 microns; after stirring at 1000RPM for 8 hours at 2 ℃ to 8 ℃, the composition has less than 500 particles with a diameter between 10 microns and 100 microns; after stirring at 1000RPM for 8 hours at 2 ℃ to 8 ℃, the composition has less than 200 particles with a diameter between 10 microns and 100 microns; at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fc region of the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal linkage after storage at 4 ℃ for 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 the immunoglobulins are disialylated via the NeuAc- α 2,6-Gal terminal linkage after 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months of storage at 4 ℃; at least 60%, 70%, 80%, 90% or 95% of the branched glycans on the Fab region of the immunoglobulin are disialylated via the NeuAc- α 2,6-Gal terminal linkage after storage at 4 ℃ for 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 months; 5% -10% of the immunoglobulins are dimers after 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months of storage at 4 ℃; at least 85% of the immunoglobulins are monomeric or dimeric after storage at 4 ℃ for 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months; at least 90% of the immunoglobulins are monomeric or dimeric after storage at 4 ℃ for 3,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 months; storage is in sealed usp type 1 glass vials; storage was in sealed glass injection vials type 2R 1.

In hsIgG, at least 50% (e.g., 60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98% up to 100% and including 100%) of the branched glycans on the Fc region of an immunoglobulin have sialic acid residues on both the α 1,3 arm and the α 1,6 arm (i.e., disialylation is via the NeuAc- α 2,6-Gal terminal linkage). In some embodiments, at least 50% (e.g., 60%, 70%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98%, or up to 100% and including 100%) of the branched glycans on the Fab region are disialylated through the NeuAc- α 2,6-Gal terminal bond in addition to Fc sialylation. In some cases, at least 85% (87%, 90%, 92%, 94%, 95%, 97%, 98%, or up to 100% and including 100%) of the total branched glycans (sum of glycans on the Fc domain and the Fab domain) are disialylated through the NeuAc- α 2,6-Gal terminal bond. 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 via the NeuAc- α 2,6-Gal terminal linkage (e.g., sialylation only on the α 1,3 arm or the α 1,6 arm). Immunoglobulins are HsIgG preparations of predominantly IgG antibodies (e.g., at least 80%, 85%, 90%, 95% by weight of the immunoglobulins are IgG antibodies of various isotypes).

As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides," each comprising an antibody constant region other than a CH1 domain. In some embodiments, an "Fc region" comprises two Fc polypeptides connected by one or more disulfide bonds, chemical linkers, or peptide linkers. "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include part or all of the flexible hinge N-terminus of these domains.

As used herein, a "glycan" is a saccharide, which may be a monomer or polymer of saccharide residues, such as at least three saccharides, and may be linear or branched. "glycans" can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexoses, arabinose, ribose, xylose, etc.) and/or modified sugars (e.g., 2' -fluororibose, 2' -deoxyribose, mannose phosphate, 6' sulfo N-acetylglucosamine, etc.). The term "glycan" includes homopolymers and heteropolymers of sugar residues. The term "glycan" also encompasses the glycan component of glycoconjugates (e.g., polypeptides, glycolipids, proteoglycans, etc.). The term also encompasses free glycans, including glycans that have been cleaved or otherwise released from the glycoconjugate.

As used herein, the term "glycoprotein" refers to a protein comprising a peptide backbone covalently linked to one or more sugar moieties (i.e., glycans). The sugar moiety may be in the form of a monosaccharide, disaccharide, oligosaccharide and/or polysaccharide. The sugar moiety may comprise a single unbranched chain of sugar residues, or may comprise one or more branched chains. The glycoprotein may comprise an O-linked sugar moiety and/or an N-linked sugar moiety.

IVIg is the preparation of pooled multivalent immunoglobulins (including 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 Gamma, (Baxter Healthcare corporation), Gamma, (Bio Products laboratory), Bivigam (Biotest Pharmaceuticals corporation), CarimmuneNF (CSL Behring AG), Gamunes-C (Grifols Therapeutics, Inc.), Glecogamama DID (institutto Grifols, SA), and Octagam (Octapharma Pharmazeutika Produgs Mbh). IVIg is approved for plasma protein replacement therapy in immunodeficient patients and other uses. The level of sialylation of IVIg Fc glycans varies between IVIg preparations, but is typically less than 20%. The disialylation level is generally much lower.

As used herein, "N-glycosylation site of an 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 comprises a dimer of Fc polypeptides, and the Fc region comprises two N-glycosylation sites, one on each Fc polypeptide.

As used herein, "percentage (%) of branched glycans" refers to moles of glycan X relative to the total moles of glycans present, wherein X represents the glycan of interest.

The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount (e.g., a dose) that is effective in treating a patient having a disease or disorder described herein. It is also understood herein that a "pharmaceutically effective amount" may be construed as an amount that imparts a desired therapeutic effect, either in a single dose or any dose or route, taken alone or in combination with other therapeutic agents.

"pharmaceutical formulations" and "pharmaceutical products" can be included in kits that contain the formulation or product and instructions for use.

"pharmaceutical preparation" and "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. To this end, the "pharmaceutical preparation" and the "pharmaceutical product" are substantially free of ST6Gal sialyltransferase and/or sialic acid donor (e.g., cytidine 5 '-monophosphate-N-acetylneuraminic acid) or by-products thereof (e.g., cytidine 5' -monophosphate).

"pharmaceutical preparations" and "pharmaceutical products" are generally substantially free of other components of the cell in which the glycoprotein is produced (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA, if recombinant).

By "purified" (or "isolated") is meant that the polynucleotide or polypeptide is removed or isolated from other components that are present in its natural environment. For example, an isolated polypeptide is a polypeptide 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 a polynucleotide that is isolated from other nuclear components (e.g., histones) and/or from upstream or downstream nucleic acids. An isolated polynucleotide or polypeptide may be at least 60% free, or at least 75% free, or at least 90% free, or at least 95% free of other components present in the natural environment of the indicated polynucleotide or polypeptide.

As used herein, the term "sialylation" refers to a glycan having a terminal sialic acid. The term "monosialylated" refers to branched glycans with one terminal sialic acid, e.g., on the α 1,3 arm or the α 1,6 arm. The term "disialylated" refers to branched glycans with terminal sialic acids on both arms (e.g., both α 1,3 and α 1,6 arms).

Drawings

Fig. 1 schematically depicts an example of branched glycans. The shallow circle is Gal; the black circle is Man; triangle is Fuc and diamond is NANA; the square is GlcNAc.

FIG. 2 left panel: schematic representation of enzymatic sialylation reaction to convert pooled immunoglobulins to hsIgG. Right panel: IgG Fc glycan profiles of the starting IVIg and hsIgG enzymatically prepared from IVIg. Glycan profiles of different IgG subclasses were obtained via glycopeptide mass spectrometry. The peptide sequences used to quantify glycopeptides of different IgG subclasses were: IgG1 was EEQYNSTYR, IgG2/3EEQFNSTFR, IgG3/4EEQYNSTFR and EEQFNSTYR.

FIG. 3 depicts a vial of hsIgG for use in a conventional formulation of IVIg that has been subjected to shear stress.

FIG. 4 depicts a vial of hsIgG in a formulation of the disclosure that has been subjected to shear stress.

Detailed Description

Immunoglobulins are glycosylated at conserved positions in the constant region of their heavy chains. For example, human IgG has a single N-linked glycosylation site at Asn297 of the CH2 domain. Each immunoglobulin type has a different kind of N-linked carbohydrate structure in the constant region. For human IgG, the core oligosaccharide is usually made up of GlcNAc with different numbers of outer residues₂Man₃GlcNAc. Differences between individual iggs can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).

The present disclosure encompasses, in part, pharmaceutical preparations comprising a pooled human immunoglobulin with an Fc region having a specific level of a branched glycan sialylated on both branched glycans in the Fc region (e.g., via a NeuAc- α 2,6-Gal terminal linkage).

The formulation of pooled multivalent human immunoglobulins, including IVIg formulations, is highly complex because they are highly heterogeneous in several respects. They include immunoglobulins pooled from hundreds or more than 1000 individuals. While at least about 90% or 95% of the immunoglobulins are of the IgG isotype (in all subclasses), other isotypes exist, including IgA and IgM. The production of immunoglobulins in IVIg and pooled multivalent human immunoglobulins differ in both specificity and glycosylation pattern.

The high sialylation of the pooled multivalent immunoglobulins alters the glycans present on the immunoglobulins. For some glycans, the alteration requires the addition of one or more galactose molecules and the addition of one or more sialic acid molecules. For other glycans, the change only requires the addition of one or more sialic acid molecules. Furthermore, while substantially all IgG antibodies (i.e., the primary immunoglobulin in the pooled multivalent immunoglobulin preparation) have glycosylation sites on each polypeptide forming the Fc region, not all IgG antibodies have glycosylation sites on the Fab domain. Altering the glycosylation of an immunoglobulin preparation alters the structure and activity of the individual immunoglobulins in the preparation and, importantly, alters the interactions between the individual immunoglobulins and the overall behavior of the immunoglobulin preparation.

The widely used preparations for IVIg preparations are completely unsuitable for pharmaceutical preparations of highly sialylated immunoglobulins (hsIgG), at least because the preparations are unstable to shear stresses occurring in normal transport of pharmaceutical preparations when used for hsIgG. When subjected to this type of shear stress, sub-visible particles are formed in the hsIgG formulation. It is known that such sub-visible particles in antibody preparations can cause serious adverse events at the injection site and lead to a target immune response. Sub-visible particles in the antibody preparation may also activate the complement system, cause embolism, and other negative immunogenic reactions. It was found that the addition of nonionic surfactant makes hsIgG formulations more stable to shear stress and greatly reduces the formation of sub-visible particles.

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 polypeptides derived from IVIg. HsIgG can be prepared as described in WO 2014/179601. The preparation of hsIgG has also been described in Washburn et al (Proc Natl Acad Sci USA, 3/17/2015; 112 (11): E1297-306). The level of sialylation in an hsIgG preparation can be measured on the Fc domain (e.g., the number of sialylated branched glycans on the a 1,3 arm, the a 1,6 arm, or both of the branched glycans in the Fc domain), or on the overall sialylation (e.g., the number or percentage of sialylated branched glycans on the a 1,3 arm, the a 1,6 arm, or both of the branched glycans, whether on the Fc domain or the Fab domain in the preparation of the polypeptide).

In some cases, pooled sera used as immunoglobulin sources for making hsIgG are isolated from a particular population of individuals, e.g., individuals who produce antibodies against one or more viruses (such as COVID-19, SARS, parainfluenza, influenza) but do not have an active infection. In some cases, the immunoglobulin is isolated from a population of individuals in which greater than 50%, 55%, 60%, 75% of the individuals produce antibodies to the selected virus.

A protein in which an N-linked oligosaccharide chain is added to the lumen of the endoplasmic reticulum. In particular, the initial oligosaccharide (typically the 14-sugar) 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. The structure of this initial oligosaccharide is common to most eukaryotes and contains three glucose residues, nine mannose residues and two N-acetylglucosamine residues. This initial oligosaccharide chain can be trimmed by specific glycosidases in the endoplasmic reticulum, resulting in a short-branched core oligosaccharide consisting of two N-acetylglucosamine residues and three mannose residues. One of the branches is referred to in the art as the "α 1,3 arm" and the second branch is referred to as the "α 1,6 arm" as shown in fig. 1.

N-glycans can be subdivided into three distinct groups called "high mannose," heterozygotes, "and" complicates, "in which a common pentasaccharide core (Man (α 1,6) - (Man (α 1,3)) -Man (β 1,4) -GlcpNAc (β 1, N) -Asn) occurs in all three groups.

After initial processing in the endoplasmic reticulum, the polypeptide is transported to the golgi apparatus where further processing can occur. If glycans are transferred to the golgi apparatus before they are completely trimmed to the core pentasaccharide structure, a "high mannose glycan" results.

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 subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains terminated with any of sialic acid, galactose, or N-acetylglucosamine residues. In addition, 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.

"hybrid glycans" comprise characteristics of both high mannose and complex glycans. For example, one branch of a hybrid glycan may contain predominantly or exclusively mannose residues, while the other branch may contain N-acetylglucosamine, sialic acid, galactose, and/or fucose.

Sialic acids are a family of 9-carbon monosaccharides that have heterocyclic structures. They are negatively charged via carboxylic acid groups attached to the ring and other chemical modifications including N-acetyl and N-glycolyl groups. The two major types of sialic acid residues present in polypeptides produced in mammalian expression systems are N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). They typically occur as terminal structures attached to galactose (Gal) residues at the non-reducing ends of both N-linked glycans and O-linked glycans. The glycosidic bond configuration of these sialic acid groups can be α 2,3 or α 2, 6.

The Fc region is glycosylated at a conserved N-linked glycosylation site. For example, each heavy chain of an IgG antibody is at C_H2 domain has a single N-linked glycosylation site at Asn 297. IgA antibodies in C_H2 and C_H3 domain with N-linked glycosylation sites, IgE antibody at C_H3 domain with N-linked glycosylation sites, and IgM antibodies at C_H1、C_H2、C_H3 and C_H4 domains have N-linked glycosylation sites.

Each antibody isotype has a different kind of N-linked carbohydrate structure in the constant region. For example, IgG is at C in each Fc polypeptide of the Fc region_H2 domain has a single N-linked double-branched carbohydrate at Asn297 which also contains the binding site for C1q and Fc γ R. For human IgG, the core oligosaccharide is usually made up of GlcNAc with different numbers of outer residues₂Man₃GlcNAc. Differences between individual iggs can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc). The glycans of the polypeptides may be assessed using any method known in the art. For example, sialylation of the glycan composition (e.g., the level of branched glycans sialylated on the α 1,3 arm and/or the α 1,6 arm) can be characterized using the methods described in WO 2014/179601.

In addition to antibody monomers, compositions containing hsIgG can include dimers and aggregates of antibodies. In some cases, pH can be used to adjust the percentage of monomers, dimers, and aggregates in the composition, as measured by size exclusion chromatography in weight% purity. In some cases, lowering the pH increases the weight% of monomer + dimer in solution. In some cases, lowering the pH increases the weight% of monomer in solution. In some cases, increasing the pH decreases the% monomer in solution. In some cases, the weight% of the aggregate is less than or equal to 3.0 weight% (e.g., less than or equal to 2.7 weight%, 2.5 weight%, 2.3 weight%, 2.0 weight%, 1.7 weight%, 1.5 weight%, 1.3 weight%, 1.0 weight%, 0.9 weight%, 0.8 weight%, 0.7 weight%, 0.6 weight%, 0.5 weight%, 0.4 weight%, 0.3 weight%, 0.2 weight%, or 0.1 weight%). In some cases, the weight% of monomer + dimer is greater than or equal to 97.0 weight% (e.g., greater than or equal to 98 weight% or 99 weight%). In some cases, the weight% of monomer is greater than or equal to 80, 83, 85, or 87 weight%. In some cases, the pH is less than or equal to 5.3 (e.g., less than or equal to 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 of the pharmaceutical composition is adjusted such that the weight% of the monomer changes. In some cases, the pH of the pharmaceutical composition is lowered such that the weight% of the monomer is increased. In some cases, the pH of the pharmaceutical composition is increased such that the wt% of dimer is increased. In some cases, the pH is such that the monomer weight% is greater than or equal to 90 weight% (e.g., greater than or equal to 91, 92, 93, 94, 95, 96, 97, 98, or 99 weight%).

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

Example 1: highly sialylated IgG formulated in 250mM Glycine

Highly sialylated IgG in which more than 60% of the branched Fc region glycans are dissialylated are typically prepared as described in WO 2014/179601.

Briefly, IVIg was exposed to a one-pot sequential enzymatic reaction using β 1,4 galactosyltransferase 1(B4-GalT) and α 2, 6-sialyltransferase (ST6-Gal 1). Galactosyltransferases selectively add galactose residues to pre-existing asparagine-linked glycans in IVIg. The resulting galactosylated glycans serve as substrates for sialyltransferases, which selectively add sialic acid residues to end-cap asparagine-linked glycan structures linked to IVIg. Thus, the total sialylation reaction employed two sugar nucleotides (UDPGal and CMP-NANA). The latter is periodically supplemented to increase the di-sialylation product relative to the mono-sialylation product. The reaction includes a cofactor manganese chloride.

Representative examples of corresponding IgG-Fc glycan profiles of the starting IVIg and reaction products are shown in the right panel of fig. 1. Glycan data are shown in IgG subclasses. Glycans from the IgG3 and IgG4 subclasses cannot be quantified individually. As shown, for IVIg, the sum of all non-sialylated glycans was greater than 80% and the sum of all sialylated glycans was less than 20%. For the reaction product, the sum of all unsialylated glycans is less than 20%, and the sum of all sialylated glycans is greater than 80%. The nomenclature of the different glycans listed in the glycoform uses the Oxford notation of N-linked glycans.

Non-highly sialylated ivigs (including commercial ivigs) are generally stable in glycine and do not generally form sub-visible particles when agitated, for example, during transport. Thus, an initial highly sialylated IgG (hsIVIg) preparation was prepared at 109mg/mL in 250mM glycine. The pH value is 4.7-5.5. The formulation was a clear to milky white, colorless to pale yellow solution. The formulation was also examined after filtration through a 0.2 micron PES filter into PETG containers. Table 1 provides the characteristics of the highly sialylated IgG preparation before and after filtration. Glycine only formulations appear to have acceptable product characteristics both before and after filtration.

Table 1: characterization of hsIgG in 250mM Glycine (pH 4.7-5.5)

Further studies with 250mM glycine (pH 4.7-5.5) formulations showed that hsIgG was subjected to agitation stress. The filtered sample was transferred to a glass vial and shaken at 1000RPM for up to 8 hours at refrigerated temperatures (2 ℃ -8 ℃). The samples were tested before stirring and after 4 and 8 hours after stirring. As can be seen from the photograph in fig. 3, stirring resulted in the formation of sub-visible particles that clouded the solution. It was found that the 250mM (pH 4.7-5.5) formulation became cloudy even when transported by airplane in the passenger compartment, indicating that the formulation would form sub-visible particles during distribution to healthcare providers and patients. Sub-visible particles can cause serious adverse events at the injection site and lead to a targeted immune response. Sub-visible particles may also activate the complement system, cause embolism, and other negative immunogenic reactions. Therefore, it is important to design formulations that are stable and do not form sub-visible particles upon stirring.

Example 2: stable formulations of hsIgG

A study was conducted to develop hsIVIG formulations that were stable to agitation stress but still retained the desired product attributes present in the 250mM glycine formulation.

It was found that low concentrations of polysorbate 20(2- [2- [3, 4-bis (2-hydroxyethoxy) oxolane-2-yl ] -2- (2-hydroxyethoxy) ethoxy ] ethyldodecanoate, polyoxyethylene (20) sorbitan monolaurate) improved the ability of hsIgG formulations to withstand the stirring stress while maintaining the desired product properties. Notably, the presence of the nonionic surfactant does not meaningfully alter the relative amounts of antibody monomers, dimers, and higher order aggregates.

Table 2: characterization of hsIgG in 250mM Glycine/0.02% Polysorbate 20(pH 4.7-5.5)

A sample of the filtered 250mM glycine/0.02% polysorbate 20(pH 4.7-5.5) formulation was transferred to a glass vial and shaken at 1000RPM for up to 8 hours at refrigerated temperatures (2-8 ℃). The samples were tested before stirring and after 4 and 8 hours after stirring. As can be seen from the photograph in fig. 4, the formulation is stable to the stirring stress.

Table 3 below provides information on the product properties of the two formulations before and after the stirring stress. It can be seen that the addition of 0.02% polysorbate greatly improved the stirring stability, but did not change the monomer/dimer ratio. The addition of 0.02% polysorbate 20 had no significant protein concentration or percentage of monomers, dimers, aggregates and low molecular weight species before or after exposure to the agitation stress.

Significantly lower sub-visible particle concentrations were observed in the polysorbate 20-containing formulation compared to the initial formulation. After exposure to the stirring stress (4 hours and 8 hours), comparable particle concentrations of both formulations compared to their respective stress-free conditions were observed.

Table 3: effect of agitation stress on stability

Example 3: effect of dilution with 5% dextrose injection

To prepare hsIgG for administration to a patient, the concentrated hsIgG formulation is sterile filtered through two 0.2 micron PES filters into pre-sterilized USP1 type glass vials to form the pharmaceutical product. The vials were transported to the clinical site for administration within 72 hours of manufacture (from the time of filtration). At the clinical site, the drug product (100mg/mL) was diluted to 60mg/mL using 5% glucose injection USP in IV bag and then administered. The diluted product is administered to the patient using standard infusion lines and systems with an optional 0.2 micron inline filter.

For success, the formulation must be stabilized by these steps, including dilution in 5% dextrose injection (USP).

In this study, a solution of 100mg/mL hsIgG in 250mM glycine/0.02% polysorbate 20 was prepared. The resulting solution was filtered through two 0.2 micron PES filter membranes into a PETG container. Transferred sample aliquots were transferred to glass vials and diluted to 60mg/mL hsIgG using 5% glucose injection USP. The samples were tested under the following conditions:

a) diluting to 60mg/mL hsIgG in 5% glucose injection USP;

b) diluted to 60mg/mL hsIgG + in 5% glucose injection USP + stored at 5 ℃ for 72 hours; and

c) dilution to 60mg/mL hsIgG in 5% glucose injection USP + storage at 5 ℃ for 72 hours + filtration using a 0.2 micron PES filter.

Under all conditions, the initial formulation, i.e., a solution of 100mg/mL hsIgG in 250mM glycine was used as a control. The results of this study are in table 4, where it can be seen that the 250mM glycine/0.02% polysorbate 20 formulation showed good stability upon dilution.

Table 4: stability on dilution

In this study, a solution of 100mg/mL (hsIgG) in 250mM glycine/0.02% (w/v) polysorbate 20 was prepared. The resulting solution was filtered through two 0.2 micron PES filter membranes into a PETG container. Samples from PETG containers were tested under the following conditions:

a) t zero (stability onset);

b) storing at 5 deg.C for 1 month;

c) storing at 5 ℃ and 25 ℃ for 3 months; and

d) stored at 5 ℃ for 7 months.

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

Table 5: stability on storage

Example 4: effect of pH on purity

In this study, three formulations were evaluated: h0: 100mg/mL hsIgG, 250mM glycine, pH 5.2; h1: 100mg/mL hsIgG, 250mM glycine, pH 5.2, 0.02% PS 20; h2: 100mg/mL hsIgG, 250mM glycine, pH 4.2; and H3: 100mg/mL hsIgG, 250mM glycine, pH 4.2, 0.02% PS 20. The resulting formulation was filtered through a 0.2 μ MPES filter into a particle-free PETG container. The formulations were then transferred to 2R1 type glass vials and tested.

Samples with lower pH were associated with higher percent purity of the monomer as determined by SEC-HPLC. Samples with lower pH were associated with higher percent purity of monomer + dimer and lower percent aggregate as determined by SEC-HPLC.

Example 5: effect of Polysorbate 20 on sub-visible particle formation

In this study, the effect of polysorbate 20 on shear stress resistance was examined. The following three formulations were prepared: h0: m254, 250mM glycine, pH 5.2, 100 mg/mL; h1: 100mg/mL M254, 250mM glycine, pH 5.2, 0.02% PS 20; h2: m254, 250mM glycine, pH 4.2, 100 mg/mL; and H3: 100mg/mL M254, 250mM glycine, pH 4.2, 0.02% PS 20. The formulation was filtered through a 0.2 μ MPES filter into a particle-free PETG container. The formulations were then transferred to 2R1 type glass vials and tested.

Example 6: effect of nonionic surfactants on shear stress

The ability of other surfactants to protect a 100mg/ml hsIgG formulation from adverse effects of shear stress was examined. It was found that 0.02%, 0.06% or 0.10% PS20 was as effective as polysorbate 80 (2-hydroxyethyl 2-deoxy-3, 5-bis-O- (2-hydroxyethyl) -6-O- {2- [ (9E) -octadec-9-enoyloxy ] ethyl } hexanfuranoside; polyoxyethylene 20 sorbitan monooleate PS80) or F68 (polyoxyethylene-polyoxypropylene block copolymer, CAS No. 9003-11-6.PubChem SID24898182) at the same concentration. In each case, vials containing 2.4ml of formulation and a control without surfactant were stirred at 1,000rpm for four hours at ambient temperature and visually analyzed by size exclusion HPLC and particle imaging analysis. After stirring, the non-surfactant samples showed slight cloudiness when compared to the static counterpart. The surfactant-containing samples were clear and contained no visible particles. SE HPLC analysis found that all samples containing surfactant showed comparable monomer percentages (88.2% -89.3%). After stirring, the non-surfactant samples showed higher particle concentrations than their static counterparts. Due to their significantly high particle concentration, the agitated non-surfactant samples could not be digitally filtered. The surfactant containing samples had a very low content of sub-visible particles compared to the surfactant free samples.

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