WO2021119234A1 - Glycosylated antibodies against insulin-like growth factor i receptor and uses thereof - Google Patents

Abstract

The disclosure provides a composition comprising a plurality of recombinant monoclonal antibodies (mAbs), identical in sequence, that bind insulin-like growth factor I receptor (IGF-IR), wherein each mAb comprises human IgG1 or IgG3 heavy-chain constant domains glycosylated with a sugar chain at Asn297, said mAbs being characterized in that 96-98% of the sugar chains have at least one fucosyl group and each sugar chain has from about 1-3% sialic acid derivatives, 1% or less α-1,3-galactose, and from about 35-40% galactose, and uses thereof.

Description

GLYCOSYLATED ANTIBODIES AGAINST INSULIN-LIKE GROWTH FACTOR I

RECEPTOR AND USES THEREOF

[0001] This application claims the benefit of United States Provisional Application No. 62/946,230, filed December 10, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to pharmaceutical compositions comprising a population of recombinant antibodies, having the same amino acid sequence, that bind insulin- like growth factor I receptor and have their respective Fc regions glycosylated with unique carbohydrate structures, and uses thereof.

BACKGROUND

[0003] Immunoglobulins or antibodies in their native form are usually tetrameric glycoproteins composed of two light and two heavy chains. Antibodies contain constant domains which assign the antibodies to different classes like IgA, IgD, IgE, IgM, and IgG, and several subclasses like IgGl, IgG2, IgG3, and IgG4. Antibodies of humans of class IgGl and IgG3 usually mediate ADCC (antibody-dependent cellular cytotoxicity).

[0004] Monoclonal antibodies elicit a number of effector functions including, for example, ADCC, phagocytosis, and complement-dependent cytotoxicity. Modifying antibody constant domains for improving or modifying effector functions is well-known in the art. ADCC and phagocytosis are mediated through the interaction of cell-bound antibodies with Fc gamma receptors (FcγRs). The canonical FcRs (type I FcRs) for human IgG (hFcγRs) including both activating (FcγRI, FcγRIIA, FcγRIIC, FcγlllRA, and FcγRIIIB) and inhibitory (FcγRIIB) receptors that mediate cytotoxicity/proinflammatory responses and inhibitory responses, respectively.

[0005] Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 (Asn297). The glycan composition of the IgG Fc domain regulate the differential engagement of FcRs. The Asn297 biantennary N-glycan is composed of a heptasaccharide core, which can be further extended with core fucose (Fuc), terminal galactose (Gal), terminal sialic acid (Sia), and bisecting N-acetylglucosamine (GlcNAc) through selective enzymatic glycosylation reactions. The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγlllRA as well as their ADCC activity. Further, the amount of afucosylated glycan in the antibody samples correlate with both FcγlllRA binding activity and ADCC activity in a linear fashion. Chung et al., mAbs 4(3):326-40, 2012. Thus, removal of the core fucose increases the Fc’s affinity for FcγlllRA and thereby augments ADCC in vitro and in vivo. On the other hand, terminal a2,6-sialylation has been shown to be critical for the anti-inflammatory activity of intravenous immunoglobulin therapy. To this end, sialylation can inversely impact ADCC in the context of core fucosylation, but not in its absence. Li et al., PNAS 114(13):3485-90, 2017.

[0006] Insulin- like growth factor I receptor (IGF-IR, EC 2.7.112, CD 221 antigen) belongs to the family of transmembrane protein tyrosine kinases. IGF-IR binds IGF-I with high affinity and initiates the physiological response to this ligand in vivo. IGF-IR also binds to IGF-II, however with slightly lower affinity. While the art describes a number of anti-IGF- IR antibodies with the potential for anti-cancer therapeutics, where ADCC-mediated anti tumor activity would be preferred, there is still a need for human antibodies against IGF-IR having convincing benefits for patients with non-cancer or non-tumor related diseases where ADCC-mediated activity is not desired.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 exemplifies a low-resolution hydrophilic interaction liquid chromatography (HILIC) chromatogram of the 2-aminobenzamide (2-AB) labelled N-glycan pool released from an anti-IGF-IR antibody sample.

[0008] FIG. 2 exemplifies a high-resolution HILIC chromatogram, annotated with glucose unit (GU) values, of the 2-AB labelled N-glycan pool released from an anti-IGF-IR antibody sample.

[0009] FIGs. 3A-E exemplify HILIC chromatograms, annotated with GU values, of the 2-AB labelled N-glycan pool from an anti-IGF-IR antibody sample following digestion with various exoglycosidase enzymes; where FIG. 3 A is undigested (UND); FIG. 3B is digested with Arthobacter ureafaciens sialidase (ABS); FIG. 3C is digested with ABS and bovine kidney a-fucosidase (BKF); FIG. 3D is digested with ABS, BKF, and Streptococcus pneumonia b-galactosidase (SPG); and FIG. 3E is digested ABS, BKF, SPG, and Streptococcus pneumonia hexosaminidase (GUH).

[0010] FIGs. 4A-B exemplify HILIC chromatograms, annotated with GU values, of the 2-AB labelled N-glycan pool from an anti-IGF-IR antibody sample following digestion with various exoglycosidase enzymes; where FIG. 4A is digested with ABS and BKF; FIG. 4B is digested with ABS, BKF, and SPG; and FIG. 4C is digested with ABS, BKF, and bovine testis b-galactosidase (BTG).

[0011] FIGs. 5A-C exemplify weak anion exchange (WAX) high performance liquid chromatography (HPLC) chromatograms of 2-AB labelled N-glycan pools; where FIG. 5A exemplifies the N-glycans from the reference standard fetuin; FIG. 5B exemplifies the N- glycans from an undigested (UND) anti-IGF-IR antibody sample; and FIG. 5C is from an antibody sample digested with ABS.

[0012] FIGs. 6A-C exemplify HILIC chromatograms, annotated with GU values, of the 2-AB labelled N-glycan pool from an anti-IGF-IR antibody sample following digestion with various exoglycosidase enzymes; where FIG. 6A is undigested; FIG. 6B is digested with recombinant sialidase (NANI); and FIG. 6C is digested with ABS.

[0013] FIGs. 7A-D exemplify HILIC chromatograms, annotated with GU values, of the 2-AB labelled N-glycan pool from an anti-IGF-IR antibody sample following WAX fractionation.

[0014] FIG. 8 exemplifies a chromatogram overlay of a DMB -labelled water negative control and sialic acid reference panel (NANA and NGNA) supplied with Ludger Kit.

[0015] FIG. 9 exemplifies a chromatogram of a DMB-labelled anti-IGF-IR antibody sample.

[0016] FIG. 10 exemplifies a HILIC chromatogram of the 2-AB labelled N-glycan pool released from Reference Lot.

[0017] FIG. 11 exemplifies a HILIC chromatogram of the 2-AB labelled N-glycan pool released from Lot F.

[0018] FIG. 12 exemplifies a HILIC chromatogram, annotated with GU values, of the 2- AB labelled N-glycan pool of Reference Lot.

[0019] FIG. 13 exemplifies a HILIC chromatogram, annotated with GU values, of the 2- AB labelled N-glycan pool of Lot F.

[0020] FIG. 14 exemplifies HILIC chromatograms, annotated with GU values, of the N- glycan pool (UND) from Reference Lot after digestion with a range of exoglycosidase enzymes*. *ABS removes all sialic acids (a2-3, -6 and -8), BKF removes α(1-6) linked core fucose and outer arm α(1-2 and 1-6) linked fucose, SPG removes β(1-4) linked galactose and GUH removes b linked GlcNAc.

[0021] FIG. 15 exemplifies HILIC chromatograms, annotated with GU values, of the N- glycan pool (UND) from Lot F after digestion with a range of exoglycosidase enzymes*. *ABS removes all sialic acids (a2-3, -6 and -8), BKF removes α(1-6) linked core fucose and outer arm α(1-2 and 1-6) linked fucose, SPG removes β(1-4) linked galactose and GUH removes β linked GlcNAc.

[0022] FIG. 16 exemplifies HILIC chromatograms of the N-glycans from Reference Lot after digestion with ABS+BKF, ABS+BKF+SPG and ABS+BKF+BTG.

[0023] FIG. 17 exemplifies HILIC chromatograms of the N-glycans from Lot F after digestion with ABS+BKF, ABS+BKF+SPG and ABS+BKF+BTG.

[0024] FIG. 18 exemplifies HILIC chromatograms of the N-glycans (UND) from Reference Lot before and after digestion with ABS and NANI.

[0025] FIG. 19 exemplifies HILIC chromatograms of the N-glycans (UND) from Lot F before and after digestion with ABS and NANI.

[0026] FIG. 20 exemplifies WAX-HPLC chromatograms of fetuin N-glycans, undigested (UND) N-glycans and NANI digested N-glycans from Reference Lot.

[0027] FIG. 21 exemplifies WAX-HPLC chromatograms of fetuin N-glycans, undigested (UND) N-glycans and NANI digested N-glycans from Lot F.

[0028] FIG. 22 exemplifies a sialic acid reference panel.

[0029] FIG. 23 exemplifies a negative control profile.

[0030] FIG. 24 exemplifies an overlay of negative control and sialic acid reference panel.

[0031] FIG. 25 exemplifies DMB labeled NANA released from Lot F.

[0032] FIG. 26 exemplifies DMB labeled NANA released from Reference Lot.

[0033] FIG. 27 shows calibration plots for NANA (top plot) and NGNA (bottom plot), as well as calibration curve data.

BRIEF DESCRIPTION OF THE SEQUENCES

[0034] SEQ ID NO:1 is a mature heavy-chain protein sequence.

[0035] SEQ ID NO:2 is a mature light-chain protein sequence.

[0036] SEQ ID NO:3 is a mature heavy-chain variable region (HCVR) protein sequence.

[0037] SEQ ID NO:4 is a mature light-chain variable region (LCVR) protein sequence.

[0038] SEQ ID NO:5 is a heavy-chain complementarity-determining region (HCDR) 1 peptide sequence.

[0039] SEQ ID NO:6 is a HCDR 2 peptide sequence.

[0040] SEQ ID NO:7 is a HCDR 3 peptide sequence.

[0041] SEQ ID NO:8 is a light-chain complementarity-determining region (LCDR) 1 peptide sequence.

[0042] SEQ ID NO:9 is a LCDR 2 peptide sequence. [0043] SEQ ID NO: 10 is a LCDR 3 peptide sequence.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0044] Thyroid eye disease (TED), also known as thyroid-associated ophthalmopathy (TAO), Graves’ ophthalmopathy or orbitopathy (GO), thyrotoxic exophthalmos, dysthyroid ophthalmopathy, and several other terms, is orbitopathy associated with thyroid dysfunction. TED is divided into two types. Active TED, which typically lasts 1-3 years, is characterized by an ongoing autoimmune/inflammatory response in the soft tissues of the orbit. Active TED is responsible for the expansion and remodeling of the ocular soft tissues. The autoimmune/inflammatory response of active TED spontaneously resolves and the condition transitions into inactive TED. Inactive TED is the term used to describe the long term/permanent sequelae of active TED.

[0045] The cause of TED is unknown. TED is typically associated with Graves’ hyperthyroidism but can also occur as part of other autoimmune conditions that affect the thyroid gland and produce pathology in orbital and periorbital tissue, and, rarely, the pretibial skin (pretibial myxedema) or digits (thyroid acropachy). TED is an autoimmune orbitopathy in which the orbital and periocular soft tissues are primarily affected with secondary effects on the eye and vision. In TED, as a result of inflammation and expansion of orbital soft tissues, primarily eye muscles and adipose, the eyes are forced forward (bulge) out of their sockets — a phenomenon termed proptosis or exophthalmos.

[0046] The annual incidence rate of TED has been estimated at 16 cases per 100,000 women and 2.9 cases per 100,000 men from a study based in one largely rural Minnesota community. There appears to be a female preponderance in which women are affected 2.5-6 times more frequently than men; however, severe cases occur more often in men than in women. In addition, most patients are aged 30-50 years, with severe cases appearing to be more frequent in those older than 50 years. Although most cases of TED do not result in loss of vision, this condition can cause vision-threatening exposure keratopathy, troublesome diplopia (double vision), and compressive dysthyroid optic neuropathy.

[0047] TED may precede, coincide with, or follow the systemic complications of dysthyroidism. The ocular manifestations of TED include upper eyelid retraction, lid lag, swelling, redness (erythema), conjunctivitis, and bulging eyes (exophthalmos or proptosis), chemosis, periorbital edema, and altered ocular motility with significant functional, social, and cosmetic consequences. [0048] Many of the signs and symptoms of TED, including proptosis and ocular congestion, result from expansion of the orbital adipose tissue and periocular muscles. The adipose tissue volume increases owing in part to new fat cell development (adipogenesis) within the orbital fat. The accumulation of hydrophilic glycosaminoglycans, primarily hyaluronic acid, within the orbital adipose tissue and the perimysial connective tissue between the extraocular muscle fibers, further expands the fat compartments and enlarges the extraocular muscle bodies. Hyaluronic acid is produced by fibroblasts residing within the orbital fat and extraocular muscles, and its synthesis in vitro is stimulated by several cytokines and growth factors, including IL-Ib, interferon-g, platelet-derived growth factor, thyroid stimulating hormone (TSH) and insulin-like growth factor I (IGF-I).

[0049] TED is commonly considered to be the autoimmune orbital manifestation of Graves’ Disease (GD). However, only approximately 30% of patients with Graves’ hyperthyroidism manifest clinically relevant ocular pathology indicating there is mechanistic heterogeneity and differentiation between the conditions. The molecular mechanisms underlying TED remain unclear. It is accepted that the generation of autoantibodies that act as agonists on the thyroid-stimulating hormone receptor (TSHR) is responsible for Graves’ hyperthyroidism. Pathogenic overstimulation of TSHR, leads to overproduction of thyroid hormones (T3 and T4) and accelerated metabolism of many tissues.

[0050] In active TED, autoantibodies trigger connective tissue and fat to expand, in part from stimulating excessive synthesis of hyaluronan. The expanded tissues are infiltrated with T- and B -cells, become inflamed, and get extensively remodeled. It has been suggested that TSHR might have some pathogenic role in the development of active TED. Indeed, a positive correlation has been found between anti-TSHR antibodies and the degree of TED activity. However, no definitive link has been established, and a proportion of TED patients remain euthyroid throughout the course of their disease.

[0051] Antibodies that activate the insulin-like growth factor I receptor (IGF-IR) have also been detected and implicated in active TED. Without being bound to any theory, it is believed that TSHR and IGF-IR form a physical and functional complex in orbital fibroblasts, and that blocking IGF-IR appears to attenuate both IGF-I and TSH-dependent signaling. It has been suggested that blocking IGF-IR using an antibody antagonist might reduce both TSHR- and IGF-I-dependent signaling and therefore interrupt the pathological activities of autoantibodies acting as agonists on either receptor.

[0052] IGF-IR is a widely expressed heterotetrameric protein involved in the regulation of proliferation and metabolic function of many cell types. It is a tyrosine kinase receptor comprising two subunits. IGF-IRα contains a ligand-binding domain while IGF-IR is involved in signaling and contains tyrosine phosphorylation sites. Monoclonal antibodies directed against IGF-IR have been developed and assessed as a therapeutic strategy for several types of solid tumors and lymphomas.

[0053] Management of hyperthyroidism due to Graves’ disease is imperfect because therapies targeting the specific underlying pathogenic autoimmune mechanisms of the disease are lacking. Even more complex is the treatment of moderate-to- severe active TED. Although recent years have witnessed a better understanding of its pathogenesis, TED remains a therapeutic challenge and dilemma. There are no approved drugs to treat active TED. Intravenous glucocorticoids (ivGCs) and oral glucocorticoids are used to treat patients with moderate-to-severe active TED, but results are seldom satisfactory. Partial responses are frequent and relapses (rebound) after drug withdrawal are not uncommon. Adverse events do occur and many patients eventually require rehabilitative surgery conducted when their condition has transitioned to inactive TED.

[0054] Recently, attention has been focused on the use of biologicals, which might specifically intervene on the pathogenic mechanisms of TED. In 2015 two small, monocenter, randomized clinical trials (RCTs) investigated the effects of rituximab, a CD20+ B cell-depleting agent, versus placebo or ivGCs, respectively. The results from the two trials were conflicting; they were negative (no differences with placebo) in the first trial, but positive (beneficial effects comparable to ivGCs) in the second one. The effectiveness of rituximab for moderate-to-severe active TED therefore remains to be determined. The recent guidelines published by the European Thyroid Association/European Group on Graves’ Orbitopathy (EUGOGO) indicate rituximab as a possible second-line treatment for patients poorly responsive to a first course of ivGCs. As with rituximab, there is no dependable evidence concerning other potential therapeutic agents, such as adalimumab, etanercept, infliximab, or monoclonals or small molecules blocking the TSH receptor. The use of the interleukin-6 receptor monoclonal antibody, tocilizumab, based on an ongoing RCT also remains to be determined.

[0055] As stated above, medical therapies for moderate-to-severe TED that have proved to be effective and safe in adequately powered, prospective, placebo-controlled trials are lacking. Previous clinical trials, which were rarely placebo-controlled, suggest that high dose glucocorticoids, alone, or with radiotherapy, can reduce inflammation-related signs and symptoms in patients with active ophthalmopathy, but only minimally affect proptosis and can cause dose-limiting adverse reactions. [0056] Immunoglobulins that activate IGF-IR signaling have been detected in patients with GD and TED. Furthermore, IGF-I synergistically enhances the actions of thyrotropin. IGF-IR, a membrane-spanning tyrosine kinase receptor with roles in development and metabolism, also stimulates immune function and thus might be targeted therapeutically in autoimmune diseases. IGF-IR is overexpressed by orbital fibroblasts and by T- and B-cells in persons with GD and TED. It forms a signaling complex with TSHR through which it is transactivated. In vitro studies of orbital fibroblasts and fibrocytes show that IGF-IR- inhibitory antibodies can attenuate the actions of IGF-I, thyrotropin, thyroid- stimulating immunoglobulins, and immunoglobulins isolated from patients with GD and TED. These observations prompted a trial of teprotumumab, a fully human IGF-IR-inhibitory monoclonal antibody, in patients with active, moderate-to-severe TED. Provided herein are antibodies against insulin-like growth factor I receptor (IGF-IR) for use in the treatment of GO or TED. [0057] Antibodies contain carbohydrate structures at conserved positions in the heavy chain constant regions, with each isotype possessing a distinct array of N-linked carbohydrate structures, which variably affect protein assembly, secretion or functional activity. The structure of the attached N-linked carbohydrate varies considerably. Antibodies of IgGl and IgG3 type are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The two complex biantennary oligosaccharides attached to Asn297 are buried between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the antibody to mediate effector functions such as ADCC. [0058] Understanding the impact of glycosylation and keeping a close control on glycosylation of monoclonal antibody-therapy product candidates is required to ensure proper safety and efficacy profiles. Depending on the expression host, glycosylation patterns in mAbs can be significantly different and impact the pharmacokinetics (PK) and pharmacodynamics (PD) of the mAbs. Liming Liu, Journal of Pharmaceutical Sciences 104:1866-84, 2015. Core fucose in the glycan structure conjugated to Fc region amino acid Asn297 reduces IgG antibody binding to FcγlllRA relative to IgG lacking fucose, resulting in decreased ADCC activities. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of mAbs.

[0059] The terminal monosaccharide of N-linked complex glycans is typically occupied by sialic acid. Presence of this sialic acid affects absorption, serum half-life, and clearance from the serum, as well as the physical, chemical and immunogenic properties of the respective glycoprotein (such as an mAb). It has been shown that increased Fc sialylation can result in decreased binding to immobilized antigens and some FcγRs, as well as decreased ADCC. From a manufacturing perspective, the degree of sialylation is important. Bork et al., Journal of Pharmaceutical Sciences 98(10):3499-508, 2009. Human serum IgG is typically less than 10% sialylated, whereas recombinant mAb generated from CHO cell lines has negligible sialylation. However, mAb produced in mouse hybridoma cells can be up to 50% sialylated. Naso et ah, mAbs 2(5):519-27, 2010.

[0060] Chung et ah, mAbs 4(3):326-40, 2012, demonstrated that even minor changes in fucosylation (in the range of 1-2%) can affect Fc receptor binding and ADCC activity. The conclusion is that even very small changes of this magnitude can have profound effect on FcγRIIIa receptor binding and ADCC activity. Given that this linear relationship was also observed among samples carrying low (< 10%) levels of afucosylated glycans, it is clear that small differences in levels of afucosylated glycans can result in considerable changes in biological activity.

[0061] As used herein, the term "Fc region of human IgG type" includes also naturally occurring allelic variants of the Fc region of an immunoglobulin (antibody) as well as variants having alterations which are substitutions, additions, or deletions but which do not affect Asn297 glycosylation. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity.

[0062] The term "antibody" encompasses the various forms of antibodies including but not being limited to whole antibodies, antibody fragments, human antibodies, humanized antibodies and genetically engineered antibodies as long as the characteristic properties according to the disclosure are retained. Therefore, an antibody according to the disclosure contains at least a functionally active (FcR binding) Fc part of IgGl or IgG3 type comprising glycosylated Asn297.

[0063] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of identical amino acid sequence. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. Further, the term “recombinant monoclonal antibody” or “recombinant human antibody” is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell according to the disclosure, using a recombinant expression vector transfected into such a host cell, preferably mammalian. [0064] The "constant domains" are not involved directly in binding of an antibody to an antigen but exhibit other functions like effector functions. Human antibody constant domains having of IgGl or IgG3 type are described in detail art. Constant domains of IgGl or IgG3 type are glycosylated at Asn297. "Asn 297" according to the present disclosure means amino acid asparagine located at about position 297 in the Fc region; based on minor sequence variations of antibodies, Asn297 can also be located some amino acids (usually not more than ± 3 amino acids) upstream or downstream.

[0065] Glycosylation of human IgGl or IgG3 occurs at Asn297 as a core fucosylated bianntennary complex oligosaccharide glycosylation terminated with up to 2 Gal residues. These structures are designated as G0, G1 (α1,6 or α1,3) or G2 glycan residues, depending from the amount of terminal Gal residues.

[0066] The "variable region" (variable region of a light chain (VL), variable region of a heavy chain (VH)) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen. The domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a b-sheet conformation and the CDRs may form loops connecting the b-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The combination of antibody heavy and light chain CDRs play a particularly important role in the binding specificity/affinity of the antibodies according to the present disclosure. [0067] The terms "hypervariable region" or "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from the CDRs. [0068] Antibody binding to IGF-IR can be investigated by a variety of in vitro, in vivo, or ex vivo assays known in the art. The affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), kd (dissociation constant), and Kp (kd/ka). The binding of IGF-I and IGF-II to IGF-IR may also be inhibited by the antibodies of the present disclosure.

[0069] The term "antibody-dependent cellular cytotoxicity (ADCC)" refers to lysis of human target cells by an antibody according to the disclosure in the presence of effector cells. ADCC is measured preferably by the treatment of a preparation of IGF-IR expressing cells with an antibody according to the disclosure in the presence of effector cells such as freshly isolated PBMC (peripheral blood mononuclear cells) or purified effector cells from buffy coats, like monocytes or NK cells (natural killer cells).

[0070] An antibody producing CHO host cell can be selected which is able to provide via recombinant expression a composition of a monoclonal antibody showing a glycosylation pattern according to the present disclosure. Such a CHO host cell comprises one or more expression vector(s) for the recombinant expression of such antibody. Preferably the host cell is stable transfected with the vector(s) and the antibody encoding nucleic acids are integrated in to the CHO host cell genome.

[0071] The term "CHO cell" encompasses the various forms of Chinese Hamster Ovary (CHO) cells. Such cells and methods for their generation are described in art. CHO cells successfully co-transfected with expression vector(s) for an antibody of human IgGl or IgG3 type. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations of cells used to express recombinant antibodies of the present disclosure, including, for example, CHO cells.

[0072] The antibodies according to the disclosure are preferably produced by recombinant means. Such methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity. For the protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression can, for example, be performed in CHO host cells and the antibody is recovered from the cells or supernatant preferably after lysis. Recombinant production of antibodies is well-known in the art.

[0073] The antibodies may be present in whole cells, in the supernant, in a cell lysate, or in a partially purified or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art.

[0074] Monoclonal antibodies can be suitably separated from a hybridoma culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies is readily isolated from the hybridoma and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA. Once identified and isolated, the DNA may be inserted into expression vectors, which are then transfected into, for example, CHO cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.

[0075] In one aspect, the present disclosure provides a composition comprising a plurality of recombinant monoclonal antibodies (mAbs), identical in sequence, that bind insulin-like growth factor I receptor (IGF-IR), wherein each mAb comprises human IgGl or IgG3 heavy- chain constant domains glycosylated with a sugar chain at Asn297, and wherein 96-98% of the sugar chains comprise at least one fucosyl group.

[0076] According to the disclosure it is possible to provide a population of antibodies where 96-98% of the N-glycan sugar chains conjugated to Asn297 are fucosylated. In some embodiments, 96% of the antibodies glycosylated at Asn297 have a fucosyl group. In some embodiments, 97% of the antibodies glycosylated at Asn297 have a fucosyl group. In some embodiments, 98% of the antibodies glycosylated at Asn297 have a fucosyl group. Further, a composition comprising a population of the fucosylated antibodies described herein display low ADCC activity as compared to a population of like-antibodies where 0% of the N-glycan sugar chains conjugated to Asn297 are fucosylated (i.e., 100% afucosylated).

[0077] In some embodiments, the mAbs of the composition, despite having fewer than 100% or 99% of the N-glycan sugar chains conjugated to Asn297 fucosylated, the mAbs still do not elicit a significant ADCC response. In some embodiments, the lower-fucosylated mAbs demonstrate about 10%, about 15%, or about 20% ADCC activity.

[0078] In some embodiments, the N-glycan sugar chains conjugated to Asn297 possess from about 1%, about 2%, or about 3% sialic acid derivatives. In some embodiments, the sialic acid derivative is N-acetylneuraminic acid (NANA). In some embodiments, about 1% to about 2% of the sugar chains are monosialylated with NANA. In some embodiments, about 0.1% to about 0.5% the sugar chains are disialylated with NANA.

[0079] In some embodiments, the N-glycan sugar chains conjugated to Asn297 possess 1% or less a-1, 3-galactose. In some embodiments, the N-glycan sugar chains conjugated to Asn297 possess from about 35%, about 40%, or about 45% galactose.

[0080] The antibody of the composition according to the disclosure is preferably a chimeric antibody, a human antibody, a humanized antibody, a non-human antibody, a single chain antibody comprising IgGl or IgG3 heavy chain constant part, or a IgGl or IgG3 heavy chain constant part.

[0081] The disclosure further comprises the use of an antibody according to the disclosure for the manufacture of a medicament. Preferably the medicament is useful for treatment of TED. In some embodiments, the composition of mAbs do not cause dose- limiting lysis of orbital fibroblasts. The disclosure further comprises a pharmaceutical composition comprising an antibody according to the disclosure.

[0082] In another aspect, the present disclosure provides a pharmaceutical composition, comprising a composition of the present disclosure, formulated together with a pharmaceutically acceptable carrier.

[0083] A composition of the present disclosure can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

[0084] To administer a compound of the disclosure by certain routes of administration, it may be necessary to coat the compound with or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0085] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

[0086] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0087] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0088] Regardless of the route of administration selected, the compounds of the present disclosure, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0089] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0090] The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.

[0091] Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

Glycan Nomenclature

[0092] All N-glycans have two core N-acetylglucosamines (GlcNAcs); F(6) at the start of the abbreviation indicates a core α(1-6) fucose linked to the inner GlcNAc; Mx, number (x) of mannose on core GlcNAcs; Ax, number of antenna (GlcNAc) on trimannosyl core; A2, biantennary with both GlcNAcs as β(1-2) linked; A3, triantennary with a GlcNAc linked b1-2 to both mannose and a third GlcNAc linked β(1-4) to the α(1-3) linked mannose; A3’, triantennary with a GlcNAc linked β(1-2) to both mannose and the third GlcNAc linked β(1- 6) to the α(1-6) linked mannose; A4, GlcNAcs linked as A3 with additional GlcNAc β(1-6) linked to α(1-6) mannose; B, bisecting GlcNAc linked β(1-4) to β(1-3) mannose; G(4)x, number (x) of b 1-4 linked galactose on the antenna; Ga(3)x number (x) of α1-3 linked galactose to β1-4 linked galactose; Lx number (x) of LacNAc (Galβ(l-4)GlcNAc) extensions linked to galactose;Fx, number (x) of linked fucose on antenna, (4) or (3) after the F indicates that the Fucose is α(1-4) or α(1-3) linked to a GlcNAc, (2) after the F indicates that the Fucose is α(1-2) linked to a galactose; Sx, number (x) of NANA sialic acids linked to galactose; Sgx, number (x) of NGNA sialic acids linked to galactose the number (3) or (6) after S or Sg indicates whether the sialic acid is in an α(2-3) or α(2-6) linkage; Sx-lAc, number of mono-acetylated sialic acids; Sx-2Ac, number of di-acetylated sialic acids.

Table 1. Glycan nomenclature.

Enumerated Embodiments

[0093] In addition the embodiments provided throughout this disclosure, provided herein are the following specific embodiments for illustrative purposes.

[0094] Provided as Embodiment 1 is a composition comprising a plurality of recombinant monoclonal antibodies (mAbs), identical in sequence, that bind insulin-like growth factor I receptor (IGF-IR), wherein each mAb comprises human IgGl or IgG3 heavy- chain constant domains glycosylated with a sugar chain at Asn297, and wherein 96-98% of the sugar chains comprise at least one fucosyl group.

[0095] Also provided are the following embodiments.

[0096] Embodiment 2. The composition of Embodiment 1, wherein 97.0-97.3% of the sugar chains comprise at least one fucosyl group. [0097] Embodiment 3. The composition according to either of Embodiments 1 and 2, wherein the plurality of mAbs exhibit less antibody-dependent cellular cytotoxicity (ADCC) as compared to an afucosylated mAb.

[0098] Embodiment 4. The composition according to Embodiment 3, wherein the plurality of mAbs exhibit between about 10% and about 20% ADCC activity.

[0099] Embodiment 5. The composition according to any of Embodiments 1-4, wherein the plurality of mAbs does not cause dose-limiting lysis of orbital fibroblasts.

[0100] Embodiment 6. The composition according to any of Embodiments 1-5, wherein the sugar chains further comprise from about 1% to about 3% sialic acid derivatives.

[0101] Embodiment 7. The composition according to Embodiment 6, wherein the sialic acid derivative is N-acetylneuraminic acid (NANA).

[0102] Embodiment 8. The composition according to Embodiment 7, wherein about 1% to about 2% of the sugar chains are monosialylated with NANA.

[0103] Embodiment 9. The composition according to Embodiment 8, wherein about 1.25% to about 1.55% of the sugar chains are monosialylated with NANA.

[0104] Embodiment 10. The composition according to Embodiment 7, wherein about 0.1% to about 0.5% of the sugar chains are disialylated with NANA.

[0105] Embodiment 11. The composition according to Embodiment 10, wherein about 0.14% to about 0.25% of the sugar chains are disialylated with NANA.

[0106] Embodiment 12. The composition according to any of Embodiments 1-11, further comprising 1% or less a-1, 3-galactose.

[0107] Embodiment 13. The composition according to Embodiment 12, further comprising 0% a- 1,3-galactose.

[0108] Embodiment 14. The composition according to any of Embodiments 1-13, further comprising from about 35-40% galactose.

[0109] Provided as Embodiment 15 is a composition comprising a plurality of recombinant monoclonal antibodies (mAb), identical in sequence, that bind insulin-like growth factor I receptor (IGF-IR), wherein each mAb comprises human IgGl or IgG3 heavy- chain constant domains glycosylated with a sugar chain at Asn297, wherein 96-98% of the sugar chains comprise at least one fucosyl group, wherein each sugar chain comprises i) from about 1-3% sialic acid derivatives; ii) 1% or less a- 1,3-galactose; and iii) from about 35-40% galactose, and wherein the mAb exhibits less antibody-dependent cellular cytotoxicity (ADCC) as compared to an afucosylated mAb.

[0110] Also provided are the following embodiments. [0111] Embodiment 16. The composition according to any of Embodiments 1-15, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO:5.

[0112] Embodiment 17. The composition according to any of Embodiments 1-16, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 2 of SEQ ID NO:6.

[0113] Embodiment 18. The composition according to any of Embodiments 1-17, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 3 of SEQ ID NO:7.

[0114] Embodiment 19. The composition according to any of Embodiments 1-15, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO:5; a HCDR2 of SEQ ID NO:6; and a HCDR3 of SEQ ID NO:7. [0115] Embodiment 20. The composition according to any of Embodiments 1-19, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 8.

[0116] Embodiment 21. The composition according to any of Embodiments 1-20, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 2 of SEQ ID NO:9.

[0117] Embodiment 22. The composition according to any of Embodiments 1-21, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 3 of SEQ ID NO: 10.

[0118] Embodiment 23. The composition according to any of Embodiments 1-22, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8; a LCDR2 of SEQ ID NO:9; and a LCDR3 of SEQ ID NO: 10. [0119] Embodiment 24. The composition according to any of Embodiments 1-15, wherein each mAb further comprises a heavy-chain variable region (HCVR) of SEQ ID NO:3.

[0120] Embodiment 25. The composition according to any of Embodiments 1-15 and 24, wherein each mAb further comprises a light-chain variable region (LCVR) of SEQ ID NO:4.

[0121] Embodiment 26. The composition according to any of Embodiments 1-15, wherein each mAb further comprises a heavy-chain of SEQ ID NO:l.

[0122] Embodiment 27. The composition according to any of Embodiments 1-15 and 26, wherein each mAb further comprises a light-chain of SEQ ID NO:2. [0123] Provided as Embodiment 28 is a recombinant monoclonal antibody (mAb) that binds insulin-like growth factor I receptor (IGF-IR) comprising human IgGl or IgG3 heavy- chain constant domains glycosylated with a sugar chain at Asn297, wherein each such sugar chain comprises from about 1% to about 3% sialic acid derivatives.

[0124] Also provided are the following embodiments.

[0125] Embodiment 29. The mAb according to Embodiment 28, wherein the sialic acid derivative is N-acetylneuraminic acid (NANA).

[0126] Embodiment 30. The mAb according to Embodiment 29, wherein about 1% to about 2% the sugar chains are monosialylated with NANA.

[0127] Embodiment 31. The composition according to Embodiment 30, wherein about 1.25% to about 1.55% of the sugar chains are monosialylated with NANA.

[0128] Embodiment 32. The mAb according to Embodiment 29, wherein about 0.1% to about 0.5% the sugar chains are disialylated with NANA.

[0129] Embodiment 33. The composition according to Embodiment 32, wherein about 0.14% to about 0.25% of the sugar chains are disialylated with NANA.

[0130] Embodiment 34. The mAb according to any of Embodiments 28-33, wherein the sialylation occurs on a galactose residue.

[0131] Embodiment 35. The mAb according to any of Embodiments 28-34, further comprising 1% or less a- 1,3-galactose.

[0132] Embodiment 36. The composition according to Embodiment 35, further comprising 0% a- 1,3 -galactose.

[0133] Embodiment 37. The mAb according to any of Embodiments 28-36, further comprising from about 35-40% galactose.

[0134] Also provided are the following embodiments.

[0135] Embodiment 37. The composition according to any of Embodiments 28-37, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO:5.

[0136] Embodiment 38. The composition according to any of Embodiments 28-38, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 2 of SEQ ID NO:6.

[0137] Embodiment 39. The composition according to any of Embodiments 28-39, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 3 of SEQ ID NO:7. [0138] Embodiment 40. The composition according to any of Embodiments 28-37, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO:5; a HCDR2 of SEQ ID NO:6; and a HCDR3 of SEQ ID NO:7. [0139] Embodiment 41. The composition according to any of Embodiments 28-40, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 8.

[0140] Embodiment 42. The composition according to any of Embodiments 28-41, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 2 of SEQ ID NO:9.

[0141] Embodiment 43. The composition according to any of Embodiments 28-42, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 3 of SEQ ID NO: 10.

[0142] Embodiment 44. The composition according to any of Embodiments 28-40, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8; a LCDR2 of SEQ ID NO:9; and a LCDR3 of SEQ ID NO: 10. [0143] Embodiment 45. The composition according to any of Embodiments 28-37, wherein each mAb further comprises a heavy-chain variable region (HCVR) of SEQ ID NO:3.

[0144] Embodiment 46. The composition according to any of Embodiments 28-37 and 45, wherein each mAb further comprises a light-chain variable region (LCVR) of SEQ ID NO:4.

[0145] Embodiment 47. The composition according to any of Embodiments 28-37, wherein each mAb further comprises a heavy-chain of SEQ ID NO:l.

[0146] Embodiment 48. The composition according to any of Embodiments 28-37 and 47, wherein each mAb further comprises a light-chain of SEQ ID NO:2.

[0147] Provided as Embodiment 49 is a method of treating or reducing the severity of thyroid eye disease (TED), or a symptom thereof, comprising administering to a subject an effective amount of a composition of any of Embodiments 1-48.

[0148] Embodiment 50: The method of Embodiment 49, wherein the symptom of TED is chosen from: proptosis; diplopia; visual appearance; visual functioning; pain (orbital or retrobulbular, e.g., on attempted eye movements), redness of conjunctiva or eyelids, and swelling of the caruncle/plica, conjunctiva, or eyelids.

[0149] Embodiment 51: The method of any one of Embodiments 49-50, wherein the antibody is administered at a dosage of about 1 mg/kg to about 5 mg/kg antibody as a first dose.

[0150] Embodiment 52: The method of any one of Embodiments 49-50, wherein the antibody is administered at a dosage of about 5 mg/kg to about 10 mg/kg antibody as a first dose.

[0151] Embodiment 53: The method of either of Embodiments 50-51, wherein the antibody is administered at a dosage of about 5 mg/kg to about 20 mg/kg antibody in subsequent doses.

[0152] Embodiment 54: The method of any one of Embodiments 49-50, wherein the antibody is administered in the following amounts: about 10 mg/kg antibody as a first dose; and about 20 mg/kg antibody in subsequent doses.

[0153] Embodiment 55: The method of any of Embodiments 49-54, wherein the subsequent doses are administered every three weeks for at least 21 weeks.

[0154] Also provided are embodiments wherein any embodiment herein may be combined with any one or more of other embodiments disclosed herein, provided the combination is not mutually exclusive. As used herein, two embodiments are “mutually exclusive” when one is defined to be something which is different than the other. The choice not to claim any particular embodiment at application filing or during pendency does not constitute a disclaimer of the embodiment for purposes of amending claims or pursuing continuing applications.

EXAMPLES

[0155] The following examples are presented only by way of illustration and to assist one of ordinary skill in using the disclosure. The examples are not intended in any way to otherwise limit the scope of the disclosure and it is understood that modifications can be made in the procedures set forth without departing from the spirit of the disclosure.

Production of Antibodies [0156] Teprotumumab may be made by methods known in the art, for example as disclosed in US20070248600.

[0157] The cell culture process is initiated by thawing a single cell vial from a working CHO cell bank (WCB) expressing teprotumumab and inoculating into culture expansion medium. Initial cell expansion is performed in standard cell culture systems, including shake- flasks and wave bags. Cell expansion is continued in single-use bioreactors (SUBs) at 100 L and 500 L scales. The cells are then transferred into a production SUB (2000 L scale) that is ran under fed batch conditions.

[0158] The cell culture process incorporates three serum and animal-component free proprietary media developed by Boehringer-Ingelheim including expansion, production and nutrient feed media. The expansion medium is animal-component free, containing a first medium powder, sodium hydrogen carbonate, ferric choline citrate and recombinant human insulin. Expansion medium is used from thawing of vials up to and including the N- 1 seed train bioreactor. The production medium is used in the production bioreactor and is animal- component free containing a second” medium powder, ferric choline citrate, sodium hydrogen carbonate, recombinant human insulin and sodium succinate dibasic hexahydrate. The production medium is used to support the growth of cells and the production of teprotumumab. The nutrient feed F is added to the production reactor at a constant feed rate and is composed of a third animal-component free feed powder, L-Cysteine HCI H2O, sodium hydrogen carbonate and recombinant human insulin. The nutrient feed is added at a continuous feed rate target of 1.55 kg/hour initiated on Day 3 and until the end of the culture. pH is controlled within the dead-band by automatic gassing with C02 and addition of 1 M sodium carbonate solution.

[0159] The cell culture broth is harvested and clarified by depth filtration followed by 0.45/0.2 μm filtration to remove cells from the culture fluid, providing cell-free culture fluid (CCF) for further purification of the product. The product present in the CCF is then purified through a series of chromatography steps. After the final ultrafiltration/diafiltration (UF/DF) step, the drug substance is formulated, 0.45/0.2 μm filtered into 2 L polycarbonate bottles and stored at -80 ± 10 °C until shipment to the drug product manufacturing site. Each harvest is considered as one cell culture batch and is purified separately to yield one batch of purified teprotumumab drug substance. [0160] The experiments below were conducted on Lots A-N, and a Reference Lot, each comprising teprotumumab.

N-Glycan Profiling of Antibodies

[0161] N-glycan profiling was conducted for six monoclonal antibody samples. N- glycans were enzymatically cleaved from the glycoproteins and labelled with 2- aminobenzamide (2-AB). The labelled N-glycans were separated using hydrophilic interaction liquid chromatography (HILIC) and weak anion exchange (WAX) high performance liquid chromatography (HPLC) with fluorescence detection. Exoglycosidase digestions were carried out to determine the sequence and linkage of the N-glycans present. N-glycan assignments were confirmed by liquid chromatography-mass spectrometry (LC- MS).

In-Solution Release of N-glycans with N-Glycosidase F (PNGase F)

[0162] National Institute for Bioprocessing Research and Training (NIBRT) SOP Number CR-2.04

[0163] 500 μg of each sample was buffer exchanged into 20 mM NaHC0₃ using

Nanosep® 10 K MWCO filters (PALL). The following was added to the filter: 90 μL of 20 mM sodium hydrogen carbonate; 10 μL of 1% Rapigest solution in 20 mM sodium hydrogen carbonate; 2 μL 400 mM DTT. Contents were mixed via pipette action and incubated on the filter at 65°C for 15 minutes. Samples were then alkylated by adding 2 μL of 80 mM iodoacetamide (IAA) and incubated in the dark at room temperature for 30 min. Samples were de-N-glycosylated by adding 2 μL PNGase F (NEB, P0709L). Contents were mixed via pipette action and incubated on the filter at 37°C overnight. Deglycosylated protein was removed by using Nanosep® 10 K MWCO filters (PALL) prior to 2-AB labelling step. Samples were then dried in a vacuum centrifuge. 20 μL of 1% formic acid was added to dried N-linked glycans and the mixture was incubated at room temperature for 20 minutes.

Samples were then dried in a vacuum centrifuge prior to further processing.

2-AB Glycan Labelling and Clean-Up

[0164] NIBRT SOP Numbers CR-3.01 and CR-4.01.

[0165] Samples were labelled by adding 5 μL of 2-AB labelling solution (Ludger Tag 2- AB labelling kit, Ludger, Abingdon, UK), vortexed, incubated for 2 hrs at 65 °C. Excess 2- AB was removed using amide resin Phytips from Phynexus.

HILIC Ultra-Performance Liquid Chromatography (UPLC) N-Glycan Method

[0166] NIBRT SOP Number CR-5.07. [0167] The UPLC system was calibrated by running an external standard of 2-AB dextran ladder (2-AB labeled glucose homopolymer) alongside the sample runs. A fifth-order polynomial distribution curve was fitted to the dextran ladder and used to allocate glucose unit (GU) values from retention times, using Empower software (Waters).

• Sample preparation: 70% acetonitrile.

• Injection volume: 10 μL.

• Column: 1.7 μm BEH glycan column (2.1 x 150 mm).

• Column temperature: 40°C.

• System: Waters Acquity UPLC equipped with a fluorescence detector.

• Software: Empower 3 (Waters).

• Solvent A: 50 mM ammonium formate pH 4.4.

• Solvent B : Acetonitrile.

• Gradient: 30 minute linear gradient with a flow rate of 0.561 mL/min (except for wash step): 30% Solvent A for 1.47 minutes, increasing to 47% Solvent A over 23.34 minutes, increasing to 70% Solvent A over 0.69 minutes; 70% Solvent A for 0.75 minutes and then for a further 0.3 minutes at a reduced flowrate of 0.4 mL/min, returning to 30% Solvent A over 0.3 minutes at a flow rate of 0.4 mL/min, then equilibrating with 30% Solvent A for 1.95 minutes with the flow rate returned to 0.561 mL/min.

• Wavelengths: Excitation 330 nm and emission 420 nm.

• Data rate: 20 pts/sec and PMT gain: 20.

• Weak Wash: 80% acetonitrile.

• Strong Wash: 20% acetonitrile.

• Sample Temperature: 5°C.

[0168] The total N-glycan pool of each antibody sample was released in triplicate from aliquots containing 500 μg of sample by PNGase F digestion and fluorescently labelled with 2-AB. Aliquots of the labelled N-glycan pools were then analysed by HILIC (see, e.g., FIG. 1). An enlarged chromatogram for each sample was also run with multiple peak identified; however, more than one glycan structure can elute at the same GU value (see, e.g., FIG. 2). WAX-HPLC Method [0169] NIBRT SOP Number CR-5.04. [0170] A reference standard of 2-AB labelled Fetuin N-links was run alongside the samples to determine the retention time of neutral, mono-, di-, tri- and tetra- charged structures.

• Sample preparation: 100% water.

• Injection volume: 95 μL.

• Column: 10 μm Waters Biosuite DEAE (7.5 mm x 75 mm).

• Column temperature: 30°C.

• System: Waters 2795 Alliance separations module equipped with a Waters 2475 fluorescence detector.

• Software: Empower 3 (Waters).

• Solvent A: 20% acetonitrile.

• Solvent B: 100 mM ammonium acetate pH 7.0 in 20% acetonitrile.

• Gradient: 30 minute linear gradient with a flow rate of 0.75 mL/min: 100% A for 5 minutes followed by 0 to 100% Solvent B over 15 minutes, 100% Solvent B for 2.5 minutes returning to 100 % Solvent A over 0.5 minutes then equilibrated with 100% Solvent A for 7 minutes.

• Wavelengths: Excitation 330 nm and emission 420 nm.

• Data rate: 10 pts/sec and PMT gain: 10.

• Sample Temperature: 5°C.

[0171] Sample N-glycans were separated according to charge by WAX-HPLC. Fetuin N- glycans were utilised as a reference standard to identify the retention times of mono- (SI), di- (S2), tri- (S3), and tetra-antennary (S4) charged structures (FIG. 5A). The neutral glycans (SO) eluted prior to the excess free 2-AB dye.

[0172] For an exemplary antibody sample, the majority of N-glycans (98.2%) present were neutral structures (FIGs. 5B-C). 1.8% of the sample consists of sialylated structures and these structures are mono- (SI) and di- charged (S2).

[0173] The relative percentage of sialylated structures, as calculated from WAX-HPLC was ca. 1.4-2.1 % across all samples (Tables 2 and 3). Further, WAX-HPLC indicated that all samples contain mono-sialylated and di-sialylated structures. Table 2. Relative percent sialic acid in the total glycan pool of each sample.

Table 3. Relative percent sialic acid in the total glycan pool of each sample.

WAX-HILIC Analysis

[0174] Each charged fraction from the WAX analyses of various antibody samples was collected, dried down, and then analysed using HILIC (FIGs. 7A-D).

Liquid Chromatography Fluorescence Detection Mass Spectrometry (LC-FLD-MS) [0175] Exoglycosidase digestions were performed on antibody samples separately and then pooled. Pooled digests were analysed by HILIC UPLC-FLD coupled with online mass spectrometric detection with a QTOF mass analyser. Samples were either undigested (UND), digested with ABS, digested with ABS and BKF and SPG, or digested with ABS and BKF and SPG and SPA. Ionization was performed on the electrospray source in negative mode and the generated ions were separated, detected and measured according to their mass-to- charge (m/z) ratio. Glycans were detected as [M-H]- and [M-2H]^2- ions.

• Sample preparation: 75% acetonitrile.

• Injection volume: 10 μL.

• Column: 1.7 μm Waters BEH glycan column (1.0 x 150 mm).

• Column temperature: 60°C.

• System: Online coupled (FLR)-mass spectrometry: Acquity UPLC equipped with fluorescence detector coupled to Waters Xevo G2.

• Software: MassLynx 4.1 software (Waters).

• Solvent A: 50 mM ammonium formate pH 4.4.

• Solvent B: Acetonitrile.

• Gradient: 40 minute linear gradient with a flow rate of 0.150 mL/min: 28% Solvent A for 1 minute, increasing to 43% Solvent A over 30 minutes, increasing to 70% Solvent A over 1 minute, 70% Solvent A for 3 minutes, returning to 28% Solvent A over 1 minute then equilibrated at 28% Solvent A for 4 minutes. To avoid contamination of Mass Spec system, flow was sent to waste for the first 1.0 minutes and after 32 minutes. • Wavelengths: Excitation 330 nm and emission 420 nm.

• Data rate: 1 pts/sec and PMT gain: 10.

• MS: Negative-sensitivity mode: capillary voltage 1.80 kV, ion source block and nitrogen desolvation gas temperatures 120°C and 400°C respectively, desolvation gas flow rate 600 L/h, cone voltage 50 V, full-scan data for glycans were acquired over m/z range of 450 to 2500.

• Wash: Weak wash solvent: 80% acetonitrile; strong wash solvent: 20% acetonitrile.

• Sample Temperature: 5°C.

[0176] The corresponding observed masses on the MS spectra for one exemplary sample, the monosaccharide composition, and the assignments are summarised in Table 6.

Exoglycosidase Digestion

[0177] NIBRT SOP Number CR-6.01.

[0178] Exoglycosidase digestions were carried out on aliquots of the 2-AB labelled N- glycan pools, where pool refers to the combined sample from the initial triplicate release of N-glycans. Digestions were carried out in accordance with methods previously described by Royle et al. (HPLC -based analysis of serum N-glycans on a 96-well plate platform with dedicated database software; Anal. Biochem. 376(1): 1-12, 2008) and according to manufacturer’s instructions. All exoglycosidase enzymes (except Streptococcus pneumonia Hexosaminidase (GUH)) were obtained from New England Biolabs, USA. GUH was obtained from Prozyme, USA.

[0179] Percent fucosylation was calculated from the sum of the total percent area of the fucosylated peaks in the ABS digested sample for all samples. For all samples, less than 3.0% of the N-glycans reported are afucosylated (i.e. no core fucose).

[0180] Percent galactosylation was calculated from the sum of the total percent area of the galactosylated peaks in the ABS+BKF digested sample for all samples. Based on the N- glycan assignments made by LC-FLD-MS, one α(1,3)-linked galactose containing peak is reported. The total amount of a-galactose was quantified from average peak area of the a- galactose containing peak in the UND profile of each sample.

Table 3. Critical features analysis.

[0181] Exoglycosidase digestions were performed in order to quantify percent fucosylation and percent galactosylation in all samples. Refer to Table 4 for glycan structures reported in the UND profile and exoglycosidase samples. Total percent fucosylation was calculated from the ABS digested samples and total percent galactosylation was calculated from the ABS+BKF digested samples (see, e.g., FIGs. 3A-E). ABS removes all sialic acids (a2-3, -6 and -8), BKF removes α(1-6) linked core fucose and outer arm α(1-2 and 1-6) linked fucose, SPG removes β(1-4) linked galactose, and GUH removes b linked GlcNAc (Table 4).

[0182] B-galactosidase linkage analysis was also performed on some samples using an alternate exoglycosidase digestion treatment. The total N-glycan pool was treated with SPG, as above, and also bovine testis b-galactosidase (BTG), which removes both β(1-4) and β(1- 3) linked galactose (see, e.g., FIGs. 4A-C). Both digestions showed similar glycan profiles, indicating that all galactose residues are β(1-4) linked.

[0183] Sialic acid linkage analysis was also performed on some samples. The total N- glycan pool of a sample was treated with different sialidases to determine the sialic acid linkages. Recombinant sialidase (NANI) removes α(2-3) linked sialic acid whereas ABS removes all sialic acids (α2-3, -6 and -8). Both digestions showed similar glycan profiles, indicating that all sialic acid residues are α(2-3) linked (see, e.g., FIGs. 6A-C).

Table 4. Structures present in the exoglycosidase digested antibody samples.

[0184] N-glycan structures, GU values and relative percentage areas of structures were identified in all samples by UPLC, exoglycosidase digestions, WAX analysis, and LC-MS (Table 5).

Sialic Acid Release, Labelling and Analysis by UPLC-FLR

[0185] Sialic acid quantification of anti-IGF-IR antibody samples was determined. To avoid large variability in observed sialic acid amounts between sample replicates, samples were provided without the excipient trehalose for DMB analysis using the LudgerTag™ DMB Sialic Acid Release and Labelling Kit (LT-KDMB-A1). Samples were diluted gravimetrically and concentrations were corrected accordingly. Sialic acids were released from the sample by acid hydrolysis using 0.1M hydrochloric acid. A portion of the hydrolysed sample was then mixed with a reduction solution of mercaptoethanol and sodium dithionite and incubated with the DMB dye for 3 hours at 50°C. Stock standards of N- acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA), which were supplied with the kit, were DMB-labelled alongside the antibody samples. Reactions were stopped with the addition of water and serial dilutions were performed of the NANA and NGNA standards. Samples and standards were analysed on a Waters Acquity UPLC with fluorescence detection using the LudgerSep UR2 HPLC column.

[0186] 200 μg of each sample was prepared by gravimetric dilution in triplicate and dried in vacuum centrifuge. The sample was treated with 25 μL of 0.1M hydrochloric acid and incubated for 1 hour in the thermomixer at 80°C. 5 μL of the cooled sample was transferred to a separate microtube for labelling. The labelling solution was prepared by adding 440 μL of the mercaptoethanol solution, LT-MERCAPTO-01, to the vial of sodium dithionite (reductant) LT-DITHIO-01 and mixing until all the reductant had completely dissolved. The entire contents of the reduction solution was added to the vial of DMB dye LT-DMB-01 and mixed until dye had dissolved. 20 μL of labelling reagent was added to each sample, including the Sialic Acid Reference Panel, CM-SRP-01, NANA standard, CM-NEUAC-01, and NGNA standard, CM-NEUGC-01, the samples are incubated at 50°C for 3 hours in the dark. The reaction was terminated with the addition of water to make a final volume of 500 μL of each sample. dH₂O was hydrolysed and labelled alongside the antibody samples, acting as a negative control. The NANA and NGNA 1000 pmol standards were serially diluted and samples were analysed by UPLC as per the following Acquity UPLC analysis protocol. Acquity UPLC Analysis

[0187] 100 μL of each sample and standard was transferred to HPLC vials for analysis.

Each standard was injected in triplicate.

• Sample preparation: 100% water.

• Injection volume: 5 μL.

• Injection Mode: PLNO (partial loop needle overfill).

• Column: LudgerSep UR2 HPLC Column. LS-UR2-2.1 x 100.

• Column temperature: 30°C.

• System: Waters Acquity UPLC system and Waters Acquity UPLC fluorescence detector.

• Software: Empower 3 (Waters).

• Mobile Phase: MeOH:ACN:H20 (7:9:84 v/v).

• Weak Wash: 5% acetonitrile.

• Strong Wash: 95% acetonitrile.

• Wavelengths: Excitation 373 nm and emission 448 nm.

• Data rate: 20 pts/sec and PMT gain: 1.0.

• Sample Temperature: 5°C.

• Run Time: 10 mins.

• Llow Rate: 0.25 mL/min.

Antibody-Dependent Cellular Cytotoxicity (ADCC)

[0188] Sample antibody ADCC activity assays are well known in the art. Here, anti-IFG- IR antibody samples were assessed for ADCC activity using a reporter assay kit obtained from Promega (Durham, North Carolina). An engineered Jurkat cell line transfected with CD16 (FcγlllRA, V158, high affinity variant) and a luciferase reporter construct with a nuclear factor of activated T-cells (NFAT) response element was used as the effector cell. A prostate cancer cell line (DU145) that overexpresses IGF-IR on the cell surface was used as the target cell. In theory, the antibody being tested binds to the IGF-IR expressed on the surface of the target cells via its Fv binding domain, and the CD 16 on the effector cell surface binds to the Fc region of the antibody. The cross-linking of the target cells and effector cells through the interaction of CD16 (FcγlllRA )-anti body-target antigen leads to the expression of the luciferase through the NFAT signaling pathway. The amount of luciferase expressed was quantified by the detecting reagent Bio-Glo™ provided in the assay kit. The kit contains a positive afucosylated control (MAK<IGF-IR>-afu-rh-IgG). The signal level therefore correlates with the effector function of the antibody sample. Full dose-response curves were fit to a 4 parametric logistical (PL) model. Results for an exemplary sample demonstrated low ADCC activity as represented by the high half-maximal effective concentration ( EC₅₀) values obtained (where the positive control EC₅₀ = 222 ng/mL) (Table 8).

Table 8. ADCC activity for anti-IGF-IR antibody.

N-Glycan Analysis of Two Monoclonal Antibodies

[0189] The following outlines the results of N-glycan characterization of two samples, Reference Lot (HZP-20-08-S01, 24 mg/mL concentration) and Lot F (HZP-19-02-02-S06, 59 mg/mL concentration). N-gl yeans were enzymatically cleaved from the glycoproteins and labelled with 2-AB. The labelled N-glycans were separated using HILIC-UPLC and WAX- HPLC with fluorescence detection. Exoglycosidase digestions were carried out to determine the sequence and linkage of the N-glycans present.

In-solution release of N-glycans with PNGase F NIBRT SOP number: CR-2.04 [0190] 500 mg of sample was buffer exchanged into 20 mM NaHCO₃ using Nanosep® 10

K MWCO filters (PALL). The following was added to the filter: 90 μL of 20 mM sodium hydrogen carbonate; 10 μL of 1 % Rapigest solution in 20 mM sodium hydrogen carbonate; 2 μL 400 mM DTT. Contents were mixed via pipette action and incubated on the filter at 65 °C for 15 minutes. Samples were then alkylated by adding 2 μL of 80 mM IAA and incubated in the dark at room temperature for 30 min. Samples were de-N-glycosylated by adding 2 μL PNGase F (NEB, P0709L). Contents were mixed via pipette action and incubated on the filter at 37 °C overnight. Deglycosylated protein was removed by using Nanosep® 10 K MWCO filters (PALL) prior to 2-AB labelling step. Samples were then dried in a vacuum centrifuge. 20 μL of 1 % formic acid was added to dried N- linked glycans and the mixture was incubated at room temperature for 20 minutes. Samples were then dried in a vacuum centrifuge prior to further processing.

2-AB Glycan labelling and clean-up

NIBRT SOP numbers: CR-3.01, CR-4.01.

[0191] Samples were labelled by adding 5 μL of 2-AB labelling solution (LudgerTag 2- AB labelling kit, Ludger, Abingdon, UK), vortexed, incubated for 2 hrs at 65 °C. Excess 2- AB was removed using amide resin Phytips from Phynexus.

HILIC UPLC N-Glycan Method

NIBRT SOP number: CR-5.07

The UPLC system was calibrated by running an external standard of 2-AB dextran ladder (2- AB labeled glucose homopolymer) alongside the sample runs. A fifth-order polynomial distribution curve was fitted to the dextran ladder and used to allocate glucose unit (GU) values from retention times, using Empower software (Waters) [1].

• Sample preparation: 70 % acetonitrile

• Injection volume: 10 μL

• Column: 1.7 μm BEH Glycan column (2.1 X 150 mm)

• Column temperature: 40°C

• System: Waters Acquity UPLC equipped with a fluorescence detector

• Software: Empower 3 (Waters)

• Solvent A: 50 mM ammonium formate pH 4.4

• Solvent B: Acetonitrile

• Gradient: 30 minute linear gradient with a flow rate of 0.561 mL/min (except for wash step): 30 % Solvent A for 1.47 minutes, increasing to 47 % Solvent A over 23.34 minutes, increasing to 70 % Solvent A over 0.69 minutes; 70 % Solvent A for 0.75 minutes and then for a further 0.3 minutes at a reduced flowrate of 0.4 mL/min, returning to 30 % Solvent A over 0.3 minutes at a flow rate of 0.4 mL/min, then equilibrating with 30 % Solvent A for 1.95 minutes with the flow rate returned to 0.561 mL/min.

• Wavelengths: Excitation 330 nm and emission 420 nm. Data rate: 20 pts/sec and PMT gain: 20.

• Weak Wash: 80 % Acetonitrile

• Strong Wash: 20 % Acetonitrile

• Sample Temperature: 5 °C

Weak Anion Exchange HPLC Method

NIBRT SOP number: CR-5.04

A reference standard of 2-AB labelled Fetuin Wl inks was run alongside the samples to determine the RT of neutral, mono-, di-, trl- and tetra- charged structures.

• Sample preparation: 100 % water

• Injection volume: 95 μL

• Column: 10 μm Waters Biosuite DEAE (7.5 mm x 75 mm)

• Column temperature: 30°C

• System: Waters 2795 Alliance separations module equipped with a Waters 2475 fluorescence detector

• Software: Empower 3 (Waters)

• Solvent A: 20 % Acetonitrile

• Solvent B: 100 mM Ammonium acetate pH 7.0 in 20 % Acetonitrile

• Gradient: 30 minute linear gradient with a flow rate of 0.75 mL/min: 100 % A for 5 minutes followed by 0 to 100 % Solvent B over 15 minutes, 100 % Solvent B for 2.5 minutes returning to 100 % Solvent A over 0.5 minutes then equilibrated with 100 % Solvent A for 7 minutes.

• Wavelengths: Excitation 330 nm and emission 420 nm. Data rate: 10 pts/sec and PMT gain: 10

• Sample Temperature: 5 °C

Exoglycosidase Digestion

NIBRT SOP number: CR-6.01 [0192] Exoglycosidase digestions were carried out on aliquots of the 2-AB labelled N- glycan pools, where pool refers to the combined sample from the initial triplicate release of N-glycans. Digestions were carried out in accordance with methods previously described by Royle et al. [1] and according to manufacturer’s instructions. All exoglycosidase enzymes (with the exception of ABS (a2-3,6,8,9 Neuraminidase A) and BKF (a-Fucosidase)) were obtained from Prozyme, San Leandro, CA, USA. ABS and BKF were obtained from New England Biolabs, Ipswich, Massachusetts, USA.

LC-FLD-MS

NIBRT SOP number: CR023

• Sample preparation: 75 % acetonitrile

• Injection volume: 10 μL

• Column: 1.7 μm Waters BEH Glycan column (1.0 x 150 mm).

• Column temperature: 60 °C

• System: Thermo Scientific Q Exactive Plus

• Software: XCalibur (Thermo Scientific)

• Solvent A: 50 mM ammonium formate pH 4.4

• Solvent B: Acetonitrile

• Gradient: 40 minute linear gradient with a flow rate of 0.15 mF/min: 28 % Solvent A for 1.0 minute, increasing to 43 % Solvent A over 30.0 minutes, increasing to 45 % Solvent A over 1 minute; returning to 28 % Solvent A over 4.0 minutes, then equilibrating with 30 % Solvent A for 4.0 minutes.

• Wavelengths: Excitation 320 nm and emission 420 nm.

• MS: Negative mode, spray voltage 3.40kV, Capillary temperature 320 °C, Aux gas heater temperature 300 °C, Sheath and sweep gas flow rate 30 and 10 E/h respectively, Scan range 450 to 2,500 m/z, Resolution 70,000.

• Sample Temperature: 5 °C

Sialylation

Table 9. Relative % sialic acid in the total glycan pool.

The relative percentage of sialylated structures, as calculated from WAX-HPLC, was ca. 1.4% for Reference Lot and ca. 1.8% for Lot F (Table 9). WAX-HPLC indicated that both samples contain mono-sialylated and di-sialylated structures. Sialidase digestions indicated that all the sialic acids are a (2-3)-linked in both samples.

Fucosylation

[0193] Percent fucosylation was calculated from the sum of the total relative % area of the fucosylated peaks in the ABS digested sample. In Reference Lot and Lot F, up to 97.2 % and 97.1% of the N-glycans identified are α(1-6) core fucosylated* respectively.

[0194] No N-glycan structures containing outer arm fucosylation were identified (Table 11). The most abundant glycan structure is F(6)A2, which consists of ca 57.8% and ca 56.5% of the total glycan structures for sample Reference Lot and sample Lot F respectively. (Table 11). *Where glycan species are co-eluting the total relative % area of the peak is taken. Galactosylation

[0195] Exoglycosidase digestion with SPG and BTG indicated that all galactose residues are β(1,4) linked. No α(1,3)-linked galactose was detected.

Table 10. Critical Features Analysis.

RESULTS

Analysis of N-glycans by HILIC-UPLC

[0196] The total N-glycan pool of Reference Lot and Lot F was released from aliquots containing 500 μg of sample by PNGase F digestion and fluorescently labelled with 2-AB. Aliquots of the labelled N-glycan pools for Reference Lot and Lot F were then analyzed by HILIC-UPLC (FIG. 10 and FIG. 11, respectively).

[0197] FIG. 12 and FIG. 13 are enlarged chromatograms showing the N-glycan profile of Reference Lot and Lot F, respectively, obtained using HILIC-UPLC. For Reference Lot, the chromatogram indicates the presence of 32 identified peaks; however, more than one glycan structure may elute at the same GU value. Further detailed analyses, such as exoglycosidase digestions and WAX-HPLC, identified additional structures. Table 11 shows all structures identified from the analyses contained within this report. Table 11. N-Glycan structures, GU values and relative percentage areas of structures identified in Reference Lot and Lot F by HILIC-UPLC, exoglycosidase digestions, WAX- HPLC and MS analysis.

N-Glycan Characterization via Exoglycosidase Digestions

[0198] A panel of exoglycosidase digestions was performed to elucidate the monosaccharide sequence and linkage of the N-glycans present in Reference Lot and Lot F (FIG. 14 and FIG. 15, respectively). Refer to Table 11 for glycan structures.

Table 12. Structures present in the exoglycosidase digested samples.

ND Species are detected by LC-MS only. No peak present in HILIC-UPLC profile. b-Galactose Linkage Analysis

[0199] The total N-glycan pool of Reference Lot and Lot F were treated with SPG which removes β(1-4) linked galactose and also BTG which removes both β(1-4) and β(1-3) linked galactose. Both digestions showed similar glycan profiles, indicating that all galactose residues are β(1-4) linked. Results are shown in FIG. 16 and FIG. 17 for Reference Lot and Lot F, respectively.

Sialic Acid Linkage

[0200] The total N-glycan pool of Reference Lot and Lot F were treated with different sialidases to determine the sialic acid linkages. NANI removes α(2-3) linked sialic acid whereas ABS removes all sialic acids (a2-3, -6 and -8). Both digestions show similar glycan profiles, indicating that all sialic acid residues are α(2-3) linked. Results are shown in FIG. 18 and FIG. 19 for Reference Lot and Lot F, respectively.

WAX-HPLC Analysis

[0201] Reference Lot N-gl yeans were separated according to charge by WAX-HPLC before treatment with NANI. Fetuin N-glycans were utilized as a reference standard to identify the retention times of mono- (SI), di- (S2), tri- (S3), and tetra-antennary (S4) charged structures. The neutral glycans (SO) elute prior to the excess free 2-AB dye. As shown in FIG. 20 and FIG. 21 for Reference Lot and Lot F, respectively, the majority of N- glycans (98.6% and 98.2% for Reference Lot and Lot F, respectively) present are neutral structures. 1.4% of sample Reference Lot and 1.8% of sample Lot F consisted of sialylated structures, which are mono- (SI) and di- charged (S2). Solely neutral glycans (SO) remain after digestion with NANI, confirming that the charge on these structures arises from sialylation. The relative percentage area of each species in the WAX-HPLC chromatogram is shown in Table 13.

Table 13. Relative % sialic acid in the total glycan pool.

by WAX-HPLC.

Sialic Acid Quantification of Two Monoclonal Antibodies [0202] The following outlines the sialic acid quantification of two monoclonal antibody samples, Reference Lot (HZP-20-09-S01, 24 mg/mL concentration) and Lot F (HZP-19-02- 02-S06 (54.1 mg/mL concentration). The method uses the LudgerTag™ DMB Sialic Acid Release and Labelling Kit (LT-KDMB-A1). The samples were diluted gravimetrically and the concentrations were corrected accordingly. Sialic acids are released from the sample by acid hydrolysis using 0.1 M Hydrochloric acid. A portion of the hydrolysed sample is then mixed with a reduction solution of mercaptoethanol and sodium dithionite and incubated with the DMB dye for 3 hours at 50°C. Stock standards of N-acetyl neuraminic acid (NANA) and N-glycolyl neuraminic acid (NGNA) are also supplied with the kit and these are DMB labelled alongside the samples. The reactions are stopped with the addition of water and serial dilutions are performed of the NANA and NGNA standards. Samples and standards were analysed on a Waters Acquity UPLC with fluorescence detection using the LudgerSep UR2 HPLC column.

Sialic Acid Release, Labelling and Analysis by UPLC-FLR

[0203] 200 μg of each sample was prepared by gravimetric dilution in triplicate and dried in vacuum centrifuge. The sample was treated with 25 μL of 0.1 M Hydrochloric acid and incubated for 1 hour in the thermomixer at 80°C. 5 μL of the cooled sample was transferred to a separate microtube for labelling. The labelling solution was prepared by adding 440 μL of the mercaptoethanol solution, LT-MERCAPTO-01, to the vial of sodium dithionite (reductant) LT-DITHIO-01 and mixing until all the reductant had completely dissolved. The entire contents of the reduction solution was added to the vial of DMB dye LT-DMB-01 and mixed until dye had dissolved. 20 μL of labelling reagent was added to each sample, including the Sialic Acid Reference Panel, CM-SRP-01, N-acetyl neuraminic acid standard, CM-NEUAC-01, and N-glycolyl neuraminic acid standard, CM-NEUGC-01, the samples are incubated at 50°C for 3 hours in the dark. The reaction was terminated with the addition of water to make a final volume of 500 μL of each sample. dH20 was hydrolyzed and labelled alongside the samples, acting as a negative control. The NANA and NGNA 1000 pmol standards were serially diluted as per Table 14. Samples were analyzed by UPLC. Results are provided in Table 15.

Acquity UPLC analysis

[0204] 100 μL of each sample and standard was transferred to HPLC vials for analysis.

Each standard was injected in triplicate.

• Sample preparation: 100 % water

• Injection volume: 5 μL

• Injection Mode: PLNO (Partial loop needle overfill)

• Column: LudgerSep UR2 HPLC Column. LS-UR2-2.1 x 100

• Column temperature: 30°C

• System: Waters Acquity UPLC system and Waters Acquity UPLC fluorescence detector

• Software: Empower 3 (Waters)

• Mobile Phase: MeOH:ACN:H2O (7:9:84 v/v)

• Weak Wash: 5 % Acetonitrile

• Strong Wash: 95 % Acetonitrile • Wavelengths: Excitation 373 nm and emission 448 nm. Data rate: 20 pts/sec and PMT gain: 1.0

• Sample Temperature: 5°C

• Run Time: 10 mins

• Flow Rate: 0.25 mL/min

Profiles

[0205] 200 mg of sample was hydrolyzed with 0.1 M HCL for 1 hour at 80°C and then

20% of the sample was DMB labelled. FIG. 22 shows the profile of the positive control; the sialic acid reference panel supplied with Ludger Kit. NANA and NGNA retention times were determined using the sialic acid reference panel (SRP). FIG. 23 shows a dH₂O blank. dH₂O was used as a negative control. No NANA or NGNA was detected. Peaks present are DMB reagent peaks; reagent peaks don’t interfere with NANA or NGNA retention times. FIG. 24 shows an overlay of the negative control and sialic acid reference sample. No NGNA or NANA was present in the negative control. FIG. 25 and FIG. 26 show DMB labeled NANA released from Lot F and Reference Lot, respectively, in triplicate.

Calibration Curves and Plots

[0206] Calibration data is provided in Tables 16 and 17, as well as FIG. 27.

Table 16. Calibration Curves for NANA and NGNA.

Table 17. Calibration Curve data for NANA and NGNA.

[0207] The detailed description set-forth above is provided to aid those skilled in the art in practicing the present disclosure. However, the disclosure described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the disclosure. Any equivalent embodiments are intended to be within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description, which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A composition comprising a plurality of recombinant monoclonal antibodies (mAbs), identical in sequence, that bind insulin-like growth factor I receptor (IGF-IR), wherein each mAb comprises human IgGl or IgG3 heavy-chain constant domains glycosylated with a sugar chain at Asn297, and wherein 96-98% of the sugar chains comprise at least one fucosyl group.

2. The composition according to claim 1, wherein the plurality of mAbs exhibit less antibody-dependent cellular cytotoxicity (ADCC) as compared to an afucosylated mAb.

3. The composition according to claim 2, wherein the plurality of mAbs exhibit between about 10% and about 20% ADCC activity.

4. The composition according to claim 1, wherein the plurality of mAbs does not cause dose-limiting lysis of orbital fibroblasts.

5. The composition according to claim 1, wherein the sugar chains further comprise from about 1% to about 3% sialic acid derivatives.

6. The composition according to claim 5, wherein the sialic acid derivative is N- acetylneuraminic acid (NANA).

7. The composition according to claim 6, wherein about 1% to about 2% of the sugar chains are monosialylated with NANA.

8. The composition according to claim 7, wherein about 0.1% to about 0.5% of the sugar chains are disialylated with NANA.

9. The composition according to any of claims 1-8, further comprising 1% or less a-1,3- galactose.

10. The composition according to any of claims 1-9, further comprising from about 35- 40% galactose.

11. A composition comprising a plurality of recombinant monoclonal antibodies (mAb), identical in sequence, that bind insulin-like growth factor I receptor (IGF-IR), wherein each mAb comprises human IgGl or IgG3 heavy-chain constant domains glycosylated with a sugar chain at Asn297, wherein 96-98% of the sugar chains comprise at least one fucosyl group, wherein each sugar chain comprises i) from about 1-3% sialic acid derivatives; ii) 1% or less a- 1,3-galactose; and iii) from about 35-40% galactose, and wherein the mAb exhibits less antibody-dependent cellular cytotoxicity (ADCC) as compared to an afucosylated mAb.

12. The composition according to any of claims 1-11, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO:5.

13. The composition according to any of claims 1-11, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 2 of SEQ ID NO:6.

14. The composition according to any of claims 1-11, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 3 of SEQ ID NO:7.

15. The composition according to any of claims 1-11, wherein each mAb further comprises a heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO:5; a HCDR2 of SEQ ID NO:6; and a HCDR3 of SEQ ID NO:7.

16. The composition according to any of claims 1-11, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8.

17. The composition according to any of claims 1-11, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 2 of SEQ ID NO:9.

18. The composition according to any of claims 1-11, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 3 of SEQ ID NO:10.

19. The composition according to any of claims 1-11, wherein each mAb further comprises a light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8; a LCDR2 of SEQ ID NO:9; and a LCDR3 of SEQ ID NO: 10.

20. The composition according to claim 15, wherein each mAb further comprises a heavy-chain variable region (HCVR) of SEQ ID NO:3.

21. The composition according to claim 19, wherein each mAb further comprises a light- chain variable region (LCVR) of SEQ ID NO:4.

22. The composition according to any of claims 1-11, wherein each mAb further comprises a heavy-chain of SEQ ID NO: 1.

23. The composition according to claims 22, wherein each mAb further comprises a light- chain of SEQ ID NO:2.

24. A recombinant monoclonal antibody (mAb) that binds insulin-like growth factor I receptor (IGF-IR) comprising human IgGl or IgG3 heavy-chain constant domains glycosylated with a sugar chain at Asn297, wherein each such sugar chain comprises from about 1% to about 3% sialic acid derivatives.

25. The mAb according to claim 24, wherein the sialic acid derivative is N- acetylneuraminic acid (NANA).

26. The mAb according to claim 25, wherein about 1% to about 2% the sugar chains are monosialylated with NANA.

27. The mAb according to claim 26, wherein about 0.1% to about 0.5% the sugar chains are disialylated with NANA.

28. The mAb according to claim 24, wherein the sialylation occurs on a galactose residue.

29. The mAb according to any of claims 24-28, further comprising 1% or less a-1,3- galactose.

30. The mAb according to any of claims 24-29, further comprising from about 35-40% galactose.

31. The composition of claim 1, wherein 97.0-97.3 % of the sugar chains comprise at least one fucosyl group

32. The composition according to claim 7, wherein about 1.25% to about 1.55% of the sugar chains are monosialylated with NANA.

33. The composition according to claim 7, wherein about 0.14% to about 0.25% of the sugar chains are disialylated with NANA.

34. The composition according to claim 9, further comprising 0% a- 1,3 -galactose.