Method of controlling antibody glycosylation profile

Abstract

The invention relates to a novel method of controlling the glycosylation profile of an antibody. In particular, the method involves the use of trace metal ions, specifically manganese and copper, in defined amounts in antibody-producing cell cultures.

Description

[0001] METHOD OF CONTROLLING ANTIBODY GLYCOSYLATION PROFILE

[0002] SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] This application contains a sequence listing, which is provided in XML format with a file name "70394W001.xml". The XML file has a size of about 5 kilobytes and was created on or about August 6, 2024. The sequence listing submitted electronically is part of the specification and is incorporated herein by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to a novel method of controlling the glycosylation profile of an antibody, in particular belimumab. The method involves the use of trace metal ions, specifically manganese and copper, in defined amounts in antibody-producing cell cultures.

[0006] BACKGROUND TO THE INVENTION

[0007] Belimumab is a human immunoglobulin G1 (IgGl) monoclonal antibody (CAS Registry Number 356547-88-1 and Drug Bank Accession Number DB08879) that inhibits B-cell activating factor (BAFF), also known as B-lymphocyte stimulator (BLys). Belimumab is an approved medicine (BENLYSTA) used in the treatment of systemic lupus erythematosus (SLE) and lupus nephritis (LN). Belimumab was approved by the U.S. Food and Drug Administration (FDA) on 9 March 2011 and the European Medicines Agency (EMA) on 13 July 2011 and was the first drug to be approved for the treatment of SLE in more than 50 years.

[0008] As with other antibody therapeutics and biological medicines, belimumab is produced in a host cell line that has been transfected with nucleic acid encoding the antibody heavy chain and the antibody light chain allowing the cells to express belimumab. Commercial production of biologies, such as belimumab, requires culturing these cells at scale followed by downstream processing and purification. In order to ensure reproducible product quality, specific raw materials, conditions and process parameters are often used. Batch release testing helps to ensure the safety and effectiveness of licensed biological medicines, including belimumab, and batches that do not meet predefined criteria cannot be released for use as medicines in patients.

[0009] Glycosylation refers to the covalent attachment of carbohydrate-based molecules, or 'glycans', to the surface of proteins and is the most prevalent and structurally complex of the post- translational chemical modifications that occur naturally in proteins. Biologies may have multiple potential glycosylation sites, not all of which will be occupied simultaneously. Accordingly, biological medicines display structural heterogeneity with respect to both the site of glycan attachment (macro-heterogeneity) and also with respect to the glycan's structure (micro-heterogeneity) leading to a population of proteins having a particular 'glycosylation profile'. The glycosylation profile of a given biologic may vary with the cell line and / or cell culturing conditions used to produce it and is important from both a safety and efficacy perspective.

[0010] In order to conserve resources, maximise efficiency, and ensure a cost-effective and stable supply of medicine that is safe and efficacious for patients with SLE, there is a need for manufacturing processes that better control the glycosylation profile of belimumab.

[0011] SUMMARY OF THE INVENTION

[0012] In a first aspect of the invention, a method of controlling the glycosylation of belimumab is provided, said method comprising culturing belimumab-producing cells in a cell culture medium comprising at least 15 ppb manganese.

[0013] In a second aspect of the invention, a method of achieving a predetermined glycosylation profile of belimumab is provided, said method comprising culturing belimumab-producing cells in a cell culture medium comprising at least 15 ppb manganese.

[0014] In a third aspect of the invention, a method for increasing the percentage of G2 and o-gal in a glycoprotein composition, and reducing the percentage of GO in said glycoprotein composition is provided, whereby cells expressing said glycoprotein are cultured in a cell culture medium comprising at least 15 ppb manganese.

[0015] In an embodiment of all three aspects of the invention, the cell culture medium additionally comprises at least 4.2 ppb copper.

[0016] DESCRIPTION OF FIGURES

[0017] FIG. 1 - Growth Profile Comparisons from Example 2. Each line represents one of the 24 runs set out in Table 2. In addition, M_ refers to manganese spike, C_ refers to copper spike and Y- or Y+ is for low copper and low manganese yeastolate (LLYO) lots or high copper and low manganese yeastolate (HHYO) lots, respectively.

[0018] FIG. 2 - % Viability Profile Comparisons from Example 2. The key for the legend is the same as in FIG. 1.

[0019] FIG. 3 - Glucose Profile Comparisons from Example 2. The key for the legend is the same as in FIG. 1.

[0020] FIG. 4 - Lactate Profile Comparisons from Example 2. The key for the legend is the same as in FIG. 1. FIG. 5 - Impact of Copper Spikes on Lactate Consumption from Example 2. The key for the legend is the same as in FIG. 1.

[0021] FIG. 6 - Base Utilisation Profile Comparisons from Example 2. The key for the legend is the same as in FIG. 1.

[0022] FIG. 7 - Titer Profile Comparisons from Example 2. The key for the legend is the same as in FIG. 1.

[0023] FIG. 8 - Impact of Total Copper and Manganese in the Cell-Culture on Critical Process and Quality Attributes.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] DEFINITIONS

[0026] "Copper" is used herein as shorthand for divalent copper ions (Cu2+) in aqueous solution. A skilled person will appreciate that any one of a number of different copper salts may be dissolved in water in order to produce copper ions, e.g., copper sulphate.

[0027] The "glycosylation profile" of a biological medicine, or biologic, such as an antibody therapeutic, e.g., belimumab, is the totality of the different types, amounts and location of glycans present in a given population of molecules of the biologic. The levels and types of glycosylation present in a therapeutic glycoprotein, such as a therapeutic antibody, has implications on the halflife, immunogenicity, efficacy, solubility and pharmacokinetics of a therapeutic glycoprotein and, as such, is a critical quality attribute (CQA).

[0028] A "glycoprotein" is a molecule containing carbohydrate (or glycan) covalently linked to protein. The carbohydrate may be in the form of a monosaccharide, disaccharide, oligosaccharide or polysaccharide, or a derivative, attached to an amino acid side chain. An antibody is an example of a glycoprotein.

[0029] "Manganese" is used herein as shorthand for divalent manganese ions (Mn2+) in aqueous solution. A skilled person will appreciate that any one of a number of different manganese salts may be dissolved in water in order to produce manganese ions, e.g., manganese chloride.

[0030] "Yeastolate" is a yeast extract derived from water-soluble portions of autolyzed yeast (e.g., Saccharomyces cerevisiae). Yeastolate is highly filterable and contains undefined mixtures of amino acids, peptides, polysaccharides, vitamins and minerals and has been commonly used as a key medium component and nutritional supplement for bacterial, insect and mammalian cell cultures for many decades. However, its activity varies from lot-to-lot due to variations in yeast fermentation conditions and downstream processes, with lots varying by almost 50% in biomass and growth promoting activity (Shen et al, Cytotechnology 54(1), 25-34, 2007). It will be understood that a drug's international nonproprietary name (INN), (e.g., belimumab), is to be interpreted as including generic, bioequivalent, follow-on biologic, and / or biosimilar versions of that drug, including but not limited to any drug that has received abbreviated regulatory approval by reference to an earlier regulatory approval of that drug. Additionally, a drug's INN optionally includes, but is not limited to glycosylation variants of belimumab, and biosimilars thereof.

[0031] The term "biosimilar", refers to a biopharmaceutical or a biologic product that is highly similar to a reference biologic product (e.g., belimumab) notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biologic product and the reference product in terms of the safety, purity, and potency of the product (Section 351(i) of the Public Health Service Act (42 U.S.C. 262(i)).

[0032] Thus, the term biosimilar refers to a biologic product that is highly similar to the reference product (e.g., belimumab) approved by a regulatory agency (e.g., the Federal Drug Administration (FDA) or the European Medicines Agency (EMA)) based on data from (a) analytical studies demonstrating that the biologic product is highly similar to the reference product notwithstanding minor differences in clinically inactive components; (b) animal studies (including the assessment of toxicity); and / or (c) a clinical study or studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity, and potency in one or more appropriate conditions of use for which the reference product is approved and intended to be used and for which approval is sought (e.g., that there are no clinically meaningful differences between the biologic product and the reference product in terms of the safety, purity, and potency of the product). In certain embodiments, the biosimilar product is an interchangeable product as determined by a regulatory agency (e.g., the FDA).

[0033] Thus, it will be generally understood that a biosimilar of belimumab will comprise the post- translational modifications that are essential for the function and efficacy of belimumab. However, in certain embodiments, the biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and / or truncation which is / are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and / or efficacy of the medicinal product. In specific embodiments, the biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in, for example, its strength, pharmaceutical form, formulation, excipients and / or presentation, providing safety and efficacy of the medicinal product is not compromised. In other embodiments, the biosimilar may comprise differences in, for example, pharmacokinetic (PK) and / or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain embodiments, the biosimilar may exhibit different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the FDA and / or EMA not to be a barrier for authorization as a similar biologic product. The term "biosimilar" is also used synonymously by other national and regional regulatory agencies.

[0034] In some embodiments, the biosimilar biologic product and reference product utilizes the same mechanism or mechanisms of action for the condition or conditions of use prescribed, recommended, or suggested in the proposed labeling, but only to the extent the mechanism or mechanisms of action are known for the reference product. In some embodiments, the condition or conditions of use prescribed, recommended, or suggested in the labeling proposed for the biologic product have been previously approved for the reference product. In some embodiments, the route of administration, the dosage form, and / or the strength of the biologic product are the same as those of the reference product. A "biosimilar" can be, e.g., a presently known antibody having the same or similar primary amino acid sequence as a marketed antibody but may be made in different cell types or by different production, purification, or formulation methods, or in a different formulation.

[0035] In certain embodiments of the methods described herein, belimumab can be replaced with a biosimilar thereof. Accordingly, it will be understood that the term "belimumab" is intended to encompass any biosimilar(s) of belimumab. Also encompassed by the term "belimumab" are antibodies which have CDRs, variable regions, and / or heavy and light chains of belimumab.

[0036] EMBODIMENTS

[0037] The inventors discovered that the levels of manganese and copper used in the manufacturing process for belimumab were highly variable in certain raw materials, in particular yeastolate, and that particular concentrations of these trace metal ions in the belimumab-producing cell culture were associated with failure with respect to the required cell growth and antibody glycan profiles.

[0038] The inventors discovered that copper levels primarily impact process attributes (e.g., peak viable cell density (VCD), peak lactate accumulation, and / or titer), whereas manganese levels primarily impact the carbohydrate or 'glycan' profile of belimumab (e.g., GO, G2, and / or o-gal).

[0039] Surprisingly, belimumab-producing cell cultures comprising at least 15 ppb manganese result in consistent belimumab product quality, in particular a consistent glycosylation profile, as determined by size-exclusion chromatography (SEC), ion-exchange chromatography (IEC) and / or capillary electrophoresis-laser-induced fluorescence (CE-LIF). The presence of manganese has been shown by the inventors to reduce GO, and increase G2 and o-gal, while having little to no impact on N-glycolylneuraminic acid (NGNA) and Gl. It has also surprisingly been determined by the inventors that the presence of at least 4.0 ppb, for example, at least 4.2 ppb, or, for example, at least 4.5 ppb, copper in belimumab-producing cell cultures results in optimal peak VCD. Further, as the concentration of copper in the cell culture increases, consumption of lactose increases, which in turn reduces glucose consumption in the culture, resulting in greater cell mass and consequently a higher titer. A saturation effect was observed for peak lactate consumption when total copper concentrations in the cell culture reach ~7-10 ppb, with lactate consumption rates plateauing thereafter.

[0040] Accordingly, the invention provides a method of controlling the glycosylation of belimumab, said method comprising culturing belimumab-producing cells in a cell culture medium comprising at least 15 ppb manganese.

[0041] The invention also provides a method of achieving a predetermined glycosylation profile of belimumab, said method comprising culturing belimumab-producing cells in a cell culture medium comprising at least 15 ppb manganese.

[0042] The invention further provides a method for increasing the percentage of G2 and o-gal in a glycoprotein composition and reducing the percentage of GO in said glycoprotein composition by culturing mammalian cells expressing said glycoprotein in a cell culture medium comprising at least 15 ppb manganese. In an embodiment, the glycoprotein is an antibody. In an embodiment, the antibody is an IgGl antibody. In an embodiment, the IgGl antibody is belimumab. In an embodiment, the IgGl antibody comprises SEQ ID NO:1 and SEQ ID NO:2. In an embodiment, the IgGl antibody comprises SEQ ID NO:1 and amino acids 1 to 108 of SEQ ID NO:2. In an embodiment, the IgGl antibody comprises SEQ ID NO:3 and SEQ ID NO:4.

[0043] In an embodiment of the disclosure, the manganese concentration of the cell culture is at least 15.0 ppb, at least 15.5 ppb, at least 16.0 ppb, at least 16.5 ppb, at least 17.0 ppb, at least 17.5 ppb, at least 18.0 ppb, at least 18.5 ppb, at least 19.0 ppb, at least 19.5 ppb, at least 20.0 ppb, at least 20.5 ppb, at least 21.0 ppb, at least 21.5 ppb, at least 22.0 ppb, at least 22.5 ppb, at least 23.0 ppb, at least 23.5 ppb, at least 24.0 ppb, at least 24.5 ppb, or at least 25.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb. In an embodiment, the manganese concentration of the cell culture is at least 21.0 ppb.

[0044] In an embodiment of the disclosure, the manganese concentration of the cell culture is no more than 65 ppb, no more than 70 ppb, no more than 75 ppb, no more than 80 ppb, no more than 85 ppb, no more than 90 ppb, no more than 95 ppb, no more than 100 ppb, no more than 110 ppb, no more than 120 ppb, no more than 130 ppb, no more than 140 ppb, no more than 150 ppb, no more than 160 ppb, no more than 170 ppb, no more than 180 ppb, no more than 190 ppb, or no more than 200 ppb. In an embodiment, the manganese concentration of the cell culture is no more than 62 ppb, no more than 63 ppb, no more than 64 ppb, no more than 65 ppb, no more than 66 ppb, no more than 67 ppb, no more than 68 ppb, no more than 69 ppb, no more than 70 ppb, no more than 71 ppb, no more than 72 ppb, no more than 73 ppb, no more than 74 ppb, no more than 75 ppb, no more than 76 ppb, no more than 77 ppb, no more than 78 ppb, no more than 79 ppb, or no more than 80 ppb. In an embodiment, the manganese concentration of the cell culture is no more than 62 ppb.

[0045] In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 200 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 190 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 180 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 170 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 160 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 150 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 140 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 130 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 120 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 110 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 15 ppb and 100 ppb manganese. In an embodiment, the cell culture medium comprises between 15 ppb and 95 ppb manganese. In an embodiment, the cell culture medium comprises between 15 ppb and 90 ppb manganese. In an embodiment, the cell culture medium comprises between 15 ppb and 85ppb manganese. In an embodiment, the cell culture medium comprises between 15 ppb and 80ppb manganese. In an embodiment, the cell culture medium comprises between 15 ppb and 75 ppb manganese. In an embodiment, the cell culture medium comprises between 15 ppb and 70 ppb manganese. In an embodiment, the cell culture medium comprises between 15 ppb and 65 ppb manganese. In an embodiment, the cell culture medium comprises between 15 ppb and 62 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 100 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 95 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 90 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 85 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 80 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 75 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 70 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 65 ppb manganese. In an embodiment, the cell culture medium comprises between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 100 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 95 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 90 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 85 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 80 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 75 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 70 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 65 ppb manganese. In an embodiment, the cell culture medium comprises between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 100 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 95 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 90 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 85 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 80 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 75 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 70 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 65 ppb manganese. In an embodiment, the cell culture medium comprises between 21 ppb and 62.0 ppb manganese.

[0046] In an embodiment, the belimumab-producing cells are cultured in a cell culture medium comprising any one of the above-identified manganese concentration ranges for at least 96 hours or at least four days.

[0047] A skilled person will appreciate that any one of a number of different manganese salts may be dissolved in water in order to produce manganese ions. In an embodiment, the manganese salt is manganese chloride. In an embodiment, the manganese salt is manganese chloride tetrahydrate. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 4.0 ppb, at least 4.1 ppb, at least 4.2 ppb, at least 4.3 ppb, at least 4.4 ppb, at least 4.5 ppb, at least 4.6 ppb, at least 4.7 ppb, at least 4.8 ppb, at least 4.9 ppb, at least 5.0 ppb, at least 5.1 ppb, at least 5.2 ppb, at least 5.3 ppb, at least 5.4 ppb, at least 5.5 ppb, at least 5.6 ppb, at least 5.7 ppb, at least 5.8 ppb, at least 5.9 ppb, at least 6.0 ppb, at least 6.1 ppb, at least 6.2 ppb, at least 6.3 ppb, at least 6.4 ppb, or at least 6.5 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 4.18 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 4.2 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 5.58 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 5.6 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 6.0 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 7.0 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 8.0 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 9.0 ppb. In an embodiment of the disclosure, the copper concentration of the cell culture is at least 10.0 ppb.

[0048] In an embodiment of the disclosure, the copper concentration of the cell culture is no more than 20 ppb, no more than 25 ppb, no more than 30 ppb, no more than 35 ppb, no more than 40 ppb, or no more than 50 ppb. In an embodiment, the copper concentration of the cell culture is no more than 15 ppb, no more than 16 ppb, no more than 17 ppb, no more than 18 ppb, no more than 19 ppb, or no more than 20 ppb. In an embodiment, the copper concentration of the cell culture is no more than 17.0 ppb.

[0049] In an embodiment of the disclosure, the cell culture medium comprises between 4.18 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.18 ppb and 20.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.18 ppb and 25.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.18 ppb and 30.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.18 ppb and 35.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.18 ppb and 40.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.18 ppb and 45.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.18 ppb and 50.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.2 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.2 ppb and 20.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.2 ppb and 25.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.2 ppb and 30.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.2 ppb and 35.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.2 ppb and 40.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.2 ppb and 45.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.2 ppb and 50.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.6 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.6 ppb and 20.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.6 ppb and 25.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.6 ppb and 30.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.6 ppb and 35.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.6 ppb and 40.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.6 ppb and 45.0 ppb copper. In an embodiment, the cell culture medium comprises between 4.6 ppb and 50.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.58 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.58 ppb and 20.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.58 ppb and 25.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.58 ppb and 30.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.58 ppb and 35.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.58 ppb and 40.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.58 ppb and 45.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.58 ppb and 50.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.6 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.6 ppb and 20.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.6 ppb and 25.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.6 ppb and 30.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.6 ppb and 35.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.6 ppb and 40.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.6 ppb and 45.0 ppb copper. In an embodiment, the cell culture medium comprises between 5.6 ppb and 50.0 ppb copper. In an embodiment, the cell culture medium comprises between 7.0 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 7.0 ppb and 20.0 ppb copper. In an embodiment, the cell culture medium comprises between 7.0 ppb and 25.0 ppb copper. In an embodiment, the cell culture medium comprises between 7.0 ppb and 30.0 ppb copper. In an embodiment, the cell culture medium comprises between 7.0 ppb and 35.0 ppb copper. In an embodiment, the cell culture medium comprises between 7.0 ppb and 40.0 ppb copper. In an embodiment, the cell culture medium comprises between 7.0 ppb and 45.0 ppb copper. In an embodiment, the cell culture medium comprises between 7.0 ppb and 50.0 ppb copper. In an embodiment, the cell culture medium comprises between 8.0 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 8.0 ppb and 50.0 ppb copper. In an embodiment, the cell culture medium comprises between 9.0 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 9.0 ppb and 50.0 ppb copper. In an embodiment, the cell culture medium comprises between 10.0 ppb and 17.0 ppb copper. In an embodiment, the cell culture medium comprises between 10.0 ppb and 50.0 ppb copper.

[0050] In an embodiment, the belimumab-producing cells are cultured in a cell culture medium comprising any one of the above-identified copper concentration ranges for at least 96 hours or at least four days.

[0051] A skilled person will appreciate that any one of a number of different copper salts may be dissolved in water in order to produce copper ions. In an embodiment, the copper salt is copper sulphate. In an embodiment, the copper salt is copper sulphate pentahydrate.

[0052] In an embodiment of the disclosure, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 4.0 ppb. In an embodiment of the disclosure, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 4.18 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 4.2 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 4.6 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 5.58 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 5.6 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 7.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 8.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 9.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 15.0 ppb and the copper concentration of the cell culture is at least 10.0 ppb.

[0053] In an embodiment of the disclosure, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 4.18 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 4.2 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 4.6 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 5.58 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 5.6 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 7.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 8.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 9.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.34 ppb and the copper concentration of the cell culture is at least 10.0 ppb.

[0054] In an embodiment of the disclosure, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 4.18 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 4.2 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 4.6 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 5.58 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 5.6 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 7.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 8.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 9.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 17.5 ppb and the copper concentration of the cell culture is at least 10.0 ppb.

[0055] In an embodiment of the disclosure, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 4.18 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 4.2 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 4.6 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 5.58 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 5.6 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 7.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 8.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 9.0 ppb. In an embodiment, the manganese concentration of the cell culture is at least 20.68 ppb and the copper concentration of the cell culture is at least 10.0 ppb.

[0056] In an embodiment of the disclosure, the cell culture medium comprises between 4.0 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 4.18 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.2 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.6 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.58 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.6 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 7.0 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 8.0 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 9.0 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 10.0 ppb and 17.0 ppb copper, and between 15.0 ppb and 62.0 ppb manganese.

[0057] In an embodiment of the disclosure, the cell culture medium comprises between 4.18 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.2 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.6 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.58 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.6 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 7.0 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 8.0 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 9.0 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 10.0 ppb and 17.0 ppb copper, and between 17.34 ppb and 62.0 ppb manganese.

[0058] In an embodiment of the disclosure, the cell culture medium comprises between 4.18 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.2 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.6 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.58 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.6 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 7.0 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 8.0 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 9.0 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 10.0 ppb and 17.0 ppb copper, and between 17.5 ppb and 62.0 ppb manganese.

[0059] In an embodiment of the disclosure, the cell culture medium comprises between 4.18 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.2 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.6 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.58 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.6 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 7.0 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 8.0 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 9.0 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 10.0 ppb and 17.0 ppb copper, and between 20.68 ppb and 62.0 ppb manganese. In an embodiment of the disclosure, the cell culture medium comprises between 4.18 ppb and 17.0 ppb copper, and between 21.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.2 ppb and 17.0 ppb copper, and between 21.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 4.6 ppb and 17.0 ppb copper, and between 21.0 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.58 ppb and 17.0 ppb copper, and between 21 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 5.6 ppb and 17.0 ppb copper, and between 21 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 7.0 ppb and 17.0 ppb copper, and between 21 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 8.0 ppb and 17.0 ppb copper, and between 21 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 9.0 ppb and 17.0 ppb copper, and between 21 ppb and 62.0 ppb manganese. In an embodiment, the cell culture medium comprises between 10.0 ppb and 17.0 ppb copper, and between 21 ppb and 62.0 ppb manganese.

[0060] In an embodiment, the belimumab-producing cells are cultured in a cell culture medium comprising any one of the above-identified copper and manganese concentration ranges for at least 96 hours or at least four days.

[0061] In an embodiment, the manganese salt is manganese chloride and the copper salt is copper sulphate. In an embodiment, the manganese salt is manganese chloride tetrahydrate and the copper salt is copper sulphate pentahydrate.

[0062] Biologies, including antibodies, may be prepared by any of a number of conventional techniques. For example, they may be purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it) or produced in recombinant expression systems. Generally, host cells are transformed with a recombinant expression vector encoding the desired protein. The expression vector may be maintained by the host as a separate genetic element or integrated into the host chromosome depending on the expression system. A wide range of host cells can be employed, including Prokaryotes (including Gram-negative or Gram-positive bacteria, for example Escherichia coii, Bacilli sp, Pseudomonas sp, and Corynebacterium sp. , Eukaryotes including yeast (for example Saccharomyces cerevisiae, Pichia pastoris , fungi (for example Aspergiius sp. , or higher Eukaryotes including insect cells and cell lines of mammalian origin (for example, CHO, NS0, SPO / 2, PER.C6, HEK293, HeLa). Most FDA- and EMA-approved biologies are mAbs expressed in mammalian systems to avoid generation of glycoepitopes giving rise to allergic reactions or severe immune responses. The most commonly used producer cells in bioprocesses for the production of recombinant antibodies are the murine myeloma NS0, murine B-lymphocyte SPO / 2, and the Chinese hamster ovary (CHO) cell lines. In the case of BENLYSTA, an NSO mouse myeloma cell line is used.

[0063] In an embodiment of the disclosure, the belimumab-producing cells are mammalian cells. In a particular embodiment, the belimumab-producing cells are NSO cells. In a particular embodiment, the belimumab-producing cells are CHO cells.

[0064] The cells are cultured under conditions that promote expression of the antibody. A variety of equipment, such as shake flasks, spinner flasks, and bioreactors, may be used. The antibody is recovered by conventional protein purification procedures. Protein purification procedures typically consist of a series of unit operations comprised of various filtration and chromatographic processes developed to selectively concentrate and isolate the antibody. The purified antibody may be formulated in a pharmaceutically acceptable composition. In an embodiment, belimumab is expressed and secreted by the cells during the cell culture process. In an embodiment, at the end of cell culture, cells and cellular debris are removed and belimumab is purified and formulated using a series of chromatographic and filtration steps.

[0065] A cell culture size can be any volume that is appropriate for production of the glycoprotein. In one embodiment, the volume of the cell culture is at least 500 liters. In other embodiments, the volume of the production cell culture is 10, 50, 100, 250, 1000, 2000, 2500, 5000, 8000, 10,000, or 12,000 liters or more, or any volume in between. For example, a cell culture can have a volume between 10 to 5,000 liters, 10 to 10,000 liters, 10 to 15,000 liters, 50 to 5,000 liters, 50 to 10,000 liters, or 50 to 15,000 liters, 100 to 5,000 liters, 100 to 10,000 liters, 100 to 15,000 liters, 500 to 5,000 liters, 500 to 10,000 liters, 500 to 15,000 liters, 1,000 to 5,000 liters, 1,000 to 10,000 liters, or 1,000 to 15,000 liters. Or a cell culture can have a volume between about 500 liters and about 30,000 liters, about 500 liters and about 20,000 liters, about 500 liters and about 10,000 liters, about 500 liters and about 5,000 liters, about 1,000 liters and about 30,000 liters, about 2,000 liters and about 30,000 liters, about 3,000 liters and about 30,000 liters, about 5,000 liters and about 30,000 liters, or about 10,000 liters and about 30,000 liters, or a cell culture have a volume of at least about 500 liters, at least about 1,000 liters, at least about 2,000 liters, at least about 3,000 liters, at least about 5,000 liters, at least about 10,000 liters, at least about 15,000 liters, or at least about 20,000 liters.

[0066] The cells may initially be cultured in a cell culture medium. In one embodiment, the cell culture medium is a basal cell medium. A basal cell medium supports initial cell growth and belimumab production and contains amino acids, glucose, and ions (e.g., calcium, magnesium, potassium, sodium, and / or phosphate) essential for cell survival and growth. In an embodiment, the basal cell medium is an Advanced Granular Technology (AGT) powder containing medium. In an embodiment, the basal cell medium is a THERMOFISHER AGT powder containing medium.

[0067] In an embodiment of the disclosure, the cell culture medium is serum-free. In an embodiment, the cell culture medium is animal source-free. In an embodiment, the cell culture medium is serum-free and animal source-free. In an embodiment, the cell culture medium is a chemically defined medium. The cell culture medium may be supplemented with specific nutrients (i.e., a 'nutrient feed' or 'feed medium7) at specific time points. A feed medium prevents depletion of nutrients and sustains the production phase of belimumab. The nutrient feed may comprise yeastolate. In an embodiment, the culture is a fed batch culture. In another embodiment, the culture is a perfusion culture.

[0068] In an embodiment, the cell culture is supplemented with a nutrient feed multiple times following inoculation. A cell culture may be supplemented with a nutrient feed at regular intervals. The regular interval may be about once a day, about once every two days, about once every three days, about once every 4 days, or about once every 5 days. In an embodiment, the cell culture is supplemented with a nutrient feed on days 3, 5, and 7 following inoculation. In an embodiment, the cell culture is supplemented with a nutrient feed 72 ± 4 hours following inoculation. In an embodiment, the cell culture is supplemented with a nutrient feed 120 ± 4 hours following inoculation. In an embodiment, the cell culture is supplemented with a nutrient feed 168 ± 4 hours following inoculation. In an embodiment, the cell culture is supplemented with a nutrient feed 72 ± 4 hours, 120 ± 4 hours, and 168 ± 4 hours following inoculation.

[0069] In an embodiment of the disclosure, the pH of the cell culture medium is between 6.6 and 7.5. In an embodiment of the disclosure, the pH of the cell culture medium is between 6.8 and 7.4. In an embodiment of the disclosure, the pH of the cell culture medium is between 7.0 and 7.2. In an embodiment, the pH of the cell culture medium is about 7.0. In an embodiment, the pH of the cell culture medium is about 7.1. In an embodiment, the pH of the cell culture medium is about 7.2.

[0070] The temperature of the cell culture can be selected based primarily on the range of temperatures at which the cell culture remains viable. For example, during the initial growth phase, CHO cells grow well at 37° C. In general, most mammalian cells grow well within a range of about 25° C to 42° C. In an embodiment of the disclosure, the cells are cultured at a temperature between

[0071] 36 °C and 38 °C. In an embodiment of the disclosure, the cells are cultured at a temperature between 36.5 °C and 37.5 °C. In an embodiment, the cells are cultured at a temperature of about

[0072] 37 °C.

[0073] In an embodiment of the disclosure, the % dissolved oxygen of the cell culture is between

[0074] 10% and 75%. In an embodiment, the % dissolved oxygen of the cell culture is between 10% and 74%. In an embodiment of the disclosure, the % dissolved oxygen of the cell culture is between 10% and 73.6%. In an embodiment, the % dissolved oxygen of the cell culture is between 20% and 70%. In an embodiment, the % dissolved oxygen of the cell culture is between 30% and 70%. In an embodiment, the % dissolved oxygen of the cell culture is between 40% and 60%. In an embodiment, the % dissolved oxygen of the cell culture is about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60%. In an embodiment, the % dissolved oxygen of the cell culture is about 50%.

[0075] In an embodiment of the disclosure, the initial viable cell density (VCD) of the cell culture is at least 1.5xl05viable cells / mL. In an embodiment, the initial VCD is between 1.5xl05viable cells / mL and 3.9xl05viable cells / mL. In an embodiment, the initial VCD is between 1.8xl05viable cells / mL and 3.05X105viable cells / mL. In an embodiment, the initial VCD is about 2.4xl05viable cells / mL.

[0076] In an embodiment of the disclosure, the peak VCD of the cell culture is at least 105xl05viable cells / mL. In an embodiment of the disclosure, the peak VCD of the cell culture is about 105xl05viable cells / mL.

[0077] The cell culture can be maintained in production phase for between about 1 day and about 30 days. In one embodiment, the cell culture is maintained in production phase for between about 1 day and about 30 days, about 1 day and about 25 days, about 1 day and about 20 days, about 1 day and about 15 days, about 1 day and about 14 days, about 1 day and about 13 days, about 1 day and about 12 days, about 1 day and about 11 days, about 1 day and about 10 days, about 1 day and about 9 days, about 1 day and about 8 days, about 1 day and about 7 days, about 1 day and about 6 days, about 1 day and about 5 days, about 1 day and about 4 days, or about 1 day and about 3 days. In another embodiment, the cell culture is maintained in production phase for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 15 days, at least about 20 days, at least about 25 days, or at least about 30 days. In a further embodiment, the cell culture is maintained in production phase for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 15 days, about 20 days, about 25 days, or about 30 days.

[0078] In an embodiment of the disclosure, the belimumab-producing cells have been cultured for between 256 hours and 272 hours at the time of harvest. In an embodiment, the belimumab- producing cells have been cultured for between 260 hours and 268 hours at the time of harvest. In an embodiment, the belimumab-producing cells have been cultured for about 264 hours at the time of harvest.

[0079] In an embodiment of the disclosure, the cell culture medium comprises between 1.70 g / L glucose and 2.30 g / L glucose. In an embodiment, the cell culture medium comprises about 1.7 g / L, about 1.8 g / L, about 1.9 g / L, about 2.0 g / L, about 2.1 g / L, about 2.2 g / L, or about 2.3 g / L glucose. In an embodiment, the cell culture medium comprises about 2.0 g / L glucose.

[0080] The skilled person will appreciate that, upon production of an antibody, such as belimumab, in a host cell, post-translational modifications may occur. For example, this may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns, non-enzymatic glycation, deamidation, oxidation, disulphide bond scrambling and other cysteine variants such as free sulfhydryl, racemized disulphides, thioethers and trisulfide bonds, isomerisation, C-terminal lysine clipping, and N-terminal glutamine cyclisation.

[0081] Glycation is a post-translational non-enzymatic chemical reaction between a reducing sugar, such as glucose, and a free amine group in the protein, and is typically observed at the epsilon amine of lysine side chains or at the N-Terminus of the protein. Glycation can occur during production and storage only in the presence of reducing sugars.

[0082] Deamidation can occur during production and storage, is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid (iso-aspartate) and aspartic acid (aspartate) (D) at approximately 3:1 ratio. This deamidation reaction is therefore related to isomerization of aspartate (D) to iso-aspartate. The deamidation of asparagine and the isomerisation of aspartate both involve the intermediate succinimide. To a much lesser degree, deamidation can occur with glutamine residues in a similar manner. Deamidation can occur in a CDR, in a Fab (non-CDR region), or in the Fc region.

[0083] Oxidation can occur during production and storage (i.e., in the presence of oxidizing conditions) and results in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation happens primarily with methionine residues, but may occur at tryptophan and free cysteine residues. Oxidation can occur in a CDR, in a Fab (non-CDR) region, or in the Fc region.

[0084] Disulphide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulphide bonds can break or form incorrectly, resulting in unpaired cysteine residues (-SH). These free (unpaired) sulfhydryls (-SH) can promote shuffling.

[0085] The formation of a thioether and racemization of a disulphide bond can occur under basic conditions, in production or storage, through a beta elimination of disulphide bridges back to cysteine residues via a dehydroalanine and persulfide intermediate. Subsequent crosslinking of dehydroalanine and cysteine results in the formation of a thioether bond or the free cysteine residues can reform a disulphide bond with a mixture of D- and L-cysteine.

[0086] Trisulphides result from insertion of a sulphur atom into a disulphide bond (Cys-S-S-S-Cys ) and are formed due to the presence of hydrogen sulphide in production cell culture. N-terminal glutamine (Q) and glutamate (glutamic acid) (E) in the heavy chain and / or light chain is likely to form pyroglutamate (pGlu) via cyclization. Most pGlu formation happens in the production bioreactor, but it can be formed non-enzymatically, depending on pH and temperature of processing and storage conditions. Cyclization of N-terminal Q or E is commonly observed in natural human antibodies.

[0087] C-terminal lysine clipping is an enzymatic reaction catalysed by carboxypeptidases and is commonly observed in recombinant and natural human antibodies. Variants of this process include removal of lysine from one or both heavy chains due to cellular enzymes from the recombinant host cell. Administration to the human subject / patient is likely to result in the removal of any remaining C-terminal lysines.

[0088] Overall, IgG antibodies are mainly biantennary N-glycosylated in their Fc-region at a single site (Asn297). Glycosylation in the Fc region affects IgG affinity for Fc y-receptors (Fc yRs), which mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), phagocytosis and cytokine secretion. The overall diversity of N- glycosylation lies primarily in the antennae of the glycan and in the presence or absence of a fucose residue on the core of the N-glycan. However, other glycosylation sites have been observed in serum IgGs in the variable Fab region. Such N-glycans have been shown to have a greater level of structural heterogeneity (Biotechnology and Bioengineering, June 2022, vol. 119, issue 6, pp. 1343- 1358). As glycosylation heterogeneity substantially impacts therapeutic antibody immunogenicity and efficacy, the control and characterization of therapeutic antibody glycosylation profiles is specified in International Conference on Harmonisation (ICH) Q6B guidance document (Specifications: Test Procedures and Acceptance Criteria for Biotechnological / Biological Products, European Medicines Agency, 1999).

[0089] Different residues, such as fucose, bisecting GIcNAc, galactose, and sialic acid, can be added to the core biantennary complex structure (GlcNAc2Man3GlcNAc2) of N-linked glycans of human IgGs, generating heterogeneity of the IgG-Fc glycans. The heterogeneous glycans can be classified into three sets (GO, Gl, and G2), depending on the number of galactose residues in the outer arms of biantennary glycans. Within each of these sets, there are different species that arise from the presence or absence of core fucose and bisecting GIcNAc (Antibodies, June 2020, doi: 10.3390 / antib9020022).

[0090] Sialic acids are present in human serum IgGs as N-acetylneuraminic acid (NANA) attached to a terminal galactose by an o-2,3 or o-2,6 linkage. Recombinant monoclonal antibodies expressed in a CHO cell line also have NANA, but it is only attached by o-2,3 linkage. Recombinant monoclonal antibodies produced in NSO and SP2 / 0 cell lines have N-glycolylneuraminic acid (NGNA), a sialic acid form produced by hydroxylation of NANA utilizing cytidine monophosphate N-acetylneuraminic acid hydroxylase enzyme which is absent in human and CHO cells under normal conditions. Recombinant monoclonal antibodies produced in NSO and SP2 / 0 cell lines may also have galactose-o-1,3- galactose (Galo(l,3)Gal or o-gal) epitopes that are not found in humans.

[0091] In an embodiment of the disclosure, the belimumab level of G1 is 40-50%. In an embodiment of the disclosure, the belimumab level of G1 is 41-49%. In an embodiment, the belimumab level of G1 is 41-46%. In an embodiment, the belimumab level of G1 is 43-46%.

[0092] In an embodiment of the disclosure, the belimumab level of GO is 15-31%. In an embodiment, the belimumab level of GO is 25-31%. In an embodiment, the belimumab level of GO is 26-28%.

[0093] In an embodiment of the disclosure, the belimumab level of G2 is 12-23%. In an embodiment, the belimumab level of G2 is 12-16%. In an embodiment, the belimumab level of G2 is 14-15%.

[0094] In an embodiment of the disclosure, the belimumab level of o-Gal is 6-15%. In an embodiment, the belimumab level of o-Gal is 6-11%. In an embodiment, the belimumab level of o- Gal is 8-11%.

[0095] In an embodiment of the disclosure, the belimumab level of NGNA is <2.1%. In an embodiment, the belimumab level of NGNA is 0.8-1.3%. In an embodiment, the belimumab level of NGNA is <1%.

[0096] In an embodiment of the disclosure, the belimumab level of GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 6-15% and / or NGNA is <2.1%. In an embodiment, the belimumab level of GO is 25-31%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3%. In an embodiment, the belimumab level of GO is 26-28%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1%.

[0097] In an embodiment of the disclosure, GO, Gl, G2, o-Gal and / or NGNA amounts are determined by capillary electrophoresis using laser-induced fluorescence detection (CE-LIF).

[0098] In an embodiment, a method of controlling the glycosylation of belimumab is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper. In an embodiment, the mammalian cells are NSO cells.

[0099] In an embodiment, a method of controlling the glycosylation of belimumab is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0100] In an embodiment, a method of controlling the glycosylation of belimumab is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 15 - 62 ppb manganese and 4.2 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0101] In an embodiment, a method of controlling the glycosylation of belimumab is provided, said method comprising culturing belimumab-producing NSO cells in a serum-free cell culture medium comprising 21- 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the belimumab producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0102] In an embodiment, a method of controlling the glycosylation of belimumab is provided, said method comprising culturing belimumab-producing NSO cells in a serum-free, AGT powder containing cell culture medium comprising 21- 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the belimumab producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0103] In an embodiment, a method of controlling the glycosylation of an antibody comprising SEQ ID NO:3 and SEQ ID NO:4 is provided, said method comprising culturing said antibody-producing NSO cells in a serum-free, AGT powder containing cell culture medium comprising 21- 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the antibody-producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0104] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper.

[0105] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper.

[0106] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 17.3 ppb manganese and at least 4.6 ppb copper.

[0107] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 17.3 ppb manganese and at least 4.6 ppb copper.

[0108] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 21 ppb manganese and at least 5.6 ppb copper.

[0109] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 21 ppb manganese and at least 5.6 ppb copper.

[0110] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 15 - 200 ppb manganese and 4.2 - 50 ppb copper.

[0111] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 15 - 200 ppb manganese and 4.2 - 50 ppb copper.

[0112] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 15 - 62 ppb manganese and 4.2 - 17 ppb copper.

[0113] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, a-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 15 - 62 ppb manganese and 4.2 - 17 ppb copper.

[0114] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 17.3 - 62 ppb manganese and 4.6 - 17 ppb copper.

[0115] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 17.3 - 62 ppb manganese and 4.6 - 17 ppb copper.

[0116] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper.

[0117] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper.

[0118] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0119] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0120] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 17.3 ppb manganese and at least 4.6 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0121] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 17.3 ppb manganese and at least 4.6 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0122] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 21 ppb manganese and at least 5.6 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0123] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 21 ppb manganese and at least 5.6 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0124] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 15 - 200 ppb manganese and 4.2 - 50 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0125] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 15 - 200 ppb manganese and 4.2 - 50 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0126] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, a-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 15 - 62 ppb manganese and 4.2 - 17 ppb copper, between 1.7 and

[0127] 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0128] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 15 - 62 ppb manganese and 4.2 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0129] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 17.3 - 62 ppb manganese and 4.6 - 17 ppb copper, between 1.7 and

[0130] 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0131] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 17.3 - 62 ppb manganese and 4.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0132] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and

[0133] 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0134] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0135] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1% is provided, said method comprising culturing belimumab-producing NSO cells in a serum- free cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the belimumab-producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0136] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper.

[0137] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 17.3 ppb manganese and at least 4.6 ppb copper.

[0138] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 21 ppb manganese and at least 5.6 ppb copper.

[0139] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 15 - 200 ppb manganese and 4.2 - 50 ppb copper.

[0140] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 15 - 62 ppb manganese and 4.2 - 17 ppb copper.

[0141] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 17.3 - 62 ppb manganese and 4.6 - 17 ppb copper.

[0142] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby -Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper.

[0143] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing mammalian cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0144] In an embodiment, a method of achieving a predetermined glycosylation profile of an antibody comprising SEQ ID NO:3 and SEQ ID NO:4, whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing said antibody-producing NS0 cells in a serum free cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the antibody-producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0145] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing NS0 cells in a serum free, AGT powder containing cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the belimumab-producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0146] In an embodiment, a method of achieving a predetermined glycosylation profile of an antibody comprising SEQ ID NO:3 and SEQ ID NO:4, whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing said antibody-producing NS0 cells in a serum free, AGT powder containing cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the antibodyproducing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0147] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby -Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing belimumab-producing NSO cells in a serum free, AGT powder and yeastolate containing cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the belimumab-producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0148] In an embodiment, a method of achieving a predetermined glycosylation profile of an antibody comprising SEQ ID NO:3 and SEQ ID NO:4, whereby GO is 25-31%, G1 is 41-46%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3% is provided, said method comprising culturing said antibody-producing NSO cells in a serum free, AGT powder and yeastolate containing cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the antibody-producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0149] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper.

[0150] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 17.3 ppb manganese and at least 4.6 ppb copper.

[0151] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 21 ppb manganese and at least 5.6 ppb copper.

[0152] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 15 - 200 ppb manganese and 4.2 - 50 ppb copper.

[0153] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 15 - 62 ppb manganese and 4.2 - 17 ppb copper. In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 17.3 - 62 ppb manganese and 4.6 - 17 ppb copper.

[0154] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper.

[0155] In an embodiment, a method of achieving a predetermined glycosylation profile of belimumab whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing belimumab-producing NSO cells in a cell culture medium comprising at least 15.0 ppb manganese and at least 4.2 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2.

[0156] In an embodiment, a method of achieving a predetermined glycosylation profile of an antibody comprising SEQ ID NO:3 and SEQ ID NO:4, whereby GO is 26-28%, G1 is 43-46%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1% is provided, said method comprising culturing said antibody producing NSO cells in a serum-free cell culture medium comprising 21 - 62 ppb manganese and 5.6 - 17 ppb copper, between 1.7 and 2.3 g / L glucose, and between 30 and 70 % dissolved oxygen, at a temperature of between 36 and 38 °C and a pH of between 7.0 and 7.2 for at least 96 hours, wherein the initial viable cell density of the antibody-producing cells is between 1.5xl05and 3.9xl05viable cells / mL.

[0157] EXAMPLES

[0158] List of Abbreviations

[0159] AGT Advanced Granular Technology

[0160] BN Batch Number

[0161] CE-LIF Capillary Electrophoresis-Laser Induced Fluorescence

[0162] CDH Chemically Defined Hybridoma

[0163] DO Dissolved Oxygen

[0164] DOE Design of Experiment

[0165] DPM Dry Powder Medium

[0166] EOR End of Run

[0167] GMP Good Manufacturing Practice HHYO High Copper, High Manganese Yeastolate

[0168] HPLC High Performance Liquid Chromatography

[0169] ICP-MS Inductively Coupled Plasma Mass Spectrometry IEC Ion-Exchange Chromatography

[0170] LLYO Low Copper, Low Manganese Yeastolate

[0171] LSM Large-Scale Manufacturing ppb Parts Per Billion ppm Parts Per Million

[0172] SEC Size Exclusion Chromatography

[0173] TXRF Total Reflection X-ray Fluorescence

[0174] VCD Viable Cell Density

[0175] VLN Vendor Lot Number

[0176] YO Yeastolate vcd Viable Cell Density wm Vessel Volume Per Minute

[0177] Example 1: Raw Material Variability: Yeastolate Analysis

[0178] Commercial manufacturing runs (batches) of belimumab have historically demonstrated intermittent issues with growth variability of belimumab-producing cells, which has been traced back to lot-to-lot variability in the raw materials used. The primary raw materials used in the manufacture of belimumab are 1) a basal growth medium (customised AGT-CDH, THERMOFISHER), 2) a nutrient feed medium (DPM), and 3) yeastolate (YO).

[0179] Yeastolate in particular is a complex mixture and its constituents vary widely from lot-to-lot. It was found that copper and manganese were the most variable components between lots of yeastolate that resulted in successful and unsuccessful batches of belimumab-producing cell cultures. In this example, twenty-two different lots of yeastolate were analysed to better understand the copper and manganese variability.

[0180] Yeastolate lot samples (20 g) were dried at 37.0 °C for 35 minutes to remove any moisture. The dried powder was used to prepare a 100 mL sample solution according to the spectrometer manufacturer's instructions (S4 T-STAR spectrometer sample preparation procedure) and samples were analysed as per manufacturer's instructions.

[0181] Vendor lot number (VLN) 8211748 was selected as a low copper, low manganese yeastolate (LLYO) lot due to its inherently low levels of both copper and manganese and VLN 8281591 was selected as a high copper, high manganese yeastolate (HHYO) lot due to its inherently high levels of both copper and manganese, as shown in Table 1.

[0182] Table 1 Selected Yeastolate Vendor Lots

[0183] Example 2: Effect of Varying Concentrations of Copper and / or Manganese on Belimumab Production in Micro-Bioreactors

[0184] Methods

[0185] Belimumab-producing cells were taken from a GMP 500 L seed train at the Large-Scale Manufacturing (LSM) facility and expanded into 2 x 3 L shake flasks. The 3 L shake flasks were used to inoculate a 15 L bench-top reactor (N-l). The N-l culture served as a seed for the 24 AMBR bioreactors and the inoculation cell density for each bioreactor was 2.4 ± 0.6 xlO5cells / mL.

[0186] Commercial production process conditions were adhered to with adjustments for scale and capabilities of the AMBR250 bioreactors.

[0187] In this example, the same VLN of basal cell medium (customised AGT-CDH) and the same VLN of feed medium (DPM) were used in all of the AMBR250 reactors. Copper and Manganese levels for these raw material lots were estimated using inductively coupled plasma mass spectrometry (ICP-MS) data. The manganese concentration in the basal cell medium and feed medium was 0.283 ppm and 0.130 ppm, respectively. The copper concentration in the basal cell medium and feed medium was 0.102 ppm and 0.208 ppm, respectively. Accordingly, the difference in overall copper and manganese concentrations in the belimumab-producing cell cultures in each run was as a result of the yeastolate batch used: HHYO or LLYO. It was assumed that there was no or negligible amounts of copper and manganese in the inoculum. In any case, all of the bioreactors used the same inoculum culture.

[0188] Feed medium (DPM), with YO added, was spiked with three different concentrations each for copper (resultant feed concentrations of 0.01, 0.02, and 0.041 ppm) and manganese (resultant feed concentrations of 0.04, 0.08, and 0.163 ppm) and evaluated for their ability to contribute to robust cell culture attributes, and consistent and acceptable product quality in AMBR250 reactors. The spiking was done at the end of feed preparation and prior to filtration. High concentration stock solutions of manganese chloride and copper sulphate were used (0.25 M and 1 M, respectively) such that volume changes were negligible.

[0189] The initial working volume in each AMBR250 was approximately 215 mL (i.e., the volume of the culture in the reactor following inoculation on day 0). Three identical feeds were administered on days 3, 5, and 7 at 9.8% of the current bioreactor volume on each day. However, it was necessary to remove volume from all reactors in order to add the day 7 feeds due to the 250 mL capacity of each AMBR250 reactor.

[0190] Glucose was replenished in the reactors starting at 144 hours, up to 2.0 g / L when the concentration was <1.7 g / L. Culture pH was controlled between 7.0 and 7.2 with sodium hydroxide for base and carbon dioxide for acid and the temperature was maintained at 37 °C. The reactors were sampled daily for pH, CO2, glucose, lactate, glutamine, glutamate, osmolality, and base usage. Cells were agitated at 372 rpm and gassed at 0.1 mL / min air and 0 to 50 mL / min oxygen with a 50% dissolved oxygen (DO) setpoint.

[0191] Twenty-four AMBR250 runs, each of a duration of 264 ± 8 hours, were carried out as shown in Table 2: this includes 2 runs (run numbers 23 and 24) evaluating high concentrations of copper and manganese spikes in addition to the highest yeastolate lot (HHYO) in order to determine possible upper limits of manganese and copper concentrations.

[0192] Table 2: AMBR250 reactions - Feed Medium with LLYO or HHYO, and with or without Copper and / or Manganese Spikes

[0193] On day 11, the culture in each bioreactor was clarified and filtered, purified using a Protein A column, and belimumab material was analysed. Analysis included carbohydrate analysis using CE- LIF, SEC for fragments, monomer and aggregates, HPLC for protein concentration, and IEC for acidic, main and basic peaks.

[0194] Statistical software (Design-Expert, STATEASE) was used to program and determine the effect and any statistical significance of different spike concentrations of copper and manganese on process performance and product quality.

[0195] Design-Expert software was also used to analyse the data and predict the range of copper and manganese spikes that would meet pre-determined process and product quality criteria (which are more stringent than commercial criteria to ensure a robust process and product quality outcome) with a 95% confidence (DOE optimisation).

[0196] Results

[0197] The effect of low overall copper concentration manifested in slower growth with lower peak viable cell density (VCD), reduced viability, higher consumption of glucose, higher peak lactate, and increased final lactate leading to an increase in base usage. The cell culture profiles (Figures 1 to 7) illustrate a marked difference between the effects of low and high copper yeastolate lots. Notably, cultures lowest in copper consumed high amounts of glucose per cell density (Figure 3) and failed to metabolically switch to lactate consumption (Figure 4) as was previously observed in several poor performing commercial runs. There was a dose response effect on lactate as the concentration of copper in the spike to the feed medium containing LLYO increased, with the levels of lactate lowering as a desirable metabolic shift to lactate consumption was induced (Figure 5).

[0198] Copper levels in the feed impacted growth characteristics to a greater extent than manganese levels. However, manganese levels had a more significant effect on product quality attributes than did copper (with the exceptions of IEC peaks where copper increased the acidic peak significantly more than manganese lowered the acidic peak and also with respect to NGNA in which copper effects were also more significant than manganese, but not of a high impact, with NGNA remaining well within product release criteria under all conditions).

[0199] Additions of copper and manganese eventually ceased to improve process attributes or carbohydrate profile any further and reached a plateau (Figure 8). The saturation effect can be observed for peak growth and titer when total copper concentrations in the cell culture reach ~6.5 ppb, and for peak lactate at ~7-10 ppb, remaining stable through 17 ppb (the highest concentration tested, i.e., spiking the feed with 0.041 ppm copper - run numbers 23 and 24). Total manganese concentrations in the cell culture plateau for %G0 and %G2 between ~30 ppb and ~35 ppb (Figure 8), remaining stable through ~61.5 ppb (the highest concentration tested, i.e., spiking the feed with 0.163 ppm manganese - run numbers 23 and 24). Increasing copper beyond ~7.6 ppb in the cell culture reduces titer slightly, but then this too plateaus (Figures 7 and 8).

[0200] The peak cell density for runs 23 and 24 was slightly lower (6.3% lower) than the control (run 17 with HHYO and no copper or manganese spikes, which equates to cell culture concentrations of copper and manganese of 6.78 ppb and 20.68 ppb, respectively). However, peak lactate was significantly reduced. These high concentrations of manganese and copper increased IEC acidic peaks slightly (but still within acceptable ranges), while having no impact on IEC main or basic peaks. SEC aggregates increased slightly without any significant impact on SEC monomer or SEC fragments. GO was significantly reduced, while G2 and o-gal were increased, with little impact on NGNA. This data provides confidence that should lots of raw materials contain unusually high levels of copper or manganese not seen to date, the addition of a mandated fixed spike of copper or manganese will not harm the process.

[0201] Accordingly, runs with high concentrations of manganese and high concentrations of copper, well above the levels needed to provide good growth attributes and product quality, still demonstrated acceptable results for both process attributes and product quality.

[0202] Based on the data compiled, the risk to the production process and product quality is greatest at the lower end of total concentrations of copper and manganese, e.g., runs Al, A15, and A19 failed to meet the pre-determined process quality criteria (as a result of too low copper) and runs A7, A19, and A21 failed to meet pre-determined product quality criteria (as a result of too low manganese). The minimum spiking amounts of manganese and copper in the feed calculated by Design-Expert (DOE optimisation) to meet the pre-determined product quality criteria were 0.054 ppm and 0.013 ppm, respectively. This equates to 17.34 ppb manganese and 4.18 ppb copper in the cell culture. The lowest experimentally tested copper concentration that ensured pre-determined process quality criteria were met was 5.58 ppb in the cell culture. The lowest experimentally tested manganese concentration that ensured pre-determined product quality criteria were met was 20.68 ppb in the cell culture.

[0203] Example 3: Effect of Varying Concentrations of Copper and Manganese on Belimumab Production in 15 L Bioreactors

[0204] Aim

[0205] This experiment was designed to verify data from Example 2 in 15 L bench-top bioreactors and confirm the selected optimal spiking concentrations for copper and manganese.

[0206] Methods

[0207] The basal media lot used was the same as in Example 2 (VLN 1765368) and the HHYO and LLYO lots were also the same as in Example 2. The feed (DPM) lot (VLN2040555) was different to that used in Example 2 due to availability.

[0208] Based on the results from Example 2, the selected spiking concentrations of copper and manganese in the feed used in the 15 L bench-top bioreactors were 0.013 ppm and 0.054 ppm, respectively.

[0209] Belimumab-producing cells were taken from the same GMP 500 L seed train in LSM used in Example 2 and expanded into 3 L shake flasks. The 3 L shake flasks (N-2) were used to inoculate 2 x 15 L bench-top reactors (N-l), with the inoculation cell density for each bioreactor being 2.4 ± 0.6 xlO5cells / mL (same as Example 2).

[0210] The initial working volume in each 15 L reactor was approximately 67% of the volume capacity, i.e., approximately 10 L. Feeding conditions were as set out in Table 3.

[0211] Table 3: Feeding conditions

[0212] Culture pH was set to 7.2 for the first 48 hours and then 7.0 until the end of the run (264 ± 8 hours) and the temperature was maintained at 37 °C. Cells were agitated at 320 rpm and gassed at 0.02 wm (max) air and oxygen was set to 50% dissolved oxygen (DO) with no upper oxygen wm. The cultures were sampled daily as per the schedule set out in Table 4.

[0213] Table 4: Sampling Schedule

[0214] Eight 15 L Bioreactor runs, each of a duration of 264 ± 8 hours, were set up as shown in

[0215] Table 5. Table 5: 15 L Bioreactor Feed Medium with LLYO or HHYO and with or without Copper and Manganese Spikes

[0216] NS = no spike

[0217] End of run samples (approximately 500 mL) were centrifuged, filtered and purified using Protein A chromatography. The purified belimumab-containing sample was analysed using CE-LIF, SEC, IEC, and HPLC titer analysis.

[0218] Results

[0219] Due to an overhead stirrer malfunction, cells in C14 did not grow and the culture was terminated at ~72 hours. Accordingly, seven of the eight 15 L bioreactor cell cultures ran to completion.

[0220] Cell growth in cultures C19 and C20 (LLYO without Cu Mn spikes) were negatively affected resulting in low peak viable cell densities less than the expected minimum of 105.0xl05cells / mL and a rapid drop in cell viability between day 5 and day 11. In addition, final cell viabilities were slightly lower than cultures with higher levels of copper and manganese (C13, C15, and C16). However, adding Cu and Mn spikes to feed containing the same LLYO lot improved cell growth as observed in C17 and C18 resulting in peak viable cell densities of 170xl05cells / mL and 190xl05cells / mL, respectively. Cell viability in these cultures appear to trend comparably with the cultures that were fed with HHYO with and without spikes (C13, C15 and C16).

[0221] Glucose consumption in C19 and C20 (LLYO without Cu Mn spikes) increased more rapidly between day 4 and day 5 as lactate consumption decreased. Low lactate consumption by the cells resulted in lactate accumulation in the culture leading to poor cell growth. This trend contrasts with C17 and C18 (LLYO with Cu Mn spikes) and C13, C15 and C16 (HHYO with and without Cu Mn spikes).

[0222] Bioreactor cultures C19 and C20 (LLYO without Cu Mn spikes) failed to meet the minimum acceptance titer criteria of 1.32 g / L. However, C17 and C18 (LLYO with Cu Mn spikes) were able to meet and exceed the minimum acceptance criteria for titer by 0.34 and 0.40 g / L, respectively. Cultures C13-C16 that were fed with the HHYO lot showed no difference in titer results when comparing spiked runs to no spike runs.

[0223] Regarding carbohydrate profiles generated from CE-LIF, the data shows that spiking the feed with manganese and copper consistently reduced GO, on average by ~ 1% when compared to no spike conditions (for both the LLYO and HHYO lots). Spiking feed containing the LLYO lot raised G1 on average ~ 1.5%, with no change observed when spiking feed containing the HHYO lot. On average G2 was lowered by ~ 1.0% when spiking LLYO feed and 0.8% when spiking HHYO feed, o- gal also increased by 1.6% on average when spiking LLYO feeds. When comparing NGNA results for spike and no spike in the feed with the LLYO lot, there was a 0.4% decrease with the spike, with no differences observed in cell cultures with HHYO in the feed when comparing spike and no spike conditions.

[0224] IEC data shows that acidic peaks in cultures with LLYO were slightly higher (~0.8% when averaged). Spiking with manganese and copper decreased the main peak slightly in cultures with LLYO, when compared to the main peaks in cultures with HHYO with and without spikes. Basic peaks in cultures with LLYO were slightly higher when spiked (~1.6% when averaged). Lysine variants were also slightly higher (~1.2% when averaged) in LLYO cultures that were spiked versus no spike. For cultures with HHYO, there was no difference in lysine variants between spiked versus no spike. SEC data showed no difference in monomer and fragment levels when comparing cultures with spikes and those with no spikes. As such supplementing copper and manganese into production cultures had no significant impact on IEC and SEC profiles.

[0225] Accordingly, the optimal spiking amounts of manganese and copper in the feed determined in Example 2 were successful in the 15 L bioreactor cell cultures indicating that the supplementation strategy is effective and scalable.

[0226] Example 4: Robustness of Copper and Manganese Supplementation on Belimumab Production in Micro-Bioreactors

[0227] Aim

[0228] This example was designed to test the robustness of the copper and manganese supplementation strategy of Example 2 and Example 3. In contrast to Example 2 and Example 3, in this example different lots of the basal growth medium (AGT-CDH) and different lots of the feed medium (DPM) were used in the AMBR250 reactors. This experiment was designed to see if specific concentrations of manganese (0.054 ppm) and copper (0.013 ppm) spiked in the feed medium could ensure critical process and product attribute responses regardless of the VLN used for yeastolate, basal cell media (AGT-CDH) and feed media (DPM) (see Table 6).

[0229] Methods

[0230] As with Example 3, the selected spiking concentrations of copper and manganese in the feed were 0.013 ppm and 0.054 ppm, respectively.

[0231] Copper and Manganese levels for the raw material lots used in this experiment were estimated using available total reflection x-ray fluorescence (TXRF) or inductively coupled plasma mass spectrometry (ICP-MS) data: YO levels used TXRF, and AGT-CDH and DPM used ICP-MS.

[0232] Belimumab-producing cells were taken from the same GMP 500 L seed train in LSM used in Example 2 and Example 3 and expanded into 3 L shake flasks. The 3 L shake flasks (N-2) were used to inoculate 2 x 15 L bench-top reactors (N-l), with the inoculation cell density for each bioreactor being 2.4 ± 0.6 xlO5cells / mL (same as Example 2 and Example 3).

[0233] The initial working volume in each AMBR250 was approximately 200 mL, i.e., 15 mL less than the initial working volume used in Example 2 in order to avoid reaching the volume capacity of AMBR250 when giving the day 7 feed.

[0234] As with Example 2, glucose was replenished in the reactors starting at 144 hours, up to 2.0 g / L when the concentration was <1.7 g / L. Culture pH was controlled between 7.0 and 7.2 with sodium hydroxide for base and carbon dioxide for acid and the temperature was maintained at 37 °C. The reactors were sampled daily for pH, CO2, glucose, lactate, glutamine, glutamate, osmolality, and base usage. Cells were agitated at 372 rpm and gassed at 0.1 mL / min air and 0 to 50 mL / min oxygen with a 50% dissolved oxygen (DO) setpoint.

[0235] Feeding conditions were as set out in Table 3, i.e., the same as in Example 3. The daily sampling schedule was the same as in Example 3 (see Table 4), except that the daily sample volume (0-264 hours) was 1.4 mL.

[0236] Twelve AMBR250 reactor runs, each of a duration of 264 ± 8 hours, were set up as shown in Table 6. AMBR250 reactors A04 and A08 contained the highest and lowest total amounts of copper in the cell cultures, respectively. AMBR250 reactors A10 and A09 contained the highest and lowest total amounts of manganese in the cell cultures, respectively. Table 6: Selected Vendor Lots and Estimated Copper and Manganese Amounts in

[0237] Belimumab-Producing Cell Culture

[0238] As shown in Table 6, the highest concentrations of manganese and copper tested were 37.97 ppb and 11.41 ppb, respectively, in the cell culture. The lowest concentrations of manganese and copper tested were 17.35 ppb and 2.14 ppb, respectively, in the cell culture.

[0239] End of run samples (approximately 200 mL) were centrifuged, filtered and purified using Protein A chromatography. The purified belimumab-containing sample was analysed using CE-LIF, SEC, IEC, and HPLC titer analysis.

[0240] Results

[0241] Spiking 0.013 ppm copper in the feed in every run (from low to high inherent copper levels in the raw materials) ensured that all runs met the minimum criteria established for acceptable process characteristics and final titer. Six reactors began with the low-copper AGT. By day 2, their growth was slower than conditions that began with AGT containing normal concentrations of copper. Two reactors in the low-Cu AGT group also received lower copper in their feeds, and these demonstrated the lowest growth overall. By day 9, however, after having received all three feeds, they were able to recover and meet the minimum commercial criteria for peak VCD of 105.0xl05cells / mL. This confirms that even with deficient raw materials, the spiking strategy promotes a robust process. Differences for glucose and lactate profiles were observed in the first few days of cell-culturing due to differences in the initial concentration of copper. Peak lactate was defined by copper levels, with the runs having the lowest concentrations of copper exhibiting the highest peak accumulation of lactate. However, all runs metabolically switched to lactate consumption within one or two days of each other, ending with very low levels of lactate. The result of the higher peak lactate in the affected reactors was a higher base usage for pH control and slightly elevated osmolality.

[0242] For all runs, the final titer met the minimum criteria of >1.3 g / L as well as the criterion used for the DOE optimisation (>1.55 g / L).

[0243] Example 5: 20,000 L Commercial Scale Bioreactor

[0244] The reproducibility of the optimal spiking concentrations from Examples 3 and 4 was also confirmed in two runs at commercial scale (20,000 L reactor).

[0245] Example 6: Analysis of Copper and Manganese in Raw Materials used in Successful Belimumab Batches

[0246] An analysis of the copper and manganese concentrations in the raw materials used in batches of belimumab was conducted. The highest concentration of manganese and copper in the cell culture of successful batches was approximately 39.1 ppb and 11 ppb, respectively. The lowest concentration of manganese and copper in the cell culture of successful batches was approximately 15.0 ppb and 4.6 ppb, respectively.

[0247] Overall Conclusions

[0248] The risk of process failure due to raw material variability with respect to low manganese and low copper levels (which manifest in poor growth, high lactate, lower titer and negative effects of GO, G2 and o-gal), can be mitigated by spiking feeds with manganese (0.054 ppm) and copper (0.013 ppm). Data generated indicates that these concentrations improve the culture lactate consumption rate, which in turn improves cell growth and titer, and also improve carbohydrate profiles (specifically decreasing GO and increasing both G2 and o-gal - CE-LIF - see Figure 8). Further, there was no significant impact on IEC (basic and acidic peaks) and SEC (monomer, fragment and aggregate amounts) profiles.

[0249] Accordingly, whilst yeastolate lot-to-lot variation is the primary driver of trace metal variation, supplementing feeds with fixed amounts of manganese and copper was robust and resulted in favourable and consistent process and quality attributes in every instance, addressing variation across all critical raw materials used in the belimumab cell culture process.

[0250] High manganese and copper concentrations of 62.0 ppb and 17.0 ppb, respectively, in the cell culture were tested and still resulted in acceptable process and quality attributes (Example 2, runs 23 and 24). As such, upper concentration limits of manganese and copper have not been established.

[0251] Taking into account results from AMBR250 (Example 2) and 15 L (Example 4) reactors where the effects of different concentrations of copper and manganese were systematically investigated, at least 15.00 ppb manganese and at least 4.2 ppb copper in belimumab-producing cell cultures is required to achieve desired product quality and process attributes.

[0252] SEQUENCE LISTING

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[0256] SEO ID NO:2

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Claims

CLAIMS1. A method of controlling the glycosylation of belimumab, said method comprising culturing belimumab-producing cells in a cell culture medium comprising at least 15.0 ppb manganese.

2. A method of achieving a predetermined glycosylation profile of belimumab, said method comprising culturing belimumab-producing cells in a cell culture medium comprising at least 15.0 ppb manganese.

3. The method of claim 1 or claim 2, wherein the cell culture medium comprises 15.0-62.0 ppb manganese.

4. The method of any preceding claim, wherein the cell culture medium comprises at least17.5 ppb manganese.

5. The method of any preceding claim, wherein the cell culture medium comprises at least20.5 ppb manganese.

6. The method of any preceding claim, wherein the cell culture medium comprises at least 4.2 ppb copper.

7. The method of claim 6, wherein the cell culture medium comprises 4.2-17.0 ppb copper.

8. The method of any preceding claim, wherein the cell culture medium comprises at least4.6 ppb copper.

9. The method of any preceding claim, wherein the cell culture medium comprises at least5.6 ppb copper.

10. The method of any preceding claim, wherein the cell culture medium is serum-free.

11. The method of any preceding claim, wherein the cell culture medium is a chemically defined medium.

12. The method of any one of claims 1 to 10, wherein the cell culture medium comprises yeastolate.

13. The method of any preceding claim, wherein the belimumab-producing cells are mammalian cells.

14. The method of any preceding claim, wherein the belimumab-producing cells are murine myeloma (NS0) cells.

15. The method of any preceding claim, wherein the belimumab-producing cells are Chinese hamster ovary (CHO) cells.

16. The method of any preceding claim, wherein the belimumab level of GO is 15-31%, G1 is 41-49%, G2 is 12-23%, o-Gal is 7-15%, and / or NGNA is <2.1%.

17. The method of any preceding claim, wherein the belimumab level of GO is 25-31%, G2 is 12-16%, o-Gal is 6-11%, and / or NGNA is 0.8-1.3%18. The method of any preceding claim, wherein the belimumab level of GO is 26-28%, G2 is 14-15%, o-Gal is 8-11%, and / or NGNA is <1%.

19. The method of any one of claims 16 to 18, wherein the belimumab level of G1 is 41- 46%, optionally 43-46%.

20. The method of any one of claims 16 to 19, wherein GO, Gl, G2, o-Gal, and / or NGNA amounts are determined by capillary electrophoresis using laser induced fluorescence detection (CE-LIF).

21. A method for increasing the percentage of G2 and the percentage of o-gal in a glycoprotein composition and reducing the percentage of GO in said glycoprotein composition by culturing mammalian cells expressing said glycoprotein in a cell culture medium comprising at least 15.0 ppb manganese, optionally 15.0-62.0 ppb manganese.

22. The method of claim 21, wherein the cell culture medium comprises at least 4.2 ppb copper, optionally 4.2-17.0 ppb copper.

23. The method of claim 21 or claim 22, wherein the glycoprotein is an antibody.

24. The method of claim 23, wherein the antibody is an IgGl antibody.

25. The method of any one of claims 21 to 24, wherein the glycoprotein comprises SEQ ID NO:1 and SEQ ID NO:2.

26. The method of claim 25, wherein the glycoprotein comprises SEQ ID NO:3 and SEQ ID NO:4.

27. The method of claim 26, wherein the IgGl antibody is belimumab.

28. The method of claim 26, wherein the IgGl antibody comprises SEQ ID NO:3 and SEQ ID NO:4.

29. The method of any one of claims 21 to 28, wherein the cells are murine myeloma (NS0) cells or Chinese hamster ovary (CHO) cells.

30. The method of any preceding claim, wherein the pH of the cell culture medium is between 7.0 and 7.2, optionally about 7.0.

31. The method of any preceding claim, wherein the cells are cultured at a temperature between 36 and 38 °C, optionally about 37 °C.

32. The method of any preceding claim, wherein % dissolved oxygen of the cell culture is between 10 and 73.6, optionally between 30 to 70, optionally about 50.

33. The method of any preceding claim, wherein the initial belimumab-producing viable cell density (VCD) of the cell culture is at least 1.5xl05viable cells / mL, optionally 1.5xl05viable cells / mL to 3.9xl05viable cells / mL, optionally 1.8xl05viable cells / mL to 3.05X105viable cells / mL, or optionally about 2.4xl05viable cells / mL.

34. The method of any preceding claim, wherein the peak belimumab-producing VCD of the cell culture is at least 105xl05viable cells / mL.

35. The method of any preceding claim, wherein the belimumab-producing cells are cultured for at least 96 hours.

36. The method of any preceding claim, wherein the cell culture medium comprises between 1.7 and 2.30 g / L glucose, optionally 2.0 g / L glucose.

37. A method of producing belimumab as set forth in any one of the preceding claims.

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