Method for producing sperolizumab

Abstract

To provide a method for producing anti-IL-36R antibodies, spesolimab.SOLUTION: To provide a method for producing spesolimab using a serum-free cell culture medium in a fed-batch culture in the presence of a reduced copper concentration and an elevated iron concentration. In addition, the present invention provides compositions comprising reduced levels of basic species of Spesolimab and / or reduced levels of Spesolimab species with high mannose structures.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for producing the anti-IL-36 receptor antibody spesolimab. More specifically, the present invention relates to a method for producing spesolimab using serum-free cell culture media in fed-batch culture in the presence of reduced copper and elevated iron concentrations. Additionally, the present invention relates to compositions comprising low levels of basic species of spesolimab and / or low levels of spesolimab species with high mannose structures. [Background technology]

[0002] background Recombinant monoclonal antibodies (mAbs) are typically expressed in mammalian cell culture. Collected antibodies contain product variants with different properties, such as charge and size, also known as product heterogeneity. The primary source of heterogeneity is post-translational modification and degradation during cell culture. Antibodies are typically purified through several filtration and chromatography steps. However, it is not practical to remove all heterogeneity throughout downstream processing. Therefore, modifications and degradation occurring during the cell culture process have the most significant impact on the quality of the final product. Heterogeneity resulting from post-translational modifications can affect the quality, safety, and efficacy of monoclonal antibody products. Key quality characteristics include glycosylation, charge variants (basic and acidic species, e.g., oxidized species, deamidated species, and C- and N-terminally modified species), aggregates, and low molecular weight species (LMVs). Upstream processes, including post-translational modifications and other modifications, have a strong impact on antibody product characteristics, which can vary between individual antibodies and cell lines. The most widely used cell lines for biopharmaceutical production were originally derived from Chinese hamster ovary (CHO cells), and most recombinant monoclonal antibodies today are produced in fed-batch cultures.

[0003] The anti-IL-36 receptor (IL-36R) antibody spesolimab reduces or blocks signaling mediated by the IL-36 ligand and is useful in treating diseases or conditions associated with such signaling.

[0004] Interleukin-36 (IL-36) is a group of cytokines within the IL-1 family that have proinflammatory effects. The IL-36 family includes four members: IL-36α (IL-1F6), IL-36β (IL-1F8), IL-36γ (IL-1F9), and IL-36Ra (IL-1F5). All of these members bind to the IL-36 receptor (previously known as IL-1Rrp2) and form heterodimers with IL-1RAcP. IL-36 receptor ligands are involved in many disease states, and the anti-IL-36 receptor antibody spesolimab is effective in treating inflammatory and autoimmune diseases, such as inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UD), atopic dermatitis (AtD), palmoplantar pustulosis (PPP), and generalized cystic psoriasis (GPP).

[0005] Upstream processes and parameters, including cell culture media and process steps, such as temperature and seeding density, have a strong influence on antibody product characteristics. Cell culture media must meet the complex nutritional requirements of mammalian cells cultured in suspension in technical systems, as opposed to those of their natural origin.

[0006] Cell culture media mostly consist of energy sources, such as carbohydrates or amino acids, lipids, vitamins, trace elements, salts, growth factors, polyamines, and non-nutritive components, such as buffers, surfactants, or antifoaming agents. Media used in fed-batch culture can be divided into two subgroups: process medium (P medium) or basal medium and feed medium (F medium). Basal medium contains all essential components at initial concentrations and is used for inoculation. Feed medium mostly provides nutrients at high concentrations during the culture process. Therefore, cell culture media are complex compositions of many different compounds, and identifying compounds that result in improved growth, productivity, or product quality is challenging. Trace metals in CHO cell cultures promote a wide variety of intracellular and extracellular functions necessary for optimal monoclonal antibody productivity and quality, including lactate consumption, energy metabolism, productivity, and product quality. Commonly used trace metals in cell culture media are iron, copper, zinc, and manganese, which are traditionally supplemented with fetal bovine serum, leading to variable results from lot-to-lot variations. In chemically defined media, trace elements and nutrients are supplied at specified concentrations, and any deficiency or excess of trace metals can affect cell culture performance, such as cell growth or viability, as well as productivity and product quality. For example, zinc deficiency has long been known to induce premature death in mammalian cells. Magnesium and calcium deficiencies have been shown to induce apoptosis in CHO cells, and copper deficiency has been shown to affect lactate metabolism in CHO cells (Graham RJ, Bhatia H., Yoon S., Biotechnology and Bioengineering, 2019, 116: 3446-3456). Furthermore, excess copper has been shown to increase monoclonal antibody productivity but also increase basic-charge variants. Therefore, there remains a need to further improve culture conditions to improve product quality without significantly affecting yield. Summary of the Invention [Problem to be solved by the invention]

[0007] Summary of the Invention In the present invention, we provide methods that allow for an efficient and effective production process for the antibody spesolimab, resulting in antibodies with specific product characteristics, such as few basic species and species with low levels of mannose 5 structures. [Means for solving the problem]

[0008] Accordingly, the present invention provides a method for producing CHO cells comprising the steps of: (a) culturing CHO cells comprising a nucleic acid encoding the antibody spesolimab using fed-batch culture in a serum-free cell culture medium, the method comprising: (i) seeding the cells in a culture medium; and (ii) culturing the cells in the culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, the cells comprising: (i) seeding the cells in a culture medium; and (ii) feeding the cells in the cell culture with a feed medium, wherein Cu 2+ The present invention provides a method for producing the antibody spesolimab in cell culture, comprising the steps of: (i) adding Cu at 0.35 to 1.2 μM and iron at 1500 μM or more to the culture medium before seeding the cells and / or within 2 days after seeding; (b) collecting a cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. Preferably, Cu is added to the culture medium before seeding the cells and / or within 2 days after seeding the cells; (b) collecting a cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. 2+ Cu and iron are added to the culture medium before and / or within one day after the inoculation in step (i). 2+ and / or iron is added to the culture medium as one or more bolus additions or continuously. The one or more bolus additions may include Cu, 2+and / or iron together with the basal medium. In a specific embodiment, the nucleic acid encoding the antibody spesolimab is stably integrated into the CHO genome. The method includes the step of feeding the cells with a feed medium. In a specific embodiment, the feed medium is added from day 0 to day 3 of culture, preferably from day 1 to day 3, more preferably from day 1 to day 2, and even more preferably on day 1. In the method of the present invention, an increased iron concentration and / or a decreased copper concentration in the culture medium results in the production of the antibody spesolimab with a reduced basic peak group (BPG) %. In a specific preferred embodiment, the antibody spesolimab produced by the method of the present invention has a BPG of 7.5% or less.

[0009] The method may further comprise the step of adapting the seeding density. In certain embodiments, the seeding density in step (a) is 0.7×10 6 cells / ml or more, preferably 0.7 x 10 6 cells / ml to 1.5 x 10 6 cells / ml, more preferably 0.8 x 10 6 cells / ml to 1.5 x 10 6 cells / ml, and even more preferably 0.9 x 10 6 cells / ml to 1.3 x 10 6cells / ml. Increased seeding density results in production of the antibody spesolimab with reduced BPG% and / or Man5 structure%. The method may further comprise adapting the culture temperature and / or dissolved oxygen concentration, wherein increased culture temperature and / or reduced dissolved oxygen (DO) results in production of the antibody spesolimab with reduced BPG% and / or Man5 structure%. In a specific embodiment, the cells are cultured at 36.0°C to 37.5°C under conditions that allow production of the antibody spesolimab, including feeding the cells with a feed medium, and / or wherein the dissolved oxygen concentration in the culture is maintained within the range of 30-60%. In certain embodiments, the antibody spesolimab has less than 5% Man5 structures, preferably less than 4% Man5 structures, more preferably less than 3% Man5 structures, and / or 7.5% or less BPG, preferably 7% or less BPG, more preferably 6.5% or less BPG, even more preferably 6% or less BPG, 7% or less BPG, preferably 6% or less BPG.

[0010] Although any CHO cells may be used in the methods described in the present invention, exemplary CHO cells are CHO-K1 cells and CHO-DG44 cells.

[0011] In another embodiment, (a)(i) cells are cultured in culture medium at a concentration of 0.7 x 10 6 and (ii) culturing the cells in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, comprising the steps of: (i) seeding the CHO cells containing the nucleic acid encoding the antibody spesolimab using fed-batch culture in a serum-free cell culture medium; and (ii) culturing the cells in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, comprising the steps of: (i) seeding the CHO cells containing the nucleic acid encoding the antibody spesolimab in a serum-free cell culture medium using a fed-batch culture in a serum-free cell culture medium; and (ii) culturing the cells in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, comprising the step of: (i) seeding the CHO cells 2+ (b) collecting a cell culture supernatant containing the antibody spesolimab; and (c) optionally, purifying the antibody spesolimab from the cell culture supernatant.

[0012] The present invention further relates to a composition comprising the antibody spesolimab, having (a) 7.5% or less BPG, preferably 7% or less BPG, more preferably 6.5% or less BPG, and even more preferably 6% or less BPG; and / or (b) less than 5% Man5 structures, preferably less than 4% Man5 structures, and more preferably less than 3% Man5 structures; and / or (c) less than 3% heavy chain (HC) lysine glycosylation variants, and / or lysines K38(HC) and K67(HC) are not glycosylated and glycosylation at K23(HC) is less than 0.3%.

[0013] The present invention further relates to a composition comprising the antibody spesolimab, wherein the antibody spesolimab is obtained by a method according to the present invention.

[0014] The composition comprising the antibody spesolimab can be a harvested cell culture fluid (HCCF), an affinity capture pool, a drug substance, or a drug product, preferably a drug substance or a drug product. Preferably, the composition is a drug product comprising the antibody spesolimab having less than 7.5% BPG and / or less than 5% Man5 structure. In a specific embodiment, the antibody spesolimab in the composition comprises 6% or less lysine glycosylation variants of the heavy chain (HC), and / or lysines K38(HC) and K67(HC) are not glycosylated, and glycosylation at K23(HC) is 0.3% or less.

[0015] The present invention further provides compositions comprising the antibody spesolimab having 3% or less heavy chain (HC) lysine glycosylation variants, and / or wherein lysines K38(HC) and K67(HC) are not glycosylated and glycosylation at K23(HC) is 0.3% or less. [Brief explanation of the drawings]

[0016] [Figure 1A]Figure 1 shows the effect of varying iron concentration (μM) and varying Cu2+ concentration (μM) on product quality as described in the design of experiments (DoE) approach of the Examples. (A) shows the effect of varying iron and copper concentrations on BPG (%) as measured by strong cation exchange HPLC chromatography (HP-SCX) for Cu2+ and iron as a contour plot. [Figure 1B] (B) shows the effect of varying copper concentrations on BPG (%) as measured by strong cation exchange HPLC chromatography (HP-SCX) as a one-factor plot. [Figure 1C] (C) shows the effect of varying iron concentrations on BPG (%) as measured by strong cation exchange HPLC chromatography (HP-SCX) as a single factor plot. The dotted lines in the single factor plot indicate the 95% confidence interval. [Figure 1D] Additionally, as a contour plot, (D) product concentration (mg / L); [Figure 1E] (E) IVC (integrated viable cell density) (1 × 10 cells / h / mL) and [Figure 1F] (F) The effect of varying iron and copper concentrations on percent survival is shown. [Figure 2A] Figure 2 shows the effect of varying inoculation density (1 x 10 cells / ml, factor 1), culture temperature (°C, factor 2), dissolved oxygen (DO%, factor 3), and feed rate (mL / L / day, factor 4) on product quality as described in the Design of Experiments (DoE) approach in the Examples. (A) Shown is the effect of varying culture temperature and inoculation density on BPG (%) as measured by strong cation exchange HPLC chromatography (HP-SCX) as a contour plot. [Figure 2B] (B) The effect of varying seeding density on BPG (%) as measured by strong cation exchange HPLC chromatography (HP-SCX) as a one-factor plot is shown. [Figure 2C](C) The effect of varying incubation temperature on BPG (%) as measured by strong cation exchange HPLC chromatography (HP-SCX) as a one-factor plot is shown. [Figure 2D] (D) The effect of varying seeding cell density as a one-factor plot on Man5 structure is shown (Oligomap Peak 3 (%)). [Figure 2E] (E) The effect of varying incubation temperature as a one-factor plot on Man5 structure is shown (Oligomap Peak 3 (%)). The dotted lines in the one-factor plot indicate the 95% confidence interval. [Figure 2F] Additionally, as a contour plot, (F) product concentration (titer, mg / L); [Figure 2G] (G) Cumulative viable cell density (1 × 106 cells / h / mL) and [Figure 2H] (H) The effect of varying iron and seeding density on percent survival is shown. [Figure 3A] Figure 3 shows the effect of varying seeding density (1 x 10 cells / ml, factor 1), culture temperature (°C, factor 2), dissolved oxygen (DO%, factor 3), and feed rate (mL / L / day, factor 4) as described in the Design of Experiments (DoE) approach in the Examples. (A) A one-factor plot shows the effect of varying dissolved oxygen concentration on BPG (%), as measured by strong cation exchange HPLC chromatography (HP-SCX). [Figure 3B] (B) The effect of varying dissolved oxygen concentration in a one-factor plot on Man5 structure is shown (Oligomap peak 3 (%)). [Figure 3C] (C) The effect of varying dissolved oxygen concentration on one-factor plots on product concentration (titer, mg / L) is shown. [Figure 3D] (D) The effect of varying dissolved oxygen concentration on the integrated viable cell density (1 × 10 cells / h / mL) is shown in a one-factor plot. The dotted lines in the one-factor plot indicate the 95% confidence interval. [Figure 4A]Figure 4 shows the heterogeneity profile of spesolimab using cation exchange chromatography (CEX). (A) The chromatographic profile of cation exchange chromatography (CEX) is shown with annotation of the acidic and basic peaks. [Figure 4B] (B) Chromatographic profile of the same cation exchange chromatography (CEX) as in (A) on an enlarged scale. [Figure 4C] (C) CEX chromatograms collected from fractions of different single acidic peaks are shown as an overlay. [Figure 5] Figure 5 shows a representative oligosaccharide pattern of spesolimab using HILIC (hydrophilic interaction liquid chromatography)-UHPLC (ultra-high performance liquid chromatography) with fluorescence detection. The major oligosaccharide peaks, labeled as peaks 1–6, are shown: Peak 1 = A1FG0 (nonsialylated, nongalactosylated core-fucosylated single chain); Peak 2 = A2FG0 (nonsialylated, nongalactosylated core-fucosylated two chains); Peak 3 = Man5 pentamannose core; Peak 4 = A2FG1 (nonsialylated, monogalactosylated core-fucosylated two chains); Peak 5 = A2FG1 (nonsialylated, monogalactosylated core-fucosylated two chains); and Peak 6 = A2FG2 (nonsialylated, digalactosylated core-fucosylated two chains). The x-axis shows the integration range (min) of the chromatogram, and the y-axis shows fluorescence as a voltage signal. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description The general embodiments "comprise" or "comprising" encompass the more specific embodiment "consisting of." Furthermore, the singular and plural forms are not used in a limiting sense.

[0018] As used herein, the term "cell culture medium" or "culture medium" refers to a medium for culturing mammalian cells that contains minimal essential nutrients and components, such as vitamins, trace elements, salts, bulk salts, amino acids, lipids, and carbohydrates, preferably in a buffered medium. Typically, cell culture media for mammalian cells have a near-neutral pH, e.g., a pH of about 6.5 to about 7.5, preferably about 6.8 to about 7.3, and more preferably about 7. Non-limiting examples of such cell culture media include commercially available media such as Ham's F12 medium (Sigma, Deisenhofen, Germany), RPMI-1640 medium (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO medium (Invitrogen, Carlsbad, CA), CHO-S medium (Invitrogen), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma), as well as proprietary media from various vendors, such as those disclosed in WO 2016 / 156476 (the entire contents of which are incorporated herein by reference). The cell culture medium may be a basal cell culture medium. The cell culture medium may also be a basal cell culture medium supplemented with a feed medium and / or additives. The cell culture medium may also be referred to as a fermentation broth when the cells are cultured in a fermentor or bioreactor.

[0019] The term "cell culture" or "cell culture" includes cell culture and fermentation processes at all scales (e.g., from microtiter plates to large-scale industrial bioreactors, i.e., from sub-mL scale to over 10,000 L), in all different process modes (e.g., batch culture, fed-batch culture, perfusion culture, continuous culture), in all process control modes (e.g., uncontrolled systems, fully automated systems, and controlled systems with, e.g., control of pH, temperature, oxygen content), and in all types of fermentation systems (e.g., single-use systems, stainless steel systems, glassware systems). According to the present invention, the cell culture is a mammalian cell culture and is a fed-batch culture. In a preferred embodiment, the cell culture is a cell culture with a volume of more than 10 L, more than 1,000 L, more than 5000 L, and more preferably more than 10,000 L.

[0020] As used herein, the term "fed-batch" refers to a cell culture in which cells are continuously or periodically fed with a nutrient-containing feed medium. Feeding can begin immediately after cell culture initiation on day 0, or more typically 1, 2, or 3 days after initiation of the culture. Feeding can follow a pre-established schedule, such as every other day, every two days, or every three days. Alternatively, the culture can be monitored for cell growth, nutrients, or toxic by-products, and feeding can be adjusted accordingly. Generally, the following parameters are determined, often daily, and include viable cell concentration, product concentration (titer), and several metabolites, such as glucose, pH, lactate, osmolality (a measure of salt content), and ammonium (a growth inhibitor that negatively affects growth rate and reduces viable biomass). Higher product titers can be achieved in fed-batch mode compared to batch cultures (cultures without feeding). Typically, the fed-batch culture is stopped at some point, the cells and / or medium are harvested, and the product of interest, e.g., the antibody spesolimab, is isolated and / or purified. The fed-batch process is typically maintained for about 2-3 weeks, e.g., about 10-24 days, about 12 to 21 days, about 12 to 18 days, preferably about 12-16 days.

[0021] As used herein, the term "basal medium" or "basal cell culture medium" refers to a cell culture medium for culturing mammalian cells, as defined below. It refers to a medium in which cells are cultured from the beginning of a cell culture operation and is not used as an additive to another medium, although various components may be added to the medium. The basal medium serves as a base, to which additional additives (or supplements) and / or feed medium may optionally be added during the culture, i.e., during the cell culture operation that produces the cell culture medium. The basal cell culture medium is provided from the beginning of the cell culture process. Typically, the basal cell culture medium provides nutrients, such as a carbon source, amino acids, vitamins, bulk salts (e.g., sodium chloride or potassium chloride), various trace elements (e.g., iron, copper, zinc, and manganese), a pH buffer, lipids, and glucose. Major bulk salts are usually provided only in the basal medium, and should not exceed a final osmolality of approximately 280-350 mOsmol / kg in the cell culture, allowing the cell culture to grow and proliferate under reasonable osmotic stress.

[0022] As used herein, the term "feed" or "feed medium" refers to a nutrient concentrate / concentrated nutrient composition used as a feed in mammalian cell culture. It is provided as a "concentrated feed medium" to minimize dilution of the cell culture. Typically, the feed medium is provided in a vessel at 10-50 ml / L / day, preferably 15-45 ml / L / day, and more preferably 20-35 ml / L / day, based on the culture starting volume (CSV, meaning the starting volume on day 0). This corresponds to a daily addition of about 1-5%, preferably about 1.5-4.5%, and more preferably about 2-3% of the culture starting volume. For cultures using high-density or ultra-high-density inoculation, faster feeding rates, such as 10-50 ml / L / day, 15-45 ml / L / day, or 25-45 ml / L / day, may be beneficial. This corresponds to a daily addition of about 1-5%, preferably about 1.5-4.5%, or about 2.5-4.5% of the culture starting volume. The feeding rate should be understood as the average feeding rate during the feeding period. The feed medium typically has higher concentrations of most, if not all, of the components of the basal cell culture medium. Generally, the feed medium substitutes nutrients consumed during cell culture, such as amino acids and carbohydrates, while salts and buffers are less important and are generally provided with the basal medium. Trace elements, typically provided primarily with the basal medium, may also be present in the feed medium. The feed medium is typically added to the (basal) cell culture medium / fermentation broth in fed-batch mode. The feed medium, added (repeatedly or continuously) to the basal medium, results in the cell culture medium. The feed may be added in different modes, such as continuous addition or bolus addition, or via perfusion-related techniques (chemostat or hybrid perfusion system). Preferably, the feed medium is added once a day, but may be added more frequently, for example twice a day, or less frequently, for example once every two days. More preferably, the feed medium is added continuously. Nutrient addition is generally carried out during the culture (i.e., from day 0 onwards).In contrast to basal media, feed media typically consist of highly concentrated nutrient solutions (e.g., greater than 6x) that provide all components similar to basal media, except for "high osmolality active compounds," such as major bulk salts (e.g., NaCl, KCl, NaHCO3, MgSO4, Ca(NO3)2). Typically, 6x concentrated or higher basal media, without or with reduced bulk salts, maintain good compound solubility and a sufficiently low osmolality (e.g., 270-1500 mOsmol / kg, preferably 310-800 mOsmol / kg) to maintain an osmolality in the cell culture of about 270-550 mOsmol / kg, preferably about 280-450 mOsmol / kg, and more preferably about 280-350 mOsmol / kg. Feed media may be added as a complete feed medium or may contain one or more feed supplements for separate addition to the cell culture. The use of one or more feed supplements may be necessary due to different feeding regimes, such as demand feeding, as is often practiced with periodic feeding and glucose addition (which is therefore also provided as a separate feed). The use of one or more feed supplements may also be necessary due to low solubility of certain compounds, solubility of certain compounds at different pHs, and / or interactions of compounds at high concentrations in the feed medium.

[0023] The term "feed supplement" as used herein refers to a concentrate of nutrients that can be added to a feed medium before use or can be added separately from the feed medium to the basal medium and / or cell culture medium. Thus, a compound can be provided with the feed medium or feed supplement, or a compound can be provided with the feed medium and feed supplement. For example, cysteine ​​can be added with the feed medium and feed supplement in a two-feed strategy. As a feed medium, a "feed supplement" is provided as a concentrate to avoid dilution of the cell culture.

[0024] The cell culture medium, i.e., both the basal medium and / or the feed medium, is preferably serum-free and chemically defined, or chemically defined and protein-free. As used herein, "serum-free medium" refers to a cell culture medium for in vitro cell culture that does not contain serum of animal origin. This is preferred because serum may contain contaminants from the animal, such as viruses, and serum has an undefined chemical composition that varies from batch to batch. The basal medium and feed medium described in this invention are serum-free.

[0025] As used herein, the term "chemically defined medium" refers to a cell culture medium suitable for in vitro cell culture, in which all components are known. More specifically, it does not contain any supplements, such as animal serum or plant, yeast, or animal hydrolysates. It may contain hydrolysates only if all components have been analyzed, their exact composition is known, and they can be reproducibly prepared. The basal medium and feed medium according to the present invention are preferably chemically defined. Chemically defined media may further contain recombinant proteins, such as recombinant growth factors, in particular insulin or insulin-like growth factors (IGFs).

[0026] As used herein, "protein-free medium" refers to a cell culture medium for in vitro cell culture that does not contain any protein, except for proteins produced by the cells to be cultured, where protein refers to a polypeptide of any length, but excludes single amino acids, dipeptides, or tripeptides. Specifically, growth factors, such as insulin and insulin-like growth factors (IGFs), are not present in the medium. Preferably, the basal medium and feed medium according to the present invention are chemically defined and protein-free.

[0027] As used herein, the term "medium platform" or "media platform" consists of a basal cell culture medium (which is provided from the beginning of the cell culture process) and a feed medium (which is added to the basal cell culture medium during cultivation). Optionally, further additives, such as glucose, may be added during the cell culture process. The feed medium may be supplied in any kind of fed-batch process mode (e.g., continuously with varying feed rates or as a bolus feed addition).

[0028] The terms "vitality" and "viability" are used synonymously and refer to the percentage of living cells in a cell culture as determined by methods known in the art, such as the trypan blue exclusion method using a Cedex instrument (Roche Diagnostics, Mannheim) based on automated microscopic cell counting. However, there are also many other methods for determining viability, such as fluorometric (e.g., propidium iodide-based), calorimetric, or enzymatic methods used to reflect the energy metabolism of living cells, such as methods using LDH lactate dehydrogenase or certain tetrazolium salts, such as Alamar Blue, MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide), or TTC (tetrazolium chloride).

[0029] The terms "polypeptide" or "protein" or "product" or "protein product" or "amino acid residue sequence" are used synonymously. These terms refer to polymers of amino acids of any length, preferably, in the context of the present invention, a monoclonal antibody, and even more preferably, the monoclonal antibody spesolimab. These terms also include proteins or antibodies that have been post-translationally modified through reactions that include, but are not limited to, glycosylation, glycation, acetylation, phosphorylation, oxidation, amidation, or protein processing.

[0030] As used herein, the terms "encode" and "encoding" broadly refer to any process that uses information within a polymeric macromolecule to direct the production of a second molecule that differs from a first molecule. The second molecule may have a chemical structure that differs from the chemical nature of the first molecule. For example, in some embodiments, the term "encode" refers to a semi-conservative DNA replication process in which one strand of a double-stranded DNA molecule is used as a template to encode a complementary sister strand that is newly synthesized by a DNA-dependent DNA polymerase. In other embodiments, a DNA molecule may encode an RNA molecule (e.g., by a transcription process using a DNA-dependent RNA polymerase enzyme). An RNA molecule may also encode a polypeptide, as in the process of translation. When used to describe the translation process, the term "encode" also extends to triplet codons that encode amino acids. In some embodiments, an RNA molecule may encode a DNA molecule, for example, by a reverse transcription process incorporating an RNA-dependent DNA polymerase. In other embodiments, a DNA molecule may encode a polypeptide, and "encode" as used herein in this context is understood to encompass both the transcription and translation processes. In the context of the present invention, the term "nucleic acid encoding the antibody spesolimab" refers to a DNA molecule or sequence encoding a polypeptide having the amino acid sequence of the antibody spesolimab, i.e., the heavy and light chains. Preferably, the nucleic acid encoding the antibody spesolimab is stably integrated into the genome of the CHO cell.

[0031] As used herein, the term "spesolimab" refers to a humanized monoclonal IgG1 anti-IL-36R antibody having the INN name spesolimab, registered under CAS Registry Number 2097104-58-8. Spesolimab has the following heavy and light chain amino acid sequences:

[0032] Heavy chain (HC) amino acid sequence: [ka]

[0033] Light chain (LC) amino acid sequence: [ka]

[0034] Heavy chain variable (VH) amino acid sequence: [ka]

[0035] Light chain variable (LC) amino acid sequence: [ka]

[0036] As used herein, the term "antibody spesolimab" preferably refers to the antibody spesolimab produced in CHO cell culture by the method of the present invention, and therefore refers to a large number of antibody molecules with a certain degree of variant heterogeneity. Therefore, those skilled in the art will understand that it refers to a mixture of various spesolimab species, including those with various post-translational modifications, including glycosylation variants, charge variants, and glycosylation variants or species.

[0037] As used herein, the term "seeding" refers to the process of collecting a sample of mammalian cells, such as CHO cells, and placing them in a medium containing nutrients necessary for growth. Typically, mammalian cells are placed in a basal medium for growth or production. This process may also be referred to as inoculation. Mammalian cells may be inoculated into a basal medium at various seeding densities. As referred to herein, the term "normal seeding" refers to a seeding density of approximately 0.7 x 10 6 cells / ml to approximately 1 x 10 6 The term "highly seeded" refers to a standard seeding density of 1 x 10 cells / ml. 6 >4 x 10 cells / ml to approximately 4 x 10 6 The term "ultra-high seeding" refers to a seeding density of 4 x 10 cells / ml. 6 >20 x 10 cells / ml to approximately 20 x 10 6cells / ml or even more, preferably about 6 x 10 6 cells / ml to approximately 15 x 10 6 cells / ml, more preferably 8 x 10 6 cells / ml to approximately 12 x 10 6 refers to a seeding density of 0.7 x 10 cells / ml. According to the present invention, CHO cells are preferably at a density of 0.7 x 10 6 In a specific embodiment, CHO cells are seeded at 0.7 x 10 cells / ml or more. 6 cells / ml to approximately 2 x 10 6 cells / ml, preferably 0.7 x 10 6 cells / ml to approximately 1.5 x 10 6 cells / ml, more preferably 0.8 x 10 6 cells / ml to approximately 1.5 x 10 6 cells / ml, and even more preferably 0.9 x 10 6 cells / ml to approximately 1.3 x 10 6 Cells are seeded at 100 cells / ml.

[0038] Iron is an essential component in mammalian cell culture media (i) as a trace element and (ii) as a transferrin substitute (e.g., iron as an iron chelate). Transferrin is typically derived from plasma and can be supplied as a lyophilized powder of human transferrin, which is partially saturated with iron. Transferrin is a glycoprotein with homologous N- and C-terminal iron-binding domains and is related to several other iron-binding proteins, including lactoferrin, melanotransferrin, and ovotransferrin. Transferrin is commercially available for use in animal cell culture (e.g., Sigma-Aldrich, CAS No. 11096-37-0). Numerous iron compounds exist that are used as transferrin substitutes in cell culture media. These exist in Form II / III, as various salts, and in hydrated / dehydrated forms. Examples include, but are not limited to, iron(III) phosphate, iron(III) pyrophosphate, iron(III) nitrate, iron(II) sulfate, iron(III) chloride, iron(II) lactate, iron(III) citrate, ammonium iron(III) citrate, iron-dextran, choline citrate(III), or sodium ethylenediaminetetraacetic acid iron salt. Preferred iron sources are iron pyrophosphate (Fe(P2O7)3), ammonium iron citrate ((NH4)5[Fe(C6H4O7)2]), iron citrate (C6H5FeO7), iron choline citrate (C 33 H 57 Fe2N3O 24 ), iron nitrate (Fe(NO3)3), iron phosphate (FePO4), iron sulfate (FeSO4), and iron chloride (FeCl3).

[0039] The term "iron choline citrate" as used herein refers to the compound iron choline citrate, which corresponds to CAS number 1336-80-7 and forms an iron choline citrate complex. Synonyms used include, for example, iron cholinate citrate, iron choline citrate, choline citrate, iron(III) choline citrate, iron choline citrate, tricholine citrate, iron choline citrate, 2-hydroxyethyl-trimethyl-ammonium, 2-hydroxypropane-1,2,3-tricarboxylate. This compound can be added as an iron carrier to both basal and feed media. Iron choline citrate with a 2:3:3 molar ratio of iron:choline:citrate (Iron Choline Citrate, CAS No. 1336-80-7, iron complex with a molecular weight Mw = 991.5 g / mol + / - 49.57 g / mol at 5% crystalline water content and an iron content of approximately 10.2-12.4%, with a molar ratio of iron:choline:citrate of 2:3:3, molecular formula C 33 H 57 Fe2N3O 24 ) is available, for example, from Dr. Paul Lohmann GmbH KG. However, other suitable iron choline citrate structures may be used in equimolar amounts based on iron concentration, such as a 1:1:1 ratio of iron:choline:citrate, molecular weight Mw=348.11 g / mol, or a (2):3:3 ratio of (iron):choline:citrate, molecular weight Mw=501.61 g / mol, C 21 H 47 N3O 10 (sum formula not including iron). Iron choline citrate may also be provided as separate compounds containing an iron source (e.g., iron chloride), a choline source (e.g., choline chloride), and a citrate source (e.g., sodium citrate), or a choline source and iron citrate, preferably in the ratios provided for iron choline citrate as described above.

[0040] Cell culture media for culturing mammalian cells contain essential nutrients, including amino acids and carbohydrates, and components such as vitamins, trace elements, salts, bulk salts, and lipids or lipid precursors, preferably in a buffered medium. Growth factors, such as recombinant insulin-like growth factors (IGFs) or recombinant insulin, may also be added to the basal cell culture medium or feed medium. Thus, in certain embodiments, the basal cell culture medium and / or feed medium are chemically defined and protein-free, except for the presence of IGFs or recombinant insulin.

[0041] As used herein, the term "amino acid" refers to the 20 naturally occurring amino acids encoded by the universal genetic code, typically in the L-form (i.e., L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine). Amino acids (e.g., glutamine and / or tyrosine) may be provided as dipeptides, preferably containing L-alanine (L-ala-x) or extensions of L-glycine (L-gly-x), such as glycyl-glutamine and alanyl-glutamine, for increased stability and / or solubility. Additionally, cysteine ​​may also be provided as L-cystine. As used herein, the term "amino acid" includes all different salts thereof, including (but not limited to) L-arginine monohydrochloride, L-asparagine monohydrate, L-cysteine ​​hydrochloride monohydrate, L-cystine dihydrochloride, L-histidine monohydrochloride dihydrate, L-lysine monohydrochloride, and hydroxyl L-proline, L-tyrosine disodium dehydrate. The exact form of the amino acid is not critical to the present invention, as long as characteristics such as solubility, osmolality, stability, purity, etc. are not impaired. Typically and preferably, L-arginine is used as L-arginine x HCl, L-asparagine as L-asparagine x HO, L-cysteine ​​as L-cysteine ​​x HCl x HO, L-cystine as L-cystine x 2HCl, L-histidine as L-histidine x HCl x HO, and L-tyrosine as L-tyrosine x 2Na x 2HO, where each preferred amino acid form may be selected independently, together, or in any combination thereof. Also encompassed are dipeptides containing one or two of the related amino acids. For example, L-glutamine is often added to cell culture media in the form of a dipeptide, such as L-alanyl-L-glutamine, to improve stability and reduce ammonium production during storage or long-term cultivation.

[0042] As used herein, the term "total amino acids in the medium" or "total amino acid content" refers to the sum of "amino acids" as defined above in mM. For dipeptides, each amino acid is counted separately, thus 1 mM alanyl-glutamine is counted as 1 mM L-alanine and 1 mM L-glutamine (molar ratio 1:1). Similarly, for L-cystine, each cysteine ​​is counted separately, thus 1 mM L-cystine is counted as 2 mM L-cysteine ​​(molar ratio 1:2). Typically, the total amino acid content in a concentrated feed medium is about 5-20 times, preferably about 7-15 times, and more preferably about 10 times or more, compared to the basal cell culture medium. The total amino acid content of the basal medium described in the present invention can be about 25-150 mM, preferably about 30-130 mM, more preferably about 35-120 mM, and even more preferably about 40-100 mM. The total amino acid content of the feed medium can be about 100-1000 mM, preferably about 200-900 mM, more preferably about 300-800 mM, and even more preferably about 400-700 mM. Other amino acids not directly encoded by the universal genetic code, such as L-ornithine, hydroxyl L-proline, or their metabolites, e.g., taurine, may also be present in the basal cell culture medium or the feed medium, but these are not counted in the total amino acid content.

[0043] Non-limiting examples of suitable vitamins include biotin (B7), calcium pantothenate, cyanocobalamin (B12), folic acid, myo-inositol, niacinamide (B3), pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin (B2), and / or thiamine (B1). Non-limiting examples of trace elements include molybdenum, vanadium, copper, nickel, selenite, silicate, and zinc, and exemplary sources of trace elements include ammonium molybdate, ammonium vanadate, copper sulfate, nickel sulfate, sodium selenite, sodium silicate, and zinc sulfate, and / or zinc chloride. Non-limiting examples of lipid precursors include choline chloride, ethanolamine, glycerol, inositol, linoleic acid, fatty acids, phospholipids, or cholesterol-related compounds.

[0044] Furthermore, the salt may be, but is not limited to, calcium chloride, calcium nitrate, magnesium chloride, magnesium sulfate, potassium chloride, and / or sodium chloride. One function of the salt is to adjust the osmolality in the medium. Preferably, the osmolality of a basal cell culture medium does not exceed an optimal range, typically between 280 and 350 mOsmol / kg. Typically, the osmolality of a concentrated feed medium is less than 2000 mOsmol / kg, preferably less than 1500 mOsmol / kg, and more preferably less than 1000 mOsmol / kg. The osmolality of the feed medium may be higher, but upon addition, it should not increase the osmolality in the cell culture beyond an optimal range of 270 to 550 mOsmol / kg, preferably 280 to 450 mOsmol / kg, and more preferably 280 to 350 mOsmol / kg.

[0045] Preferably, the feed medium has a reduced or low salt content. Reduced or low salt content refers, for example, to a total salt concentration of about 100 mM or less, preferably about 50 mM or less (e.g., a feed medium without sodium chloride and with a reduced concentration of potassium chloride). The most important contributors to osmolality are sodium ions, chloride ions, and bicarbonate, as well as glucose and other carbon sources, such as amino acids. Furthermore, in a typical fed-batch process, the feed medium needs to be concentrated to minimize the culture volume during the cultivation period. The size of the bioreactor can actually cause feeding constraints, allowing a total feed volume of only about 35% (30-40%) or about 45% (40-50%) of the starting culture volume.

[0046] Carbohydrates can be, but are not limited to, glucose, mannose, galactose, fructose, sucrose, or glucosamine. These carbohydrates can be added directly to the basal cell culture medium and / or feed medium or added separately to the cell culture. Other energy sources include, but are not limited to, sodium pyruvate.

[0047] Mammalian cells should be cultured at a neutral pH, for example, about pH 6.5 to about pH 7.5, preferably about pH 6.6 to about pH 7.3, and more preferably about pH 7. Therefore, a buffering agent should be added to the basal cell culture medium. In the feed medium, the pH can be slightly outside this range as long as the addition of the feed medium does not cause the pH of the cell culture to fall outside this range, since the feed medium is added as a concentrate. A preferred range for the pH of the feed medium is about 6 to about 8. Suitable buffering agents include, but are not limited to, HEPES, phosphate buffer (e.g., potassium phosphate monobasic and potassium phosphate dibasic and / or sodium phosphate dibasic anhydrate and sodium phosphate monobasic), phenol red, sodium bicarbonate, and / or sodium bicarbonate.

[0048] Generally, the feed medium contains nutrients consumed during cell culture, such as amino acids and carbohydrates, but salts and buffers are less important, and therefore some salts may be omitted entirely from the feed medium.

[0049] The basal cell culture medium and / or feed medium should meet the cell-specific requirements and metabolic needs of mammalian cell cultures over the time course of the cell culture. In other words, it meets (i) the cell-specific needs of the mammalian cells, (ii) in the cell culture system, and (iii) throughout the life cycle of the culture operation (which is approximately 10-20 days). Mammalian cells in culture have different nutritional requirements at different times during the cell culture process.

[0050] Cell culture may further include cell growth, which is required for sequential inoculation in a scale-up procedure. For example, the scale of the culture is increased stepwise from a thawed cell bank (mL scale) to a production scale (greater than 10,000 L). The better the growth in each Nx stage (N stage refers to the final production scale, and Nx refers to the cell growth stage before the final production stage, usually in batch mode), the faster and better the transition to the next stage can occur. Specifically, better cell growth and higher viable cell concentration allow the Nx culture to be performed with fewer manipulations (and therefore more quickly). Better cell growth and higher viable cell concentration also result in improved transition, resulting in overall improved performance. For example, if a particular Nx stage should be inoculated at a specific inoculation cell density and the viable cell concentration is high, a relatively small volume of cell culture needs to be transferred from one stage to the next (the transition of inoculation volume per culture starting volume (CSV) is defined as the split ratio, and is typically 1:5 to 1:20). This simultaneously means that only a reduced volume of "spent" cell culture medium is transferred from one stage to the next, and a maximum volume of "fresh" medium can be added to the next stage (constant total culture volume), which also results in improved overall cell culture performance (e.g., increased product titer) in the final N stages.

[0051] In most cell cultures, a non-ideal nutrient composition for the primary carbon may be determined by overflow metabolism. This means that the primary carbon source, glucose, is inefficiently utilized, thereby causing an increase in organic acids, such as lactic acid. Elevated levels of lactic acid cause a decrease in pH below 6.65, which will negatively affect the buffering capacity of the culture medium and therefore the viability of the culture. For this reason, the carbon dioxide concentration in the culture atmosphere is reduced at the beginning of the exponential growth phase to minimize acid levels in the culture medium.

[0052] The antibody spesolimab is purified from other recombinant proteins, host cell proteins, and contaminants in downstream processing. Samples obtained and / or analyzed at various purification stages are also referred to as in-process control (IPC) samples or process intermediates. Harvesting typically involves centrifugation and / or filtration, for example, to produce harvested cell culture fluid. Further process steps may include affinity chromatography, particularly Protein A column chromatography, to separate the antibody spesolimab from contaminants. Further process steps may include acid treatment to inactivate viruses, preferably followed by clarification of the product pool by depth filtration to remove cellular contaminants such as host cell-derived proteins and DNA. Further process steps may include the following, in this order or in any other order as may be appropriate in a particular case: ion exchange chromatography, particularly anion exchange chromatography, to further remove contaminating cellular components, and / or cation exchange chromatography to remove product-associated contaminants such as aggregates. Additionally, the following process steps may preferably include nanofiltration to further remove viruses, and ultrafiltration and diafiltration to concentrate the recombinant protein and exchange the buffer, respectively.

[0053] A sample containing the antibody spesolimab purified from harvested cell culture fluid (HCCF) after, for example, Protein A column, acid treatment, depth filtration, anion exchange chromatography, and / or cation exchange chromatography may also be referred to as a "purified antibody product pool." The purified antibody product pool may be further purified and may, but need not, be formulated with excipients. Thus, the purified antibody product pool may be identical to the drug substance, but may also be at a different concentration and / or in a different buffer system.

[0054] As used herein, the term "harvested cell culture fluid (HCCF)" refers to a fluid containing a harvested recombinant protein, in this case the antibody spesolimab. Typically, the host cells used for production are engineered to secrete the polypeptide into the cell culture medium, and thus the cell culture supernatant will be collected. However, theoretically, the cells may be lysed prior to collection. Harvesting involves centrifugation and / or filtration to produce a harvested cell culture fluid. Thus, harvested cell culture fluid may also be referred to as clarified harvested cell culture fluid. It does not contain viable cells, and cellular debris and cellular components have been removed. Typically, it refers to a clarified cell culture supernatant, where clarification refers to centrifugation or filtration, preferably by filtration using a 0.1 μm filter, for example. HCCF is, in some embodiments, a clarified HCCF. In further or alternative embodiments, the HCCF contains about 1.8 g / L to about 5 g / L of antibody spesolimab, preferably about 2.0 g / L to about 5.0 g / L of antibody spesolimab, and more preferably about 2.5 g / L to about 4.5 g / L of antibody spesolimab. The methods described herein are large-scale purification methods for HCCF containing more than 20 kg, or even more than 30 kg of spesolimab, and / or for HCCF from 2,000 L or larger fermentors, preferably 12,000 L or larger fermentors.

[0055] The term "product pool" as used herein refers to a solution containing the product, i.e., the antibody spesolimab, at the end of a process step. Therefore, the term "product pool" is used synonymously with the term "antibody-containing product pool." This may be the eluate, the flow-through, or the filtrate, as long as it contains the major fraction of the product. Therefore, the term "product pool sample" refers to a sample of a solution containing the antibody spesolimab, particularly after one of the method steps described in the methods of the present invention.

[0056] The antibody spesolimab is produced in a large scale, i.e., at a scale of 12,000 L or more, in a CHO cell line. The titer of HCCF is about 1.8 g / L to about 5 g / L. Thus, the process described in the present invention provides starting material (HCCF) for purification containing more than 20 kg, or even more than 30 kg, of spesolimab.

[0057] As used herein, the term "contaminating" or "contaminant" refers to the presence of undesired and / or unintended substances, such as host cell proteins, host cell DNA, and / or at least one protein or substance with hydrolytic activity.

[0058] The term "drug substance (DS)" refers to an active pharmaceutical ingredient (API) formulated with excipients. The API has a therapeutic effect in the body, as opposed to the excipients, which aid in the delivery of the API. In the case of biotherapeutics, the API formulated with excipients typically refers to the API in the final formulation buffer at a concentration of at least the highest concentration used in the final dosage form, also referred to as the drug product.

[0059] The term "drug product," also abbreviated as DP, as used herein refers to the final marketed dosage form of a drug substance, such as a tablet or capsule, or, in the case of biological products, typically an injectable solution in a suitable container such as a vial or syringe. The drug product may also be in a lyophilized dosage form. Spesolimab is provided as an aqueous formulation at 60 mg / mL in a glass vial or at 150 mg / mL in a glass syringe.

[0060] Antibodies typically have oligosaccharides attached to Asn297 of the CH2 domain of the immunoglobulin heavy chain (IgG). Most of these oligosaccharides have a bichain structure, which means that they have a core structure (Man(α1-4)GlcNAc(β1-4)GlcNAc→Asn) with an optional Fuc(α1-6) linkage to the terminal GlcNAc residue, and two outer arms (Gal(β1-4)GlcNAc(β1-2)Man(α1-6)→Man and Gal(β1-4)GlcNAc(β1-2)Man(α1-3)→Man; the terminal galactose (Gal) residue is optional) (Man = mannose, GlcNAc = N-acetylglucose, Gal = galactose, Fuc = fucose) connected to the terminal mannose of the core structure. The terminal galactose on each outer arm is optional, resulting in the G(0), G(1), and G(2) isoforms, where the G(2) isoform has terminal galactose residues on each of the outer arms of the oligosaccharide structure, the G(1) isoform has terminal galactose residues only on either the (α1-6)- or (α1-3)-linked outer arms, and the G(0) isoform has no galactose residues on either outer arm.

[0061] The terms "Man5 structure" or "mannose pentasaccharide structure" are used synonymously herein and refer to an oligomannose structure that comprises or consists of five mannose residues and two N-acetylglucose core residues, forming a three-chain structure, linked to an Asn residue of a polypeptide.

[0062] The introduction of sugar structures into polypeptides, such as antibodies, is a post-translational modification. Due to incomplete glycosylation, every cell expresses a polypeptide, such as an antibody, with a glycosylation pattern or profile containing different sugar structures. The total or individual sugar structures are referred to as the glycosylation pattern or profile. The oligosaccharides of purified spesolimab or Protein A pools can be determined by labeling the released oligosaccharides with 2-aminopyridine and analyzing them using hydrophilic interaction chromatography (HILIC-HPLC), preferably hydrophilic interaction ultra-high performance liquid chromatography (HILIC-UPLC). The chromatographic profile shows six major peaks, among which peak 3 is the Man5 peak (Figure 5), also referred to as (oligomap peak 3). As used herein, the term "Man5 structure %" refers to the relative peak area % (peak 3) to the total peak area.

[0063] As used herein, the term "about" refers to a 10% variation of the specified numerical value, for example, about 50% has a variation of 45-55%.

[0064] The present invention provides a method for producing CHO cells comprising a nucleic acid encoding the antibody spesolimab using fed-batch culture in a serum-free cell culture medium, the method comprising: (a) culturing the cells in the culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, the method comprising: (i) inoculating the cells in the culture medium; and (ii) feeding the cells in the cell culture with a feed medium, wherein copper(II) (Cu 2+ (b) collecting a cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. Preferably, Cu is added to the culture medium at 0.35 to 1.2 μM and iron at 1,500 μM or more before and / or within two days after seeding the cells in step (i); (b) collecting a cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. 2+ and iron are added to the culture medium before and / or within one day after seeding in step (i), more preferably Cu2+ and iron are added to the culture medium prior to seeding or at the time of inoculation (i.e., at the time of cell seeding) in step (i). In one embodiment, Cu 2+ and / or iron is added to the culture medium as one or more bolus additions or continuously. 2+ and / or iron with the basal medium. Thus, in certain embodiments, step (i) comprises providing Cu 2+ The method includes a step of seeding the cells in a basal medium containing 0.35 to 1.2 μM of ATP and 1,500 μM or more of iron. The culture medium in step (i) may also be referred to as a basal medium.

[0065] Copper is added to the culture medium at 0.35-1.2 μM prior to seeding the cells in step (i) and / or within 2 days or 1 day after seeding. Those skilled in the art will appreciate that the means by which copper is added to the culture medium is irrelevant, and that addition prior to seeding the cells in step (i) includes the copper being present in or part of the culture medium described in step (i) and / or the addition of a copper-containing supplement to the ready-to-mix culture medium prior to seeding in step (i). In a preferred embodiment, Cu 2+ Copper(II) is added to the culture medium at 0.4-1.0 μM, more preferably 0.5-0.8 μM, prior to seeding the cells in step (i) and / or within two days, preferably one day, after seeding. Copper(II) is typically provided as a salt or hydrate thereof, a suitable salt, including, but not limited to, CuSO or CuCl. Preferably, copper is provided as CuSO.

[0066] Iron is added to the culture medium at 1,500 μM or greater before seeding the cells in step (i) and / or within two or one days after seeding. Those skilled in the art will appreciate that the means by which iron is added to the culture medium is irrelevant; addition before seeding the cells in step (i) includes iron being present in or part of the culture medium described in step (i) and / or adding an iron-containing supplement to the ready-mix culture medium prior to seeding in step (i). In preferred embodiments, iron is added at 2,000 μM or greater, more preferably 2,500 μM or 3,000 μM or greater. Iron may be added up to 10,000 μM, preferably 5,000 μM, although lower concentrations are typically used, primarily due to solubility and precipitation of medium components in the presence of high iron concentrations, but also due to potential toxicity issues with very high concentrations of iron. Thus, in certain embodiments, the iron concentration is 1,500 μM to 10,000 μM, 2,000 μM to 10,000 μM, 2,500 μM to 10,000 μM, or 3,000 μM to 10,000 μM, preferably 1,500 μM to 5,000 μM, 2,000 μM to 5,000 μM, 2,500 μM to 5,000 μM, or 3,000 μM to 5,000 μM. Iron is typically provided as a salt and / or chelate from a suitable iron source, including, but not limited to, iron pyrophosphate (Fe(P2O7)3), ferrous ammonium citrate ((NH4)5[Fe(C6H4O7)2]), iron citrate (C6H5FeO7), iron choline citrate, iron(III) nitrate, iron phosphate, iron sulfate, and iron chloride. Preferred iron sources are iron citrate, iron choline citrate, and iron chloride. Further suitable iron sources are known in the art, and those skilled in the art will be aware that the addition of certain chelating agents, such as citrate and / or choline, can increase iron uptake by cells, and that the chelating agents may be added separately to the iron source.

[0067] In the context of the present invention, it is important that copper and iron are added (and / or present) at specific concentrations at the beginning of the growth phase, i.e., before inoculation and / or within 2 or 1 days after inoculation, whereas the concentrations of copper and iron during the culture, especially during the production phase, are less important. In particular, due to the synergistic effect of copper reduction and iron increase on PBG and Man5 content, further reductions can be achieved, or the same reductions can be achieved at slightly higher copper concentrations, thereby avoiding the negative effects of too low copper concentrations.

[0068] In a particular embodiment, the nucleic acid encoding the antibody spesolimab is stably integrated into the CHO genome. Methods for transfecting or transducing CHO cells with antibodies and selecting antibody-producing clones are known in the art.

[0069] The method according to the present invention comprises the step of feeding the cells with a feed medium. In a particular embodiment, the feed medium is added starting from day 0 to day 3 of the culture, preferably starting from day 1 to day 3, more preferably starting from day 1 to day 2, and even more preferably starting on day 1. Although not required, the feed medium may further contain Cu 2+ For example, the feed medium may contain less than 15 nM, less than 12 nM, less than 10 nM Cu once a day, preferably less than 7 nM Cu once a day, more preferably less than 6 nM Cu once a day. 2+ The feed medium may further contain iron ions. For example, the feed medium may be supplemented with up to 100 μM iron once a day, preferably up to 50 μM, 40 μM, 30 μM, or 20 μM iron once a day. The feed medium may further contain Cu at a specific concentration. 2+and iron ions. The feed medium used in the methods of the present invention is to be added to cells cultured in a basal cell culture medium, wherein (a) the feed medium is added at about 10-50 ml / L / day, preferably 20-35 ml / L / day, based on the culture starting volume, (b) the feed medium is added starting on day 0, 1, 2, or 3, and / or (c) the feed medium is added continuously or as a bolus several times a day, twice a day, once a day, once every two days, or once every three days.

[0070] In the method of the present invention, an increased iron concentration and / or a decreased copper concentration in the culture medium results in the production of the antibody spesolimab with a reduced BPG%. The term "reduced BPG%" in the context of the present invention should be understood as a comparison with the antibody spesolimab produced by the same method using a lower iron concentration and / or a higher copper concentration, either of which is within the range described in the method of the present invention, or a comparison with the same method using an iron concentration lower than the specified range and / or a copper concentration higher than the specified claimed range. In a specific preferred embodiment, the antibody spesolimab produced by the method of the present invention has a BPG% of 7.5% or less, preferably 7% or less, more preferably 6.5% or less, and even more preferably 6% or less. According to the present invention, gradually increasing the iron concentration reduces the BPG% and can therefore compensate for a slightly higher copper concentration that cannot be further reduced due to other factors.

[0071] Accordingly, the present invention also provides a method for producing an antibody, spesolimab, in a cell culture, comprising the steps of: (a) culturing cells in a culture medium under conditions that allow the production of the antibody spesolimab in the cell culture, the method comprising the steps of: (i) seeding cells in a culture medium; and (ii) feeding the cells in the cell culture with a feed medium, wherein BPG% is the Cu content of the cell culture medium provided in the culture medium prior to seeding the cells in step (i) and / or within two days after seeding. 2+(b) reducing the BPG% of the antibody spesolimab by decreasing the copper concentration and increasing the iron concentration, wherein the copper concentration provided in the culture medium is 0.35 to 1.2 μM and the iron concentration provided in the culture medium is 1500 μM or more; (b) collecting a cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. Preferably, the BPG% of the antibody spesolimab is reduced by increasing the copper concentration and increasing the iron concentration, wherein the copper concentration provided in the culture medium is 0.35 to 1.2 μM and the iron concentration provided in the culture medium is 1500 μM or more. 2+ and iron are added to the culture medium before and / or within one day after the inoculation in step (i), more preferably Cu 2+ and iron are added to the culture medium prior to or at the time of inoculation (i.e., at the time of cell inoculation) in step (i). In one embodiment, Cu 2+ and / or iron is added to the culture medium as one or more bolus additions or continuously. The one or more bolus additions may include Cu, 2+ and / or iron with the basal medium. Thus, in certain embodiments, step (i) comprises providing Cu at 0.35 to 1.2 μM. 2+ and seeding the cells in a basal medium containing 1500 μM or more iron. The term "reduced BPG%" in the context of the present invention should be understood as a comparison with antibody spesolimab produced by the same method using a lower iron concentration and / or a higher copper concentration, either of which is within the range described in the method of the present invention, or a comparison with the same method using an iron concentration lower than the specified range and / or a copper concentration higher than the specified claimed range. In a specific preferred embodiment, the antibody spesolimab produced by the method of the present invention has a BPG of 7.5% or less, preferably 7% or less, more preferably 6.5% or less, and even more preferably 6% or less.

[0072] As used herein, the term "basic peak group (BPG) %" refers to the relative peak area % of the total peak area (relative % of the total antibody) in an HPLC chromatogram of the spesolimab profile, as determined by cation exchange chromatography (CEX). The term "BPG" may also refer to the basic species or basic variants of the antibody spesolimab.

[0073] BPG can be determined using cation exchange chromatography (CEX HPLC). More specifically, the basic species herein corresponds to a peak eluting after the main peak in the HPLC chromatogram of the profile of spesolimab. In one embodiment, the HPLC chromatogram is generated using a first mobile phase of 10 mM MOPS (3-(N-morpholino)propanesulfonic acid, 4-morpholinepropanesulfonic acid), pH 7.6, and a second mobile phase of 10 mM MOPS, 100 mM potassium chloride, pH 7.6, wherein the HPLC chromatogram is generated using detection at 280 nm.

[0074] The level of BPG is primarily determined by upstream processes and is largely unaffected by downstream processes. Thus, the % BPG in the HCCF of spesolimab is similar to the % BPG in the drug substance. This is in contrast to, for example, high molecular weight species (HMW) or low molecular weight species (LMW), which are typically reduced in downstream processes.

[0075] The method according to the present invention may further comprise the step of adopting a seeding density. An increased seeding density results in the production of the antibody spesolimab with a reduced BPG% and / or Man5 structure%. Thus, in a particular embodiment, the seeding density in step (a) is 0.7×10 6 cells / ml or more, preferably 0.7 x 10 6 cells / ml to 1.5 x 10 6 cells / ml, more preferably 0.8 x 10 6 cells / ml to 1.5 x 10 6cells / ml, and even more preferably 0.9 x 10 6 cells / ml to 1.3 x 10 6 The term "reduced BPG% and / or Man5 structure%" in the context of the present invention should be understood as being compared to the same method for producing the antibody spesolimab using a lower seeding density, either of which is within the range described in the method of the present invention, or compared to the same method using a lower seeding density within the specified range.

[0076] The methods described herein may further comprise the step of adjusting the culture temperature. In certain embodiments, the cells are cultured at 36.0°C to 37.5°C under conditions that allow the production of the antibody spesolimab, including the step of feeding the cells with a feed medium. Preferably, the culture temperature is within the range of 36.0°C to 37.3°C, or 36.5°C to 37.0°C. The methods may also comprise the step of adjusting the dissolved oxygen (DO) concentration. In certain embodiments, the dissolved oxygen (DO) concentration in the culture is maintained within the range of 30 to 60%, preferably within the range of 40 to 55%, and more preferably within the range of 40 to 45%. Elevated culture temperature and / or reduced dissolved oxygen result in the production of the antibody spesolimab with a reduced BPG% and / or reduced Man5 structure%. The term "reduced BPG% and / or reduced Man5 structure%" in this context should be understood as a comparison with the antibody spesolimab produced by the same method using a lower culture temperature and / or a higher dissolved oxygen concentration, either within the claimed range described in the method of the present invention, or compared with the same method using a culture temperature lower than the specified range or a dissolved oxygen concentration higher than the specified range. In certain embodiments, the antibody spesolimab has less than 5% Man5 structures, preferably less than 4% Man5 structures, more preferably less than 3% Man5 structures, and / or 7.5% or less BPG, preferably 7% or less BPG, more preferably 6.5% or less BPG, and even more preferably 6% or less BPG. The culture temperature may also depend on the cell line used. For example, CHO-K1 cells tend to have a lower culture temperature optimum (e.g., 33-36°C) compared to CHO-DG44 cells, with the optimum culture temperature being approximately 36.0-37.5°C, preferably 36.5-37°C.

[0077] As used herein, the term "% Man5 structure" refers to the % mannose-5 peak area (peak 3) relative to the total or combined area of ​​all glycosylated peaks as determined by hydrophilic interaction chromatography (HILIC-HPLC), preferably hydrophilic interaction ultra-high performance liquid chromatography (HILIC-UPLC) (Figure 5). The Man5 structure is represented by the % peak area of ​​peak 3.

[0078] High mannose structures, particularly Man5 structures, can be determined using hydrophilic interaction chromatography (HILIC), preferably hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC). In one embodiment, the Man5 fraction is determined after enzymatic oligosaccharide release using N-glycosidase F (PNGaseF) and 2-aminobenzamide (2-AB) labeling using HILIC-UPLC.

[0079] Similar to BPG levels, the level of high mannose, particularly the level of Man5 structures, is primarily determined by upstream processes and only slightly affected by downstream processes. Thus, the % Man5 structures in the HCCF of spesolimab is similar to the % Man5 structures in the drug substance. This is in contrast to, for example, high molecular weight species (HMW) or low molecular weight species (LMW), which are typically reduced in downstream processes.

[0080] The mammalian cells used in the methods of the present invention are Chinese hamster ovary (CHO) cells, such as CHO-K1 cells, CHO-DG44 cells, DuxB11 cells, or CHO GS-deficient cells, preferably CHO-DG44 cells or CHO-K1 cells. To enable efficient cell line development processes, CHO cells are metabolically engineered, such as by glutamine synthase (GS) knockout and / or dihydrofolate reductase (DHFR) knockout, to facilitate selection with methionine sulfoximine (MSX) or methotrexate, respectively. CHO cells used in the methods of the present invention include CHO-K1 cells, CHO-DXB11 cells (also referred to as CHO-DUKX cells or DuxB11 cells), CHO-DUKX B1 cells, CHO-S cells, CHO-DG44 cells, and CHO glutamine synthase (GS)-deficient cells, or derivatives / progeny of any of these cell lines. Preferred are the CHO-DG44 cell line, the CHO-DUKX cell line, the CHO-K1 cell line, the CHO-S cell line, and the CHO-DG44 GS-deficient cell line, with CHO-DG44 cells and CHO-K1 cells being particularly preferred, and CHO-DG44 cells being even more preferred. According to the present invention, CHO cells are cultured as suspension cells. Non-limiting examples of mammalian cells that can be used with the medium of the present invention are summarized in Table 1.

[0081] [Table 1]

[0082] The CHO producer cells are preferentially cultured under conditions that allow cell growth. Furthermore, the CHO producer cells are preferentially cultured under conditions that favor expression of the antibody spesolimab. The antibody spesolimab is then isolated from the cells and / or cell culture supernatant. Preferably, the antibody spesolimab is recovered from the culture medium as a secreted polypeptide.

[0083] The antibody spesolimab is purified from HCCF, other recombinant proteins, host cell proteins, and contaminants in downstream processing. Samples obtained and / or analyzed at various purification steps are also referred to as in-process control (IPC) samples and / or process intermediates. Harvesting typically involves centrifugation and / or filtration, for example, to produce harvested cell culture fluid. Harvested cell culture fluid may therefore also be referred to as clarified harvested cell culture fluid. It contains no viable cells, and cell debris and most cellular components have been removed. Clarified typically refers to centrifugation or filtration, preferably filtration. Further process steps may include affinity chromatography, particularly protein A column chromatography, to separate the antibody spesolimab from contaminants. Further process steps may include clarification of the product pool by depth filtration, preferably acid treatment to inactivate viruses, to remove cellular contaminants such as host cell-derived proteins and DNA. Further process steps may include the following, in this order or in any other order as may be appropriate in the individual case: ion exchange chromatography, in particular anion exchange chromatography, to further remove contaminating cellular components, and / or cation exchange chromatography to remove product-associated contaminants such as aggregates. Furthermore, preferably the following process steps may include nanofiltration to further remove viruses, and ultrafiltration and diafiltration to concentrate the recombinant protein and exchange the buffer, respectively.

[0084] A sample containing the antibody spesolimab purified from HCCF after, for example, a Protein A column, acid treatment, depth filtration, anion exchange chromatography, and / or cation exchange chromatography may also be referred to as a "purified antibody product pool." The purified antibody product pool may be further purified and may, but need not, be formulated with excipients. Thus, the purified antibody product pool may be identical to the drug substance but at a different concentration and / or in a different buffer system.

[0085] In another embodiment, (a)(i) cells are cultured in culture medium at a concentration of 0.7 x 10 6 (ii) culturing the CHO cells comprising the nucleic acid encoding the antibody spesolimab using fed-batch culture in serum-free cell culture medium, the CHO cells comprising the nucleic acid encoding the antibody spesolimab being cultured in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, the cell culture comprising the steps of: (i) seeding the CHO cells at a cell density of at least 10 ... 2+ A method for producing the antibody spesolimab in cell culture is provided, comprising the steps of: (i) adding 0.35 to 1.2 μM of ATP and 1500 μM or more of iron to the culture medium prior to and / or within two days after seeding the cells in step (i); (b) collecting a cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. Increased seeding density results in the production of the antibody spesolimab with a reduced BPG% and / or Man5 structure%. In a specific embodiment, the seeding density in step (a) is 0.7×10 6 cells / ml to 1.5 x 10 6 cells / ml, more preferably 0.8 x 10 6 cells / ml to 1.5 x 10 6 cells / ml, and even more preferably 0.9 x 10 6 cells / ml to 1.3 x 10 6The average cell density is 1000 cells / ml. The term "reduced BPG% and / or Man5 structure%" in this context should be understood as a comparison with the production of the antibody spesolimab by the same method using a lower seeding density, optionally within the range described in the method of the present invention, or compared with the same method using a seeding density lower than the specified range, and optionally using a lower iron concentration and / or a higher copper concentration, optionally within the range described in the method of the present invention, or compared with the same method using an iron concentration lower than the specified range and / or a copper concentration above the specified claimed range. The embodiments and modifications or disclosures regarding the previous aspect equally apply to the method described in this aspect. Gradually increasing the culture temperature can further reduce BPG and / or Man5 structure. The exact culture temperature may depend on the CHO cells used and is preferably 36.0°C to 37.5°C, but may be 33°C to 36°C for other CHO cells. Furthermore, gradual reduction of dissolved oxygen (while maintaining dissolved oxygen at 30-60%) can further reduce BPG and / or Man5 structures. Thus, in certain embodiments, elevated culture temperature and / or reduced dissolved oxygen results in the production of antibody spesolimab with reduced BPG% and / or Man5 structure%. In a preferred embodiment, the produced antibody spesolimab has less than 5% Man5 structures, preferably less than 4% Man5 structures, more preferably less than 3% Man5 structures, and / or 7.5% or less BPG, preferably 6.5% or less BPG.

[0086] In another aspect, the present invention provides a composition comprising the antibody spesolimab, wherein the antibody spesolimab is obtained by a method described in the present invention. In a specific embodiment, the composition comprises the antibody spesolimab having (a) 7.5% or less BPG, preferably 7% or less BPG, more preferably 6.5% or less BPG, even more preferably 6% or less BPG, less than 7% BPG, preferably less than 6% BPG; and / or (b) less than 5% Man5 structures, preferably less than 4% Man5 structures, more preferably less than 3% Man5 structures, preferably less than 2% Man5 structures. The composition may be a harvested cell culture fluid (HCCF), an affinity capture tool, a drug substance, or a drug product. Preferably, the composition is a drug substance or a drug product.

[0087] In yet another aspect, there is provided a composition comprising the antibody spesolimab, having (a) 7.5% or less BPG, preferably 7% or less BPG, more preferably 6.5% or less BPG, even more preferably 6% or less BPG; and / or (b) less than 5% Man5 structures, preferably less than 4% Man5 structures, more preferably less than 3% Man5 structures, preferably less than 2% Man5 structures. The composition may be a harvested cell culture fluid (HCCF), an affinity capture tool, a drug substance, or a drug product. Preferably, the composition is a drug substance or a drug product.

[0088] The antibody spesolimab in the composition according to the present invention can further be characterized by low overall glycosylation, and does not contain any glycosylation at critical lysines, i.e., lysines close to the antibody's CDRs (complementarity determining regions), e.g., within 3 amino acids of the CDRs. Thus, in one embodiment, the antibody spesolimab has 6% or less heavy chain (HC) lysine glycosylation variants, preferably 3% or less HC lysine glycosylation variants, and / or heavy chain (HC) lysines K38 and K67 are not glycosylated and glycosylation at K23 is 0.3% or less. In a particular embodiment, the antibody spesolimab further has 2% or less light chain (LC) lysine glycosylation variants, preferably 1% or less light chain (LC) lysine glycosylation variants. The resulting light chains and deglycosylated heavy chains were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and analyzed online by ESI (electrospray ionization) Q-TOF (quadrupole time-of-flight) MS (mass spectrometry) (Xevo G2 Q-TOF). Protein subunits and corresponding glucose adducts (1M glucose adduct = 162 Da) were analyzed, and the acquired data were decoded using the MaxEnt™ algorithm.

[0089] As used herein, the term "% heavy chain lysine glycosylation variants" refers to the ratio of glycosylated heavy chains (heavy chains + glucose) to total heavy chains (glycosylated heavy chains and non-glycosylated heavy chains). As used herein, the term "% light chain lysine glycosylation variants" refers to the ratio of glycosylated light chains (light chains + glucose) to total light chains (glycosylated light chains and non-glycosylated light chains). Lysine glycosylation variants and non-glycosylated variants of light and / or heavy chains can be determined by separation of reduced and deglycosylated (e.g., N-glycosidase F-treated) light and heavy chains by reverse-phase high-performance liquid chromatography (RP-HPLC) and analysis by ESI Q-TOF MS.

[0090] Single lysine glycation can be analyzed by reversed-phase liquid chromatography (LC-MS) and electrospray ionization-mass spectrometry (ESI-MS) in chymotrypsin-digested peptides after denaturation of spesolimab and alkylation with iodoacetic acid. The relative amount of glycated peptides is quantified based on extracted ion chromatograms of wild-type peptides and peptides with a glucose adduct (+162 Da).

[0091] Also provided herein are compositions comprising the antibody spesolimab having 6% or less heavy chain lysine glycosylation variants, preferably 3% or less heavy chain lysine glycosylation variants, and / or wherein lysines K38 (heavy chain) and K67 (heavy chain) are not glycosylated and glycosylation at K23 (heavy chain) is 0.3% or less. In certain embodiments, the antibody spesolimab further has 2% or less light chain (LC) lysine glycosylation variants, preferably 1% or less light chain (LC) lysine glycosylation variants.

[0092] Also provided herein are compositions comprising the antibody spesolimab, which comprise the APG subfractions AP4 and AP3, less than 1% and less than 4%, in particular less than 1% AP4 and less than 1% AP3b fractions.

[0093] In view of the above, it will be understood that the present invention also encompasses the following items: 1. (a)(i) seeding cells in a culture medium; and (ii) culturing the cells in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, including feeding the cells in the cell culture with a feed medium. Culturing CHO cells comprising a nucleic acid encoding the antibody spesolimab using fed-batch culture in a serum-free cell culture medium, comprising: where Cu 2+ is added to the culture medium at 0.35 to 1.2 μM and iron at 1500 μM or more before seeding the cells in step (i) and / or within 2 days after seeding; (b) collecting the cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. 1. A method for producing the antibody spesolimab in cell culture, comprising:

[0094] 2. (a)(i) seeding the cells in a culture medium; and (ii) culturing the cells in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, including feeding the cells in the cell culture with a feed medium, where BPG% is the Cu content of the culture medium provided prior to and / or within two days after seeding the cells in step (i). 2+ by decreasing the copper concentration and increasing the iron concentration, preferably wherein the copper concentration provided in the culture medium is 0.35-1.2 μM and the iron concentration provided in the culture medium is 1500 μM or more; (b) collecting the cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. A method for reducing the BPG% of the antibody spesolimab, comprising:

[0095] 3. Cu 2+ and iron is added to the culture medium before the inoculation in step (i) and / or within one day after the inoculation.

[0096] 4. Cu 2+ and iron is added to the culture medium as one or more bolus additions or continuously.

[0097] 5. The method of any one of the preceding items, wherein the nucleic acid encoding the antibody spesolimab is stably integrated into the CHO genome.

[0098] 6. The method of any one of the preceding items, wherein the feed medium is added starting from day 0 to day 3 of the culture.

[0099] 7. Optionally, the feed medium contains less than 15 nM Cu once daily.2+ and / or up to 100 μM iron is added.

[0100] 8. The method of any one of the preceding items, wherein increased iron concentration and / or decreased copper concentration in the culture medium results in production of the antibody spesolimab with a decreased BPG%.

[0101] 9. The method of any one of the preceding items, wherein the antibody spesolimab has a BPG of 7.5% or less, preferably 7% or less, more preferably 6.5% or less, and even more preferably 6% or less.

[0102] 10. The method of item 8, wherein BPG is determined using cation exchange chromatography (CEX HPLC).

[0103] 11. The seeding density in step (a) is 0.7 x 10 6 cells / ml or more, preferably 0.7 x 10 6 cells / ml to 1.5 x 10 6 cells / ml, more preferably 0.8 x 10 6 cells / ml to 1.5 x 10 6 cells / ml, and even more preferably 0.9 x 10 6 cells / ml to 1.3 x 10 6 10. The method of any one of the preceding items, wherein the total number of cells / ml is 100.

[0104] 12. (a)(i) Cells in culture medium at 0.7 × 10 6 seeding at a cell density of 100 cells / ml; (ii) culturing the cells in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, including feeding the cells in the cell culture with a feed medium. Culturing CHO cells comprising a nucleic acid encoding the antibody spesolimab using fed-batch culture in a serum-free cell culture medium, comprising: Here, in some cases, Cu 2+is added to the culture medium at 0.35 to 1.2 μM and iron at 1500 μM or more before seeding the cells in step (i) and / or within 2 days after seeding; (b) collecting the cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant. 1. A method for producing the antibody spesolimab in cell culture, comprising:

[0105] 13. The method of any one of the preceding items, wherein increased seeding density results in production of the antibody spesolimab with reduced BPG% and / or Man5 structure%.

[0106] 14. The method of any one of the preceding items, wherein the cells are cultured at 36.0°C to 37.5°C under conditions that allow for production of the antibody spesolimab, including the step of feeding the cells with a feed medium, and / or wherein the dissolved oxygen (DO) concentration in the culture is maintained within the range of 30-60%.

[0107] 15. The method of any one of the preceding items, wherein increased incubation temperature and / or reduced dissolved oxygen results in production of the antibody spesolimab with reduced BPG% and / or Man5 structure%.

[0108] 16. The method of any one of the preceding items, wherein the antibody spesolimab has less than 5% Man5 structures, preferably less than 4% Man5 structures, more preferably less than 3% Man5 structures, and / or 7.5% or less BPG, preferably 6.5% or less BPG.

[0109] 17. The method of any one of the preceding items, wherein the CHO cells are CHO-K1 cells or CHO-DG44 cells.

[0110] 18. (a) 7.5% BPG or less, preferably 7% BPG or less, more preferably 6.5% BPG or less, even more preferably 6% BPG or less; and / or (b) less than 5% Man5 structures, preferably less than 4% Man5 structures, more preferably less than 3% Man5 structures A composition comprising the antibody spesolimab having the formula:

[0111] 19. A composition comprising the antibody spesolimab, wherein the antibody spesolimab is obtainable by the method according to any one of items 1 to 17.

[0112] 20. The composition of item 18 or 19, wherein the composition is a harvested cell culture fluid (HCCF), an affinity capture pool, a drug substance or a drug product, preferably a drug substance or a drug product.

[0113] 21. The composition of any one of items 18 to 20, wherein the composition is a drug product comprising spesolimab, an antibody having a BPG of less than 7.5% and / or a Man5 structure% of less than 5%.

[0114] 22. The composition of any one of items 18 to 21, wherein the antibody spesolimab comprises 6% or less lysine glycosylation variants in the heavy chain (HC), and / or lysines K38 (heavy chain) and K67 (heavy chain) are not glycosylated and glycosylation at K23 (heavy chain) is 0.3% or less.

[0115] A composition comprising the antibody spesolimab, which contains 23.6% or less lysine glycosylation variants in the heavy chain (HC) and / or lysines K38 (heavy chain) and K67 (heavy chain) are not glycosylated and glycosylation at K23 (heavy chain) is 0.3% or less.

[0116] 24. A composition comprising the antibody spesolimab, which comprises the APG subfractions AP4 and AP3, less than 1% and less than 4%, in particular AP4 and AP3b, less than 1%. [Example]

[0117] cell line A CHO cell line (CHO-DG44) was adapted to serum-free conditions and transfected with DNA to produce the recombinant monoclonal antibody spesolimab. Specifically, a BI HEX (Boehringer Ingelheim High Expression) CHO-DG44-derived CHO cell line (designated HEX II) that had been independently adapted to serum-free medium was used. These cells are DHFR (dihydrofolate reductase) deficient, and methotrexate was used as a selection marker.

[0118] Analysis method Cell concentration and cell viability were determined by trypan blue exclusion using a CEDEX Hires (version 2.2.3) automated cell analyzer (Roche Diagnostics, Mannheim, Germany). The concentration of recombinant monoclonal antibodies produced in the culture medium was quantified using a photometrically based Konelab 60i (Thermo Scientific, Dreieich, Germany) analyzer. The Konelab 60i was also used to quantify metabolites such as glucose, lactic acid (lactate), glutamine, glutamate, and ammonium in the cell culture supernatant. Osmolality profiles were analyzed using an Osmomat automated device (Gonotec, Berlin, Germany). This method is based on the freezing point depression of a particular solution, which is proportional to the amount of dissolved particles. Dissolved carbon dioxide (pCO2), dissolved oxygen (pO2), and pH were determined once daily using a Rapidlab 248 / 348 blood gas analyzer (Siemens Healthcare Diagnostics, Eschborn, Germany). These instruments and the required methods are well known in the art and are used in biopharmaceutical process development and manufacturing for process monitoring and control.

[0119] Batch and fed-batch modes For antibody production, fed-batch processes are typically used in the final production stage, while batch cultures are primarily implemented in the cell expansion stage prior to the final production stage. A series of batch cultures is also called a seed train during cell expansion, meaning that cells from each expansion step are transferred to a culture vessel with a larger culture volume. Batch processes in the final production stage generally do not result in high productivity and are therefore rarely used for recombinant protein production. In fed-batch processes, concentrated feed medium is added during cultivation to supplement nutrients along with fresh medium. These processes achieve higher productivity and are therefore primarily used for recombinant protein production. In contrast to batch mode, nutrient replenishment by adding concentrated feed medium also reduces inhibition of cell growth by undesirable metabolic by-products such as lactate or ammonium. Typically, fed-batch processes are initiated with a volume much smaller than the maximum capacity of the stirred tank, so that concentrated nutrient solutions can be added over the course of the bioreactor cultivation period. The fed-batch cultivation was performed for 14 days.

[0120] Bioreactor cultivation Bioreactor experiments were performed in a controlled 48-mini-bioreactor system with a maximum volume of 15 ml (ambr15 bioreactor system) or a controlled 24-mini-bioreactor system with a nominal volume of 250 ml (ambr250 bioreactor system) using a proprietary basal medium and feed medium. Fully controlled bioreactors were run in fed-batch mode. Concentrated feed solution was added continuously by a feed pump at a feeding rate of 33 ml / L / day from day 1 onwards, or varied in the experiment as indicated (based on the culture starting volume). The seeding density was 1.0 x 10 6The pH was set at 100 cells / ml or varied as indicated during the experiment. The dissolved oxygen concentration was maintained at 60% or varied as indicated during the experiment. Cell growth over longer time frames followed a standard seed train protocol for cell growth and culture splitting to ensure phenotypic stability. This procedure ensured comparisons between different experimental setups at various time points. The standard process format consisted of a pH range of 7.10 to 6.95 (+ / - 0.25) and a constant agitation speed of 1000 to 1150 rpm in the 48-mini bioreactor system and 614 rpm in the 24-mini bioreactor system. The temperature was maintained at 36.5°C or varied during the experiment. Glucose was fed on demand to maintain an actual glucose concentration of 2 to 4 g / L during the culture period. Using analytical methods such as those described above, key culture parameters, such as cell number, cell viability, and concentrations of major carbon metabolites, were determined to provide an optimal nutrient supply to the cell culture. In the bioreactor system, pH and pO2 were monitored online. Offline process parameters and set points were fully controlled by the control software, which used an automated closed-loop system to monitor, for example, pH control, nutrient feed addition, temperature control, agitation, and gas supplementation. This culture process can be and has been successfully scaled up to 2,000 and 12,000 L.

[0121] Charge variant detection Cation exchange chromatography (CEX HPLC) allows the separation of proteins and protein populations according to their charge, while the salt content in the gradient increases at a constant pH. Depending on their functional groups and their microheterogeneity, proteins can have either a positive or negative charge. CEX HPLC uses these pH-dependent charges to facilitate the separation of different proteins / protein modifications.

[0122] Samples containing approximately 1 mg / mL of antibody in mobile phase A were prepared and analyzed using a gradient elution HPLC system with a temperature-controlled autosampler and a UV detector using a MAbPac SCX-10 4 x 250 nm, 10 μm HPLC column. HPLC chromatograms were generated using a first mobile phase (eluent A) of 10 mM MOPS (3-(N-morpholino)propanesulfonic acid, 4-morpholinepropanesulfonic acid) (pH 7.6) and a second mobile phase (eluent B) of 10 mM MOPS, 100 mM potassium chloride (pH 7.6). Elution was performed with a 40-minute linear gradient of 15% to 85% eluent A at a flow rate of 0.8 mL / min. Detection was performed with a UV detector at a wavelength of 280 nm.

[0123] The APG (acidic peaks), main peak, and BPG (basic peaks) were separated by splitting using a continuous baseline. The integration range started at approximately 2 minutes and ended at approximately 44 minutes. The acidic peaks could be further separated into seven subspecies, eluting from the main peak in the following order: AP1 (a, b, c), AP2, AP3 (a, b), and AP4 (Table 2) at the following retention times. The integration of the single peaks was split by using the following retention times, while the main peak was the reference peak. The integration was performed according to the reference chromatograms shown in Figures 4A-B.

[0124] [Table 2]

[0125] Detection of glycosylation variants Analytical samples and reference standards were prepared in duplicate using 0.2 mg of protein per sample. Oligosaccharides were enzymatically released from the samples / reference standards using PNGaseF (NEB P0704L, according to the manufacturer's instructions) and labeled with 2-aminobenzamide (2-AB (Ludger; LT-KAB-A2)) according to the manufacturer's instructions. Purified water was used as a blank control, prepared in parallel with the samples / reference standards. The labeled oligosaccharides were then transferred to a HILIC column (Glycan Analysis was performed using UPLC (e.g., Nexera, Fa, Shimadzu) with a BEH amide column (130 Å, 1.7 μm, 2.1 × 150 mm) and a fluorescence detector (FLD). This method was used to determine the oligosaccharide profile (oligomap) and quantify the oligosaccharide structures of spesolimab drug substance or Protein A pools. After N-glycan release and 2-AB labeling, aliquots were purified using aminopropyl cartridges (e.g., SepPak; Waters; WAT020840) using a vacuum chamber or automated solid-phase extraction system. The eluted fractions were dried using an evaporator and resuspended in 80 μl. HPLC analysis was then performed using a flow rate of 0.7 mL / min and a fluorescence detector (excitation wavelength 330 nm; emission wavelength 420 nm). Mobile phase A (0.05 M ammonium formate, pH 4.5 / 50% acetonitrile (ACN)) and mobile phase B (acetonitrile HPLC grade (ACN)) with the following gradient: Elution was performed by running a linear gradient of 50% to 80% eluent A over 25 min.

[0126] The integration range of the chromatograms of the reference standards and samples is approximately 4 to 20 minutes. Retention times vary slightly depending on the instrument and mobile phase used. The relative peak area of ​​peak 3 (Figure 5, also referred to as (Oligo Map Peak 3)) is calculated according to the following formula:

number

[0127] The relative peak area for all peaks from one injection is referred to as the sum of the peaks (absolute peak area). Peak 3 contains high mannose (Man5) structures.

[0128] Example 1: Effect of iron and copper on BPG and high mannose The effects of various concentrations of iron and copper, and their interactions, on cell culture performance and product quality parameters, including acidic and basic charge variants (acidic peak group (APG) and basic peak group (BPG)) and high mannose species, were investigated using a design of experiments (DOE) approach. DOE studies are data collection and analysis tools that allow for the alteration of multiple input factors and determine their combined and independent effects on various output parameters. Thus, this type of study can identify the interactions of multiple factors within a process by simultaneously varying the levels of multiple inputs within the process.

[0129] Twenty-four culture runs in fully automated 15 mL mini-bioreactors were carried out using the process conditions described above. Cells were cultured at 1.0 x 10 in basal medium. 6 Cells were seeded at 100 cells / ml. The iron concentration in the basal medium was varied from 1.4 to 6.0 mM using iron choline citrate (Dr. Paul Lohmann GmbH KG) as the iron source. Iron choline citrate (0.7, 1.4, 2.1, and 3.0 g / L iron choline citrate) was added directly to the medium formulation containing 5.8 μM iron (iron nitrate and iron sulfate) before sterile filtration. Copper was provided as CuSO4 and varied between 0.36 and 1.71 μM. Additional copper solution (CuSO4) was supplemented by adding 0.139 μM CuSO4 to the bioreactor directly after sterilization before inoculation. Feed medium containing 1.13 mM iron (provided as iron choline citrate; 565 μM) and 0.43 mM CuSO4 was added once daily at 30 mL / L / day.

[0130] Output parameters evaluated using a statistical software suite (Design Expert, Stat Ease) were the spesolimab product titer, integrated viable cell density, viability at day 14, and product quality parameters, APG, BPG measured by cation exchange chromatography (CEX), and high mannose species indicated through mannose 5 structures determined by HILIC UHPLC (Table 3).

[0131] [Table 3]

[0132] The results shown in Figure 1A-F demonstrate that the lowest BPG is reached using high iron and low copper concentrations (Figure 1A, B, and C). Similarly, higher iron and lower copper concentrations appear to be beneficial for titer (Figure 1D), viable cell density (Figure 1E), and viability (Figure 1F).

[0133] Example 2: Effect of process parameters on BPG and high mannose The cell culture process parameters seeding cell density, culture temperature, and dissolved oxygen concentration were evaluated using a design of experiments approach to investigate their effects on the product quality parameters BPG and high-mannose species. Seeding cell densities ranged from 0.5 million to 1.5 million cells / ml, temperatures varied from 35°C to 38°C, dissolved oxygen concentrations in the bioreactor varied from 40% to a maximum of 80%, and feed rates varied from 29.7 to a maximum of 36.3 mL per L of culture starting volume per day. Twenty-four parallel runs were performed in a fully automated 24-mini-bioreactor system in fed-batch mode for 14 days. Other culture parameters and analytical methods were performed as described above. Output parameters evaluated using a statistical software suite (Design Expert, Stat Ease) were the spesolimab product titer, integrated viable cell density, viability at day 14, and product quality parameters APG, BPG measured by CEX, and high mannose 5 species determined by HILIC-UHPLC (Table 4).

[0134] [Table 4]

[0135] The results shown in Figures 2A–H demonstrate that the lowest BPG is achieved at high seeding densities and higher temperatures (Figures 2A, B, and C), and the lowest high-mannose (5Man) structures are similarly achieved at high seeding densities and lower temperatures (Figures 2D and E). Similarly, titer (Figure 2F) and viable cell density (Figure 2G) increase with higher seeding densities and higher culture temperatures. Viability after 14 days of culture appears to decrease slightly at high seeding densities and higher culture temperatures (Figure 2H), but this does not appear to negatively affect titer. The results shown in Figures 3A–D demonstrate that reduced dissolved oxygen also has a beneficial effect on BPG (Figure 3A), high-mannose structures (Figure 3B), titer (Figure 3C), and viable cell density (Figure 3D).

[0136] Example 3: Binding of IL36 and fetal Fc receptors to various allogeneic variants (APG / BPG) To determine the potency of the collected CEX fractions for spesolimab, binding to the IL36 receptor (surface plasmon resonance) and IL36 receptor bioassays were performed (Table 5).

[0137] The majority of the isolated CEX fractions exhibit potencies comparable to those of the source material of the drug substance. Reduced potency was observed in the majority acidic fraction AP4 and, to a lesser extent, in the acidic fraction AP3b (the same applies to binding to fetal Fc receptors (surface plasmon resonance)). Both fractions, and especially fraction AP4, exhibited rather unclear profiles (Figures 4A and B), with relatively low abundances of less than 1% (Table 5).

[0138] Similarly, the most basic fraction, BP3, also exhibits a rather unclear elution profile (Tables 4A and B), with a relatively low abundance of less than 1.5% (Table 5). This fraction exhibits reduced recovery of binding to the IL36 receptor (surface plasmon resonance), implying reduced binding to the Protein A / G sensor chip, consistent with the increased oligomers observed within this fraction shown in Figure 4C.

[0139] [Table 5]

[0140] BPG seeds The basic peaks contain, inter alia, different N- and C-terminal charge variants. The observed N-terminal charge variant is spesolimab containing an N-terminal glutamine instead of an N-terminal pyro-glutamine (predominantly observed at the N-terminus of the light chain in fraction BP2, but less frequently at the N-terminus of the heavy chain in fraction BP1). Furthermore, spesolimab containing an additional amino acid VHS at the N-terminus of the heavy chain is enriched in fraction BP3 (the remainder of the N-terminal signal peptide). The observed C-terminal charge variant is spesolimab containing an additional lysine at the C-terminus of one (fraction BP1) or both (fraction BP3) heavy chains. Furthermore, spesolimab containing a proline amide at the C-terminus of one (fraction BP1) or both (fraction BP3) heavy chains is enriched (generated by removal of the C-terminal glycine and amidation of the adjacent proline).

[0141] APG seeds The acidic peaks include, inter alia, spesolimab with N-glycans containing N-acetylneuraminic acid (NANA) and / or deamidation at N386+N391+N392 (heavy chain). Additionally, moderate enrichment for a putative fragment consisting of spesolimab lacking one light chain is observed within the more acidic peaks.

[0142] Example 4: Lysine glycation variants Glycation results from the formation of various types of covalent adducts, where glucose can react with primary amines at either lysine residues or the N-terminus, resulting in the formation of acidic variants. Glycation of spesolimab occurs during upstream manufacturing because glucose is contained in the culture medium. Glycated species can be generated in cell culture, where, upon harvest, the cells and spesolimab molecules are exposed to higher hexose levels. Reducing sugars are not applied in any downstream manufacturing processes, but can be present or formed in the pharmaceutical formulation, i.e., the drug product.

[0143] The glycosylation susceptibility of a lysine residue is determined by its solvent accessibility (tertiary structure) and the chemical environment of its side chain (primary and secondary structure). In IgG, the majority of glycosylation is distributed over more than 30 lysine residues (Miller AK, Hambly DM, Kerwin BA, Treuheit MJ, Gadgil HS, Journal of Pharmaceutical Sciences, 2011, 100(7): 2543-2550). Spesolimab has 11 lysine residues in the light chain (LC; SEQ ID NO: 2) and 33 lysine residues in the heavy chain (HC; SEQ ID NO: 1) that can be glycosylated. Because there are three lysine residues near the CDRs of the heavy chain (potentially important lysines: HC-K23, K38, K67; SEQ ID NO: 1), glycosylation may be important for antibody efficacy / potency.

[0144] Sum (reduction) of glycation via relative quantification using LC-MS (liquid chromatography-mass spectrometry) To determine the relative quantification of total reduced glycosylation using LC-MS, a 100 μL sample of spesolimab diluted to 1 mg / mL was treated with 1 μL (1 U / μL) of N-glucosidase (Roche PO11365193001 or equivalent) to remove N-linked oligosaccharides and then reduced with 1 μL of 1 M dithiothreitol. Reduction was performed at 57°C for 20 minutes. The resulting light chain and deglycosylated heavy chain were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and analyzed online by ESI Q-TOF MS (Xevo G2 Q-TOF). Protein subunits and corresponding glucose adducts (1 M glucose adduct = 162 Da) were analyzed, and the acquired spectra were analyzed using the MaxEnt™ algorithm (Table 6).

[0145] [Table 6]

[0146] Relative quantification (single glycation site) using LC-MS To determine the relative quantification of single glycosylation sites using LC-MS, samples were denatured and buffer exchanged into Tris guanidinium hydrochloride buffer (samples were diluted to 1 mg / mL with 7 M guanidinium hydrochloride / 100 mM Tris / HCl, pH 8.3), reduced with dithiothreitol (DTT, final concentration: 10 mM) for 20 min at 57 °C, and alkylated with iodoacetic acid (IAA, final concentration: 10 mM) for 20 min at room temperature in the dark. The reaction was then quenched by the addition of 50 mM dithiothreitol. After reduction and alkylation, the samples were again exchanged into 100 mM ammonium bicarbonate buffer and enzymatically digested using chymotrypsin in the presence of detergent. The reaction was stopped after 30 min at 37 °C by the addition of formic acid (1:120, v:v). The peptides were separated by reversed-phase liquid chromatography and analyzed by electrospray ionization (ESI)-MS (mass spectrometry). The relative amounts of glycated peptides were quantified based on the extracted ion chromatograms of the wild-type peptide and the peptide with a glucose adduct (+162 Da) (Table 7).

[0147] [Table 7]

[0148] Glycated lysine variants for the heavy and light chains were detectable at low levels of 3% and 1%, respectively (Table 6), typically found in the lower range of 5-15% in the literature for recombinant IgG (Eon-Duval A et al, J Pharm Sci., 2012, 101(10): 3604-3618). Even more surprisingly, glycosylation on critical residues was found to be below the detection limit, or less than 0.3% for K23 (Table 7, critical residues are highlighted in bold).

[0149] While specific aspects and embodiments of the invention have been described, they are offered by way of example only and are not intended to limit the scope of the invention. Indeed, the novel methods and systems described herein may be embodied in a wide variety of other forms without departing from the spirit thereof. The accompanying claims and their equivalents are intended to cover such forms or modifications as fall within the scope and spirit of the invention.

[0150] All patents and / or publications (including journal articles) cited in this disclosure are hereby expressly incorporated by reference.

Claims

1. (a)(i) seeding the cells in a culture medium; and (ii) culturing the cells in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, including feeding the cells in the cell culture with a feed medium. Culturing CHO cells comprising a nucleic acid encoding the antibody spesolimab using fed-batch culture in a serum-free cell culture medium comprising: Here, Cu ２＋ is added to the culture medium at 0.35 to 1.2 μM and iron at 1500 μM or more before and / or within 2 days after seeding the cells in step (i); (b) collecting the cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant.

1. A method for producing the antibody spesolimab in cell culture, comprising:

2. Cu ２＋ 2. The method of claim 1, wherein iron is added to the culture medium before and / or within one day after inoculation in step (i).

3. 3. The method of claim 1 or 2, wherein the increased iron concentration and / or decreased copper concentration in the culture medium results in the production of the antibody spesolimab with a reduced Basic Peak Group % (BPG%).

4. 10. The method of any one of the preceding claims, wherein the antibody spesolimab has a BPG of 7.5% or less, preferably a BPG of 7% or less, more preferably a BPG of 6.5% or less, and even more preferably a BPG of 6% or less.

5. The seeding density in step (a) is 0.7 × 10 ６ cells / ml or more, preferably 0.7 x 10 ６ cells / ml to 1.5 x 10 ６ cells / ml, more preferably 0.8 x 10 ６ cells / ml to 1.5 x 10 ６ cells / ml, and even more preferably 0.9 x 10 ６ cells / ml to 1.3 x 10 ６ 10. The method of any one of the preceding claims, wherein the cell count is 100 cells / ml.

6. (a)(i) Cells were cultured in culture medium at 0.7 × 10 ６ seeding at a cell density of 0.1 cells / ml or more; and (ii) culturing the cells in a culture medium under conditions that allow for the production of the antibody spesolimab in the cell culture, including feeding the cells in the cell culture with a feed medium. Culturing CHO cells comprising a nucleic acid encoding the antibody spesolimab using fed-batch culture in a serum-free cell culture medium comprising: Wherein, optionally, Cu ２＋ is added to the culture medium at 0.35 to 1.2 μM and iron at 1500 μM or more before and / or within 2 days after seeding the cells in step (i); (b) collecting the cell culture supernatant containing the antibody spesolimab; and (c) optionally purifying the antibody spesolimab from the cell culture supernatant.

1. A method for producing the antibody spesolimab in cell culture, comprising:

7. 10. The method of any one of the preceding claims, wherein the increased seeding density results in the production of the antibody spesolimab with a reduced Basic Peak Group % (BPG %) and / or Man5 structure %.

8. 10. The method of any one of the preceding claims, wherein the cells are cultured at 36.0°C to 37.5°C under conditions that allow the production of the antibody spesolimab, comprising the step of feeding the cells with a feed medium, and / or wherein the dissolved oxygen (DO) concentration in the culture is maintained within the range of 30-60%.

9. 10. The method of any one of the preceding claims, wherein increased culture temperature and / or reduced dissolved oxygen results in the production of the antibody spesolimab with reduced BPG % and / or Man5 structure %.

10. 10. The method of any one of the preceding claims, wherein the antibody spesolimab has less than 5% Man5 structures, preferably less than 4% Man5 structures, more preferably less than 3% Man5 structures, and / or 7.5% or less BPG, preferably 6.5% or less BPG.

11. (a) a BPG of 7.5% or less, preferably 7% or less, more preferably 6.5% or less, and even more preferably 6% or less; and / or (b) less than 5% Man5 structure, preferably less than 4% Man5 structure, more preferably less than 3% Man5 structure A composition comprising the antibody spesolimab having the formula:

12. A composition comprising the antibody spesolimab, wherein the antibody spesolimab is obtainable by a method according to any one of claims 1 to 10.

13. 13. The composition of claim 11 or 12, wherein the composition is a drug product comprising antibody 4, spesolimab, having less than 7.5% BPG and / or less than 5% Man5 structures.

14. The composition of any one of claims 11 to 13, wherein the antibody spesolimab comprises 6% or less lysine glycosylation variants in the heavy chain (HC), and / or lysines K38 (HC) and K67 (HC) are not glycosylated, and glycosylation at K23 (HC) is 0.3% or less.

15. A composition comprising the antibody spesolimab, which contains 6% or less lysine glycosylation variants in the heavy chain (HC) and / or lysines K38(HC) and K67(HC) are not glycosylated and glycosylation at K23(HC) is 0.3% or less.

16. A composition comprising the antibody spesolimab, which comprises subfractions of the acidic peak group (APG) of less than 1% AP4 and less than 4% AP3 fractions, in particular less than 1% AP4 and less than 1% AP3b fractions.

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