Methods of producing fcf-containing proteins

Abstract

Provided herein are improved methods for producing Fc-containing proteins. The methods generally involve culturing cells expressing Fc-containing proteins in a production bioreactor for a first period of time, followed by a first nutrient feed, and culturing the cells for a second period of time, followed by a second nutrient feed. The methods can also involve culturing the cells for a third period of time, followed by a third nutrient feed.

Description

Reference to sequence list

[0001] This application is submitted together with a sequence list in ST.26 XML format. The sequence list is provided as a file named "30591_WO.xml" created on December 4, 2024, and is 4.67 kilobytes in size. The full text of the ST.26 XML format sequence list information is incorporated herein by reference. Background Technology

[0002] The production of recombinant Fc-containing proteins for therapeutic purposes typically involves protein expression in cultured cells. Cell culture conditions can affect the yield and / or quality of Fc-containing proteins produced from cultured cells. In particular, suboptimal cell culture conditions may lead to increased protease cleavage and / or increased undesirable glycosylation of the resulting Fc-containing proteins.

[0003] Therefore, there is a need for improved cell culture methods to produce recombinant Fc-containing proteins that minimize problems such as protease cleavage and / or the generation of undesirable glycosylation, while maximizing protein yield and quality. Summary of the Invention

[0004] This disclosure provides improved methods for producing Fc-containing proteins. These methods are particularly advantageous because they reduce protease cleavage and undesirable glycosylation of Fc-containing proteins. As demonstrated in the embodiments herein, these methods result in efficient, high-titer production of Fc-containing proteins with the desired quality profile. The method generally involves culturing cells expressing Fc-containing proteins (e.g., mammalian cells) in a production bioreactor for a first time period, followed by feeding a first nutrient, and culturing the cells for a second time period, followed by feeding a second nutrient. In some embodiments, the method further involves culturing the cells for a third time period, followed by feeding a third nutrient.

[0005] Therefore, in one aspect, this disclosure provides a method for producing duraglutide, the method comprising culturing mammalian cells expressing duraglutide in a production bioreactor for a total culture time, wherein after a first portion of the total culture time, a first nutrient is supplied to the production bioreactor; after a second portion of the total culture time, a second nutrient is supplied to the production bioreactor; and after a third portion of the total culture time, a third nutrient is supplied to the production bioreactor, such that duraglutide is produced from the mammalian cells.

[0006] In one embodiment, the first nutrient feed, the second nutrient feed, and / or the third nutrient feed comprises L-tyrosine. In another embodiment, each of the first nutrient feed, the second nutrient feed, and the third nutrient feed comprises L-tyrosine.

[0007] In one embodiment, the total culture time is 12 to 16 days. In another embodiment, the total culture time is approximately 14 days. In one embodiment, the first part of the total culture time is 2 to 4 days after the start of culture. In one embodiment, the first nutrient is fed approximately 3 days after the start of culture. In one embodiment, the second part of the total culture time is 5 to 7 days after the start of culture. In one embodiment, the second nutrient is fed approximately 6 days after the start of culture. In one embodiment, the third part of the total culture time is 8 to 10 days after the start of culture. In one embodiment, the third nutrient is fed approximately 9 days after the start of culture. In one embodiment, the first nutrient is fed approximately 3 days after the start of culture, the second nutrient is fed approximately 6 days after the start of culture, and the third nutrient is fed approximately 9 days after the start of culture.

[0008] In one implementation, the maximum viable cell density (VCD) in the production bioreactor is between 8 × 10⁻⁶. 6 Cells / mL and 14 × 10 6 Between cells per milliliter.

[0009] In one embodiment, the mammalian cells are selected from the group consisting of: COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HeLa cells, NSO cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. In one embodiment, the mammalian cells are CHO cells.

[0010] In one embodiment, less than about 4% of the generated duraglutide is an N-terminal cleaved variant. In one embodiment, the N-terminal cleaved variant lacks an N-terminal histidine residue or both an N-terminal histidine residue and a glycine residue. In one embodiment, between about 1.5% and 2.6% of the generated duraglutide comprises G2F glycan. In one embodiment, between about 10.6% and 15.4% of the generated duraglutide comprises G1F glycan. In one embodiment, between about 74.7% and 80% of the generated duraglutide comprises G0F glycan. In one embodiment, no more than about 1% of the generated duraglutide comprises Man-5 glycan. In one embodiment, 1.5% to 2.6% of the generated duraglutide comprises G2F glycan, 10.6% to 15.4% of the generated duraglutide comprises G1F glycan, 74.7% to 80% of the generated duraglutide comprises G0F glycan, and no more than about 1% of the generated duraglutide comprises Man-5 glycan.

[0011] On the other hand, this article provides duraglutide produced by any of the methods described herein. Attached Figure Description

[0012] Figure 1 The updated duraglutide manufacturing protocol according to various aspects of this disclosure is shown to produce an increased duraglutide titer on day 14 compared to the initial duraglutide manufacturing protocol. A 1.5X nutrient feed concentration was supplied to mammalian cell cultures in two test vessels on day 6, followed by a 20 mM glucose feed on day 10 (“Initial Feed Parameters”), or a 0.7X nutrient feed concentration was supplied twice on days 3 and 6, followed by three 30 mM glucose feeds on days 8, 10, and 12 (“Updated Feed Parameters”). The titers after a total culture time of 14 days are depicted, expressed in g / L of culture.

[0013] Figure 2 An updated method for manufacturing duraglutide according to various aspects of this disclosure is shown to result in increased protease cleavage. For example... Figure 1 The feed was mammalian cell culture. On day 14, protease cleavage of duraglutide was assessed by measuring the level of the des H / HG variant. The amount of des H / HG under each condition is plotted as a percentage of total duraglutide produced. The dashed line at 3.5% represents the upper limit of des H / HG %.

[0014] Figure 3An optimized duraglutide manufacturing protocol according to various aspects of this disclosure is shown to result in reduced protease cleavage. Mammalian cell cultures were grown under the conditions described in Example 2 and Table 3 herein, and the protease cleavage of duraglutide was assessed by measuring the levels of the desH / HG variant. The amount of desH / HG under each condition is depicted as a percentage of the total duraglutide produced. The asterisks for conditions 2 and 3 indicate repetitions. The dashed line at 3.5% indicates the upper limit of the desH / HG percentage. Detailed Implementation

[0015] This disclosure provides an improved method for producing Fc-containing proteins. The method generally involves culturing mammalian cells expressing Fc-containing proteins in a production bioreactor for a first time period, followed by feeding a first nutrient, and then culturing the mammalian cell line for a second time period, followed by feeding a second nutrient. In some embodiments, the method further involves culturing the mammalian cell line for a third time period, followed by feeding a third nutrient. These methods are particularly advantageous because they promote efficient, high-titer production of Fc-containing proteins while reducing protease cleavage and undesirable glycosylation of the resulting Fc-containing proteins. I. Definition

[0016] As used herein, the term "Fc-containing protein" refers to a protein that contains an Fc region. In one embodiment, the Fc-containing protein contains a variant Fc region that includes one or more amino acid substitutions, additions, and / or deletions relative to a naturally occurring Fc region. In one embodiment, the Fc-containing protein is an antibody. In one embodiment, the Fc-containing protein is not an antibody.

[0017] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDR, VH region, and / or VL region. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, heteroconjugated antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camel-derived antibodies, affinity molecules, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above.

[0018] As used herein, when referring to values ​​or parameters, the term "about" includes a fluctuation of ±5% of that value or parameter. For example, when referring to the amount of a protein having a particular characteristic (e.g., N-terminal splicing), "about" means a range including values ​​less than 5% and values ​​more than 5% of the mentioned value. Thus, about 10% of multiple proteins being N-terminal splicing variants means covering multiple proteins that are N-terminal splicing variants, ranging from 9.5% to 10.5%. II. Methods for culturing mammalian cells

[0019] When producing Fc-containing proteins, optimizing the nutrient feed conditions of mammalian cell cultures in the production bioreactor is important for maintaining the quality of the Fc-containing protein product. This document discloses a method for producing Fc-containing proteins, which includes culturing mammalian cells expressing Fc-containing proteins in a production bioreactor for a first time period, followed by feeding a first nutrient, and culturing the mammalian cell lines for a second time period, followed by feeding a second nutrient. In some embodiments, the method also involves culturing the mammalian cell lines for a third time period, followed by feeding a third nutrient. The methods disclosed herein are particularly advantageous because they can reduce protease cleavage and undesirable glycosylation of Fc-containing proteins in mammalian cell cultures, thereby increasing the amount of intact Fc-containing proteins. In one embodiment, the Fc-containing protein is a duraglutide protein, as described in further detail below.

[0020] In one aspect, the method disclosed herein includes culturing mammalian cells expressing Fc protein in a production bioreactor for a total culture time, wherein a) after a first portion of the total culture time, a first nutrient is supplied to the production bioreactor; b) after a second portion of the total culture time, a second nutrient is supplied to the production bioreactor; and c) after a third portion of the total culture time, a third nutrient is supplied to the production bioreactor, such that the mammalian cells produce Fc protein.

[0021] In one aspect, the method disclosed herein includes culturing mammalian cells expressing duraglutide protein in a production bioreactor for a total culture time, wherein a) after a first portion of the total culture time, a first nutrient feed is supplied to the production bioreactor; b) after a second portion of the total culture time, a second nutrient feed is supplied to the production bioreactor; and c) after a third portion of the total culture time, a third nutrient feed is supplied to the production bioreactor, such that duraglutide protein is produced from the mammalian cells.

[0022] Exemplary cell culture methods and conditions applicable to the foregoing methods are described in detail below.

[0023] In the methods disclosed herein, one or more nutrient feeds supplied to cell cultures may comprise any nutrient feed composition known to those skilled in the art for culturing mammalian cells. Typically, the nutrient feed composition will comprise a single nutrient feed component or a mixture of nutrient feed components. Nutrient feeds may include simultaneously supplying one or more nutrient feed components to the cell culture (e.g., as a single mixture or as one or more separate mixtures simultaneously supplied to the cell culture). In one embodiment of the methods provided herein, the first nutrient feed and the second nutrient feed comprise the same nutrient feed composition. In one embodiment, the first nutrient feed and the second nutrient feed comprise different nutrient feed compositions. In one embodiment, the first nutrient feed, the second nutrient feed, and / or the third nutrient feed comprise the same nutrient feed composition. In one embodiment, the first nutrient feed, the second nutrient feed, and / or the third nutrient feed comprise different nutrient feed compositions.

[0024] In one embodiment, the nutrient feed that can be used in the methods provided herein comprises a nutrient feed composition or nutrient feed component that has been used in the previously described methods. For example, the nutrient feed may comprise a commercially available nutrient feed component or mixture, or the nutrient feed may comprise a nutrient feed component or mixture used in previously developed methods for producing Fc-containing proteins (e.g., duraglutide).

[0025] In one embodiment of the method provided herein, the nutrient feed comprises a nutrient feed component present at a concentration reduced relative to the concentration of the component described in the previously described method. For example, when the nutrient component is commercially available, it may be used in the method provided herein at a concentration reduced relative to the concentration recommended in the manufacturer's scheme. As another example, when the nutrient feed component is used at a specific concentration in a previously developed method for producing Fc-containing proteins (e.g., duraglutide), the nutrient feed component may be used in the method provided herein at a concentration reduced relative to said specific concentration. In one embodiment of the method provided herein, the nutrient feed comprises a nutrient feed component present at a concentration of about 0.05X to 0.95X (e.g., 0.1X, 0.15X, 0.2X, 0.25X, 0.3X, 0.35X, 0.4X, 0.45X, 0.5X, 0.55X, 0.6X, 0.65X, 0.7X, 0.75X, 0.8X, 0.85X, or 0.9X) relative to the concentration of the component in the previously described method.

[0026] In one embodiment of the method provided herein, the nutrient feed comprises a nutrient feed component present at a concentration increased relative to the concentration of the component described in the previously described method. In one embodiment, the nutrient feed comprises a nutrient feed component at a concentration of approximately 1.05X to 10X (e.g., 1.1X, 1.15X, 1.2X, 1.25X, 1.3X, 1.35X, 1.4X, 1.45X, 1.5X, 1.55X, 1.6X, 1.65X, 1.7X, 1...) relative to the concentration of the component in the previously described method. .75X, 1.8X, 1.85X, 1.9X, 2X, 2.1X, 2.15X, 2.2X, 2.25X, 2.3X, 2.35X, 2.4X, 2.45X, 2 .5X, 2.55X, 2.6X, 2.65X, 2.7X, 2.75X, 2.8X, 2.85X, 2.9X, 3X, 3.1X, 3.15X, 3.2X, 3. 25X, 3.3X, 3.35X, 3.4X, 3.45X, 3.5X, 3.55X, 3.6X, 3.65X, 3.7X, 3.75X, 3.8X, 3.85X , 3.9X, 4X, 4.1X, 4.15X, 4.2X, 4.25X, 4.3X, 4.35X, 4.4X, 4.45X, 4.5X, 4.55X, 4.6X, The concentrations of 4.65X, 4.7X, 4.75X, 4.8X, 4.85X, 4.9X, 5X, 5.25X, 5.5X, 5.75X, 6X, 6.25X, 6.5X, 6.75X, 7X, 7.25X, 7.5X, 7.75X, 8X, 8.25X, 8.5X, 8.75X, 9X, 9.25X, 9.5X, or 9.75X) are present.

[0027] In one embodiment, the nutrient feed comprises one or more nutrient feed components, which are present in a weight defined relative to the total weight of the cell culture to which the nutrient feed is supplied. For example, the nutrient feed components may be present in a rate of about 1 gram of nutrient feed component per kilogram of cell culture (g / kg) to about 200 g / kg (e.g., about 1 g / kg, about 2 g / kg, about 4 g / kg, about 6 g / kg, about 8 g / kg, about 10 g / kg, about 12 g / kg, about 14 g / kg, about 16 g / kg, about 18 g / kg, about 20 g / kg, about 22 g / kg, about 24 g / kg, about 26 g / kg, about 28 g / kg, about 30 g / kg, about 32 g / kg, about 34 g / kg, about 36 g / kg, about 38 g / kg, about 40 g / kg, about 42 g / kg, about 44 g / kg). g, approximately 46g / kg, approximately 48g / kg, approximately 50g / kg, approximately 52g / kg, approximately 54g / kg, approximately 56g / kg, approximately 58g / kg, approximately 60g / kg, approximately 62g / kg, approximately 64g / kg, approximately 66g / kg, approximately 68g / kg, approximately 70g / kg, approximately 72g / kg, approximately 74g / kg, approximately 76g / kg, approximately 78g / kg, approximately 80g / kg, approximately 82g / kg, approximately 84g / kg, approximately 86g / kg, approximately 88g / kg, approximately 90g / kg, approximately 92g / kg, approximately 94g / kg, approximately 96g / kg, approximately 98g / kg, approximately 100g / kg, approximately 1 0.2g / kg, approximately 104g / kg, approximately 106g / kg, approximately 108g / kg, approximately 110g / kg, approximately 112g / kg, approximately 114g / kg, approximately 116g / kg, approximately 118g / kg, approximately 120g / kg, approximately 122g / kg, approximately 124g / kg, approximately 126g / kg, approximately 128g / kg, approximately 130g / kg, approximately 132g / kg, approximately 134g / kg, approximately 136g / kg, approximately 138g / kg, approximately 140g / kg, approximately 142g / kg, approximately 144g / kg, approximately 146g / kg, approximately 148g / kg, approximately 150g / kg, approximately 152g / kg Approximately 154g / kg, 156g / kg, 158g / kg, 160g / kg, 162g / kg, 164g / kg, 166g / kg, 168g / kg, 170g / kg, 172g / kg, 174g / kg, 176g / kg, 178g / kg, 180g / kg, 182g / kg, 184g / kg, 186g / kg, 188g / kg, 190g / kg, 192g / kg, 194g / kg, 196g / kg, 198g / kg, or 200g / kg) are present in the nutrient feed.

[0028] In one embodiment, the method provided herein includes supplying L-tyrosine to mammalian cell cultures. In one embodiment, one or more nutrient feeds contain L-tyrosine as a nutrient feed component (i.e., the nutrient feed composition contains L-tyrosine, or L-tyrosine is supplied to the cell culture separately from the nutrient feed). In one embodiment, a first nutrient feed and / or a second nutrient feed contains L-tyrosine. In one embodiment, a first nutrient feed and a second nutrient feed contain L-tyrosine. In one embodiment, a first nutrient feed, a second nutrient feed, and / or a third nutrient feed contain L-tyrosine. In one embodiment, a first nutrient feed, a second nutrient feed, and a third nutrient feed contain L-tyrosine.

[0029] In one embodiment, L-tyrosine is present as having a content of about 1% to about 10% (e.g., about 1%, about 1.2%, about 1.4%, about 1.6%, about 1.8%, about 2%, about 2.2%, about 2.4%, about 2.6%, about 2.8%, about 3%, about 3.2%, about 3.4%, about 3.6%, about 3.8%, about 4%, about 4.2%, about 4.4%, about 4.6%, about 4.8%, about 5%, about 5.2%, about 5.4%, about...). A portion of an L-tyrosine solution with an L-tyrosine concentration of 5.6%, approximately 5.8%, approximately 6%, approximately 6.2%, approximately 6.4%, approximately 6.6%, approximately 6.8%, approximately 7%, approximately 7.2%, approximately 7.4%, approximately 7.6%, approximately 7.8%, approximately 8%, approximately 8.2%, approximately 8.4%, approximately 8.6%, approximately 8.8%, approximately 9%, approximately 9.2%, approximately 9.4%, approximately 9.6%, approximately 9.8%, or approximately 10%) is supplied to mammalian cell cultures. In one embodiment, the L-tyrosine solution has a concentration of approximately 3.5%. In one embodiment, the L-tyrosine solution has an L-tyrosine concentration of 3.5% or 35 g / L dissolved in water.

[0030] In one embodiment, the L-tyrosine solution is used at a concentration of approximately 1 g of 3.5% L-tyrosine solution per 1 kg of bioreactor weight (i.e., 1 g / kg) to approximately 12 g / kg (e.g., approximately 1 g / kg, approximately 1.2 g / kg, approximately 1.4 g / kg, approximately 1.6 g / kg, approximately 1.8 g / kg, approximately 2 g / kg, approximately 2.2 g / kg, approximately 2.4 g / kg, approximately 2.6 g / kg, approximately 2.8 g / kg, approximately 3 g / kg, approximately 3.1 g / kg, approximately 3.2 g / kg, approximately 3.3 g / kg, approximately 3.4 g / kg, approximately 3.5 g / kg, approximately 3.6 g / kg, approximately 3.7 g / kg, approximately 3.8 g / kg, etc.). Approximately 3.9g / kg, approximately 4g / kg, approximately 4.1g / kg, approximately 4.2g / kg, approximately 4.3g / kg, approximately 4.4g / kg, approximately 4.5g / kg, approximately 4.6g / kg, approximately 4.7g / kg, approximately 4.8g / kg, approximately 4.9g / kg, approximately 5g / kg, approximately 5.1g / kg, approximately 5.2g / kg, approximately 5.3g / kg, approximately 5.4g / kg, approximately 5.5g / kg, approximately 5.6g / kg, approximately 5.7g / kg, approximately 5.8g / kg, approximately 5.9g / kg, approximately 6g / kg, approximately 6.1g / kg, approximately 6. 2g / kg, approximately 6.3g / kg, approximately 6.4g / kg, approximately 6.5g / kg, approximately 6.6g / kg, approximately 6.7g / kg, approximately 6.8g / kg, approximately 6.9g / kg, approximately 7g / kg, approximately 7.1g / kg, approximately 7.2g / kg, approximately 7.3g / kg, approximately 7.4g / kg, approximately 7.5g / kg, approximately 7.6g / kg, approximately 7.7g / kg, approximately 7.8g / kg, approximately 7.9g / kg, approximately 8g / kg, approximately 8.1g / kg, approximately 8.2g / kg, approximately 8.3g / kg, approximately 8.4g / kg, approximately 8.5g / kg The L-tyrosine solution is supplied to mammalian cell cultures at concentrations of approximately 8.6 g / kg, 8.7 g / kg, 8.8 g / kg, 8.9 g / kg, 9 g / kg, 9.2 g / kg, 9.4 g / kg, 9.6 g / kg, 9.8 g / kg, 10 g / kg, 10.2 g / kg, 10.4 g / kg, 10.6 g / kg, 10.8 g / kg, 11 g / kg, 11.2 g / kg, 11.4 g / kg, 11.6 g / kg, 11.8 g / kg, or 12 g / kg. In one embodiment, the L-tyrosine solution is supplied to mammalian cell cultures at a concentration of approximately 4.1 g / kg. In one embodiment, the L-tyrosine solution is supplied to the mammalian cell culture at a concentration of approximately 7.7 g / kg.In one embodiment, an L-tyrosine solution is supplied to the mammalian cell culture at a concentration of 7.7 g / kg.

[0031] In one embodiment, the method provided herein includes supplying glucose to mammalian cell cultures. In one embodiment, glucose levels in the cell cultures within a bioreactor are monitored, and glucose is supplied when glucose levels fall below a threshold. In one embodiment, glucose is supplied when glucose levels are below about 20 mM (e.g., below about 19 mM, below about 18 mM, below about 17 mM, below about 16 mM, below about 15 mM, below about 14 mM, below about 13 mM, below about 12 mM, below about 11 mM, or below about 10 mM). In one embodiment, glucose is supplied when glucose levels are below about 15 mM. In one embodiment, glucose is supplied when glucose levels are below 15 mM.

[0032] In one embodiment, glucose is supplied to the mammalian cell culture on a predetermined number of days during the total culture time. In one embodiment, glucose is supplied on any one of days 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and / or 20. In one embodiment, glucose is supplied once during the total culture time. In one embodiment, glucose is supplied more than once during the total culture time (e.g., two, three, four, five, or more than five times). In one embodiment, glucose is supplied on day 10. In one embodiment, glucose is supplied on days 8 and 11, days 8 and 13, days 11 and 13, or days 8, 11, and 13. In one embodiment, glucose is supplied on days 8 and 10, days 8 and 12, days 10 and 12, or days 8, 10, and 12.

[0033] In one embodiment, glucose is supplied to the mammalian cell culture at concentrations of about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM. In one embodiment, glucose is supplied at a concentration of about 20 mM. In one embodiment, glucose is supplied at a concentration of 20 mM. In one embodiment, glucose is supplied at a concentration of about 30 mM. In one embodiment, glucose is supplied at a concentration of 30 mM. In one embodiment, glucose is supplied at a concentration of 20 mM on day 10. In one embodiment, glucose is supplied at 20 mM on day 8 and 30 mM on day 11, 20 mM on day 8 and 30 mM on day 13, 30 mM on day 11 and 30 mM on day 13, or 20 mM on day 8, 30 mM on day 11 and 30 mM on day 13. In another embodiment, glucose is supplied at 30 mM on days 8 and 10, 30 mM on days 8 and 12, 30 mM on days 10 and 12, or 30 mM on days 8, 10 and 12.

[0034] In one implementation of the method provided herein, the total culture time is at least 8 days (e.g., at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days).

[0035] In one implementation, the total culture time is less than 20 days (e.g., less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, or less than 8 days).

[0036] In one embodiment, the total culture time is 8 to 20 days (e.g., 9 to 19 days, 10 to 18 days, 11 to 17 days, 12 to 16 days, 13 to 15 days, 8 to 12 days, 8 to 16 days, 10 to 20 days, 10 to 16 days, 10 to 14 days, 12 to 20 days, 12 to 18 days, 12 to 14 days, 14 to 20 days, 14 to 18 days, 14 to 16 days, 16 to 20 days, 16 to 18 days, or 18 to 20 days). In one embodiment, the total culture time is about 12 to about 16 days. In one embodiment, the total culture time is 12 to 16 days. In one embodiment, the total culture time is about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days. In one implementation, the total culture time is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In another implementation, the total culture time is approximately 14 days. In yet another implementation, the total culture time is 14 days.

[0037] In one embodiment of the method provided herein, the first portion of the total culture time is about 10% to about 40% of the total culture time (e.g., about 15% to about 35%, about 20% to about 30%, about 10% to about 30%, about 10% to about 20%, about 15% to about 30%, about 20% to about 35%, about 20% to about 40%, about 25% to about 40%, or about 30% to about 40%). In one embodiment of the method provided herein, the first portion of the total culture time is about 20% to about 30% of the total culture time. In one embodiment of the method provided herein, the first portion of the total culture time is 20% to 30% of the total culture time. In one embodiment, the first portion of the total culture time is 1 to 5 days (e.g., 1 to 4 days, 1 to 3 days, 1 to 2 days, 2 to 5 days, 2 to 4 days, 2 to 3 days, 3 to 5 days, 3 to 4 days, or 4 to 5 days). In one embodiment, the first portion of the total culture time is 2 to 4 days. In one implementation, the first part of the total culture time is about 1 day, about 2 days, about 3 days, about 4 days, or about 5 days. In one implementation, the first part of the total culture time is 1 day, 2 days, 3 days, 4 days, or 5 days. In one implementation, the first part of the total culture time is about 3 days. In one implementation, the first part of the total culture time is 3 days.

[0038] In one embodiment of the method provided herein, the second portion of the total culture time is approximately 30% to approximately 60% of the total culture time (e.g., approximately 35% to approximately 55%, approximately 40% to approximately 50%, approximately 30% to approximately 50%, approximately 30% to approximately 40%, approximately 35% to approximately 50%, approximately 40% to approximately 55%, approximately 40% to approximately 60%, approximately 45% to approximately 60%, or approximately 50% to approximately 60%). In one embodiment of the method provided herein, the second portion of the total culture time is approximately 40% to approximately 50% of the total culture time. In one embodiment of the method provided herein, the second portion of the total culture time is 40% to 50% of the total culture time. In one embodiment, the second portion of the total culture time is 4 to 8 days (e.g., 4 to 7 days, 4 to 6 days, 4 to 5 days, 5 to 8 days, 5 to 7 days, 5 to 6 days, 6 to 8 days, 6 to 7 days, or 7 to 8 days). In one embodiment, the second portion of the total culture time is 5 to 7 days. In one embodiment, the second part of the total culture time is about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days. In one embodiment, the second part of the total culture time is 4 days, 5 days, 6 days, 7 days, or 8 days. In one embodiment, the second part of the total culture time is about 6 days. In one embodiment, the second part of the total culture time is 6 days.

[0039] In one embodiment of the method provided herein, a third portion of the total culture time is approximately 50% to approximately 80% of the total culture time (e.g., approximately 55% to approximately 75%, approximately 60% to approximately 70%, approximately 50% to approximately 70%, approximately 50% to approximately 60%, approximately 55% to approximately 70%, approximately 60% to approximately 75%, approximately 60% to approximately 80%, approximately 65% ​​to approximately 80%, or approximately 70% to approximately 80%). In one embodiment of the method provided herein, a third portion of the total culture time is approximately 60% to approximately 70% of the total culture time. In one embodiment of the method provided herein, a third portion of the total culture time is 60% to 70% of the total culture time. In one embodiment, a third portion of the total culture time is 7 to 11 days (e.g., 7 to 10 days, 7 to 9 days, 7 to 8 days, 8 to 11 days, 8 to 10 days, 8 to 9 days, 9 to 11 days, 9 to 10 days, or 10 to 11 days). In one embodiment, a third portion of the total culture time is 8 to 10 days. In one implementation, the third portion of the total culture time is approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, or approximately 11 days. In one implementation, the third portion of the total culture time is 7 days, 8 days, 9 days, 10 days, or 11 days. In one implementation, the third portion of the total culture time is approximately 9 days. In one implementation, the third portion of the total culture time is 9 days.

[0040] In one embodiment, the first portion of the total culture time is about 20% to about 30% of the total culture time, and the second portion of the total culture time is about 40% to about 50% of the total culture time. In another embodiment, the first portion of the total culture time is 20% to 30% of the total culture time, and the second portion of the total culture time is 40% to 50% of the total culture time. In another embodiment, the first portion of the total culture time is 2 to 4 days, and the second portion of the total culture time is 5 to 7 days. In yet another embodiment, the first portion of the total culture time is 3 days, and the second portion of the total culture time is 6 days.

[0041] In one embodiment, the first portion of the total culture time is about 20% to about 30% of the total culture time, the second portion is about 40% to about 50% of the total culture time, and the third portion is about 60% to about 70% of the total culture time. In another embodiment, the first portion of the total culture time is 20% to 30% of the total culture time, the second portion is 40% to 50% of the total culture time, and the third portion is 60% to 70% of the total culture time. In another embodiment, the first portion of the total culture time is 2 to 4 days, the second portion is 5 to 7 days, and the third portion is 8 to 10 days. In yet another embodiment, the first portion of the total culture time is 3 days, the second portion is 6 days, and the third portion is 9 days.

[0042] In one embodiment, the method provided herein includes culturing mammalian cells at a first temperature for 1 to 5 days, followed by culturing mammalian cells at a second temperature for 9 to 14 days. In one embodiment, the method further includes culturing mammalian cells at a first temperature for about 1 day, followed by culturing mammalian cells at a second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days. In one embodiment, the method further includes culturing mammalian cells at a first temperature for about 2 days, followed by culturing mammalian cells at a second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days. In one embodiment, the method further includes culturing mammalian cells at a first temperature for about 3 days, followed by culturing mammalian cells at a second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days. In one embodiment, the method further includes culturing mammalian cells at a first temperature for about 4 days, followed by culturing mammalian cells at a second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days. In one embodiment, the method further includes culturing mammalian cells at a first temperature for about 5 days, and then culturing mammalian cells at a second temperature for about 9, about 10, about 11, about 12, about 13, or about 14 days.

[0043] In one embodiment, the second temperature is lower than the first temperature. In another embodiment, the second temperature is about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, or about 6°C lower than the first temperature.

[0044] In one embodiment, the first temperature is 34°C to 40°C. In one embodiment, the first temperature is about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In one embodiment, the first temperature is about 36°C.

[0045] In one embodiment, the second temperature is 30°C to 36°C. In one embodiment, the second temperature is about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, or about 36°C. In one embodiment, the second temperature is about 33°C.

[0046] In one embodiment, mammalian cells are cultured at a first temperature for 3 to 4 days. In another embodiment, mammalian cells are cultured at a first temperature for about 3 days. In one embodiment, mammalian cells are cultured at a second temperature for 11 to 12 days. In another embodiment, mammalian cells are cultured at a second temperature for about 11 days.

[0047] In one embodiment of the method provided herein, mammalian cells are cultured in a bioreactor. In one embodiment, the mammalian cells are cultured in a batch feed mode. Examples of bioreactors include, but are not limited to, plug flow bioreactors, continuous stirred tank bioreactors, fixed bed bioreactors, airlift bioreactors, and bioreactor bags. In one embodiment, the maximum viable cell density (VCD) in the production bioreactor is between approximately 4 × 10⁻⁶. 6 18 × 10⁻⁶ cells / mL (cells / mL) 6 Between 6 × 10⁶ cells / mL (e.g., between approximately 6 × 10⁶ cells 6 With approximately 16×10 6 Between, between approximately 8×10 6 With approximately 14 × 10 6 Between, between approximately 10×10 6 With approximately 12×10 6 Between, between approximately 4×10 6 With approximately 14 × 10 6 Between, between approximately 4×10 6 With approximately 10×10 6 Between, between approximately 6×10 6 With approximately 18×10 6 Between, between approximately 6×10 6 With approximately 14 × 10 6 Between, between approximately 6×106 With approximately 10×10 6 Between, between approximately 8×10 6 With approximately 18×10 6 Between, between approximately 8×10 6 With approximately 14 × 10 6 Between, between approximately 8×10 6 With approximately 10×10 6 Between, between approximately 10×10 6 With approximately 18×10 6 Between, between approximately 10×10 6 With approximately 14 × 10 6 Between, between approximately 12×10 6 With approximately 18×10 6 Between, between approximately 12×10 6 With approximately 14 × 10 6 Between or between approximately 14 × 10 6 With approximately 18×10 6 (between). In one implementation, the maximum VCD in the production bioreactor is between approximately 8 × 10⁻⁶. 6 cells / mL and approximately 14 × 10 6 Between [number] cells / mL. In one embodiment, the maximum VCD in the production bioreactor is between 8 × 10 [units / mL]. 6 cells / mL and 14 × 10 6 Between cells / mL.

[0048] In some embodiments, the mammalian cells are selected from the group consisting of: COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HeLa cells, NSO cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells. In one embodiment, the mammalian cell is a CHO cell.

[0049] The cell culture medium used in the methods disclosed herein may comprise any culture medium known to those skilled in the art suitable for culturing mammalian cells.

[0050] In one embodiment, less than about 4% of the generated Fc-containing protein is N-terminally cleaved. In one embodiment, less than about 4%, about 3.9%, about 3.8%, about 3.7%, about 3.6%, about 3.5%, about 3.4%, about 3.3%, about 3.2%, about 3.1%, about 3%, about 2.9%, about 2.8%, about 2.7%, about 2.6%, about 2.5%, about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, or about 1.0% of the generated Fc-containing protein is N-terminally cleaved.

[0051] In one embodiment, less than about 4% of the generated duraglutide is N-terminally cleaved. In one embodiment, less than about 4%, about 3.9%, about 3.8%, about 3.7%, about 3.6%, about 3.5%, about 3.4%, about 3.3%, about 3.2%, about 3.1%, about 3%, about 2.9%, about 2.8%, about 2.7%, about 2.6%, or about 2.5% of the generated duraglutide is N-terminally cleaved. In one embodiment, the N-terminally cleaved duraglutide does not have the N-terminal H1 or G2 residues of duraglutide.

[0052] In one embodiment, less than about 5% (e.g., less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1%, or less than about 0.5%) of the generated Fc-containing protein (e.g., duraglutide) contains G2F glycans. In one embodiment, between about 0.5% and about 4% (e.g., between about 1% and about 3.5%, between about 1.5% and about 3%, between about 1.5% and about 2.6%, or between about 2% and about 2.5%) of the generated Fc-containing protein (e.g., duraglutide) contains G2F glycans. In one embodiment, between about 1.5% and about 2.6% of the generated Fc-containing protein (e.g., duraglutide) contains G2F glycans. In one embodiment, between 1.5% and 2.6% of the generated Fc-containing protein (e.g., duraglutide) contains G2F glycans.

[0053] In one embodiment, less than about 20% (e.g., less than about 19.5%, less than about 19%, less than about 18.5%, less than about 18%, less than about 17.5%, less than about 17%, less than about 16.5%, less than about 16%, less than about 15.5%, less than about 15%, less than about 14.5%, less than about 14%, less than about 13.5%, less than about 13%, less than about 12.5%, less than about 12%, less than about 11.5%, less than about 11%, less than about 10.5%, or less than about 10%) of the generated Fc-containing protein (e.g., duraglutide) contains G1F glycans. In one embodiment, the Fc-containing protein (e.g., duraglutide) produced at between about 8% and about 18% (e.g., between about 9% and about 17%, between about 10% and about 16%, between about 10.6% and about 15.4%, between about 11% and about 15%, or between about 12% and about 14%) contains G1F glycans. In one embodiment, the Fc-containing protein (e.g., duraglutide) produced at between about 10.6% and about 15.4% contains G1F glycans. In another embodiment, the Fc-containing protein (e.g., duraglutide) produced at between 10.6% and 15.4% contains G1F glycans.

[0054] In one embodiment, less than about 90% (e.g., less than about 88%, less than about 86%, less than about 84%, less than about 82%, less than about 80%, less than about 78%, less than about 76%, less than about 74%, or less than about 72%) of the generated Fc-containing protein (e.g., duraglutide) contains GOF glycans. In one embodiment, between about 70% and about 85% (e.g., between about 71% and about 84%, between about 72% and about 83%, between about 73% and about 82%, between about 74% and about 81%, between about 74.7% and about 80%, between about 75% and about 80%, or between about 74% and about 79%) of the generated Fc-containing protein (e.g., duraglutide) contains GOF glycans. In one embodiment, between about 74.7% and about 80% of the generated Fc-containing protein (e.g., duraglutide) contains GOF glycans. In one embodiment, between 74.7% and 80% of the generated Fc-containing protein (e.g., duraglutide) contains G0F glycans.

[0055] In one embodiment, less than about 4% (e.g., less than about 4%, less than about 3.8%, less than about 3.6%, less than about 3.4%, less than about 3.2%, less than about 3%, less than about 2.8%, less than about 2.6%, less than about 2.4%, less than about 2.2%, less than about 2%, less than about 1.8%, less than about 1.6%, less than about 1.4%, less than about 1.2%, less than about 1%, less than about 0.8%, less than about 0.6%, less than about 0.4%, or less than about 0.2%) of the generated Fc-containing protein (e.g., duraglutide) contains Man-5 glycans. In one embodiment, less than about 1% of the generated Fc-containing protein (e.g., duraglutide) contains Man-5 glycans. In one embodiment, less than 1% of the generated Fc-containing protein (e.g., duraglutide) contains Man-5 glycans. III. Contains Fc protein

[0056] The method disclosed herein is used to produce Fc-containing proteins by culturing mammalian cells expressing Fc-containing proteins.

[0057] In one embodiment, the Fc-containing protein comprises one or more amino acid sequences as shown in Table 1 below.

[0058] In one embodiment, the Fc-containing protein comprises a glucagon-like peptide-1 (GLP-1) analog that includes one or more modifications compared to the wild-type GLP-1 amino acid sequence (SEQ ID NO: 1).

[0059] In one embodiment, the Fc-containing protein comprises a GLP-1 analog containing the amino acid sequence of SEQ ID NO: 2.

[0060] In one embodiment, the Fc-containing protein includes a peptide linker. In one embodiment, the C-terminal amino acid of the GLP-1 analog moiety containing the Fc protein is fused to the N-terminus of the Fc moiety of the immunoglobulin via the peptide linker. In one embodiment, the peptide linker comprises 1 to 10 G4S units (SEQ ID NO: 3).

[0061] In one embodiment, the Fc-containing protein comprises: a GLP-1 analog comprising the amino acid sequence of SEQ ID NO: 2; a peptide linker comprising 1 to 10 G4S units (SEQ ID NO: 3); and the Fc moiety of an immunoglobulin. In one embodiment, the N-terminal residue of the peptide linker is directly fused to the C-terminal residue of the GLP-1 analog, and the C-terminal residue of the peptide linker is directly fused to the N-terminal residue of the Fc moiety.

[0062] In one embodiment, the Fc-containing protein comprises the amino acid sequence of SEQ ID NO: 4. In another embodiment, the Fc-containing protein is a homodimer comprising two identical amino acid chains, each amino acid chain comprising the amino acid sequence of SEQ ID NO: 4.

[0063] In one implementation, the Fc-containing protein is duraglutide.

[0064] Duraglutide is a human GLP-1 receptor agonist comprising a dimer of a GLP-1 analog fused at its C-terminus to the N-terminus of an immunoglobulin Fc fragment analog via a (G4S)3 peptide linker. This duraglutide is identified by CAS Registry No. 923950-08-7 and is provided with the following chemical name: 7-37-glucagon-like peptide I [8-glycine, 22-glutamic acid, 36-glycine] (synthetic human) fusion protein with peptide (synthetic 16-amino acid linker) fusion protein with immunoglobulin G4 (synthetic human Fc fragment) dimer. Each monomer of duraglutide has the amino acid sequence shown in SEQ ID NO: 4.

[0065] The two monomers are attached to each other via a disulfide bond between cysteine ​​residues at positions 55 and 58 of SEQ ID NO: 4 to form a dimer. The structure, function, production, and use of duraglutide in the treatment of type 2 diabetes mellitus are described in more detail in U.S. Patent No. 7,452,966 and U.S. Patent Application No. US20100196405. Duraglutide agonizes the GLP-1 receptor, leading to stimulation of insulin synthesis and secretion, and has been shown to provide improved glycemic control in patients with type 2 diabetes mellitus.

[0066] When used herein, the term “duraglutide” refers to any GLP-1 receptor agonist protein dimer having two monomers having the amino acid sequence of SEQ ID NO: 4, including any protein that is the subject of a regulatory submission seeking approval of a GLP-1 receptor agonist product, which is wholly or partially dependent on data relating to duraglutide submitted to a regulatory agency by Eli Lilly and Company, regardless of whether the party seeking approval of the protein actually identifies the protein as duraglutide or uses some other terminology.

[0067]

[0068] In one implementation, the Fc protein is etanercept, afascept, abatacept, linascept, romiscrine, berazip, aflibercept, conbercept, efmoroctocog α, eftrenonacog α, asfotase α, or rotezcept.

[0069] In one embodiment, the Fc protein-containing component is an antibody. In another embodiment, the Fc protein-containing component is not an antibody.

[0070] In one respect, this article provides a method for producing an Fc-containing protein using any of the methods disclosed herein.

[0071] In one respect, this article provides duraglutide produced by any of the methods disclosed herein. Example

[0072] The following examples are provided in an illustrative rather than limiting manner. Example 1: Optimization of the initial duraglutide manufacturing protocol

[0073] This embodiment describes a study evaluating changes in feed parameters during the production bioreactor phase of a duraglutide manufacturing protocol. In the initial duraglutide manufacturing protocol, the feed parameters used in the production bioreactor phase (referred to herein as "initial feed parameters") included a 1.5X concentration nutrient feed on day 6 and a 20 mM glucose feed on day 10. This initial duraglutide manufacturing protocol was optimized to identify upstream conditions and feed parameters that increase viable cell density (VCD) and maintain optimal product quality (e.g., minimal protease shear, favorable glycosylation profile, etc.). When upstream conditions were optimized to increase VCD, the use of the initial feed parameters in the production bioreactor resulted in nutrient depletion prior to protein harvesting, and thus a decrease in duraglutide titer.

[0074] Several follow-up studies assessed the impact of changing the feed parameters used in the production bioreactor phase, resulting in an updated duraglutide manufacturing protocol, comprising two nutrient feeds (0.7X concentration nutrient feeds on days 3 and 6) and three glucose feeds (30 mM glucose feeds on days 8, 10, and 12). The updated feed parameters for the duraglutide manufacturing protocol are referred to as the "updated feed parameters" in this example. These studies demonstrated an increase in VCD and duraglutide titer compared to the initial duraglutide manufacturing protocol. Figure 1 However, it also demonstrates increased protease cleavage of duraglutide (e.g., cleavage of H1 residues or both H1 and G2 residues from the N-terminus, resulting in the des H / HG variant of duraglutide). Figure 2The undesirable shift in glycoform distribution was observed (as shown in Table 2). Based on this, it was suspected that certain nutrients might be limited near the end of the culture period in the updated duraglutide manufacturing protocol. Further analysis showed that L-tyrosine levels were depleted by day 14. In the context of this example and Example 2 below, “X” with respect to L-tyrosine refers to the addition of 5 g of a 3.5% L-tyrosine solution per kilogram of mass in the bioreactor, or “5 g / kg”. Thus, for example, adding 1.5X of an L-tyrosine-containing nutrient feed means adding 7.5 g / kg, and 0.7X means adding 3.5 g / kg.

[0075] Example 2: Optimizing feed parameters to maximize product quality

[0076] As described in Example 1, optimization of the initial duraglutide manufacturing protocol resulted in increased protease cleavage and an undesirable shift in glycoform distribution. This example describes a study evaluating further changes to the feed parameters of the updated duraglutide manufacturing protocol. Specifically, the effects of a higher L-tyrosine feed and the addition of a third nutrient feed during the production bioreactor phase on VCD, duraglutide titer, and product quality (e.g., protease cleavage level, glycosylation profile, etc.) were evaluated. Separate studies were conducted, and it was shown that adjusting the feed concentration from 0.7X to 0.8X resulted in a more optimized glucose profile without significantly affecting the titer. Therefore, the control nutrient and tyrosine feed were adjusted from 0.7X (e.g., as described in Example 1) to 0.8X. As mentioned above, the "X" in L-tyrosine refers to adding 5g of 3.5% L-tyrosine solution per kg of mass in the bioreactor. Therefore, adding 0.7X L-tyrosine feed means adding 3.5g / kg, and adding 0.8X L-tyrosine feed means adding 4g / kg.

[0077] For all conditions, when the glucose level was measured to be less than 15 mM, the glucose feed was set at 30 mM. The tested feed parameters are shown in Table 3 below.

[0078]

[0079] Similar growth and vigor were observed across all three conditions, indicating that the added nutrient feed and higher tyrosine concentrations did not affect growth. Similarly, no significant differences in titers were observed across all three conditions.

[0080] Product quality was measured using multiple assays across three conditions. The protease cleavage of duraglutide was assessed by measuring the des H / HG level. Figure 3Compared to control conditions, the des H / HG levels decreased by approximately 10% and 17%, respectively, with the addition of tyrosine and nutrients. Measurements of glycoform levels shown in Table 4 indicate that the added nutrient feed conditions resulted in acceptable levels of glycoform for all measurements.

[0081]

[0082] The scope of this invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications are intended to fall within the scope of the appended claims.

[0083] Other embodiments are within the scope of the following claims.

Claims

1. A method for producing duraglutide, the method comprising culturing mammalian cells expressing said duraglutide in a production bioreactor for a total culture time, wherein: a) After the first part of the total culture time, a first nutrient feed is supplied to the production bioreactor; b) After the second part of the total culture time, a second nutrient feed is supplied to the production bioreactor; as well as c) After the third portion of the total culture time, a third nutrient feed is supplied to the production bioreactor. This causes the production of the duraglutide by the mammalian cells.

2. The method according to claim 1, wherein the first nutrient feed, the second nutrient feed and / or the third nutrient feed comprises L-tyrosine.

3. The method according to claim 1 or 2, wherein each of the first nutrient feed, the second nutrient feed and the third nutrient feed comprises L-tyrosine.

4. The method according to claim 2 or 3, wherein the L-tyrosine is supplied as a 3.5% solution.

5. The method according to any one of claims 2 to 4, wherein the L-tyrosine is supplied to the production bioreactor at a concentration of about 4 g / kg.

6. The method according to any one of the preceding claims, wherein the total culture time is 12 to 16 days.

7. The method according to any one of the preceding claims, wherein the total culture time is about 14 days.

8. The method according to any one of the preceding claims, wherein the first portion of the total culture time is 2 to 4 days after the start of the culture.

9. The method according to any one of the preceding claims, wherein the first nutrient is supplied approximately 3 days after the start of the culture.

10. The method according to any one of the preceding claims, wherein the second portion of the total culture time is 5 to 7 days after the start of the culture.

11. The method according to any one of the preceding claims, wherein the second nutrient is supplied approximately 6 days after the start of the culture.

12. The method according to any one of the preceding claims, wherein the third portion of the total culture time is 8 to 10 days after the start of the culture.

13. The method according to any one of the preceding claims, wherein the third nutrient is supplied approximately 9 days after the start of the culture.

14. The method according to any one of the preceding claims, wherein the first nutrient is supplied approximately 3 days after the start of the culture, the second nutrient is supplied approximately 6 days after the start of the culture, and the third nutrient is supplied approximately 9 days after the start of the culture.

15. The method according to any one of the preceding claims, wherein the maximum viable cell density (VCD) in the production bioreactor is between 8 × 10⁻⁶. 6 Cells / mL and 14 × 10 6 Between cells per milliliter.

16. The method according to any one of the preceding claims, wherein the mammalian cells are selected from the group consisting of: COS cells, CHO cells, BHK cells, MDCK cells, HEK293 cells, HEK293T cells, HeLa cells, NSO cells, PER.C6 cells, VERO cells, CRL7O3O cells, HsS78Bst cells, NIH 3T3 cells, HepG2 cells, SP210 cells, R1.1 cells, BW cells, LM cells, BSC1 cells, BSC40 cells, YB / 20 cells, and BMT10 cells.

17. The method according to any one of the preceding claims, wherein the mammalian cell is a CHO cell.

18. The method according to any one of the preceding claims, wherein less than about 4% of the generated duraglutide is an N-terminal cleaved variant.

19. The method of claim 16, wherein the N-terminal cleaved variant lacks an N-terminal histidine residue or an N-terminal histidine residue and a glycine residue.

20. The method according to any one of the preceding claims, wherein the duraglutide produced at a concentration between 1.5% and 2.6% comprises G2F polysaccharide.

21. The method according to any one of the preceding claims, wherein the duraglutide produced at a concentration between 10.6% and 15.4% comprises G1F glycans.

22. The method according to any one of the preceding claims, wherein the duraglutide produced at a concentration between 74.7% and 80% comprises G0F glycans.

23. The method according to any one of the preceding claims, wherein less than 1% of the produced duraglutide comprises Man-5 polysaccharide.

24. Duraglutide, said duraglutide being produced by the method according to any one of the preceding claims.

25. A composition comprising duraglutide, wherein the duraglutide is prepared by a process comprising the method according to any one of the preceding claims.

26. The composition of claim 25, wherein less than about 4% of the generated duraglutide is an N-terminal cleaved variant.

27. The composition according to claim 25 or 26, wherein the duraglutide produced in amounts between 1.5% and 2.6% comprises G2F polysaccharide.

28. The composition according to any one of claims 25 to 27, wherein the duraglutide produced in an amount between 10.6% and 15.4% comprises G1F polysaccharide.

29. The composition according to any one of claims 25 to 28, wherein the duraglutide produced in amounts between 74.7% and 80% comprises GOF polysaccharides.

30. The composition according to any one of claims 25 to 29, wherein less than 1% of the generated duraglutide comprises Man-5 polysaccharide.

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