Processes for recombinant protein production

Abstract

The present invention relates to the field of recombinant production of proteins in host cells. In particular, the present invention relates to a process for producing recombinant proteins in host cells, which process reduces the misincorporation of norleucine in place of methionine.

Description

[Technical Field]

[0001] The present invention relates to the field of recombinant production of proteins in host cells. In particular, the present invention relates to a process for culturing host cells for producing recombinant proteins to reduce the misincorporation of norleucine in place of methionine. [Background technology]

[0002] In the pharmaceutical field, the use of biological entities such as proteins, e.g., antibodies or antibody-derived molecules, continues to grow in presence and importance. This use creates a need for controlled manufacturing processes. The commercialization of proteins for medical applications requires their large-scale production, and much effort has been expended on improving the cultivation of recombinant host cells expressing the desired proteins and their processing. This has resulted in increased product titers, but in many cases, large amounts of undesired by-products and product heterogeneity are observed. Removal of these undesired by-products or product variants can be difficult, so it is preferable to optimize the manufacturing process to minimize their formation.

[0003] One undesirable product variation results from the misincorporation of norleucine into proteins instead of methionine. Norleucine is an unnatural amino acid synthesized by enzymes in the leucine biosynthetic pathway of Escherichia coli (E. coli). It is a structural analog of methionine, and methionyl-tRNA synthetase (MetRS) uses norleucine as a substrate. Although less efficient than methionine, it can substitute for methionine residues in proteins by charging methionyl-tRNA during the translation process.

[0004] It has been known since the 1950s that many heterologous proteins, when expressed in Escherichia coli (E. coli), have misincorporated norleucine residues where methionine residues should occur (Munier and Cohen 1956 and Nisman and Hirsch 1958). Norleucine misincorporation is undesirable because it results in the production of denatured proteins, i.e., proteins with different primary amino acid sequences and potentially unknown properties. It has been shown that misincorporation of unnatural amino acids can alter the 3D structure of proteins and lead to aggregation. Norleucine misincorporation occurs to varying degrees in production batches, resulting in product batch heterogeneity.

[0005] Although increasing the methionine concentration in the cell culture medium can reduce norleucine misincorporation (Tsai et al., Biochem Biophys Res Comm 156:733, 1988; Bogosian et al., J Biol Chem 264:531, 1989; U.S. Patent No. 5,599,690 and WO 2007 / 103521), this has various drawbacks, including increased operational complexity and production costs (Veeravalli and Laird, Bioengineered 6:132, 2015). Additional methods have been developed to reduce norleucine incorporation into recombinant proteins, such as expressing norleucine degrading enzymes (U.S. Patent No. 8,603,781) or deleting genes involved in norleucine biosynthesis (Bogosian et al., J Biol Chem 264:531, 1989).

[0006] However, genetic modification of host cell lines can be tedious and can have other unexpected or unidentified effects. Modification of cell culture media can result in increased formation of other undesirable by-products and / or have other negative impacts on cell culture performance. Therefore, there remains a need for new processes to prevent or reduce norleucine misincorporation in proteins during production. This need is addressed by the present invention. Summary of the Invention

[0007] In a first embodiment, the present invention relates to a process for producing a recombinant protein, comprising: a) providing a host cell capable of producing a recombinant protein; b) providing a quantity of liquid medium containing 0 to 1 g of methionine per kg of liquid medium; c) culturing the host cells in a liquid medium; d) inducing the production of the recombinant protein in the liquid medium; and e) adding a certain amount of methionine to the liquid medium during or after induction while culturing the host cells to produce the recombinant protein, The amount of methionine added per kg of the liquid medium in step (e) is greater than the amount of methionine contained in the liquid medium per kg of the liquid medium in step (b).

[0008] In a second embodiment, the present invention relates to a process for reducing norleucine misincorporation during the production of a recombinant protein, comprising: a) providing a host cell capable of producing a recombinant protein; b) providing a quantity of liquid medium containing 0 to 1 g of methionine per kg of liquid medium; c) culturing the host cells in a liquid medium; d) inducing the production of the recombinant protein in the liquid medium; and e) adding a certain amount of methionine to the liquid medium during or after induction while culturing the host cells to produce the recombinant protein, The amount of methionine added per kg of the liquid medium in step (e) is greater than the amount of methionine contained in the liquid medium per kg of the liquid medium in step (b).

[0009] In a still further embodiment, the present invention relates to a recombinant protein preparation obtainable or obtained by a process according to any one of the preceding claims. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the growth profiles of various batches of Escherichia coli (E. coli) as determined by optical density at 600 nm (OD600) during fermentation in a 200 L vessel as described in Example 1 without added methionine, with added methionine before induction [step (c)] and at or after induction [step (e)], or only at or after induction [step (e)]. [Figure 2] FIG. 1 shows the cell viability of different batches of Escherichia coli (E. coli) fermenting in a 200 L vessel as described in Example 1 without methionine, with methionine added before induction [step (c)] and either before or after induction [step (e)], or only during or only after induction [step (e)]. [Figure 3] Figure 1 shows the Fab' titers in various batches after harvest of E. coli fermentation in a 200 L vessel as described in Example 1 without methionine, with methionine added before induction [step (c)] and during or after induction [step (e)] or only during or only after induction [step (e)]. [Figure 4] 1 shows the average norleucine content per methionine residue of Fab′ produced in batches of E. coli fermentation in a 200 L vessel described in Example 1 without methionine, with methionine added before induction [step (c)] and during or after induction [step (e)], or only during or after induction [step (e)]. [Figure 5] Figure 1 shows the growth profiles as determined by OD600 for different batches of E. coli fermentation in a 15,000 L vessel as described in Example 2 without added methionine, with added methionine before induction [step (c)] and either before induction or after induction [step (e)], or only during induction or only after induction [step (e)]. [Figure 6]1 shows the average norleucine content per methionine residue of Fab′ produced in batches of E. coli fermentation in a 15,000 L vessel described in Example 2 without methionine, with methionine added before induction [step (c)] and during or after induction [step (e)], or only during or after induction [step (e)]. [Figure 7] The average norleucine content per methionine residue is shown for three different manufacturing processes (Process A, Process B, and Process C) resulting in different amounts of product, all operated at the same scale to produce the same Fab'. Process A is a low productivity process in which no methionine is added to the feed, while Processes B and C are improved high productivity processes. Process B does not add methionine to the feed, while Process C is performed as described in this invention. [Figure 8] The average amount of norleucine per methionine residue for processes A and C is shown. DETAILED DESCRIPTION OF THE INVENTION

[0011] It has surprisingly been found by the inventors of the present invention that the addition of methionine to cell culture media during the growth and expansion phase of cell culture reduces or inhibits cell proliferation.

[0012] Based on this surprising finding, the present inventors have devised a new and improved manufacturing process that overcomes the problems associated with processes known in the art for reducing norleucine misincorporation during protein manufacturing.

[0013] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, the terms "including," "includes," "having," "has," "with," or variations thereof, wherever used in either the detailed description and / or claims, are intended to be inclusive, just like the term "comprising." The transitional phrases (and any grammatical variations thereof) "comprising," "comprises," and "comprise" include the phrases "consisting essentially of," "consists essentially of," "consisting," and "consists," which may be used interchangeably. The phrase "consisting essentially of" indicates that the claim includes embodiments containing specified materials or steps, and those that do not materially affect the basic and novel characteristics of the claim.

[0014] The present invention relates to a process for culturing cells to produce recombinant proteins, in which methionine is added to the culture medium in a specific manner to minimize norleucine misincorporation, thereby minimizing the negative effects of methionine on cell growth.

[0015] Therefore, in a first embodiment, the present invention relates to a process for producing a recombinant protein, comprising: a) providing a host cell capable of producing a recombinant protein; b) providing a quantity of liquid medium containing 0 to 1 g of methionine per kg of liquid medium; c) culturing the host cells in a liquid medium; d) inducing the production of the recombinant protein in the liquid medium; and e) adding a certain amount of methionine to the liquid medium during or after induction while culturing the host cells to produce the recombinant protein, The amount of methionine added per kg of the liquid medium in step (e) is greater than the amount of methionine contained in the liquid medium per kg of the liquid medium in step (b).

[0016] In a second embodiment, the present invention relates to a process for reducing norleucine misincorporation during the production of a recombinant protein, comprising: a) providing a host cell capable of producing a recombinant protein; b) providing a quantity of liquid medium containing 0 to 1 g of methionine per kg of liquid medium; c) culturing the host cells in a liquid medium; d) inducing the production of the recombinant protein in the liquid medium; and e) adding a certain amount of methionine to the liquid medium during or after induction while culturing the host cells to produce the recombinant protein, The amount of methionine added per kg of the liquid medium in step (e) is greater than the amount of methionine contained in the liquid medium per kg of the liquid medium in step (b).

[0017] In step (a), a host cell is provided that is capable of producing a recombinant protein upon induction. The host cell used in the process of the present invention can be any host cell that is suitable for the recombinant production of proteins and that is capable of growing under certain conditions. Suitable host cells include bacterial host cells and other cells that may exhibit misincorporation of norleucine for methionine.

[0018] In a third embodiment, the host cell in the process according to any one of the first, second or other embodiments of the present invention is a bacterial host cell, such as an E. coli cell or another Gram-negative bacterial cell or a Gram-positive bacterial cell, such as Staphylococcus aureus. In a more preferred embodiment of the third embodiment, the host cell is an E. coli host cell, even more preferably strain HB101, strain B7, strain K12, strain RV308, strain DH1, strain HMS174, strain W3110 or strain BL21.

[0019] Typically, a nucleic acid sequence encoding a recombinant protein under the control of an inducible promoter is introduced into a host cell. Suitable vectors for expressing such nucleic acid constructs in host cells and processes for transforming host cells are well known in the art. Suitable inducible promoters are also well known in the art, and some non-limiting examples are mentioned herein below.

[0020] In step (c) of the process according to any one of the embodiments of the invention described herein, the host cells are cultured. Processes and media for culturing various types of host cells are well known in the art. The media vary depending on the organism, but may include components such as a carbon source, a nitrogen source, amino acids, vitamins, essential metal ions, and trace elements. Step (c) preferably involves fed-batch culture, more preferably fed-batch culture in a bioreactor. A fed-batch phase may follow the batch phase. Inoculation may be performed directly from a working cell bank or via a seed culture, such as in a shake flask.

[0021] In a fourth embodiment of the present invention, step (c) of the process according to any one of the first, second, third or other embodiments of the present invention comprises growing the culture to an OD600 (optical density at a wavelength of 600 nm) of at least 20, such as at least 25, at least 35, at least 50, at least 55, at least 60, at least 70 or at least 80.

[0022] In a fifth embodiment, the liquid medium in step (b) and / or step (c) of the process according to any one of the first, second, third, fourth or other embodiments of the present invention contains less than 1 g of methionine per kg of liquid medium, for example less than 1 g / kg, such as less than 0.5 g / kg, less than 0.25 g / kg, less than 0.20 g / kg, less than 0.15 g / kg, less than 0.10 g / kg, in each case per kg of liquid medium. Preferably, the methionine concentration in the liquid medium in step (b) and / or step (c) is 0 to 0.25 g / kg or 0 to 0.5 g / kg.

[0023] In a sixth embodiment, the liquid medium in step (b) and / or step (c), meaning before induction, of the process according to any one of the first, second, third, fourth, fifth or other embodiments of the present invention does not contain methionine.

[0024] In a seventh embodiment, the liquid medium in step (b) and / or step (c), meaning before induction, of the process according to any one of the first, second, third, fourth, fifth, sixth or other embodiments of the present invention does not contain isoleucine.

[0025] In an eighth embodiment, the liquid medium in step (b) and / or step (c), meaning before induction, of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh or other embodiments of the present invention does not contain leucine.

[0026] In a ninth embodiment, step (c) of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth or other embodiments of the present invention comprises: i) culturing the host cells in a batch culture to an OD600 of 20-55, optionally with the addition of a bolus amount (i.e., a single dose added at once) of a magnesium salt, such as magnesium sulfate; ii) further culturing the bacterial host cells so that the dissolved oxygen (DO) increases to 50% or greater air saturation; iii) culturing the cells in a fed-batch culture until the OD of the liquid culture medium increases to at least 15, 20, 25, 35, 40, or 50 units greater than the OD of step (c)(i).

[0027] In a tenth embodiment, a feed containing a carbon source is added to initiate step (c)(iii) of the process of the present invention described in the ninth embodiment. Preferably, the amount of carbon source added to the liquid medium per unit time is lower in step (e) than in step (c)(iii), for example by reducing the amount of feed or reducing the concentration of the carbon source in the feed. In a preferred embodiment, no methionine is present in the liquid medium or is added to the liquid medium before the addition of the feed comprising a carbon source according to the tenth embodiment of the present invention.

[0028] In an eleventh embodiment, induction of recombinant protein production as described in step (d) of the process of any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or other embodiments of the present invention is initiated when dissolved oxygen (DO) increases to 50% air saturation in the liquid medium or when a predetermined OD600 is reached as described in step (c)(iii) of the ninth embodiment. DO may be measured by any standard means, such as an online polarographic dissolved oxygen sensor, an optical dissolved oxygen sensor or any suitable oxygen sensing technique.

[0029] In a preferred embodiment, the process according to any one of the embodiments of the present invention produces no recombinant protein or less than 0.1 g of recombinant protein per kg of liquid medium prior to the induction according to step (d).

[0030] Induction of recombinant protein production can be achieved by any suitable method. In one embodiment, in the process described in any one of the embodiments of the present invention, the gene encoding the recombinant protein is under the control of an inducible promoter. Inducible promoters are known in the art. A well-known bacterial expression system using an inducible promoter is a system in which the gene encoding the recombinant protein is placed under the control of a lac-type promoter that can be induced by IPTG (isopropyl β-Dl-thiogalactopyranoside). Other known bacterial expression systems include, for example, the arabinose promoter system (e.g., Guzman et al., J Bacteriol 177:4121, 1995) or the T7 system (e.g., Rosenberg et al., Gene 56:125, 1987). These and other systems are reviewed, for example, in Rosano and Ceccarelli, Front Microbiol 5:172, 2014.

[0031] In a preferred embodiment of the process of the present invention, the host cell of the process according to any one of the embodiments of the present invention comprises a nucleic acid sequence encoding a recombinant protein under the control of an IPTG-inducible promoter, thereby producing the recombinant protein upon induction with IPTG. In one such embodiment, step (d) comprises the addition of IPTG.

[0032] Step (e) of the process according to any one of the embodiments of the present invention typically involves fed-batch cultivation in a bioreactor. In one embodiment, the duration of step (e) of the process according to any one of the embodiments of the present invention is from about 12 to about 96 hours, such as from about 20 to about 72 hours, for example from about 25 to about 55 hours, such as from about 24 to 48 hours, or 30 to about 50 hours, or 35 to 45 hours, or 36 to 48 hours. In the context of hours, the term "about" is intended to include ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, or ±10 hours.

[0033] In a twelfth embodiment of the present invention of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or other embodiments of the present invention, step (e) is followed by step (f) of recovering the host cells. Preferably, the amount of methionine added in step (e) is such that the concentration of methionine in the liquid medium at or immediately before the recovery in step (f) is at least 0.25 g / kg, such as 0.25 g / kg to 1.5 g / kg, preferably at least 0.40 g / kg, such as 0.40 g / kg to 1.2 g / kg. More preferably, the amount added in step (e) is such that the concentration of methionine in the liquid medium at or immediately before the recovery in step (f) is 0.45 g / kg to 1.10 g / kg, for example 0.50 g / kg to 0.9 g / kg.

[0034] In a thirteenth embodiment of the present invention, the amount of methionine added in step (e) of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or other embodiments of the present invention is at least 0.25 g / kg, such as 0.25 g / kg to 2.0 g / kg, of the liquid medium provided in step (b), preferably at least 0.50 g / kg, such as 0.50 g / kg to 1.2 g / kg of the liquid medium provided in step (b). More preferably, the amount is 0.52 g to 1.10 g per kg of the liquid medium of step (b), for example 0.55 g to 1.05 g per kg of the liquid medium provided in step (b).

[0035] In a fourteenth embodiment, leucine is not added during step (e) of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or other embodiments of the present invention.

[0036] In a fifteenth embodiment, isoleucine is not added during step (e) of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or other embodiments of the present invention.

[0037] In this disclosure, ranges are abbreviated to avoid the need to specify and describe each and every value within a range. Any appropriate value within the range can be selected, and where appropriate, this can be selected as the upper, lower, or terminus of the range. For example, a range of 0.1 to 1.0 represents the end values ​​of 0.1 and 1.0, as well as intermediate values ​​of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9, and all intermediate ranges subsumed within 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, and 0.7 to 1.0. When ranges are used herein, specific embodiments of different combinations and subcombinations of these ranges (e.g., subranges within the disclosed ranges) are intended to be expressly included.

[0038] carbon source The process of the present invention typically involves the addition of one or more organic carbon sources. The carbon source used can be a single type of carbon source or a mixture of different carbon sources. Suitable carbon sources include, for example, glucose, lactose, arabinose, glycerol, sorbitol, galactose, xylose, or mannose. As an example, in the liquid medium in step (b), more than 75% of the carbon source, for example, at least 90% of the carbon source, consists of glycerol. In another preferred embodiment, more than 75% of the carbon source, for example, at least 90% of the carbon source, in the liquid medium in step (e) of the process of the present invention consists of glycerol. As another example, more than 75% of the carbon source, for example, at least 90% of the carbon source, in the liquid medium in step (c) consists of glycerol. In another preferred embodiment, more than 75% of the carbon source, for example, at least 90% of the carbon source, in the liquid medium in step (e) consists of glycerol. As a further example, more than 75% of the carbon source, for example, at least 90% of the carbon source, in the liquid medium in step (c) consists of lactose. In another preferred embodiment, more than 75%, such as at least 90%, of the carbon source in the liquid medium in step (e) consists of lactose.

[0039] pH The pH of the cell culture medium during fermentation is very important for product yield and processability of the cell culture slurry. It has been described that struvite formation is affected by pH (see, for example, Perez-Garcia et al., 1989). In one embodiment of the process of the present invention, the pH of step (c) of the process according to any of the embodiments of the present invention is greater than 6.5, such as 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, or greater than about 6.5, such as about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, or about 7.2, and the pH of the culture in step (e) is greater than 6.5, such as 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, or greater than about 6.5, such as about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, or about 7.2. In another embodiment, the pH in step (c) is 6.5 to 7.5, for example, 6.6 to 7.4, or 6.7 to 7.3, for example, 6.8 to 7.2, and the pH in step (e) is 6 to 8, for example, 6.5 to 7.5, for example, 6.6 to 7.4, or 6.7 to 7.3, for example, 6.8 to 7.2. In another embodiment, the pH in step (c) is about 6 to about 8, for example, about 6.5 to about 7.5, for example, about 6.6 to about 7.4, or about 6.7 to about 7.3, for example, about 6.8 to about 7.2, and the pH in step (e) is about 6 to about 8, for example, about 6.5 to about 7.5, for example, about 6.6 to about 7.4, or about 6.7 to about 7.3, for example, about 6.8 to about 7.2. In the context of pH, the term "about" is intended to include ±0.1, ±0.2, or ±0.3 pH units.

[0040] temperature Temperature is typically kept as constant as possible throughout the entire fermentation process. In certain embodiments, the temperature is maintained at a constant temperature of 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C. In other embodiments, the temperature may be maintained at a constant temperature of about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, or about 35°C. In the context of temperature, the term "about" is intended to include ±1°C, ±2°C, or ±3°C, or the set temperature (e.g., a range of ±0°C to ±3°C around the set temperature).

[0041] Recombinant proteins The recombinant protein produced in the process of the invention is typically a heterologous protein derived from another organism, for example, the recombinant protein may be an antibody, cytokine, growth factor, hormone or other peptide or polypeptide, or a fusion protein derivative of any of the foregoing.

[0042] In a preferred embodiment, the recombinant protein is an antibody. As used herein, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies produced by recombinant techniques known in the art. "Antibody" refers to antibodies of any species, particularly mammalian species; e.g., IgG1, IgG 2a , IgG 2bhuman antibodies of any isotype, including antibodies produced as dimers of this basic structure, including IgG, IgG3, IgG4, IgE, IgD, and IgGA1, IgGA2, or pentamers such as IgM, and modified variants thereof; non-human primate antibodies, for example from chimpanzee, baboon, rhesus, or cynomolgus monkey; rodent antibodies, for example from mouse or rat; rabbit, goat, or horse antibodies; camelid antibodies (e.g., from camel or llama, such as Nanobodies™) and derivatives thereof; antibodies of avian species, such as chicken antibodies; or antibodies of fish species, such as shark antibodies. The term "antibody" also refers to "chimeric" antibodies, in which a first portion of at least one heavy and / or light chain antibody sequence is derived from a first species and a second portion of the heavy and / or light chain antibody sequence is derived from a second species. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences. "Humanized" antibodies are chimeric antibodies comprising sequences derived from a non-human antibody. In most cases, humanized antibodies are human antibodies (recipient antibodies) in which residues from the recipient's hypervariable regions are replaced by residues from the hypervariable regions [or complementarity-determining regions (CDRs)] of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken, or non-human primate, possessing the desired specificity, affinity, and activity. In most cases, residues from the human (recipient) antibody outside the CDRs, i.e., within the framework regions (FRs), are additionally replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the application of antibodies to the treatment of human diseases. Humanized antibodies and several different techniques for producing them are well known in the art. The term "antibody" also refers to human antibodies, which can be produced as an alternative to humanization. For example, it is possible to produce transgenic animals (e.g., mice) that, upon immunization, can produce a full repertoire of human antibodies in the absence of endogenous mouse antibody production.Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies, such as phage display or ribosome display, which use recombinant DNA libraries generated at least in part from artificial or donor immunoglobulin variable (V) domain gene repertoires. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be generated from isolated human B cells that are ex vivo immunized with an antigen of interest and subsequently fused to generate hybridomas, which can then be screened for optimal human antibodies. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. Furthermore, as used herein, the term "antibody" refers not only to full-length antibodies but also to antibody fragments. Antibody fragments contain at least one heavy or light chain immunoglobulin domain known in the art and bind to one or more antigens. Examples of antibody fragments according to the invention include Fab, modified Fab, Fab', modified Fab', F(ab'), Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv, and Bis-scFv fragments. The fragments may also be diabodies, tribodies, triabodies, tetrabodies, minibodies, single domain antibodies (dAbs), such as sdAbs, VL, VH, VHH, or camelid antibodies (e.g., derived from camels or llamas, such as Nanobodies™), and VNAR fragments. Antigen-binding fragments according to the invention may also comprise a Fab linked to one or two scFvs or dsscFvs, each of which binds to the same or different targets (e.g., one scFv or dsscFv that binds a therapeutic target and one scFv or dsscFv that increases half-life, e.g., by binding to albumin). Examples of such antibody fragments are FabdsscFv (also called BYbe®) or Fab-(dsscFv)2 (also called TrYbe, see e.g. WO 2015 / 197772). Antibody fragments as defined above are known in the art.In a preferred embodiment, the recombinant protein produced is a Fab or Fab' fragment. In a further preferred embodiment, the recombinant protein is certolizumab pegol, dapirolizumab pegol, ranibizumab, abciximab, blinatumomab, idarucizumab, moxetumomab pasudotox, caplacizumab, or brolucizumab.

[0043] The process according to any one of the embodiments of the present invention can in principle be carried out in any suitable vessel, such as a shake flask or a bioreactor, which may or may not be operated in fed-batch mode, depending, for example, on the scale of production required.

[0044] In a sixteenth embodiment, at least steps (c), (d), and (e) of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, or other embodiments of the present invention are carried out in a bioreactor, preferably an industrial-scale bioreactor. The bioreactor may be, for example, a stirred tank or airlift reactor. The bioreactor may be a reusable reactor made of glass or metal, for example, stainless steel, or a single-use bioreactor made of synthetic materials, such as plastic.

[0045] In a seventeenth embodiment, at least step (e) of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth or other embodiments of the present invention comprises: The reaction is carried out in a bioreactor having a volume of 100 L or more, 500 L or more, 1,000 L or more, 2,000 L or more, 5,000 L or more, 10,000 L or more, or 20,000 L or more, 1,000 to 30,000 L, 5,000 to 30,000 L, 10,000 to 30,000 L, 1,000 to 20,000 L, 5,000 L to 20,000 L, 10,000 L to 20,000 L, or 10,000 to 25,000 L.

[0046] In the eighteenth embodiment, step (b), step (c) of the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or other embodiments of the present invention are carried out. ), step (d) or step (e), the liquid medium has a volume of 100 L or more, 500 L or more, 1,000 L or more, 2,000 L or more, 5,000 L or more, 10,000 L or more, 20,000 L or more, 1,000 to 30,000 L, 5,000 to 30,000 L, 10,000 to 30,000 L, 1,000 to 20,000 L, 5,000 to 20,000 L, 10,000 to 20,000 L, or 10,000 to 25,000 L. In a further preferred embodiment of the process according to any one of the embodiments of the present invention, in all of steps (b), (c), (d), and (e), the culture has a volume of 100 L or more, 500 L or more, 1,000 L or more, 2,000 L or more, 5,000 L or more, 10,000 L or more, or 20,000 L or more, 1,000 to 30,000 L, 5,000 to 30,000 L, 10,000 to 30,000 L, 1,000 to 20,000 L, 5,000 to 20,000 L, 10,000 to 20,000 L, or 10,000 to 25,000 L.

[0047] The process according to any one of the embodiments of the present invention may comprise one or more further steps after step (e). For example, the process may comprise the further step of recovering the recombinant protein, which may involve first separating the cells from the supernatant or inclusion bodies. Once recovered, the recombinant protein can be isolated and purified. Isolation and purification processes are well known to those skilled in the art. These typically consist of a combination of various chromatography and filtration steps. The process of the present invention may further comprise the step of formulating the recombinant protein into a pharmaceutical composition that is suitable for medical use, e.g., therapeutic or prophylactic use. In one embodiment, the recombinant protein is modified, such as by conjugation to another molecule, before being formulated into a pharmaceutical composition.

[0048] In a nineteenth embodiment, the process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth or other embodiments of the invention comprises lyophilizing a composition comprising a recombinant antibody produced according to any of the process embodiments of the invention.

[0049] In a further embodiment of the present invention, the antibody preparation is a recombinant protein preparation such as a preparation comprising certolizumab pegol, dapirolizumab pegol, ranibizumab, abciximab, blinatumomab, idarucizumab, moxetumomab pasudotox, caplacizumab, brolucizumab, wherein the antibody preparation is obtained or obtainable according to a process according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth or other embodiments of the present invention.

[0050] Detection and quantification of norleucine misincorporation Methods for detecting norleucine misincorporation are known in the art and are reviewed in Steele et al., Proteomes 9(1):2, 2021. A preferred method for analyzing and quantifying norleucine misincorporation is mass spectrometry. [Example]

[0051] Example 1 A frozen cell bank vial containing Escherichia coli (E. coli) W3110 host cells expressing antibody A (Fab' fragment with a pI ranging from 8.8 to 9.3) was used to inoculate a shake flask containing 6x peptone yeast extract (6xP-Y) medium plus tetracycline. The shake flask was incubated at 30°C and 200-250 rpm. At the required OD range, the shake flask was used to inoculate a seed fermentor containing chemically defined medium (derived from MD medium by Durany et al. 2004) plus tetracycline with a carbon source. The cell culture in the seed fermentor was maintained at 30°C. At the required OD range, the seed culture was used to inoculate a production fermentor (175 kg liquid medium) containing the same chemically defined medium used in the seed fermentor. The production fermentor was maintained under the same conditions as the seed fermentor and grown in batch phase until the carbon source was exhausted. During this time, a bolus of MgSO4 was added to avoid depletion of this metabolite. At the end of the batch phase (signaled by a spike in the measured DO), the culture was switched to an exponential carbon source feed [containing various amounts of methionine equivalent to 0.70 g per kg of liquid medium provided in step (b)], varying depending on the batch, to obtain an OD600 greater than 50 units. At this point, the carbon source feed was switched from the exponential phase feed to a production phase feed [containing various amounts of methionine equivalent to 0.50 g to 1.5 g per kg of liquid medium provided in step (b)], and expression was induced by the addition of IPTG. Cells (containing expressed antibody A) were harvested more than 40 hours after induction.

[0052] Cells were harvested by continuous centrifugation. The concentrated cell slurry was resuspended back to the original cell harvest concentration by adding deionized water and concentrated Tris-EDTA extraction buffer to obtain the required buffer concentration. For heat removal, cells were maintained at an elevated temperature with mixing for a specified period of time.

[0053] Fab' concentration: The recovered Fab' concentration was determined using Protein G HPLC analysis in 20 mM phosphate buffer. Elution was by a pH gradient decreasing from pH 7.4 at injection to pH 2.7.

[0054] Protein L purification: Extract samples were purified by Protein L affinity chromatography using a 600 μL Capto L® column to purify the cell extract prior to norleucine misincorporation analysis. The column was prepared by flushing with phosphate / sodium chloride buffer (Buffer A), washing with sodium hydroxide solution, and equilibration with Buffer A. After sample loading, washing with Buffer A was performed before elution with glycine buffer. Elution fractions were appropriately collected, and a representative Fab' sample was recovered for norleucine misincorporation analysis.

[0055] Analysis of norleucine misincorporation: To perform the analysis, samples were enzymatically digested with trypsin to fragment peptides, which were then separated using liquid chromatography prior to online analysis using electrospray ionization by mass spectrometry. Mass spectrometry measures the mass-to-charge ratio of peptides, from which mass can be inferred. Peptide mass is a highly specific characteristic of a peptide's sequence. The retention time and mass of each observed peptide are unique to the peptide's amino acid sequence, allowing comparison of the observed peptide mass and retention time with those of a theoretical sequence. The high sensitivity of mass spectrometry allows for the detection of low-abundance protein modifications.

[0056] Substitution of a methionine residue with a norleucine residue reduces the mass of the peptide by 17.9564 Da. The mass shift combined with tandem mass spectrometry (MSMS fragmentation) allows for the identification of peptides containing norleucine substitutions. A semiquantitative assessment of the amount of norleucine misincorporation can be determined by measuring the peak areas in the extracted ion chromatogram (EIC) of norleucine and methionine containing peptides.

[0057] A peptide mapping method using liquid chromatography (LC) and mass spectrometry (MS) using an Orbitrap Q-Exactive Plus mass spectrometer was used to semiquantitatively determine the amount of norleucine misincorporation in protein samples.

[0058] Cell viability measurement: Cell viability was monitored using a FACSCalibur flow cytometer. First, cells were stained with BOX and PI dyes.

[0059] DO Measurement: Dissolved oxygen (DO) was measured using an online polarographic dissolved oxygen sensor.

[0060] In this example, fermentations were performed with no methionine added at all, with methionine included in the feed used before and during or after induction (i.e., for steps (c) and (e) of the process), and with methionine included only in the feed used during or after induction (i.e., for step (e) of the process). When methionine was included in the feed used in steps (c) and (e), cell growth (Figure 1), cell viability (Figure 2), and Fab' concentration (Figure 3) were all reduced compared to when methionine was not added to the process or was only added to the feed used in step (e). These figures show that there were no significant differences in these parameters (cell growth, cell viability, and titer) between fermentations grown without methionine and those grown with methionine in the feed used in step (e). Figure 4 shows that adding methionine to the feed in step (e) was sufficient to reduce the average amount of norleucine misincorporation per methionine residue (without affecting other process parameters as described above).

[0061] Example 2 Fermentation was carried out to obtain approximately 10,000 kg of the liquid medium of step (b). The process described in Example 1 was scaled up or down accordingly to accommodate the increased starting volume. All scale-independent parameters (e.g., temperature, pH, DO set point) were maintained the same as in Example 1.

[0062] Figure 5 shows that adding methionine to the feed before induction and during or after induction (i.e., steps (c) and (e)) reduced cell growth, whereas adding methionine only to the feed used during or after induction (i.e., step (e)) had no effect on growth compared to when methionine was not added. Figure 6 shows that adding methionine to the feed in step (e) was sufficient to reduce the average amount of norleucine misincorporation.

[0063] Example 3 Figure 7 shows the average amount of norleucine misincorporation per methionine residue for three representative batches of all three processes run using approximately 10,000 kg of liquid medium provided in step (b). Process A is a low-productivity fermentation process with no added methionine to the feed. Process B was developed as a high-productivity process without added methionine to the feed to produce the same Fab', which, as can be seen in Figure 7, resulted in much higher norleucine misincorporation when compared to Process A. Process C is a further development of Process B, including application of the present invention, which resulted in lower amounts of norleucine misincorporation than the original process (see Figure 8).

Claims

1. 1. A process for producing a recombinant protein, comprising: a) providing a host cell capable of producing a recombinant protein; b) providing a quantity of liquid medium containing 0-1 g methionine per kg of liquid medium; c) culturing the host cells in the liquid medium; d) inducing production of the recombinant protein in the liquid medium; and e) adding a certain amount of methionine to the liquid medium during or after induction while culturing the host cells to produce the recombinant protein. wherein the amount of methionine added per kg of liquid medium in step (e) is greater than the amount of methionine contained in the liquid medium per kg of liquid medium in step (b).

2. 1. A process for reducing norleucine misincorporation during recombinant protein production, comprising: a) providing a host cell capable of producing a recombinant protein; b) providing a quantity of liquid medium containing 0-1 g methionine per kg of liquid medium; c) culturing the host cells in the liquid medium; d) inducing production of the recombinant protein in the liquid medium; and e) adding a certain amount of methionine to the liquid medium during or after induction while culturing the host cells to produce the recombinant protein. wherein the amount of methionine added per kg of liquid medium in step (e) is greater than the amount of methionine contained in the liquid medium per kg of liquid medium in step (b).

3. 3. The process of claim 1 or 2, wherein the host cells are harvested after step (e), and the amount of methionine added in step (e) is such that the concentration of methionine in the liquid medium at or immediately before harvesting is between 0.25 g / kg and 1.5 g / kg.

4. 3. The process of claim 1 or 2, wherein the amount of methionine added in step (e) is from 0.25 g to 2.0 g per kg of the liquid medium provided in step (b).

5. 5. The process according to claim 1, wherein the amount of methionine contained in the liquid medium in step (b) and / or step (c) is less than 0.5 g / kg, such as less than 0.25 g / kg, for example less than 0.20 g / kg, such as less than 0.15 g / kg, or less than 0.10 g / kg.

6. 6. The process of claim 5, wherein methionine is not contained in the liquid medium in step (b) and / or step (c).

7. 6. The process of any one of claims 1, 2, 3, 4 or 5, wherein step (c) comprises growing the culture to an OD600 of at least 50, such as at least 55, for example at least 60, such as at least 70, for example at least 80.

8. 8. The process of any one of claims 1 to 7, wherein step (c) and / or step (e) comprises culturing the host cells in fed-batch culture.

9. 9. The process according to any one of claims 1 to 8, wherein a feed containing a carbon source is added during steps (c) and (e), and the amount of carbon source added to the liquid medium per unit time in step (e) is less than the amount in step (c).

10. 10. The process of any one of claims 1 to 9, wherein step (d) is initiated when a 50% increase in dissolved oxygen occurs in the liquid medium or when a predetermined OD600 is reached.

11. The process according to any one of claims 1 to 10, wherein the host cell is a bacterial cell, such as an E. coli cell.

12. The process according to any one of claims 1 to 11, wherein leucine and / or isoleucine are not contained in the liquid medium in step (b) and / or step (c) and / or step (e).

13. 13. The process of any one of claims 1 to 12, wherein the host cell produces the recombinant protein upon induction with IPTG, and optionally step (d) comprises the addition of IPTG.

14. 14. The process according to any one of claims 1 to 13, wherein the duration of step (e) is from 12 to 96 hours, such as from 20 to 72 hours, for example from 25 to 55 hours, such as from 30 to 50 hours, or the duration of step (e) is from about 12 to about 96 hours, such as from about 20 to about 72 hours, for example from about 25 to about 55 hours, such as from about 30 to about 50 hours.

15. 15. The process according to any one of claims 1 to 14, wherein more than 75%, such as more than 90%, of the carbon source consists of glycerol.

16. The process according to any one of claims 1 to 15, wherein the recombinant protein is an antibody, such as a Fab' fragment.

17. 17. The process of any one of claims 1 to 16, wherein at least step (e) is carried out in a bioreactor preferably having a volume of at least 100 L, at least 500 L, at least 1,000 L, at least 2,000 L, at least 5,000 L, at least 10,000 L or at least 20,000 L.

18. 18. The process of any one of claims 1 to 17, wherein the process comprises the step of recovering the recombinant protein, the further step of purifying the recombinant protein, and optionally the further step of formulating the recombinant protein.

19. 19. The process of any one of claims 1 to 18, wherein the process comprises lyophilizing the recombinant protein.

20. A recombinant protein preparation obtainable or obtained by the process of any one of claims 1 to 19.

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