Method for producing recombinant proteins

Abstract

The present invention relates to the field of recombinant production of proteins in host cells. In particular, the present invention relates to a method for producing a recombinant protein in a host cell that reduces the misincorporation of n-leucine substituted methionine.

Description

Field of the Invention

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

[0003] In the medical field, the use of biological entities such as proteins (e.g., antibodies or antibody-derived molecules) has been increasing in popularity and importance. With this trend, the demand for controlled manufacturing methods is also increasing. The commercialization of proteins for medical use requires that they be produced in large quantities, and a lot of effort has been invested in improving the cultivation of recombinant host cells expressing the desired proteins and their processing. This has led to an increase in product titer, but more undesirable by-products and increased product heterogeneity are usually observed. Since removing such undesirable by-products or product variants may be very laborious, it is best to optimize the manufacturing method to minimize their formation.

[0004] An undesirable product variation is due to the misincorporation of norleucine into proteins instead of methionine. Norleucine is a non-natural amino acid synthesized by enzymes of the leucine biosynthetic pathway in Escherichia coli (E. coli). It is a structural analog of methionine and can replace methionine residues in proteins because, although less efficient than methionine, methionyl-tRNA synthetase (MetRS) can use norleucine as a substrate to give charge to methionyl-tRNA during translation.

[0005] It has been known since the 1950s that many heterologous proteins, when expressed in E. coli, misincorporate norleucine where a methionine residue should be (Munier and Cohen 1956 and Nisman and Hirsch 1958). Norleucine misincorporation is undesirable because it leads to the production of altered proteins, i.e. proteins with a different primary amino acid sequence, with potentially unknown characteristics. 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 manufacturing batches and thus leads to heterogeneity in product batches.

[0006] Even though norleucine misincorporation can be reduced by increasing the concentration of methionine in the cell culture medium (Tsai et al., Biochem Biopsy's Res Comm 156:733, 1988, Bogosian et al., J Biol Chem 264:531, 1989, US 5,599,690 and WO 2007 / 103521), this also has various disadvantages, including increased operational complexity and manufacturing costs (Veeravalli and Laird, Bioengineered 6:132, 2015). Other methods have been developed to reduce norleucine incorporation in recombinant proteins, such as expressing norleucine degrading enzymes (US 8,603,781) or deleting genes involved in the biosynthesis of norleucine (Bogosian et al., J Biol Chem 264:531, 1989).

[0007] However, genetic modification of host cell lines is cumbersome and may have other unexpected or unidentified effects. Changing the cell culture medium may lead to increased formation of other undesirable byproducts and / or have other negative effects on cell culture performance. Therefore, there remains a need for new methods to prevent or reduce the misincorporation of norleucine into proteins during manufacturing. The present invention addresses this need. SUMMARY OF THE INVENTION

[0009] In a first embodiment, the present invention relates to a method for producing a recombinant protein comprising the following steps:

[0010] a) providing a host cell capable of producing a recombinant protein,

[0011] b) providing a certain amount of liquid culture medium containing 0 to 1 g of methionine per kg of liquid culture medium,

[0012] c) culturing the host cell in a liquid culture medium,

[0013] d) inducing the production of the recombinant protein in liquid culture medium, and

[0014] e) adding a certain amount of methionine to the liquid culture medium during or after induction, while culturing the host cell to produce the recombinant protein,

[0015] The amount of methionine per kg of liquid culture medium added in step (e) is greater than the amount of methionine per kg of liquid culture medium contained in the liquid culture medium in step (b).

[0016] In a second embodiment, the present invention relates to a method for reducing norleucine misincorporation during the production of a recombinant protein, comprising the steps of:

[0017] a) providing a host cell capable of producing a recombinant protein,

[0018] b) providing a certain amount of liquid culture medium containing 0 to 1 g of methionine per kg of liquid culture medium,

[0019] c) culturing the host cell in a liquid culture medium,

[0020] d) inducing the production of the recombinant protein in liquid culture medium, and

[0021] e) adding a certain amount of methionine to the liquid culture medium during or after induction, while culturing the host cell to produce the recombinant protein,

[0022] The amount of methionine per kg of liquid culture medium added in step (e) is greater than the amount of methionine per kg of liquid culture medium contained in the liquid culture medium in step (b).

[0023] In a further embodiment, the present invention relates to a recombinant protein preparation obtainable by or obtained by a method according to any one of the preceding claims.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown are growth curves of different batches determined by the optical density at 600 nm (OD600) of E. coli during fermentation in a 200 L vessel as described in Example 1, wherein no methionine was added, methionine was added before induction [step (c)] and during or after induction [step (e)] or only during or after induction [step (e)].

[0026] Figure 2 Shown are the cell viabilities of E. coli in different batches of fermentation in a 200 L vessel as described in Example 1, without methionine, with addition of methionine before induction [step (c)] and during or after induction [step (e)] or only during or after induction [step (e)].

[0027] Figure 3 Shown are the titers of Fab′ in different batches after harvesting E. coli fermentations in 200 L vessels 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 after induction [step (e)].

[0028] Figure 4Shown are the average norleucine levels per methionine residue in Fab′ produced by batch E. coli fermentation in a 200 L vessel as described in Example 1, wherein no methionine was added, methionine was added before induction [step (c)] and during or after induction [step (e)] or only during or after induction [step (e)].

[0029] Figure 5 Shown are growth curves as determined by OD600 in different batches of E. coli fermentations in a 15,000 L vessel as described in Example 2, wherein no methionine was added, methionine was added before induction [step (c)] and at or after induction [step (e)] or only at or after induction [step (e)].

[0030] Figure 6 Shown are the average norleucine levels per methionine residue in Fab' produced by batch E. coli fermentation in a 15,000 L vessel as described in Example 2, wherein no methionine was added, methionine was added before induction [step (c)] and during or after induction [step (e)], or only during or after induction [step (e)].

[0031] Figure 7 Shown are the average norleucine levels per methionine residue for 3 different manufacturing methods (Methods A, B and C) producing different amounts of product, all run at the same scale to produce the same Fab'. Method A is a low yield method with no methionine added to the feed, while Methods B and C are improved higher yield methods. Method B does not involve the addition of methionine to the feed, while Method C is performed according to the present invention.

[0032] Figure 8 The average norleucine level per methionine residue for methods A and C is shown. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present inventors have unexpectedly discovered that adding methionine to a cell culture medium during the growth and expansion phases of a cell culture reduces or inhibits cell growth or expansion.

[0035] Based on this unexpected discovery, the present inventors have designed new and improved manufacturing methods that overcome the problems associated with methods known in the art for reducing norleucine misincorporation during protein manufacturing.

[0036] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, wherever the terms "comprises", "including", "having", "having", "with", or variations thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to "comprising". The transitional terms / phrases (and any grammatical variations thereof) "comprising", "comprises", "comprise", including the phrases "consisting essentially of", "consists essentially of", "consisting", and "consists of" may be used interchangeably. The phrases "consisting essentially of", "consists essentially of" indicate that the claim covers embodiments containing the specified materials or steps as well as embodiments that do not materially affect the basic and novel features of the claim.

[0037] The present invention relates to a method of culturing cells for producing recombinant proteins. In the method of the present invention, methionine is added to the culture medium in a specific manner to minimize misincorporation of norleucine, thereby minimizing the negative effects of methionine on cell growth.

[0038] Therefore, in a first embodiment, the present invention relates to a method for producing a recombinant protein comprising the following steps:

[0039] a) providing a host cell capable of producing a recombinant protein,

[0040] b) providing a certain amount of liquid culture medium containing 0 to 1 g of methionine per kg of liquid culture medium,

[0041] c) culturing the host cell in a liquid culture medium,

[0042] d) inducing the production of the recombinant protein in liquid culture medium, and

[0043] e) adding a certain amount of methionine to the liquid culture medium during or after induction, while culturing the host cell to produce the recombinant protein

[0044] The amount of methionine per kg of liquid culture medium added in step (e) is greater than the amount of methionine per kg of liquid culture medium contained in the liquid culture medium in step (b).

[0045] In a second embodiment, the present invention relates to a method for reducing norleucine misincorporation during the production of a recombinant protein, comprising the steps of:

[0046] a) providing a host cell capable of producing a recombinant protein,

[0047] b) providing a certain amount of liquid culture medium containing 0 to 1 g of methionine per kg of liquid culture medium,

[0048] c) culturing the host cell in a liquid culture medium,

[0049] d) inducing the production of the recombinant protein in liquid culture medium, and

[0050] e) adding a certain amount of methionine to the liquid culture medium during or after induction, while culturing the host cell to produce the recombinant protein

[0051] The amount of methionine per kg of liquid culture medium added in step (e) is greater than the amount of methionine per kg of liquid culture medium contained in the liquid culture medium in step (b).

[0052] In step (a), a host cell capable of producing a recombinant protein after induction is provided. The host cell used in the method of the present invention can be any host cell suitable for recombinant production of proteins and capable of growing under specified conditions. Suitable host cells include bacterial host cells and other cells that can exhibit misincorporation of norleucine in place of methionine.

[0053] In a third embodiment, the host cell in the method according to any one of the first, second or any other embodiments of the present invention is a bacterial host cell, such as an Escherichia coli cell or another Gram-negative bacterial cell or a Gram-positive bacterial cell, such as Staphylococcus aureus. In a more preferred embodiment according to the third embodiment, the host cell is an Escherichia coli host cell, even more preferably strains HB101, B7, K12, RV308, DH1, HMS174, W3110 or BL21.

[0054] Usually, the nucleic acid sequence encoding recombinant protein has been introduced into the host cell under the control of an inducible promoter. Suitable vectors for expressing such nucleic acid constructs in host cells and methods 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.

[0055] In the step (c) of the method according to any one of the embodiments of the present invention described herein, the host cell is cultivated. Methods and culture media for cultivating various types of host cells are well known in the art. Culture media vary according to organisms, but may include ingredients such as carbon sources, nitrogen sources, amino acids, vitamins, essential metal ions and trace elements. Step (c) preferably includes fed-batch culture, more preferably in a bioreactor. A batch phase may be carried out before the fed-batch phase. Inoculation can be carried out directly from a working cell bank or by seed culture, for example in a shake bottle.

[0056] In a fourth embodiment of the invention, step (c) of the method according to any one of the first, second, third or any other embodiments of the invention comprises growing the culture to an OD600 (optical density at a wavelength of 600 nm) of at least 20, e.g., at least 25, at least 35, at least 50, at least 55, at least 60, at least 70 or at least 80.

[0057] In a fifth embodiment, the liquid culture medium of step (b) and / or step (c) of the method according to any one of the first, second, third, fourth or any other embodiments of the invention contains less than 1 g of methionine per kg of liquid culture medium, such as 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 culture medium. Preferably, the concentration of methionine in the liquid culture medium of step (b) and / or (c) is between 0 and 0.25 g / kg or between 0 and 0.5 g / kg.

[0058] In a sixth embodiment, methionine is absent in the liquid culture medium of step (b) and / or step (c) (ie prior to induction) of the method according to any of the first, second, third, fourth, fifth or any other embodiments of the invention.

[0059] In a seventh embodiment, isoleucine is not present in the liquid culture medium of step (b) and / or step (c) (ie before induction) of the method according to any one of the first, second, third, fourth, fifth, sixth or any other embodiments of the invention.

[0060] In an eighth embodiment, leucine is absent in the liquid culture medium of step (b) and / or step (c) (i.e. prior to induction) of the method according to any one of the first, second, third, fourth, fifth, sixth, seventh or any other embodiments of the invention.

[0061] In a ninth embodiment, step (c) of the method according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth or any other embodiments of the invention comprises:

[0062] i) culturing the host cell in batch culture to an OD600 of 20 to 55, wherein

[0063] optionally adding a bolus amount (i.e. a single dose added all at once) of a magnesium salt (e.g. magnesium sulfate),

[0064] ii) further culturing the bacterial host cells, whereby the OD increases until the dissolved oxygen (DO) increases to ≥50% of air saturation, and

[0065] iii) cultivating the host cell in a fed-batch culture until the OD600 in the liquid medium of the culture increases by at least 15, 20, 25, 35, 40 or 50 units compared to the OD600 in step (c)(i),

[0066] In a tenth embodiment, the feed containing the carbon source is added starting from step (c)(iii) of the method of the present invention according to the ninth embodiment. Preferably, the amount of carbon source added to the liquid culture medium per unit time in step (e) is lower than during step (c)(iii), for example by reducing the feed rate or by reducing the concentration of the carbon source in the feed. In a preferred embodiment, according to the tenth embodiment of the present invention, methionine is not present in the liquid culture medium or is not added to the liquid culture medium before adding the feed with the carbon source.

[0067] In an eleventh embodiment, inducing the production of recombinant protein according to step (d) of the method according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or any other embodiments of the invention is initiated when dissolved oxygen (DO) in the liquid medium of the culture increases to 50% of air saturation, or when a predefined OD600 as defined in step (c)(iii) of the ninth embodiment is reached. DO can be measured by any standard means, such as an online polarographic dissolved oxygen sensor, an optical dissolved oxygen sensor or any other suitable oxygen sensing technology.

[0068] In a preferred embodiment, in the method according to any of the embodiments of the present invention, no recombinant protein or less than 0.1 g of recombinant protein is produced per kg of liquid culture medium before the induction according to step (d).

[0069] Induction of recombinant protein production can be achieved by any suitable method. In one embodiment, in the method according to any one of the embodiments of the present invention, the gene encoding the recombinant protein is controlled by an inducible promoter. Inducible promoters are known in the art. The 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, which can be induced by IPTG (isopropyl β-Dl-thiogalactopyranoside). Other known bacterial expression systems include, for example, arabinose promoter systems (see, for example, Guzman et al., J Bacteriol 177:4121, 1995) or T7 systems (see, for example, Rosenberg et al., Gene 56:125, 1987). These and other systems are reviewed in, for example, Rosano and Ceccarelli, Front Microbiol 5:172, 2014.

[0070] In a preferred embodiment of the method of the present invention, the host cell of the method 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, and thereby produces a recombinant protein after induction with IPTG. In this embodiment, step (d) comprises adding IPTG.

[0071] The step (e) in the method according to any one of the embodiments of the present invention is generally included in fed-batch culture in a bioreactor. In one embodiment, the duration of the step (e) of the method according to any one of the embodiments of the present invention is about 12 to about 96 hours, such as about 20 to about 72 hours, such as about 24 to about 48 hours or about 25 to about 55 hours, such as 30 to about 50 hours or 35 to 45 hours or 36 to 48 hours. In the context of time, the term "about" is intended to include ± 1, ± 2, ± 3, ± 4, ± 5, ± 6, ± 7, ± 8, ± 9 or ± 10 hours.

[0072] In a twelfth embodiment of the invention, step (e) of the method according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh or any other embodiments of the invention is followed by a step (f) of harvesting the host cells. Preferably, the amount of methionine added in step (e) is such that the concentration of methionine in the liquid culture medium at the time of harvesting or immediately before harvesting in step (f) is at least 0.25 g / kg, for example, between 0.25 g / kg and 1.5 g / kg, and preferably at least 0.40 g / kg, for example, between 0.40 g / kg and 1.2 g / kg. More preferably, the amount added in step (e) is such that the concentration of methionine in the liquid culture medium at the time of harvesting or immediately before harvesting in step (f) is between 0.45 g / kg and 1.10 g / kg, for example, between 0.50 g / kg and 0.9 g / kg.

[0073] In a thirteenth embodiment of the invention, the amount of methionine added in step (e) of the method according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or any other embodiments of the invention is at least 0.25 g / kg, such as between 0.25 g / kg and 2.0 g / kg, and preferably at least 0.50 g / kg, such as between 0.50 g / kg and 1.2 g / kg of the liquid culture medium provided in step (b). More preferably, the amount is between 0.52 g and 1.10 g per kg of the liquid culture medium in step (b), such as between 0.55 g and 1.05 g per kg of the liquid culture medium provided in step (b).

[0074] In a fourteenth embodiment, no leucine is added during step (e) of the method according to any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth or any other embodiments of the invention.

[0075] In a fifteenth embodiment, isoleucine is not added during step (e) of the method according to any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or any other embodiments of the invention.

[0076] In the present disclosure, scope is represented in shorthand form, to avoid the need to elaborate and describe each value in the scope. Where appropriate, any appropriate value in the scope can be selected as the upper limit, lower limit or endpoint of the scope. For example, the scope of 0.1-1.0 represents the endpoint values ​​of 0.1 and 1.0, and the intermediate values ​​of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges covered in the scope of 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0 etc. When using scope in this article, it is intended to clearly include the specific embodiments of the different combinations and sub-combinations (for example, the sub-ranges in the disclosed range) of these scopes.

[0077] Carbon source

[0078] The method of the present invention generally comprises adding 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, more than 75%, for example, at least 90% of the carbon source in the liquid culture medium in step (b) is composed of glycerol. In another preferred embodiment, more than 75%, for example, at least 90% of the carbon source in the liquid culture medium in step (e) of the present invention is composed of glycerol. As another example, more than 75%, for example, at least 90% of the carbon source in the liquid culture medium in step (c) is composed of glucose. In another preferred embodiment, more than 75%, for example, at least 90% of the carbon source in the liquid culture medium in step (e) is composed of glucose. As a further example, more than 75%, for example, at least 90% of the carbon source in the liquid culture medium in step (c) is composed of lactose. In another preferred embodiment, more than 75%, for example, at least 90% of the carbon source in the liquid culture medium in step (e) is composed of lactose.

[0079] pH

[0080] During fermentation, the pH of the cell culture medium is very important for the processability of product yield and cell culture slurry. The formation of magnesium ammonium phosphate is described as being affected by pH (see Pérez-García et al., 1989). In an embodiment of the inventive method, the pH of the culture in step (c) of the method according to any one of the embodiments of the present invention is higher than 6.5, such as 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, or higher 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 higher than 6.5, such as 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, or higher 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 between 6 and 8, such as 6.5 and 7.5, such as 6.6 and 7.4, such as 6.7 and 7.3, such as 6.8 and 7.2, and the pH in step (e) is between 6 and 8, such as 6.5 and 7.5, such as 6.6 and 7.4, such as 6.7 and 7.3, such as 6.8 and 7.2. In another embodiment, the pH in step (c) is between about 6 and about 8, such as about 6.5 and about 7.5, such as about 6.6 and about 7.4, such as about 6.7 and about 7.3, such as about 6.8 and about 7.2, and the pH in step (e) is between about 6 and about 8, such as about 6.5 and about 7.5, such as about 6.6 and about 7.4, such as about 6.7 and about 7.3, such as about 6.8 and about 7.2. In the context of pH, the term "about" is intended to include ± 0.1, ± 0.2 or ± 0.3 pH units.

[0081] temperature

[0082] In the whole fermentation process, the temperature is usually kept constant as much as possible. 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 can 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 a set temperature (e.g., a range of ±0°C to 3°C around the set temperature).

[0083] Recombinant protein

[0084] The recombinant protein produced in the method of the present invention is generally a heterologous protein derived from another organism. For example, the recombinant protein can be an antibody, a cytokine, a growth factor, a hormone or other peptide or polypeptide or a derivative of any fusion protein of the above.

[0085] 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. "Antibodies" include antibodies of any species, particularly antibodies of mammalian species; for example, human antibodies of any isotype, including IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, IgG19, IgG21, IgG22, IgG23, IgG24, IgG25, IgG26, IgG3 2a IgG 2b , IgG3, IgG4, IgE, IgD and antibodies produced as dimers of this basic structure, including IgGA1, IgGA2 or pentamers such as IgM and modified variants thereof; non-human primate antibodies, such as from chimpanzees, baboons, rhesus monkeys or cynomolgus monkeys; rodent antibodies, such as from mice or rats; rabbit, sheep or horse antibodies; camelid antibodies (e.g. from camels or alpacas, such as Nanobodies TM) and derivatives thereof; bird species antibodies such as chicken antibodies; or antibodies of fish species such as shark antibodies. The term "antibody" also refers to a "chimeric" antibody, in which at least one first portion of a heavy chain and / or light chain antibody sequence is from a first species, and a second portion of the heavy chain and / or light chain antibody sequence is from a second species. Chimeric antibodies of interest herein include "primatization" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, such as baboons, rhesus monkeys, or cynomolgus monkeys) and human constant region sequences. "Humanized" antibodies are chimeric antibodies containing sequences derived from non-human antibodies. In most cases, humanized antibodies are human antibodies (receptor antibodies) in which residues from a hypervariable region of a receptor are replaced by residues from a hypervariable region [or complementary determining region (CDR)] of a non-human species (donor antibody), such as mice, rats, rabbits, chickens, or non-human primates, which have the desired specificity, affinity, and activity. In most cases, residues of the human (acceptor) antibody outside the CDR; i.e., residues in the framework region (FR) are additionally replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues not found in the acceptor antibody or the donor antibody. These modifications are made to further optimize the properties of the antibody. Humanization reduces the immunogenicity of non-human antibodies in the human body, thereby promoting the use of antibodies in the treatment of human diseases. Humanized antibodies and several different techniques for generating them are well known in the art. The term "antibody" also refers to human antibodies that can be produced as a substitute for humanization. For example, transgenic animals (e.g., mice) can be produced that are able to produce a complete human antibody library without producing endogenous murine antibodies after immunization. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies such as phage display or ribosome display technology, in which a recombinant DNA library that is at least partially artificially generated or generated by a donor's immunoglobulin variable (V) domain gene library is used. 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 immunized ex vivo with an antigen of interest and subsequently fused to generate hybridomas, which can then be screened for the best human antibodies. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. In addition, the term "antibody" as used herein refers not only to full-length antibodies, but also to antibody fragments. Antibody fragments contain at least one heavy chain or light chain immunoglobulin domain as known in the art and bind to one or more antigens. Examples of antibody fragments according to the present invention include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and double scFv fragments.The fragment may also be a diabody, a tribody, a triabody, a tetrabody or a minibody, a single domain antibody (dAb) such as an sdAb, a VL, VH, VHH or a camelid antibody (e.g. from a camel or alpaca, such as a Nanobody. TM ) and VNAR fragments. The antigen-binding fragment according to the present invention may also include a Fab connected to one or two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binding to a therapeutic target and one scFv or dsscFv increasing half-life by binding to, for example, albumin). Examples of such antibody fragments are FabdsscFv (also known as BYbe) or Fab-(dsscFv)2 (also known as TrYbe, see, for example, WO2015 / 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, brolucizumab.

[0086] The method according to any of the embodiments of the invention may in principle be carried out in any suitable container, such as a shake flask or a bioreactor, which may or may not be operated in fed-batch mode, depending on e.g. the desired production scale.

[0087] In a sixteenth embodiment, at least steps (c), (d) and (e) of the method according to any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth or any other embodiments of the invention are carried out in a bioreactor, preferably in an industrial scale bioreactor. The bioreactor can be, for example, a stirred tank or an airlift reactor. The bioreactor can be a reusable reactor made of glass or metal (e.g., stainless steel) or a disposable bioreactor made of a synthetic material (e.g., plastic).

[0088] In a seventeenth embodiment, at least step (e) of the method according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth or any other embodiments of the invention is carried out in a bioreactor having a volume of equal to or greater than 100 L, equal to or greater than 500 L, equal to or greater than 1,000 L, equal to or greater than 2,000 L, equal to or greater than 5,000 L, equal to or greater than 10,000 L, or equal to or greater than 20,000 L, 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.

[0089] In an eighteenth embodiment, in step (b), (c), (d) or (e) of the method according to any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth or any other embodiments of the invention, the liquid culture medium of the culture has a volume equal to or greater than 100 L, equal to or greater than 500 L, equal to or greater than 1,000 L, equal to or greater than 2,000 L, equal to or greater than 5,000 L, equal to or greater than 10,000 L, or equal to or greater than 20,000 L, 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 method according to any of the embodiments of the invention, in all steps (b), (c), (d) and (e), the culture has a volume of equal to or greater than 100 L, equal to or greater than 500 L, equal to or greater than 1,000 L, equal to or greater than 2,000 L, equal to or greater than 5,000 L, equal to or greater than 10,000 L or equal to or greater than 20,000 L, 1,000 to 30,000 L, 5,000 to 30,000 L, 10 ,000 to 30,000L, 1,000 to 20,000L, 5,000 to 20,000L, 10,000 to 20,000L or 10,000 to 25,000L, 1,000 to 30,000L, 5,000 to 30,000L, 10,000 to 30,000L, 1,000 to 20,000L, 5,000 to 20,000L, 10,000 to 20,000L or 10,000 to 25,000L.

[0090] The method according to any one of the embodiments of the present invention may include one or more further steps after step (e). For example, the method may include a further step of recovering the recombinant protein, which may include first separating cells from the supernatant or from inclusion bodies. Once recovered, the recombinant protein can be separated and purified. Separation and purification methods are well known to those skilled in the art. They are generally composed of a combination of various chromatographic and filtration steps. The method of the present invention may further include the step of formulating the recombinant protein into a pharmaceutical composition suitable for medical use (e.g., treatment or preventive use). In one embodiment, the recombinant protein is modified before being formulated into a pharmaceutical composition, e.g., conjugated to another molecule.

[0091] In a nineteenth embodiment, the method according to any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth or any other embodiments of the invention comprises lyophilizing a composition comprising a recombinant antibody produced according to any of the embodiments of the method of the invention.

[0092] A further embodiment of the present invention is a recombinant protein preparation, such as an antibody preparation, preferably a preparation comprising becelimumab, dapilocizumab, ranibizumab, abciximab, belintoumab, idarucizumab, pacitumomab, caplucizumab, buclizumab, which is obtained or obtainable according to the method according to any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth or any other embodiments of the present invention.

[0093] Detection and quantification of norleucine misincorporation

[0094] 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

[0095] Example 1

[0096] Frozen cell bank vials containing E. coli W3110 host cells expressing antibody A (Fab' fragments with pI in the range of 8.8-9.3) were inoculated with a shake flask containing 6x peptone yeast extract (6xP-Y) culture medium and tetracycline. The shake flask was incubated at 30°C and 200-250rpm. Within the required OD range, a shake flask was inoculated with a seed fermenter containing a chemically defined culture medium (derived from Durany et al., MD culture medium of 2004) and tetracycline and containing a carbon source. The cell culture in the seed fermenter was maintained at 30°C. Within the required OD range, a seed culture was used to inoculate a production fermenter (175kg liquid culture medium), which contained the same culture medium as the chemically defined culture medium used in the seed fermenter. The production fermenter was maintained under the same conditions as the seed fermenter and grown in the batch stage until the carbon source was exhausted. During this period, a push injection of MgSO4 was added to avoid the exhaustion of the metabolite. At the end of the batch phase (marked by the peak DO value), the exponential carbon source feed was switched to [containing different amounts of methionine corresponding to 0 to 0.70 g per kg of the liquid medium provided in step (b) according to the batch], and a specific amount of carbon source was fed to the culture to achieve an OD600 greater than 50 units. At this point, the carbon source feed was switched from the exponential phase feed to the production phase feed [containing different amounts of methionine corresponding to 0.50 g to 1.5 g per kg of the liquid medium provided in step (b)], and the expression of antibody A was induced by adding IPTG. The cells (containing expressed antibody A) were harvested more than 40 hours after induction.

[0097] Cells are harvested by continuous centrifugation. The concentrated cell paste is resuspended back to the original cell harvest concentration by adding deionized water and concentrated Tris EDTA extraction buffer to achieve the desired buffer concentration. For heat extraction, cells are kept mixed at elevated temperature for a defined period of time.

[0098] Fab′ concentration : Harvest Fab' concentrations were determined using Protein G HPLC analysis in 20 mM phosphate buffer. Elution was performed by a pH gradient from pH 7.4 at injection down to pH 2.7

[0099] Protein L purification :Use 600μL Capto The column was used to purify the extract sample by protein L affinity chromatography in order to purify the cell extract prior to analysis of the level of norleucine misincorporation. The column was prepared by washing with phosphate / sodium chloride buffer (buffer A), cleaning with sodium hydroxide solution, followed by an equilibration step with buffer A. After sample loading, washing with buffer A was performed, followed by elution with glycine buffer. A portion of the eluate was appropriately collected to recover a representative Fab' sample for norleucine misincorporation analysis.

[0100] Analysis of norleucine misincorporation levels : To perform this analysis, the sample is enzymatically digested with trypsin into fragment peptides. These are subsequently separated using liquid chromatography and then analyzed online using electrospray ionization mass spectrometry. Mass spectrometry measures the mass-to-charge ratio of the peptides, from which their masses can be inferred. Peptide mass is a highly specific feature of the peptide sequence. The retention time and mass observed for each peptide are unique to the amino acid sequence of the peptide, which allows the mass and retention time of the observed peptides to be compared with the mass and retention time of the theoretical sequence. The high sensitivity of mass spectrometry allows low levels of protein modifications to be detected.

[0101] Substitution of methionine residues for norleucine residues will reduce the mass of the peptide by 17.9564 Da. This mass shift, combined with tandem mass spectrometry (MSMS fragmentation), allows the identification of peptides containing norleucine substitutions. By measuring the peak areas of the extracted ion chromatograms (EICs) of peptides containing norleucine and methionine, a semi-quantitative assessment of the level of norleucine misincorporation can be determined.

[0102] A peptide mapping approach using liquid chromatography (LC) and mass spectrometry (MS) with an Orbitrap Q-Exactive plus mass spectrometer was used to semi-quantitatively determine the level of norleucine misincorporation in protein samples.

[0103] Cell viability measurement : Cell viability was monitored using a FACSCalibur flow cytometer. Cells were first stained with BOX and PI dyes.

[0104] DO measurement : Dissolved oxygen (DO) is measured using an online polarographic dissolved oxygen sensor.

[0105] In this example, fermentation was performed without any addition of methionine, methionine was included in the feed used before induction and at or after induction [i.e., in steps (c) and (e) of the process], and methionine was included only in the feed at or after induction [i.e., in step (e) of the process]. Cell growth ( ) was increased when methionine was included in the feed used in steps (c) and (e) compared to when methionine was not added to the process or was added only to the feed used in step (e). Figure 1 ), cell viability ( Figure 2 ) and Fab′ concentration ( Figure 3 ) were all reduced. From the same figures it can be seen that there were no significant differences in these parameters (cell growth, viability and titer) between the growth fermentations without methionine and with methionine in the feed used in step (e). Figure 4 It was shown that the addition of methionine to the feed in step (e) was sufficient to reduce the average norleucine misincorporation level per methionine residue (while not affecting the other process parameters as described above).

[0106] Example 2

[0107] Fermentation was carried out to provide approximately 10,000 kg of liquid culture medium for step (b). The method described in Example 1 was appropriately scaled up to adjust for the increased starting volume. All parameters independent of scale (e.g. temperature, pH, DO set point) remained the same as in Example 1.

[0108] from Figure 5 It can be seen that the addition of methionine to the feed before and during or after induction [i.e. steps (c) and (e)] resulted in reduced cell growth, whereas the addition of methionine to the feed used only during or after induction [i.e. in step (e)] did not affect growth compared to when no methionine was added. Figure 6 It was shown that the addition of methionine to the feed in step (e) was sufficient to reduce the average norleucine misincorporation level.

[0109] Example 3

[0110] Figure 7 The average norleucine misincorporation level per methionine residue is shown for three representative batches of three processes, all run with approximately 10,000 kg of liquid culture medium provided in step (b). Process A is a lower yield fermentation process that does not add methionine to the feed. Process B was developed as a higher yield process that does not add methionine to the feed to produce the same Fab' and from Figure 7 As can be seen in Figure 2, method B results in much higher norleucine misincorporation than method A. Method C is a further development of method B, which includes the application of the present invention and results in lower norleucine misincorporation levels than the original method (see Figure 8 ).

Claims

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

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

3. The method according to 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 culture medium at or immediately before harvest is 0.25 g / kg to 1.5 g / kg.

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

5. The method of claim 1, 2, 3 or 4, wherein the amount of methionine contained in the liquid culture medium in step (b) and / or step (c) is less than 0.5 g / kg, 0.25 g / kg, such as less than 0.20 g / kg, such as less than 0.15 g / kg or less than 0.10 g / kg.

6. The method according to claim 5, wherein the liquid culture medium in step (b) and / or step (c) does not contain methionine.

7. The method according to 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, such as at least 60, such as at least 70, such as at least 80.

8. The method according to any one of the preceding claims, wherein step (c) and / or step (e) comprises the step of culturing the host cell in a fed-batch culture.

9. The method according to any one of the preceding claims, wherein a feed containing a carbon source is added during step (c) and step (e), and the amount of carbon source added to the liquid culture medium per unit time in step (e) is lower than in step (c).

10. The method according to any one of the preceding claims, wherein step (d) is initiated when a 50% increase in dissolved oxygen occurs in the liquid culture medium or when a predefined OD600 is reached.

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

12. The method according to any one of the preceding claims, wherein the liquid culture medium in step (b) and / or step (c) and / or step (e) does not contain leucine and / or isoleucine.

13. A method according to any one of the preceding claims, wherein the host cell produces the recombinant protein upon induction with IPTG, and optionally wherein step (d) comprises adding IPTG.

14. The method according to any one of the preceding claims, wherein the duration of step (e) is 12 to 96 hours, such as 20 to 72 hours, such as 25 to 55 hours, such as 30 to 50 hours, or the duration of step (e) is about 12 to about 96 hours, such as about 20 to about 72 hours, such as about 25 to about 55 hours, such as about 30 to about 50 hours.

15. A method according to any one of the preceding claims, wherein more than 75%, such as more than 90%, of the carbon source consists of glycerol.

16. A method according to any preceding claim, wherein the recombinant protein is an antibody, such as a Fab' fragment.

17. A method according to any of the preceding claims, wherein at least step (e) is carried out in a bioreactor, preferably having a volume equal to or greater than 100 L, equal to or greater than 500 L, equal to or greater than 1,000 L, equal to or greater than 2,000 L, equal to or greater than 5,000 L, equal to or greater than 10,000 L or equal to or greater than 20,000 L.

18. The method according to any one of the preceding claims, wherein the method 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. The method according to any one of the preceding claims, wherein the method of claim 18 comprises lyophilizing the recombinant protein.

20. A recombinant protein preparation obtainable or obtainable by a method according to any one of the preceding claims.

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