Methods for producing recombinant proteins

Abstract

The present invention relates to the field of recombinant production of proteins in bacterial host cells. In particular, the present invention relates to a method for culturing bacterial host cells for the production of recombinant proteins, in which the formation of struvite is reduced by keeping the amount of magnesium added during the production phase within a specific range.

Description

[Technical field]

[0001] The present invention relates to the field of recombinant production of proteins in bacterial host cells. In particular, the present invention relates to a method of culturing bacterial host cells for the production of recombinant proteins, in which the formation of struvite is reduced. [Background technology]

[0002] In the field of medicine, the use of biological entities such as antibodies or antibody-derived molecules is constantly growing in presence and importance. This has created a need for controlled production methods. The commercialization of proteins for medical applications has necessitated their production in large quantities, and much effort has been focused on improving the cultivation of recombinant host cells expressing the desired proteins and their processing. This has resulted in increased product titers, but as a consequence, higher amounts of biomass / debris and contaminants are also observed at the cell culture level. Removal of such contaminants can be laborious, and it is therefore preferable to optimize the methods to minimize the formation of undesired contaminants.

[0003] Struvite is a phosphate material with the formula NH4MgPO4·6H2O. Struvite can form in the presence of high concentrations of magnesium, ammonium, and phosphate. Struvite formation often occurs in wastewater treatment plants (see, for example, Kim et al., 2007). Struvite formation can also occur during bacterial fermentation (Beavon 1962). Struvite formation can result in undesirable precipitate formation and / or membrane fouling, which is problematic in the purification of recombinant proteins from host cells, especially in industrial-scale production of recombinant proteins.

[0004] While the use of struvite precipitation for waste removal or nutrient recovery, for example from wastewater, has been widely studied, little has been reported regarding the formation of struvite during the cultivation of host cells for recombinant protein production as to how to prevent or minimize struvite formation while still maintaining good cell growth and protein production.

[0005] This is addressed by the present invention. Summary of the Invention

[0006] In a first aspect, the present invention provides a method for producing a recombinant protein comprising the steps of: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing the bacterial host cells in a liquid medium; d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, For the entire method, a total amount of magnesium between 0.17 g and 0.28 g per kg of liquid medium in step (b) is provided; wherein the total amount of magnesium is provided stepwise during the culture from the start of step (b) to the end of step (e). In a further aspect, the present invention provides a method for reducing struvite formation, or reducing the risk of struvite formation, during the production of a recombinant protein in a bacterial host cell, comprising the steps of: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing the bacterial host cells in a liquid medium; d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, For the entire method, a total amount of magnesium between 0.17 g and 0.28 g per kg of liquid medium in step (b) is provided; wherein the total amount of magnesium is provided stepwise during the culture from the start of step (b) to the end of step (e). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Detailed Description of the Invention The present invention relates to a method for culturing cells to produce recombinant proteins. The inventors have surprisingly found that stepwise addition of magnesium to cell culture medium during fermentation, such that the concentration of magnesium during fermentation is maintained below a critical level, significantly reduces the risk of struvite precipitate formation while maintaining good cell growth and recombinant protein production. Depending on the magnesium concentration in the initial medium, the total amount of magnesium added during the production phase of the culture method should be maintained within a certain range to reduce the risk of struvite precipitate formation while maintaining good culture performance. Figure 1 is a non-limiting schematic diagram of an embodiment according to the present invention.

[0008] Thus, in a first embodiment, the present invention provides a method for producing a recombinant protein comprising the steps of: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing the bacterial host cells in a liquid medium; d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, For the entire method, a total amount of magnesium between 0.17 g and 0.28 g per kg of liquid medium in step (b) is provided; wherein the total amount of magnesium is provided stepwise during the culture from the start of step (b) to the end of step (e).

[0009] In a second embodiment, the present invention provides a method for reducing struvite formation or reducing the risk of struvite formation during the production of a recombinant protein in a bacterial host cell, comprising: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing the bacterial host cells in a liquid medium; d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, For the entire method, a total amount of magnesium between 0.17 g and 0.28 g per kg of liquid medium in step (b) is provided; wherein the total amount of magnesium is provided stepwise during the culture from the start of step (b) to the end of step (e).

[0010] In a third embodiment, the present invention relates to a method for producing a recombinant protein according to the first and second embodiments, wherein a total amount of magnesium between 0.17 g and 0.28 g is added in three or more stages during the culture from the start of step (b) to the end of step (e).

[0011] In a fourth embodiment, the present invention provides a method for producing a recombinant protein, comprising: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing the bacterial host cells in a liquid medium; d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, For the entire method, a total amount of magnesium between 0.17 g and 0.28 g per kg of liquid medium in step (b) is provided; wherein, of the total amount of magnesium provided, a first amount of magnesium is in the liquid medium in step (b), a second amount of magnesium is added as a supplement during step (c), and a third amount of magnesium is added as a supplement during step (e).

[0012] In a fifth embodiment, the present invention provides a method for reducing struvite formation or reducing the risk of struvite formation during the production of a recombinant protein in a bacterial host cell, comprising: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing the bacterial host cells in a liquid medium; d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, For the entire method, a total amount of magnesium between 0.17 g and 0.28 g per kg of liquid medium in step (b) is provided; wherein, of the total amount of magnesium provided, a first amount of magnesium is in the liquid medium in step (b), a second amount of magnesium is added as a supplement during step (c), and a third amount of magnesium is added as a supplement during step (e).

[0013] In a sixth embodiment, the total amount of magnesium provided for the method according to any of the first to fifth embodiments is between 0.18g and 0.27g per kg of liquid medium of step (b), or between 0.19g and 0.26g per kg of liquid medium of step (b). In a further embodiment, the total amount of magnesium provided for the method according to any of the embodiments of the invention is about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26 and about 0.27g (and any intermediate values ​​therein) per kg of liquid medium of step (b).

[0014] In a seventh embodiment, step (c) of the method according to any of the first to sixth embodiments comprises the addition of a second amount of magnesium of 0.04 to 0.22 g per kg of the liquid medium of step (b). As a further non-limiting embodiment, step (c) comprises the addition of a second amount of magnesium of 0.06 to 0.20 g per kg of the liquid medium of step (b). As an additional non-limiting embodiment, step (c) comprises the addition of a second amount of magnesium of 0.08 to 0.18 g per kg of the liquid medium of step (b). In other non-limiting embodiments, step (c) comprises adding a second amount of magnesium of about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, or 0.18 g (and any intermediate values ​​therein) per kg of liquid medium of step (b). In any of the embodiments of the method according to the invention, the magnesium added as the second amount during step (c) may be added, for example, as a bolus amount (i.e., a single dose added at once) and / or may be added as part of a fed-batch phase feed.

[0015] In one embodiment of the method according to any of the embodiments described herein, step (c) consists of a batch phase followed by a fed-batch phase, during which a bolus of magnesium is added and during which additional magnesium is added.

[0016] In an eighth embodiment, the total amount of ammonium provided in the method according to any of the first to seventh embodiments is at least 2 g ammonium per kg of liquid medium of step (b), but preferably not more than 20 g, for example 4-20 g, 5-15 g or 6-12 g ammonium.

[0017] In a ninth embodiment, the total amount of phosphate provided in the method according to any of the first to eighth embodiments is at least 1 g phosphate per kg of liquid medium of step (b), but preferably not more than 20 g, such as between 3 and 15 g, for example between 5 and 12 g or between 5 and 10 g phosphate.

[0018] The bacterial host cell of step (a) of the method according to any of the first to eighth embodiments is capable of producing a recombinant protein upon induction. The bacterial host cell used in the context of the present invention as a whole may be any suitable bacterial host cell that is suitable for recombinant production of a protein and capable of growing under the specified conditions. In a preferred embodiment, the bacterial host cell is an E. coli cell or a Bacillus cell. In a more preferred embodiment, the host cell is an E. coli host cell, such as an E. coli host cell of the K12, HB101, B7, RV308, DH1, HMS174, W3110 or BL21 strain, or a protease-deficient E. coli strain. Typically, a nucleic acid sequence encoding a recombinant protein under the control of an inducible promoter is introduced into the bacterial host cell. Suitable vectors for expressing such nucleic acid constructs in a host cell and methods for transformation or transfection of host cells are well known in the art. Suitable inducible promoters are also well known in the art, and some non-limiting examples are described herein below.

[0019] In step (c) of the method according to any of the first to ninth embodiments, the bacterial host cells are cultured in a liquid medium. Methods and media for culturing various types of bacterial host cells are well known in the art. The media vary depending on the organism, but typically contain components such as carbon source, nitrogen source, phosphorus source, essential metal ions and possibly trace elements (minimal medium). They may further contain additional components such as amino acids and vitamins (rich medium, see e.g. Elbing et al., 2019). Step (c) of the method according to any of the embodiments of the present invention typically involves a fed-batch culture in a bioreactor. The fed-batch phase may be preceded by a batch phase. Inoculation may be performed, for example, in a shake flask, directly from a working cell bank or via a seed culture. The main objective of step (c) of the method according to any of the embodiments of the present invention is to obtain sufficient biomass for the subsequent protein production phase. In one embodiment, step (c) 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, preferably to an OD600 of 20-80, more preferably 20-55 or 25-50.

[0020] The addition of magnesium to bacterial host cell cultures generally promotes growth. Magnesium has many roles in cells, including participating in the stabilization of membrane phospholipids, lipopolysaccharides, polyphosphate compounds such as DNA and RNA, and ribosomes. Magnesium is also required to make ATP biologically active and participates in the catalysis of certain enzymatic reactions through either direct or indirect mechanisms. In the context of the present invention, sufficient magnesium should be added for optimal growth and viability, but magnesium levels should not exceed a certain threshold concentration to avoid or minimize struvite formation. Therefore, according to the present invention, magnesium needs to be added stepwise during the fermentation process. Magnesium is typically added in the form of a magnesium salt.

[0021] In a tenth embodiment, the present invention provides a method for producing a recombinant protein, comprising: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing the bacterial host cells in a liquid medium; i) adding a bolus of magnesium when the OD600 is between 20 and 55; ii) further culturing the bacterial host cells, thereby increasing the dissolved oxygen (OD) until the DO is increased to 50% or greater than air saturation; iii) adding additional magnesium and culturing the host cells until the OD600 increases by at least 15, 20, 25, 35, 40 or 50 units, preferably 15 to 50 units, more preferably 20 to 40 units, above the OD600 mentioned in step (c)(i); d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, For the entire method, a total amount of magnesium between 0.17 g and 0.28 g per kg of liquid medium in step (b) is provided; wherein the total amount of magnesium is provided stepwise during the culture from the start of step (b) to the end of step (e).

[0022] In an eleventh embodiment, the present invention relates to a method for reducing struvite formation or reducing the risk of struvite formation during the production of a recombinant protein in a bacterial host cell, comprising: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing bacterial host cells in liquid medium to an OD600; i) adding a bolus of magnesium when the OD600 is between 20 and 55; ii) further culturing the bacterial host cells, thereby increasing the dissolved oxygen (OD) until the DO is increased to 50% or greater than air saturation; iii) adding additional magnesium and culturing the host cells until the OD600 increases by at least 15, 20, 25, 35, 40 or 50 units, preferably 15 to 50 units, more preferably 20 to 40 units, above the OD600 mentioned in step (c)(i); d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, For the entire method, a total amount of magnesium between 0.17 g and 0.28 g per kg of liquid medium in step (b) is provided; wherein the total amount of magnesium is provided stepwise during the culture from the start of step (b) to the end of step (e).

[0023] It is well known that an increase in DO is associated with the depletion of carbon sources.

[0024] As a non-limiting embodiment, magnesium can be added as a bolus in step (c)(i) of the method in an amount of 0.03 g to 0.12 g per kg of liquid medium of step (b), and the total amount of magnesium added as supplement in step (c)(iii) can correspond to 0.01 g to 0.10 g of magnesium per kg of liquid medium of step (b). As a further non-limiting embodiment, magnesium can be added as a bolus in step (c)(i) in an amount of 0.04 g to 0.11 g per kg of liquid medium of step (b), and the total amount of magnesium added as supplement in step (c)(iii) can correspond to 0.02 g to 0.09 g of magnesium per kg of liquid medium of step (b). As additional non-limiting embodiments, magnesium can be added as a bolus in step (c)(i) in an amount of 0.05 g to 0.10 g per kg of liquid medium of step (b), and the total amount of magnesium added as supplement in step (c)(iii) can correspond to 0.02 g to 0.08 g of magnesium per kg of liquid medium of step (b). In some non-limiting embodiments, magnesium can be added as a bolus in step (c)(i) at about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, or about 0.12 g of magnesium (as well as any intermediate values ​​therein) per kg of liquid medium of step (b), and the total amount of magnesium added as supplement in step (c)(iii) can correspond to about 0.01, 0.02, 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10 g of magnesium (as well as any intermediate values ​​therein) per kg of liquid medium of step (b).

[0025] In another embodiment, the magnesium added as supplement in step (c)(iii) is added as part of the main feed (i.e. the feed containing the carbon source). Alternatively, the magnesium may be added as a supplemental feed, either simultaneously with or separately from the main feed. The feed may be an intermittent feed or a continuous (uninterrupted) feed.

[0026] In one embodiment, step (d) is initiated when the DO increases to 50% of air saturation in the culture or when the pre-determined OD600 defined in step (c)(iii) 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 technique.

[0027] In step (d) of the method, production of the recombinant protein is induced. In one embodiment, no recombinant protein is produced or not significantly produced prior to step (d).

[0028] Induction of recombinant protein production can be achieved by any suitable method. In one embodiment, the gene encoding the recombinant protein is under the control of an inducible promoter. Many such inducible promoters are known in the art. A well-known bacterial expression system that uses an inducible promoter is one 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-D-1-thiogalactopyranoside). Other known bacterial expression systems include, for example, the araBAD promoter system (see, for example, Guzman et al., 1995) or the T7 / lac system (see, for example, Rosenberg et al., 1987). These and other systems are reviewed, for example, in Rosano and Ceccarelli (2014).

[0029] In one embodiment of the method of the invention, the bacterial host cell comprises a nucleic acid sequence encoding a recombinant protein under the control of an IPTG-inducible promoter and thus produces the recombinant protein upon induction with IPTG. In such an embodiment, step (d) of the method comprises the addition of IPTG. Alternatively, the bacterial host cell may comprise a nucleic acid sequence encoding a recombinant protein under the control of, for example, an arabinose-inducible promoter (e.g., an araBAD promoter), a tryptophan-inducible promoter (e.g., a trp promoter) or a phosphate-inducible promoter (e.g., a phoA promoter) and thus produce the recombinant protein upon induction with arabinose, tryptophan or phosphate, respectively. In such an embodiment, step c) comprises the addition of arabinose, tryptophan or phosphate.

[0030] In step (e) of the method of the invention, the bacterial host cells are cultured to produce the recombinant protein.

[0031] Step (e) typically comprises a fed-batch culture in a bioreactor. In one embodiment, the duration of step (e) of the method according to any of the embodiments of the present invention is from about 10 to about 96 hours, such as from about 12 to about 72 hours, such as from about 15 to about 55 hours, such as from 1 to about 8 to about 50 hours.

[0032] In a non-limiting embodiment of the method according to any of the embodiments of the present invention, the total amount of magnesium added as supplement during step (e) of the method is 0.02 g to 0.08 g per kg of the liquid medium of step (b). In a further non-limiting embodiment of the method according to any of the embodiments of the present invention, the total amount of magnesium added as supplement during step (e) is 0.02 g to 0.07 g per kg of the liquid medium of step (b). In an additional non-limiting embodiment, the total amount of magnesium added as supplement during step (e) is 0.03 to 0.06 g per kg of the liquid medium of step (b). In some specific non-limiting examples, the total amount of magnesium added as supplement during step (e) of the method according to any of the embodiments of the present invention is about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07 or about 0.08 g (as well as any intermediate values ​​therein) per kg of the liquid medium of step (b).

[0033] According to the present invention, the total amount of magnesium provided in the method to reduce or prevent struvite precipitation is 0.17g-0.28g per kg of liquid medium in step (b). If magnesium is deficient or very low in the liquid medium, the total amount of magnesium added in the method [i.e. steps (c) and (e)] can be 0.17g-0.28g per kg of liquid medium in step (b). Since most commonly used liquid media contain magnesium, the total amount of magnesium added in steps (c) and (e) needs to be adapted since it depends on the magnesium content in the liquid medium. Recipes for liquid media are well known. For example, a person skilled in the art will know that M9 minimal medium contains about 0.002g / kg magnesium, while Durany medium contains about 0.01g / kg magnesium. As a non-limiting embodiment, if the total amount of magnesium is targeted to 0.20 g / kg of liquid medium in step (b), starting with M9 minimal medium, one skilled in the art can add a total of 0.198 g magnesium / kg of liquid medium during steps (c) and (e), for example by adding a bolus of 0.1 g / kg, an exponential feed providing 0.05 g / kg, and a production phase feed providing 0.048 g / kg. In a further non-limiting embodiment, if the total amount of magnesium is targeted to 0.18 g / kg of liquid medium, starting with Durany minimal medium, one skilled in the art can add a total of 0.17 g magnesium / kg of liquid medium during steps (c) and (e), for example by adding a bolus of 0.1 g / kg, an exponential feed providing 0.025 g / kg, and a production phase feed providing 0.045 g / kg.

[0034] In a further embodiment, a feed containing a carbon source (also referred to herein as "main feed") is added at the beginning of step (c)(iii) of the method of the invention. Preferably, the amount of carbon source added per unit time to the culture is less in step (e) than in step (c)(iii), by reducing the feed rate or by reducing the concentration of the carbon source in the feed.

[0035] As described herein, the methods of the invention involve culturing a bacterial host cell in the presence of a magnesium source, such as a magnesium salt.

[0036] As mentioned above, magnesium plays an important role in the growth and metabolic functions of microbial and animal cells, and the Mg content in cell culture and fermentation media is 2+ The availability of can dramatically affect cell growth and metabolism.

[0037] The magnesium salt provided by the present invention may consist of a mixture of magnesium salts or a single magnesium salt. Any magnesium salt suitable for the growth of microorganisms or animal cells may be used, such as, but not limited to, magnesium sulfate, magnesium chloride or even magnesium boride, either on its own or in their hydrated form. In one embodiment, the magnesium salt provided does not contain a significant amount of magnesium phosphate. Preferably, the magnesium salt does not contain any salt of magnesium and phosphate. In a preferred embodiment, the magnesium salt comprises or consists of magnesium sulfate or magnesium chloride, either on its own or in their hydrated form.

[0038] The liquid medium may be a minimal or rich medium such as (but not limited to) M9, M63, Durany, LB, TNT or their induction media (see, e.g., Elbing et al., 2019).

[0039] The liquid medium typically comprises magnesium. In one embodiment, the liquid medium of step (b) of the method of the present invention comprises a total amount of magnesium between 0.001g and 0.25g per kg of liquid medium, such as between 0.01g and 0.25g, 0.02-0.25g, 0.02-0.2g, 0.02-0.1, such as (about) 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 or 0.09g per kg of liquid medium of step (b) of the method according to any of the embodiments of the present invention.

[0040] The liquid medium typically also contains ammonium. Ammonium salts are an important source of nitrogen. Any ammonium salt or mixture of ammonium salts suitable for growing or growing microbial or animal cells may be used, including but not limited to ammonium sulfate, ammonium phosphate, ammonium chloride and ammonium carbonate. Preferably, the ammonium salt does not include a salt of ammonium and phosphate. In one embodiment, the total amount of ammonium provided for the method is at least about 2 g of ammonium per kg of the liquid medium of step (b) of the method of the invention, but is preferably 20 g or less, such as 4-20 g, 5-15 g or 6-12 g, such as (about) 6.0, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12 g of ammonium per kg of the liquid medium of step (b).

[0041] The liquid medium of step (b) typically also comprises phosphate. In one embodiment the liquid medium corresponds to at least 1 g phosphate per kg of liquid medium of step (b), but preferably contains up to 20 g / kg, such as 3-15 g / kg or 5-12 g / kg, for example about 5, 6, 7, 8, 9, 10, 11 or 12 g phosphate per kg.

[0042] The method of the present invention typically includes the addition of one or more organic carbon sources. The carbon source used may 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%, such as at least 90%, of the carbon source in the culture medium of step b) consists of glycerol. In another preferred embodiment, more than 75%, such as at least 90%, of the carbon source in the culture medium of step (e) of the method of the present invention consists of glycerol. As another example, more than 75%, such as at least 90%, of the carbon source in the culture medium of step (c) consists of glucose. In another preferred embodiment, more than 75%, such as at least 90%, of the carbon source in the culture medium of step (e) consists of glucose. As a further example, more than 75%, such as at least 90%, of the carbon source in the culture medium of step (c) consists of lactose. In another preferred embodiment, more than 75%, such as at least 90%, of the carbon source in the culture medium in step (e) consists of lactose.

[0043] The formation of struvite has been described to be influenced by pH (see, for example, Perez-Garcia et al., 1989). In one embodiment of the method of the present invention, the pH of the culture in step (c) of the method 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, 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. In another embodiment, the pH of step (c) is between 6 and 8, such as between 6.5 and 7.5, for example between 6.6 and 7.4, such as between 6.7 and 7.3, for example between 6.8 and 7.2, and the pH of step (e) is between 6 and 8, such as between 6.5 and 7.5, for example between 6.6 and 7.4, such as between 6.7 and 7.3, for example between 6.8 and 7.2. The temperature is typically kept as constant as possible throughout the entire fermentation process.

[0044] The recombinant protein produced in the method of the invention is typically a heterologous protein derived from another organism. For example, the recombinant protein may be an antibody, a cytokine, a growth factor, a hormone or other peptide or polypeptide.

[0045] In a preferred embodiment, the recombinant protein is an antibody. The term "antibody" as used herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies produced by recombinant techniques known in the art. "Antibody" includes antibodies of any species, particularly mammalian species, such as IgG1, IgG 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, e.g. from chimpanzees, baboons, rhesus monkeys or cynomolgus monkeys, rodent antibodies, e.g. from mice or rats, rabbit, goat or horse antibodies, camelid antibodies (e.g. from camels or llamas such as Nanobodies™) and derivatives thereof, avian antibodies, e.g. chicken antibodies, or antibodies of fish species, e.g. 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 that contain 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. A "humanized" antibody is a chimeric antibody that contains sequences derived from a non-human antibody. In most cases, humanized antibodies are human antibodies (recipient antibody) 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 that have the desired specificity, affinity, and activity. In most cases, residues of the human (recipient) antibody outside the CDRs, i.e., in the framework regions (FR), are further replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine the 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 technologies, using recombinant DNA libraries that are at least partially generated artificially or from a donor immunoglobulin variable (V) domain gene repertoire. 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 that can then be screened for optimal human antibodies. The term "antibody" refers to both glycosylated and non-glycosylated antibodies. Furthermore, the term "antibody" as used herein refers not only to full-length antibodies, but also to antibody fragments. Fragments of antibodies 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')2, Fv, Fab-Fv, Fab-dsFv, Fab-Fv-Fv, scFv and Bis-scFv fragments. Fragments can also be diabodies, tribodies, triabodies, tetrabodies, minibodies, single domain antibodies (dAbs), such as sdAbs, VL, VH, VHH or camelid antibodies (e.g. from camels or llamas, such as Nanobodies™) and VNAR fragments. Antigen-binding fragments according to the invention can also comprise a Fab linked to one or two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g. one scFv or dsscFv that binds to a therapeutic target and one scFv or dsscFv that extends 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 for example 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.

[0046] The method according to any 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 required production scale. In a preferred embodiment, at least steps (c), (d) and (e) are carried out in a bioreactor, preferably an industrial scale bioreactor. The bioreactor may for example be a stirred tank or an airlift type reactor. The bioreactor may be a reusable reactor made of glass or metal, for example stainless steel, or a disposable bioreactor made of synthetic materials such as plastic. In a preferred embodiment, at least step (e) of the method of the present invention 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 to 20,000 L, 10,000 to 20,000 L, or 10,000 to 25,000 L. In a preferred embodiment, in step (b), (c), (d) or (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. In a further preferred embodiment, 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 method of the present invention may include one or more further steps after step (e). For example, the method may include the further step of recovering the recombinant protein, which may include first separating the cells from the supernatant or inclusion bodies. Once recovered, the recombinant protein can be isolated and purified. Isolation and purification methods are well known to those skilled in the art. They typically consist 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, for example therapeutic or prophylactic use. In one embodiment, the recombinant protein is modified (e.g., conjugated to another molecule) before being formulated into a pharmaceutical composition. [Brief description of the drawings]

[0048] [Figure 1] Schematic diagram of the phases of the method according to the invention. During the batch phase, cells are grown on a submerged carbon source. Once a defined OD600 is reached, a magnesium bolus is added. Once the batched carbon source is exhausted, feeding is initiated (exponential feeding) and the cells continue to grow. The batch and exponential feeding phases correspond to step (c) described herein. Then, after induction of expression of the heterologous protein [corresponding to inducer addition - step (d)], the cells continue to be cultivated while the heterologous protein is produced [corresponding to step (e)]. [Diagram 2] FIG. 2a shows pellets taken from a fermentation bioreactor in which struvite was produced (struvite can be seen as white spots indicated by arrows) and FIG. 2b shows those from a fermentation in which no struvite was present. [Diagram 3] FIG. 1 shows the magnesium concentration (grams of magnesium per kg of broth from step (b)) in supernatant samples taken during the fermentation process from experiments performed with varying levels of magnesium in the bolus. [Figure 4] FIG. 1 shows magnesium concentrations in supernatant samples taken during the fermentation process described in Example 4. EXAMPLES

[0049] Materials and Methods In the following examples, unless otherwise stated, the methods are carried out as follows.

[0050] Cultivation of cells. Frozen cell bank vials containing E. coli W3110 host cells expressing antibody A (Fab' fragment with pI in the range of 8.8-9.3) were used to inoculate shake flasks (700 mL total volume) containing 6x peptone-yeast extract (6xPY) medium + tetracycline. The shake flasks were incubated at 30°C and 200-250 rpm. At the required OD range, the shake flasks were used to inoculate a seed fermentor (20 L total volume) containing synthetic medium (derived from MD medium of Durany et al., 2004, containing approximately 0.05 g / kg Mg) + tetracycline with a carbon source. The cell culture in the seed fermentor was maintained at 30°C, the dissolved oxygen concentration (DO) was maintained above 20% of air saturation, and the pH was controlled at approximately 7.0. At the required OD range, the seed culture was used to inoculate a production fermenter (175 L liquid medium) containing the same synthetic medium used in the seed fermenter. The production fermenter was maintained under the same conditions as the seed fermenter and grown in batch phase until the carbon source was exhausted. During this period, bolus additions of MgSO4 were performed to avoid exhaustion of this metabolite (see all examples). At the end of the batch phase (signaled by a spike in the measured DO), the exponential carbon source feed (containing magnesium at a level of about 0.06 g / kg liquid medium in step (b)) was switched on and the culture was fed with a specific amount of carbon source to achieve an OD600 of more than 50 units. At this point, the carbon source feed was switched from exponential phase to production phase feed (see all examples) and expression of Antibody A was induced by addition of IPTG. The cells (containing expressed Antibody A) were harvested more than 40 hours after induction.

[0051] Struvite analysis. For each sampling point, triplicate 1 mL broth cultures were centrifuged in 2 ml Eppendorf tubes, the supernatant discarded, and the cell pellets dried by inverting the tubes according to standard methods. The pellets were further dried in a 110°C oven for >24 h and then visually inspected to qualitatively assess the presence of struvite by comparison with reference images of pellets with (shown in Figure 2a) and without (shown in Figure 2b).

[0052] Magnesium analysis: The concentration of magnesium in the samples was determined using a Quantichrom Magnesium Assay kit (BioAssay Systems, Cat. No. DIMG-250) and a FLUOstar OPTIMA microplate reader (BMG LABTECH) according to the manufacturer's instructions.

[0053] DO Measurement. Dissolved oxygen (DO) was measured using an online polarographic dissolved oxygen sensor.

[0054] Example 1 Method A Fermentations were carried out as described above. Samples were taken during the fermentation and analyzed for magnesium levels as well as the presence of struvite. The amounts of magnesium added via the bolus and production phase feeds are shown in the table below. [Table 1]

[0055] In this example, the total amount of magnesium fed to the cells throughout the fermentation process (via broth, bolus, exponential feed and linear feed) was greater than 0.3 g / kg broth in step (b).

[0056] Struvite is clearly visible in the pellets resulting from the samples taken at the harvest point (data not shown), and therefore it can be concluded that the bolus and feed concentrations used are not appropriate.The disadvantage of struvite precipitation in the bioreactor required the method to be modified.The molar amount of magnesium provided to the method is at least 5 times lower than the respective molar amounts of phosphate and ammonium, so the inventors hypothesized that struvite precipitation is most significantly affected by the total amount of magnesium provided to the fermentation method (i.e., the amount of magnesium contained in the liquid medium plus the magnesium added as a bolus in the exponential feed addition and the production phase feed).

[0057] Example 2. Effect of reducing Mg in the production phase feed and varying the Mg bolus amount Since we did not intend to change the composition of the broth or the exponential feed, we decided to study the effect of magnesium added as a bolus and in the production phase feed. Fermentations were carried out as described in Materials and Methods. Samples were taken during the fermentation and analyzed for magnesium levels as well as the presence of struvite. The amount of magnesium in the production phase feed was set at about 0.04 g / kg of broth in step (b) for all three conditions. This is about half the amount used in the production phase feed described in Example 1. The level of magnesium added as a bolus was varied as shown in the table below. [Table 2]

[0058] In this example, the total amount of magnesium fed to the cells throughout the fermentation process (via broth, bolus, exponential feed and linear feed) was approximately 0.2-0.25 g per kg of broth in step (b).

[0059] FIG. 3 shows that adaptations to the amounts used in the production phase feed and bolus had the expected effect on the resulting magnesium levels in the fermentation broth.

[0060] Visual inspection of pellets obtained from samples taken at the harvest points clearly indicates that none of the conditions evaluated led to the formation of struvite. Moreover, cell growth was not affected (data not shown).

[0061] Example 3. Effect of further bolus reduction Fermentations were carried out as described in the Materials and Methods section. Samples were taken during the fermentation and analyzed for magnesium levels and the presence of struvite. The concentrations of magnesium added to the bolus and feed are shown in the table below. [Table 3]

[0062] The amount of magnesium added in the bolus in this example is reduced by 83% compared to the midpoint of the study reported in Example 2. The total amount of magnesium supplied to the cells throughout the fermentation process was less than about 0.16 g / kg of broth in step (b).

[0063] Visual inspection of pellets from samples taken at harvest points clearly indicates that these concentrations did not result in the formation of struvite (data not shown). Figure 4 shows that magnesium concentrations dropped to low levels and remained low for a significant period of time, which had a detrimental effect on cell growth and led to a subsequent increase in magnesium levels (as the cells were no longer utilizing much of the magnesium that was being added via the feed due to the reduced growth).

[0064] Overall conclusion It was the inventors' surprising discovery that by maintaining the total amount of magnesium provided to the culture at about 0.17 to about 0.28 g / kg of liquid medium in step (b) and adding magnesium in a stepwise manner, struvite precipitation can be reduced or avoided while maintaining good culture performance.

[0065] References JPEG2024516892000004.jpg60138

Claims

1. 1. A method for producing a recombinant protein, comprising: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing said bacterial host cells in said liquid medium; d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, A total amount of magnesium of 0.17 g to 0.28 g per kg of liquid medium of step (b) is provided throughout the process; and The above method, wherein the total amount of magnesium is provided stepwise during the culture from the start of step (b) to the end of step (e).

2. 1. A method for reducing struvite formation or reducing the risk of struvite formation during recombinant protein production in a bacterial host cell, comprising: a) providing a bacterial host cell capable of producing a recombinant protein upon induction; b) providing a volume of liquid medium; c) culturing said bacterial host cells in said liquid medium; d) inducing production of the recombinant protein; e) further culturing the bacterial host cells in liquid medium to produce the recombinant protein; Including, A total amount of magnesium of 0.17 g to 0.28 g per kg of liquid medium of step (b) is provided throughout the process; and The above method, wherein the total amount of magnesium is provided stepwise during the culture from the start of step (b) to the end of step (e).

3. 3. The method of claim 1 or 2, wherein of the total amount of magnesium provided, a first amount of magnesium is present in the liquid medium in step (b), a second amount of magnesium is added as a supplement during step (c), and a third amount of magnesium is added as a supplement during step (e).

4. 3. The method of claim 1 or 2, wherein step (c) comprises growing the culture to an OD600 of at least 35.

5. 3. The method of claim 1 or 2, wherein step (d) is initiated when dissolved oxygen increases to 50% or greater of air saturation in the culture.

6. 3. The method of claim 1 or 2, wherein the magnesium supplement added during step (c) comprises the addition of a total amount of 0.04 to 0.22 g magnesium per kg of the liquid medium of step (b).

7. Step (c) is i) culturing a bacterial host cell; ii) adding a magnesium supplement equivalent to 0.03 g to 0.12 g of magnesium per kg of the liquid medium of step (b) when the OD600 is between 20 and 55; iii) culturing the bacterial host cells until the dissolved oxygen increases to 50% or greater than air saturation; iv) adding a total amount of magnesium of 0.01 g to 0.10 g per kg of the liquid medium of step (b) and culturing the host cells until the OD600 of the culture increases by at least 20 units above the OD600 mentioned in step (ii); The method of claim 1 or 2, comprising:

8. 3. The method according to claim 1 or 2, wherein the total amount of magnesium added as supplement during step (e) is between 0.02 and 0.08 g magnesium per kg of the liquid medium of step (b).

9. 3. The method according to claim 1 or 2, wherein magnesium is added in the form of a salt.

10. The method of claim 1 or 2, wherein the bacterial host cell is an E. coli cell.

11. The method according to claim 1 or 2, wherein the pH of the culture in step (c) is from 6 to 8 and / or the pH of the culture in step (e) is from 6 to 8.

12. The method according to claim 1 or 2, wherein the duration of step (e) is from 10 to 96 hours.

13. The method of claim 1 or 2, further comprising a step of recovering the recombinant protein.

14. 14. The method of claim 13, further comprising the step of purifying the recombinant protein and, optionally, the further step of formulating the recombinant protein.

15. 3. The process according to claim 1 or 2, which is an industrial scale process.

16. 3. The method of claim 1 or 2, wherein step (e) is carried out in a bioreactor having a volume of 100 L or more.

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