Microbial culture medium

Abstract

To provide a method for reducing precipitation in bacterial fermentation processes. [Solution] A method comprising the steps of a) culturing host cells for bacterial growth in a batch phase using a batch medium, and b) culturing host cells in a fed batch phase to produce a fermentation product using a feed medium, wherein the feed medium and the batch medium are each non-precipitated medium solutions, i) the batch medium comprises calcium salts, magnesium salts, phosphates, and at least one chelating agent which is at least 5 mM citrate and / or citric acid and / or at least 1 mM EDTA, ii) the feed medium contains at least 1 mM of calcium salts which is at least 5 times higher than that of the batch medium, iii) the feed medium contains at least 3 mM of magnesium salts which is at least 2 times higher than that of the batch medium, and iv) the feed medium contains less than 90% of the amount of phosphates contained in the batch medium.

Description

Technical Field

[0001] The present invention relates to a method for reducing precipitation in the fermentation process of bacteria, and a medium system for use in the fermentation process of bacteria including a batch medium and a feed medium.

Background Art

[0002] One of the most efficient methods for recombinant protein production in bacterial cells such as Escherichia coli is fed-batch. During the batch phase, cells can grow to a very high cell density. This batch phase is followed by a fed-batch phase, during which the formation of the fermentation product is switched on and the cells are supplied to produce the product.

[0003] Culturing Escherichia coli cells to a high cell density is an essential prerequisite for a high yield of the fermentation product. For this purpose, the cells have grown unrestrictedly in the batch phase to achieve a high cell density (μ = μ max ), and during the subsequent fed-batch phase, the carbon source (e.g., glucose or glycerol) is usually metered under substrate-limiting conditions. High cell density fermentation of bacterial cells is an attractive biotechnological means to achieve a high space-time yield for the production of fermentation products, such as heterologous proteins.

[0004] Fed-batch high density fermentation of Escherichia coli has the drawback of moderate to severe precipitation in cell culture. The occurrence of precipitation may already start when preparing the cell culture medium, for example, when preparing the medium at a low pH, after heat sterilization, or when setting the pH to the desired value of about pH 7.0. So far, even if the medium compound precipitates, it is assumed that the compound will redissolve when it becomes limited.

[0005] Korz et al. (J. Biotechnol. 1995, 39:59-65) describe a fed-batch technique for high-cell-density culture of E. coli using glucose or glycerol as a carbon source. By using the feed medium at a predetermined feed rate, carbon-limited growth in the fed-batch process is maintained at a growth rate that does not cause the formation of acetic acid, a toxic byproduct resulting from incomplete substrate oxidation.

[0006] Wilms et al. (Biotechnology and Bioengineering 2001,73(2):95-103) describes high-cell-density fermentation for heterologous protein production in Escherichia coli.

[0007] Calleja et al. (Biotechnology and Bioengineering 2016, 113(4):772-782) describe the simulation and prediction of protein production in fed-batch E. coli cultures. Different specified minimal media using glucose as the sole carbon source were used for various strains. A feed medium composition containing a phosphate solution was used to avoid co-precipitation with magnesium salts.

[0008] Hardiman et al. (J. Biotechnol. 2007, 132:359~374) describe the culture of E. coli strains in fed batches, as well as the use of batch and fed-batch media. Phosphate was used to degrade any precipitates.

[0009] European Patent No. 1584680 discloses a defined cell culture medium for use in a fed-batch fermentation process for producing plasmid DNA. The feed medium contains at least the same amount or more of phosphate compared to the batch medium.

[0010] International Publication No. 95 / 29986 discloses a method for controlling metallophosphate precipitation in high-cell-density bacterial fermentation using phosphate glass as a phosphorus source.

[0011] International Publication No. 2018011242 discloses a fermentation medium containing a specific chelating agent.

[0012] Shiloach et al. (Biotechnology Advances 2005, 23:345-357) outline the development of a method for growing E. coli to high cell density.

[0013] Riesenberg et al. (J Biotechnol. 1991, 20:17-28) describe a fed-batch process for culturing E. coli at a controlled, specific growth rate and high cell density using glucose supplemented with magnesium sulfate.

[0014] Precipitates in bacterial cell cultures can affect the robustness of the bacterial fermentation process. Compounds in the precipitate are unavailable to the cells. Variations in the amount and composition of precipitate due to different handling procedures or upscaling can lead to variations in the nutrient availability of the cells. Furthermore, this can create problems for analysis (e.g., optical density (OD) measurements disrupted by particles), downstream processing (DSP, e.g., filter clogging), or when a filtration step of the culture medium is required before use (e.g., packed containers for small-volume, high-throughput fermentation systems after pH setting). Therefore, reducing precipitates in bacterial cell cultures is desirable. [Overview of the Initiative]

[0015] The object of the present invention is to provide a fed-batch bacterial cell culture process that avoids precipitation in cell culture media, and a corresponding non-precipitating medium system. A further object is to provide batch and feed media for high cell density fermentation by bacterial cells such as E. coli, wherein precipitation of the medium compound is prevented within a reasonable pH range for growth (pH ± 6.7 to 7.3).

[0016] This objective is addressed by the subject matter claimed and further described herein.

[0017] The present invention provides a method for reducing precipitation in a bacterial fermentation process for producing fermentation products, and the fermentation process is a) A step of culturing host cells for bacterial growth in a batch phase using batch medium, and b) The step of culturing host cells using feed medium to produce a fermentation product in a fed batch phase, The feed medium and batch medium are both non-precipitated medium solutions. Here, i) The batch medium comprises a certain amount of calcium salt, magnesium salt, phosphate, and at least one chelating agent, which is at least 5 mM of a certain amount of citrate and / or citrate and / or at least 1 mM of a certain amount of EDTA. ii) The feed medium is at least 1 mM and contains at least 5 times a higher amount of calcium salt (M / M) compared to the batch medium. iii) The feed medium is at least 3 mM and contains at least 2 times, or at least 3 times, or at least 5 times, or at least 10 times, or at least 15 times higher amounts of magnesium salts compared to the batch medium (M / M), iv) The feed medium contains less than 90% of the amount contained in the batch medium, or less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, or less than one of the following amounts of phosphate: 9, 8, 7, 6, 5, 4, 3, 2, or 1% (M / M).

[0018] According to a particular aspect, a) The amount of calcium salt is a maximum of 1 mM in batch medium, a maximum of 5 mM or a maximum of 4 mM in feed medium, and / or b) The amount of magnesium salt is 1–3 mM in batch medium, 3–100 mM or 30–60 mM in feed medium, and / or c) The amount of phosphate is 30 - 120 mM in the batch medium, and in the feed medium, it is less than 90% (m / m) of the amount contained in the batch medium, preferably less than or equal to 10 mM in the feed medium, preferably at most 9 mM, at most 8 mM, at most 7 mM, at most 6 mM, at most 5 mM, or at most 4 mM, or at most 3 mM, and preferably there is no phosphate in the feed medium.

[0019] The chelating agent may contain, or consist of, any one of citrate salts such as sodium citrate, citric acid, or EDTA, or may be a mixture of sodium citrate and citric acid, for example, to obtain the total amount of citrate ions, or may be a mixture of EDTA and any one or both of citrate salts and citric acid.

[0020] Specifically, the preferred amount of the chelating agent is any one of at least 5, 6, 7, 8, 9, or 10 mM, and any one of a maximum of 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 mM of citrate ions (total), preferably within the molar range of 6 to 70 mM of citrate ions (total), and contains or consists of citrate salts such as sodium citrate and / or citric acid.

[0021] Specifically, the preferred amount of the chelating agent contains, or consists of, EDTA such as sodium EDTA, in any one of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM, and any one of a maximum of 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 mM, preferably within the molar range of 1.5 to 22 mM.

[0022] According to a specific embodiment, the batch medium further contains all nutrients, supplements, and excipients as required for growing bacterial cells at high density.

[0023] Specifically, the batch medium contains an organic carbon source such as glucose and / or glycerol, or any complex carbohydrate appropriately used to grow bacterial cells.

[0024] Specifically, the batch medium further contains an ammonium salt.

[0025] According to certain embodiments, the feed medium further contains all nutrients, supplements, and excipients as required to produce fermentation products in a culture of high-density bacterial cells.

[0026] Specifically, the feed medium contains an organic carbon source such as glucose and / or glycerol, or any chemically defined carbohydrate appropriately used in a mineral medium. Specifically, the feed medium is added to the culture of cells during the fed-batch phase in a growth-limiting mode.

[0027] According to certain embodiments, either or both of the batch medium and the feed medium further contain trace elements.

[0028] According to certain embodiments, either or both of the batch medium and the feed medium have a pH in the range of 6.7 to 7.3.

[0029] According to certain embodiments, the fermentation process is carried out at a pH in the range of 6.7 to 7.3, and preferably ammonia water or an alkaline solution or compound is metered for pH adjustment in order to obtain the desired pH throughout the culture of cells or at least during the fed-batch phase.

[0030] Typically, the fed batch phase begins approximately 8–24 hours after the cells have been cultured in the batch phase, and this depends on various individual factors such as temperature, medium composition, medium concentration, reactor size, and especially the properties of the bacterial strain used. Advantageously, the synthesis of the fermentation product switches on approximately 0–15 hours after the start of the fed batch phase. However, the exact time may depend on the cell density of the culture already reached at this point. By achieving the desired cell density before the production phase, the volume yield of the desired fermentation product can be maximized.

[0031] According to a particular embodiment, host cells are grown in a batch phase to at least one of the following densities: 5, 10, 20, 30, 40, or 50 g cell dry weight / liter, and the host cells are cultured in a fed batch phase under growth-restricted conditions. Cell densities between 10 and 80 g / l, preferably between 20 and 60 g / l, are particularly preferred.

[0032] At the start of the production phase, it is preferable that the cell density reaches approximately 10-60% of the maximum cell density.

[0033] In a particular embodiment, the bacterial host cell is a producible host cell of a species selected from the group consisting of the genera Escherichia, Pseudomonas, Bacillus, Lactococcus, Corynebacterium, Clostridium, Micrococcus, and Streptomyces.

[0034] Any suitable Gram-negative bacteria can be used as host cells for producing the fermentation products described herein. Suitable Gram-negative bacteria include Escherichia coli, Salmonella typhimurium, fluorescent bacteria, Erwinia carotovora, Shigella, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, and Acinetobacter baumanni. Preferably, the production host cells are Escherichia coli, in particular recombinant Escherichia coli engineered to produce the fermentation products in high yield.

[0035] Examples of E. coli include strains derived from E. coli K12, specifically HMS174, HMS174(DE3), XL1-Blue, C600, DH1, HB101, JM101, JM105, JM109, RV308, DH5α, XL10-Gold, TOP10, MG1655, DH10B, W3110, Origami, and BW25113, as well as strains derived from strain B, specifically BL-21, BL21(DE3), Rosetta, and C41(DE3).

[0036] Preferred bacterial host cells are recombinant cells that have been genetically engineered to express gene constructs for producing fermentation products not produced by wild-type (unengineered) host cells, or to express such gene constructs in higher yields compared to such wild-type host cells.

[0037] The fermentation product may be a heterologous product, or a product naturally produced by wild-type host cells but in a lower yield.

[0038] In certain embodiments, the fermentation product is one of the following: the protein of interest (POI), a metabolic pathway, RNA (such as siRNA), or a recombinant DNA molecule such as plasmid DNA or a plasmid vector. In certain embodiments, the fermentation product is not plasmid DNA.

[0039] For example, a bacterial host cell may be a recombinant host cell genetically engineered to introduce a heterologous expression cassette containing a gene of interest (GOI) encoding a protein of interest (POI). The bacterial host cell can then produce the POI by expressing the GOI and isolating the POI from the cell culture. According to a particular embodiment, the bacterial host cell is engineered to express the POI by incorporating one or more genes encoding helper proteins to facilitate protein folding.

[0040] In another example, bacterial host cells can be genetically engineered to produce RNA or DNA molecules, including covalently closed-ring (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors, and hybrids thereof.

[0041] The host cells may produce fermentation products into the cell culture medium, or they may be destroyed to release fermentation products into the cell culture medium. The fermentation products are appropriately isolated from the cell culture medium and, if necessary, purified.

[0042] Specifically, the fermentation process includes a step that induces the production phase. This induction step may involve changes in culture techniques, such as changes in the culture medium or temperature shifts.

[0043] Certain embodiments utilize host cells engineered to incorporate an inductive substance, such as an inductive promoter. An inductive promoter, which is activated immediately upon application of an inductive stimulus, can be used to direct gene transcription under its control. Under growth conditions with an inductive stimulus, cells typically grow more slowly than under normal conditions.

[0044] In a particular embodiment, a bacterial host cell contains a gene construct, particularly a heterogeneous gene construct such as an expression cassette, for producing and / or expressing a fermentation product containing an inducible promoter, wherein the expression of the gene construct is induced by depletion or restriction of the cell culture components or by the addition of an inducing substance.

[0045] Protein synthesis can be initiated by switching on a controllable promoter system. Depending on the system used, this switching is, in principle, done by adding a substance or changing its physical quantity.

[0046] Various inducible promoters can be used, such as the bacterial alkaline phosphatase (phoA) promoter, tac, lac, pac, T7, T5, A1, A3, lpp, Sp6, npr, trc, syn, σ70, pL, cspA, thrC, trp, or any of the mannose, melibiose, rhamnose, or arabinose promoters.

[0047] To give a specific example, expression is induced in the event of phosphate depletion or restriction.

[0048] Specifically, the feed medium does not contain phosphate. When the phoA promoter is used for heterologous protein expression in bacterial host cells, cells induced for phoA promoter activity typically become phosphate-deficient by culturing them in a phosphate-depleted medium.

[0049] In another specific example, expression is induced using a lac system (promoter, operator, and inducer), and the switch is turned on by adding IPTG (isopropylthiogalactopyranoside).

[0050] In another specific example, plasmid-containing bacterial host cells are grown at low temperatures during a portion of the fed batch phase where the growth rate is limited, followed by a temperature increase to induce plasmid production and continued growth at higher temperatures to accumulate plasmids. The temperature shift at a limited growth rate improves plasmid yield and purity. Such a process takes advantage of the temperature sensitivity of high copy number plasmids.

[0051] In another specific example, expression is induced by the addition of sugars (arabinose, rhamnose, melibiose, lactose, or mannose).

[0052] The present invention further provides a cell culture medium system for use in a bacterial fermentation process, comprising a batch medium and a feed medium for culturing bacterial cells. Both the batch medium and the feed medium are non-precipitated medium solutions. Specifically, the mediums can be suitably used in a fed-batch fermentation process as further described herein. Specifically, the mediums are provided as a partial kit or as a combination of the batch medium and the feed medium, and are characterized by one or more of the features described herein with respect to the methods of the present invention.

[0053] in particular, i) The batch medium comprises a certain amount of calcium salt, magnesium salt, phosphate, and at least one chelating agent, which is at least 5 mM of a certain amount of citrate and / or citrate and / or at least 1 mM of a certain amount of EDTA, ii) The feed medium contains at least 1 mM of a certain amount of calcium salt, and at least 5 times higher (M / M) the amount of calcium salt compared to the batch medium, iii) The feed medium contains at least 3 mM of a certain amount of magnesium salt, and at least 10 times higher than the amount of magnesium salt (M / M) compared to the batch medium, iv) The feed medium contains a certain amount of phosphate (M / M) that is less than 90% of the amount contained in the batch medium.

[0054] Specifically, the culture medium system has the following characteristics, namely a) The amount of calcium salt is a maximum of 1 mM in batch medium, a maximum of 5 mM, or a maximum of 4 mM in feed medium. b) The amount of magnesium salt is 1–3 mM in batch medium and 3–100 mM or 30–60 mM in feed medium. c) The amount of phosphate in the batch medium is 30-120 mM, and in the feed medium it is less than 90% (M / M) of the amount contained in the batch medium. d) The amount of chelating agent in the batch medium is a maximum of 100 mM citrate and / or citric acid, and / or a maximum of 30 mM EDTA. e) Batch media and feed media further contain organic carbon sources and trace elements, f) The batch medium is characterized by one or more of the following: it further contains a nitrogen source.

[0055] Specifically, the culture medium is a sterile solution with a pH in the range of 6.7 to 7.3.

[0056] To give a specific example, a) Batch medium is (i) A calcium salt, preferably CaCl2, 0-1 mM, (ii) A magnesium salt, preferably MgSO4, 1-3 mM, (iii) 10-30 g / L glucose and / or glycerol (iv) For example, 50 to 125 mM ammonium, preferably one or more of (NH4)2SO4, NH4Cl, NH4H2PO4, (NH4)2HPO4, (NH4)2-H-citrate or NH3, (v) Preferably one or more of (NH4)2SO4, MgSO4, or K2SO4, 5-50 mM sulfate, (vi) Preferably one or more of KH2PO4, NH4H2PO4, or (NH4)2HPO4 or H3PO4, or NaH2PO4, Na2HPO4 or K2HPO4, in a concentration of 30-120 mM or 30-100 mM phosphate. (vii) 5-100 mM citrate and / or citric acid, and / or 1-30 mM EDTA, (viii) Trace elements such as one or more of copper, manganese, sodium, boron, zinc, and iron salts, (ix) and, if applicable, an antifoaming agent, comprising or consisting thereof Also, b) The feed medium is (i) A calcium salt, preferably CaCl2, 1-5 mM, (ii) A magnesium salt, preferably MgSO4, in a concentration of 3-100 mM or 30-60 mM. (iii) Less than 90% of the phosphate content (M / M) in the batch medium, preferably one or more of KH2PO4, NH4H2PO4, or (NH4)2HPO4 or H3PO4, or NaH2PO4, Na2HPO4 or K2HPO4. (iv) 500-800 g / L, preferably 550-700 g / L of glucose and / or glycerol (e.g., total amount of glucose and glycerol), (v) Trace elements such as one or more of copper, manganese, sodium, boron, zinc, and iron salts, (vi) and, if applicable, a chelating agent which is citrate and / or citric acid in up to 20 mM, or EDTA in up to 10 mM.

[0057] The phosphate concentration in batch media may vary, particularly between 30 and 120 mM, or between approximately 30 and 100 mM. When using an expression system that can be induced upon phosphate depletion or restriction, the phosphate concentration may be lower than approximately 100 mM, for example, to reduce the phosphate concentration to at least one of the following: 10, 20, 30, 40, 50, 60, 70, 8, or 90%.

[0058] The phosphate concentration in the feed medium may vary, particularly to less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than one of the following: 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (M / M) of the concentration in the batch medium. Specifically, the amount of phosphate concentration in the feed medium is reduced so that the feed medium does not precipitate before, during, and / or after autoclaving.

[0059] The amount, concentration, and range of any substance contained in any of the culture media described herein are understood to be "about" a number of ±10% or ±5% of a given value.

[0060] The batch medium contains a certain amount of chelating agent, but the feed medium may or may not contain a chelating agent; therefore, the chelating agent is only optionally present in the feed medium composition.

[0061] The present invention further provides the use of the culture medium system described herein in a method for culturing bacterial host cells in a fed-batch fermentation process.

[0062] The present invention further provides a method for producing fermentation products in a bacterial fermentation process, a) Batch phase for growing host cells, followed by, b) A fed batch phase for producing fermentation products from the host cells, and c) Including isolation of fermentation products, This specification provides a method using the culture medium system described further herein.

[0063] This method is further characterized by its features in the context of the fermentation process and culture medium system, as will be further described herein.

[0064] It was surprising that precipitation was effectively prevented when chelating agents were used in batch media, even though calcium salts were absent or present in very small amounts, and magnesium salts were present in very small amounts. Supplying calcium and magnesium salts during the production phase effectively avoided precipitation in the medium and / or cell cultures. Furthermore, supplying small amounts of phosphate, or not containing phosphate in the supply, effectively avoided precipitation in the medium.

[0065] The methods, fermentation processes, and culture media systems described herein have significantly improved fermentation consistency, particularly for phosphate-depleted, phosphate-restricted, or stationary-phase-inducible systems (such as phosphate-depleted systems), within a reasonable pH range. In particular, phosphate precipitation is effectively avoided, thereby improving fermentation capacity and robustness. While slight changes in pH adjustment can more or less result in precipitation and thus increase or decrease the amount of phosphate precipitate and phosphate available to cells, the present invention provides non-precipitating conditions across the entire pH range of at least 6.7 to 7.3, even in high-cell-density fermentation, resulting in high titer. [Modes for carrying out the invention]

[0066] Unless otherwise indicated or defined, all terms used herein have their ordinary meanings in the art, as would be obvious to those skilled in the art. See, for example, standard manuals, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); or Lewin, "Genes IV", Oxford University Press, New York, (1990). Certain terms used throughout this specification have the following meanings:

[0067] As used herein, the terms “comprise,” “contain,” “have,” and “include” may be used synonymously and are understood as open definitions that allow for further members, parts, or elements. “Consisting” is considered the closest definition, without any further constituent defining features. Thus, “comprising” broadly encompasses the definition of “consisting.”

[0068] As used herein, the term “approximately” refers to the same value, or a value that differs from a given value by + / - 10% or + / - 5%.

[0069] As used herein, the term “cell” in relation to “host cell” means a single host cell, a single cell clone, or a cell line.

[0070] As used herein, the term “cell line” refers to an established clone of a particular cell type that has acquired the ability to proliferate over a long period of time. Cell lines are typically used to express endogenous or recombinant nucleic acid molecules or genes, to produce fermentation products, such as RNA or DNA nucleic acid molecules, metabolic pathway products such as cellular metabolites, or to produce polypeptides or proteins.

[0071] Where used herein, the term “host cell” shall apply specifically to any bacterial cell that is appropriately used for recombinant purposes to produce fermentation products. It is well understood that the term “host cell” does not include human cells. A “production host cell line” or “production cell line” is generally understood to be a cell line that is ready for use in cell culture in a bioreactor to obtain the products of a fermentation process.

[0072] Specifically, the recombinant host cells described herein are derivatives of artificial organisms and natural (wild-type) host cells. It should be fully understood that the host cells, methods, and uses described herein, for example, specifically including one or more gene modifications, the heterologous expression cassette or construct, the transfected or transformed host cells, and recombinant proteins, are not naturally occurring, “artificial,” or synthetic, and therefore cannot be considered the result of “natural laws.” Gene modifications described herein can be performed using tools, methods, and techniques known in the art, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2001).

[0073] As used herein, the terms “culture of cells,” “culturing,” or “culturing” mean maintaining cells in an active or quiescent state in an in vitro artificial environment, under conditions that promote growth, differentiation, continued viability, and productivity, particularly in a bioreactor controlled according to methods known in the industry.

[0074] As used herein, the term “cell culture medium” refers to a medium for culturing cells that contains substrates and nutrients that maintain cell viability and support proliferation, growth and / or production of fermentation products, such as through the biotransformation of carbon sources. A cell culture medium may contain any of the following in appropriate combination: substrates (carbon / energy sources (e.g., glycerol, succinates, lactates, and sugars, e.g., glucose, lactose, sucrose, and fructose)), nitrogen sources, precursors, and nutrients such as vitamins and minerals, salts, buffers, amino acids, antibiotics, serum or serum substitutes, and other components such as peptide growth factors.

[0075] The terms “expression” or “expression cassette” are understood herein to refer to a nucleic acid molecule containing a desired coding sequence and a control sequence of manipulable linked states, as a result of a host transformed or transfected with these molecules being able to incorporate the respective sequences and produce the encoded protein or host cell metabolite. Where used herein, the terms “gene expression,” “expressing a polynucleotide,” or “expressing a nucleic acid molecule” mean encompassing at least one step selected from the group consisting of DNA transcription into mRNA, mRNA processing, mRNA maturation, mRNA export, translation, protein folding, and / or protein transport.

[0076] One or more expression cassettes are also understood herein as “expression systems.” Expression systems may be contained within expression constructs such as vectors. However, the associated DNA may also be incorporated into host cell chromosomes. Expression may refer to secretory or non-secretory expression products, including polypeptides or metabolites.

[0077] Expression cassettes are conveniently provided as expression constructs, for example, in the form of “vectors” or “plasmids,” and are typically DNA sequences necessary for the transcription of cloned recombinant nucleotide sequences, i.e., the transcription of recombinant genes and the translation of their mRNA in a suitable host organism. Expression vectors or plasmids typically include a locus for autonomous replication or genomic integration in a host cell, a selection marker (e.g., an amino acid synthesis gene, or a gene conferring resistance to antibiotics such as zeosin, kanamycin, G418, hygromycin knowureotricin, ampicillin, chloramphenicol, or tetracycline), several restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator, with these components operably linked to one another. As used herein, the terms “plasmid” and “vector” include autonomously replicating nucleotide sequences, as well as genomes incorporating nucleotide sequences, such as artificial chromosomes, e.g., yeast artificial chromosomes (YACs).

[0078] The host cells used herein can be obtained by introducing a vector or plasmid containing the gene of interest into the cells. Techniques for transforming prokaryotic cells are well known in the art. These may include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation.

[0079] Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, and specifically designed plasmids. Preferred expression vectors described herein are those suitable for the expression of recombinant genes in eukaryotic host cells and are selected according to the host organism. Suitable expression vectors typically contain regulatory sequences suitable for expressing POI-encoding DNA in eukaryotic host cells. Examples of regulatory sequences include promoters, operators, enhancers, ribosome binding sites, and sequences that control the initiation and termination of transcription and translation. Regulatory sequences are typically operably ligated to the DNA sequence to be expressed.

[0080] Examples of plasmids that utilize Gram-negative bacteria such as E. coli as their host include pBR322, pUC18, pUC19, pUC118, pVC119, pSP64, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf(-), pET, pQE, pACYC, pBAD, and pBluescript KSTM (Stratagene). Examples of plasmids suitable for expression in E. coli include pAS, pKK223 (Pharmacia), pMC1403, pMC931, and pKC30.

[0081] To enable the expression of recombinant nucleotide sequences in host cells, promoter sequences typically regulate and initiate the transcription of downstream nucleotide sequences to which they are operably ligated. Expression cassettes or vectors typically include promoter nucleotide sequences adjacent to the 5' end of a coding sequence, for example, upstream and adjacent to a coding sequence (e.g., encoding a helper factor) or a gene of interest (GOI), or, if a signal or leader sequence is used, upstream and adjacent to the signal or leader sequence, respectively, to promote the expression and secretion of the expression product (e.g., a helper factor or POI).

[0082] The specific expression constructs described herein include a promoter operably ligated to a nucleotide sequence encoding a helper factor or POI under the transcriptional control of the promoter. Specifically, promoters that do not naturally associate with the encoding sequence can be used.

[0083] In certain embodiments, a multicloning vector may be used, which is a vector having multiple cloning sites. Specifically, an expression vector can be prepared by integrating or incorporating desired heterologous polynucleotides into the multicloning sites. In the case of a multicloning vector, the promoter is typically located upstream of the multicloning sites.

[0084] As used herein, the term “endogenous” means molecules and sequences, particularly endogenous genes or proteins, that are present in wild-type (natural) host cells before modification to reduce the expression of each endogenous gene and / or the production of endogenous proteins. In particular, endogenous nucleic acid molecules (e.g., genes) or proteins that are present in (and can be obtained from) certain host cells found in nature are understood to be “host cellular endogenous” or “endogenous to the host cell.” Furthermore, a cell that “endogenously expresses” a nucleic acid or protein expresses that nucleic acid or protein as well as the same particular type of host cell found in nature. Furthermore, a host cell that “endogenously produces” or “endogenously produces” a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as well as the same particular type of host cell found in nature.

[0085] Where used herein with respect to nucleotide sequences, constructs such as expression cassettes, amino acid sequences, or proteins, the term “heterogeneous” refers to a compound that is exogenous to a given host cell, i.e., “exogenous,” for example, a compound not found in nature in that host cell, or a compound that is naturally found in a given host cell, i.e., “endogenous,” but in the context of a heterogeneous construct, or incorporated into such a heterogeneous construct, for example, using a heterogeneous nucleic acid fused with or combined with an endogenous nucleic acid, thereby making the construct heterogeneous. Endogenously found heterogeneous nucleotide sequences may also be produced in cells in unnatural amounts, e.g., in amounts greater than expected or greater than naturally found. Heterogeneous nucleotide sequences, or nucleic acids containing heterogeneous nucleotide sequences, may have a different sequence from an endogenous nucleotide sequence, but encode the same protein found endogenously. Specifically, heterogeneous nucleotide sequences are those not found in nature in the same relationship as in the host cell. Any recombinant or artificial nucleotide sequence is understood to be heterogeneous. Examples of heterogeneous polynucleotides are, as described herein, nucleotide sequences that do not naturally associate with a promoter or are operably ligated to a coding sequence to obtain, for example, a hybrid promoter. As a result, hybrid or chimeric polynucleotides may be obtained. Further examples of heterogeneous compounds are transcriptional regulatory elements in which endogenous, naturally occurring POI coding sequences are not normally operably ligated, such as POI-coding polynucleotides operably ligated to a promoter.

[0086] The “fermentation process” as described herein is understood as a cell culture that is a fed-batch process. Specifically, host cells are cultured in a growth phase (“batch mode”) and then transitioned to a production phase (“fed-batch mode”) in order to produce a desired fermentation product.

[0087] The "batch phase" or "batch mode" should be understood as a cell culture process in which a small amount of cell culture medium is added to the culture medium, and the cells are grown without adding any additional medium or draining the culture medium during the culture.

[0088] The term "Fed Batch Phase" or "Fed Batch Mode" refers to a culture technique that begins with cell growth in a batch phase, followed by a "Fed" phase, which is a continuous mode of cell culture where cell culture medium is continuously added ("Fed") to a bioreactor. The term "Fed Batch" also includes repeated Fed Batch and semi-continuous Fed Batch fermentation processes.

[0089] In a fed-batch process, no fermentation medium compounds, either in part or in part, are added to the culture medium before the start of fermentation; instead, all or the remainder of the compounds are supplied during the fermentation process. The compounds selected for supply may be supplied together during the fermentation process or separated from each other.

[0090] In a repeated fed-batch process, a portion of the fermentation broth containing biomass is removed at regular time intervals.

[0091] In a semi-continuous fed batch, a complete starting medium is supplied during fermentation. This replenishes the fermentation process with a portion of the fresh medium corresponding to the amount of fermentation broth extracted.

[0092] The growth medium used in the batch phase typically allows for biomass accumulation and specifically includes a carbon source, a nitrogen source, a sulfur source, and a phosphate source. Typically, such a medium also contains trace elements and vitamins, and may further contain amino acids, peptones, or yeast extracts.

[0093] Preferred nitrogen sources include NH4H2PO4, (NH4)2HPO4, or NH4Cl or (NH4)2-H-citrate or NH3 or (NH4)2SO4.

[0094] Preferred sulfur sources include MgSO4, or (NH4)2SO4 or K2SO4.

[0095] Preferred phosphate sources include NH4H2PO4, or (NH4)2HPO4 or H3PO4, or NaH2PO4, KH2PO4, Na2HPO4 or K2HPO4.

[0096] Further typical culture medium components include KCl, CaCl2, NaCl, and trace elements such as Fe, Co, Cu, Ni, Zn, Mo, Mn, I, and B.

[0097] Preferably, vitamin B1 is added to the culture medium.

[0098] During the production phase, the production medium is used specifically with only a limited amount of supplemental carbon sources. For example, the supplemental carbon source added to fermentation may include a carbon source containing up to 50% by weight of available sugar or up to 100% of available alcohol.

[0099] Specifically, the host cells described herein are cultured in a mineral medium containing a suitable carbon source, thereby further significantly simplifying the isolation process. Examples of preferred mineral mediums include those containing available carbon sources (e.g., glucose, glycerol, sorbitol, methanol, ethanol, or combinations thereof), macroelements (potassium, magnesium, calcium, ammonium, chlorides, sulfates, phosphates) and trace elements (copper salts, iodide salts, manganese salts, molybdate salts, cobalt salts, zinc salts, and iron salts, as well as boric acid), and optionally containing vitamins or amino acids to complement nutritional requirements, or complex compounds, such as peptones, yeast extracts, casein, and caustic amino acids.

[0100] The fermentation processes described herein specifically enable fermentation on a pilot or industrial scale in a bioreactor. “Bioreactor” can include a fermentation tank or fermentation unit, or any other suitable reaction vessel. The fermentation process can utilize a bioreactor suitable for industrial-scale production. Industrial-scale fermentation processes typically involve 100L, 500L, or 1000L or larger, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100m³ 3 At least one of the above, and more, up to 5000m 3 It is understood to encompass a volume-scale fermentation process.

[0101] Typical fermentation times are approximately 24 to 120 hours at temperatures ranging from 16°C to 42°C, preferably from 25°C to 37°C.

[0102] As used herein, the term “fermentation product” refers to a product produced by culturing a cell line in the manner disclosed herein. The fermentation product may be a nucleic acid molecule, such as a polypeptide or protein containing a POI, particularly a heterologous protein, or a cellular metabolite, such as a primary or secondary metabolite, a pharmaceutical protein or peptide, or an industrial enzyme, or an industrial enzyme.

[0103] Primary metabolites are biomolecules essential for growth, development, or reproduction and shared by many species. They can be intermediates in major metabolic pathways such as glycolysis or the TCA cycle. Examples of primary metabolites include amino acids and nucleic acids. Secondary metabolites are not essential for growth, development, or reproduction, but instead serve ecological functions. Examples of secondary metabolites include antibiotics or β-lactam compounds.

[0104] Where used herein with respect to fermentation products that may be the isolated compound of interest, the terms “isolated” or “isolated” mean a compound that has been sufficiently separated from the environment in which it would be associated if it were natural, particularly from the culture supernatant of cells, and that has been brought into existence in a “purified” or “substantially pure” form. However, “isolated” does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that may be present, for example, due to incomplete purification, as they do not interfere with the basic activity. The isolated compound may be further formulated to produce its preparations, and may be further isolated for practical purposes, for example, when used for diagnostic or therapeutic purposes, the compound of interest may be mixed with a pharmaceutically acceptable carrier or excipient.

[0105] As used herein, the term “nucleic acid” refers to either a DNA or RNA molecule. “Polynucleotide” refers to a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5' end to the 3' end. These include expression cassettes, self-replicating plasmids, infectious polymers of DNA or RNA, and non-functional DNA or RNA.

[0106] As used herein, the term “operatably linked” refers to the association of nucleotide sequences in a single nucleic acid molecule, such as a vector or expression cassette, in such a manner that the function of one or more nucleotide sequences is influenced by at least one other nucleotide sequence present on the nucleic acid molecule. By operatably linked, nucleic acid sequences are positioned in a functional relationship with another nucleic acid sequence on the same nucleic acid molecule. For example, a promoter is operatably linked to the coding sequence of a recombinant gene when it can express the coding sequence. As a further example, a nucleic acid encoding a signal peptide is operatably linked to a nucleic acid sequence encoding a POI when it can express a secreted form of the protein, such as a preform of a mature protein or a mature protein. Specifically, such nucleic acids operatably linked to each other can be linked immediately, i.e., there are no further elements or nucleic acid sequences between the nucleic acid encoding the signal peptide and the nucleic acid sequence encoding the POI.

[0107] In the context of cell culture media, the term “precipitate” or “sediment” is understood as follows: Some components present in high concentrations in cell culture media may precipitate during preparation, autoclaving, or storage, especially when the medium's pH is near neutral. Precipitation of medium components is highly undesirable as it adds an element of uncertainty. When medium components precipitate, the relative concentrations of the medium components in the solution to those in the precipitate are unknown. This is a particularly undesirable element of uncertainty in commercial culture processes where culture conditions are carefully controlled, as the concentrations of various medium components can affect the quantity and quality of fermentation products.

[0108] A "non-precipitating" medium is prepared as a clear solution that does not contain granular material (especially, no dispersion of solids that cause turbidity), has storage stability for at least 6 weeks to 3 months, and shows no visible precipitate formation or change in optical density.

[0109] As described herein, non-precipitating batch media (also called growth media) and / or non-precipitating feed media (also called production media) may be used. The non-precipitating media systems described herein include at least batch media and feed media, each of which is a non-precipitating medium.

[0110] Non-precipitating media are provided in particular as aqueous solutions containing each of the components described herein in a mixture, which may be autoclaved.

[0111] Specifically, the culture medium described herein is non-precipitating at a pH of approximately 7, particularly in the pH range of 6.7 to 7.3.

[0112] Specifically, the culture media described herein are non-precipitating before and after the addition of the medium to the bacterial cell culture, thereby obtaining a cell culture free from any non-organic turbidity or precipitate.

[0113] The non-precipitating media described herein may be provided as aqueous media containing all of the respective components described herein, or as kits of parts containing at least two different media components, which may be provided for preparing a mixture of the media components before adding the mixture to a cell culture, or which may be used separately, in combination, subsequently, or in parallel to obtain a mixture of the media components in a cell culture during at least one phase of a fermentation process. One or more, for example, all of the media components may be provided as solids or mixtures of solid materials.

[0114] Specifically, all the components of the batch medium are supplied to the bacterial cell culture in the batch phase, and all the components of the feed medium are supplied to the bacterial cell culture in the fed batch phase.

[0115] A "promoter" sequence is typically understood to be operably ligated to a coding sequence if the promoter controls the transcription of that sequence. If a promoter sequence does not naturally associate with a coding sequence, its transcription is either not controlled by the promoter in natural (wild-type) cells, or the sequence is recombined with a different contiguous sequence.

[0116] This specification describes promoters for initiating, regulating, or otherwise mediating or controlling the expression of protein-coding polynucleotides (DNA), such as POI-coding DNA. The promoter DNA and coding DNA may originate from the same or different genes, and may originate from the same or different organisms.

[0117] Promoter can be either an "inducible promoter" or a "constitutive promoter." The term "inducible promoter" refers to a promoter that can be induced by the presence or absence of a particular compound or factor. Promoter sequences suitable for use in bacterial host cells such as E. coli include the T7 promoter, T5 promoter, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan / lactose (tac) promoter, lipoprotein (lpp) promoter, and λ phage PL promoter, as well as plasmid sugar-inducible promoters (arabinose, or rhamnose, or mannose, or melibiose, or lactose).

[0118] As used herein, the term “Protein of Interest (POI)” refers to a polypeptide or protein produced in a host cell by recombinant technology. More specifically, the protein may be a polypeptide not naturally present in the host cell, i.e., a heterologous protein, or it may be native to the host cell, i.e., a homologous protein, but is produced when it is integrated, for example, by transformation with a self-replicating vector containing a nucleic acid sequence encoding the POI, or by recombinant technology of one or more copies of the nucleic acid sequence encoding the POI into the host cell genome, or by recombinant modification of one or more regulatory sequences that control the expression of a gene encoding the POI, such as a promoter sequence. In some cases, the term POI as used herein also refers to any metabolites produced by the host cell mediated by recombinantly expressed proteins.

[0119] There are no restrictions regarding POIs. POIs can be eukaryotic or prokaryotic polypeptides, their variants, or derivatives. Proteins can be naturally secreted proteins or intracellular proteins, i.e., proteins not naturally secreted.

[0120] POIs can be therapeutic or diagnostic products. Specifically, POIs are therapeutic proteins that function in mammals. More specifically, POIs are peptides or proteins selected from the group consisting of antigen-binding proteins, therapeutic proteins, enzymes, peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine antigens, growth factors, hormones, cytokines, process enzymes, and metabolic enzymes.

[0121] Specifically, antigen-binding proteins are, a) Antibodies or antibody fragments such as chimeric antibodies, humanized antibodies, bispecific antibodies, Fab, Fd, scFv, diabody, triabody, Fv tetramer, minibody, VH, VHH, IgNAR, or V-NAR single-domain antibodies, b) Antibody mimes, e.g., Adnectins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, Monobodies, or NanoCLAMPS, or c) Selected from the group consisting of a fusion protein comprising one or more immunoglobulin-fold domains, antibody domains, or antibody mimics.

[0122] POIs can be eukaryotic proteins, preferably mammalian-derived or related proteins, such as human proteins or proteins containing human protein sequences, or bacterial proteins or bacterial-derived proteins. Any such mammalian, bacterial, or artificial protein that does not naturally exist in bacterial host cells is understood to be heterologous to the host cell.

[0123] As used herein, the term “purified” refers to a preparation containing at least 50% (mol / mol), preferably at least 60%, 70%, 80%, 90%, or 95% of the compound (e.g., POI). Purity is measured by a method appropriate to the compound (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.). Isolated and purified compounds can be obtained by purifying cell culture supernatant to reduce impurities.

[0124] The following standard methods are preferred: separation and washing of cells (remnants) by microfiltration or tangential flow filter (TFF) or centrifugation; purification of POI by precipitation or heat treatment; activation of POI by enzymatic digestion; purification of POI by chromatography, e.g., ion exchange (IEX), hydrophobic interaction chromatography (HIC), affinity chromatography, size exclusion (SEC) or HPLC chromatography; and washing by concentrated POI precipitation and ultrafiltration.

[0125] Highly purified products are essentially free of contaminating proteins and have a purity of at least 90%, more preferably at least 95%, or even further at least 98%, and up to 100%. Purified products can be obtained by purifying cell culture supernatant or cell debris.

[0126] Methods for isolating and purifying recombinant polypeptides or protein products include methods utilizing differences in solubility, such as salting out and solvent precipitation; methods utilizing differences in molecular weight, such as ultrafiltration and gel electrophoresis; methods utilizing differences in charge, such as ion exchange chromatography; methods utilizing specific affinity, such as affinity chromatography; methods utilizing differences in hydrophobicity, such as reverse-phase high-performance liquid chromatography; and methods utilizing differences in isoelectric point, such as isoelectric focusing electrophoresis.

[0127] Isolated and purified POIs can be identified by conventional methods such as Western blotting, HPLC, activity assays, or ELISA.

[0128] As used herein, the term “recombinant” means “prepared by genetic engineering or as a result of genetic engineering.” “Recombinant cell” or “recombinant host cell” is understood herein as a cell or host cell that has been genetically engineered or modified to contain a non-natural nucleic acid sequence. A recombinant host may be engineered to delete and / or inactivate one or more nucleotides or nucleotide sequences, and may particularly include expression vectors or cloning vectors containing recombinant nucleic acid sequences, using nucleotide sequences that are foreign to the host. Recombinant proteins are produced by expressing the respective recombinant nucleic acid in a host. As used herein, the term “recombinant” with respect to POIs includes POIs prepared, expressed, created or isolated by recombinant means, for example, POIs isolated from host cells transformed or transfected to express a POI. According to the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of the skills of the art can be used. Such techniques are well described in the literature. For example, see Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor, (1982). Certain recombinant host cells are “engineered” host cells, understood as host cells that have been manipulated using genetic engineering, i.e., by human intervention. When a host cell is engineered to express a given gene or its respective protein, the host cell is engineered to have a greater ability to express such gene and protein, respectively, compared to a host cell under the same conditions before the engineering, or compared to a host cell that has not been engineered to express the gene or protein.

[0129] The above description will be better understood by referring to the following examples. However, such examples are merely representative examples of ways of carrying out one or more embodiments of the present invention and should not be read as limiting the scope of the invention. Examples Example 1: Method Preparation of culture medium, setting of pH, and observation of precipitation.

[0130] First, the culture medium was prepared and autoclaved. Precipitation could be observed during medium preparation, before and after autoclaving, and after adjusting the pH to 7.0. The medium that did not precipitate was used to test cell growth in a shaking flask. The presence or absence of precipitate was measured using a spectrophotometer (OD 600 The determination was made by measuring the optical density at a wavelength of 600 nm using (). Shaking flask culture

[0131] To test the growth capacity of media that did not precipitate during preparation, shaken flask cultures were performed using E. coli W3110 (e.g., from DSM 5911). The media that allowed cells to grow in the shaken flask cultures were further tested on a bioreactor scale. Bioreactor culture

[0132] E. coli W3110 and LB2.0 (from BL21) cells were cultured in bioreactors containing media that did not precipitate during preparation, autoclaving, and pH adjustment, while simultaneously growing cells on a shaking flask scale. Cell growth and product formation were monitored over time through the bioreactor cultures. Fed-batch cultures were performed in different fermentation systems and scales ranging from 15 mL (ambr® 15f, Sartorius) and 250 mL (ambr® 250, Sartorius) to 1 L (DASGIP®, Eppendorf). Apart from precipitation, cell growth and maximum OD were monitored. 600 The effects of different batch media on DCW and titer were investigated. Example 2: Novel culture medium system, NPM

[0133] Several fermentations were performed to investigate the effects of culture media and their variants on growth and product formation. Surprisingly, it was found that reducing the calcium and magnesium salts in the batch medium and increasing them in the feed medium significantly improved the capacity of the fermentation process and reduced the tendency for undesirable medium precipitation. Furthermore, despite the reduction in the amounts of calcium and magnesium salts in the batch medium, chelating agents such as citrate unexpectedly improved the batch medium, allowing it to be used at neutral pH down to pH 7.3, and even higher pH levels, without precipitation before, during, and after autoclaving, and even during fermentation. Chelating agents selected from sodium citrate, citrate, and EDTA were used. Although citrate is an additional carbon source, it was found that E. coli could not absorb or consume its molecules and was therefore as inactive as EDTA. Cell growth was proven to be unaffected by the novel culture media.

[0134] The new culture medium system is called the NPM media system. NPM culture medium system (including various modifications): Composition of NPM batch medium: (i) 0-1 mM CaCl2; (ii) 1-3 mM MgSO4, (iii) 10-30 g / L of glycerol; (iv) 50–125 mM (NH4)2SO4 and NH4Cl (total); (v) 5-50 mM (NH4)2SO4; (vi) KH2PO4 at 30-120 mM; (vii) 5-100 mM citrate and / or citric acid; (viii) Trace elements including copper chloride, manganese sulfate, sodium molybdate, boric acid, zinc sulfate, and iron sulfate; (ix) and antifoaming agents; Composition of NPM feed medium: (i) 1-5 mM CaCl2; (ii) 3-100 mM, preferably 30-60 mM MgSO4; (iii) 550-700 g / L of glycerol; (iv) 0-10 mM KH2PO4; (v) Trace elements including copper chloride, manganese sulfate, sodium molybdate, boric acid, zinc sulfate, and iron sulfate; (vi) and, if applicable, chelating agents: up to 20 mM citrate and / or citric acid. Example 3: NPM, Capability

[0135] NPM medium was compared to precipitation medium, which has been shown to yield unrestricted growth and high productivity levels. Precipitation medium contained a complex source that ensured the presence of all components required by cells for growth and protein production. Therefore, comparing NPM medium to similar capabilities, except for avoiding precipitation, provided a good reference. Testing of batch and feed medium combinations of NPM using three different reporter molecules expressed under a rhamnose-inducible promoter.

[0136] E. coli W3110 strains expressing three different reporter molecules under the rhamnose promoter were cultured in a bioreactor to evaluate the reproducibility of NPM medium capabilities (Table 1). NPM medium was referenced using a complex precipitate medium ("precipitate") in terms of growth and productivity. The selected reporter molecules were a single-domain antibody (sdAb), an antibody fragment (Fab), and eGFP. Novel NPM media obtained from selected batch and feed combinations were also tested with a batch complex compound (yeast extract).

[0137] In terms of productivity, the NPM medium system yielded the best product yield, regardless of whether or not the complex compounds, yeast extracts ("NPM" and "NPM+YE"), were added, and it also had the advantage of not precipitating at pH 7.0.

[0138] In all cases, cell growth using NPM medium showed no limitations and was carried out in the same manner as with complex precipitation medium. [Table 1] Use of NPM medium systems with phosphate restriction promoters in fermentation

[0139] We further investigated whether the results obtained with the rhamnose-inducing system were similar to those obtained with other promoter systems in E. coli. In this case, the effect of the phosphate-depletion system was examined in the W3110 strain producing a single-domain antibody (sdAb) (Table 2). As a result, the growth profile and final biomass content were comparable to those of the compound precipitation medium. All cultures using the NPM medium system showed slightly higher single-domain antibody titers and similar growth. [Table 2] Use of NPM medium in E. coli B strains that produce antibody fragments under fermentation conditions

[0140] The performance of *E. coli* strain B cultured in NPM medium was investigated. In this case, the LB2.0 strain, which produces antibody fragments (Fab) under a rhamnose-inducible system, was examined under fermentation conditions. As in the previous example, the capabilities of NPM medium were compared with those of complex precipitation medium in terms of growth and product formation (Table 3). [Table 3] Example 4: Comparison of NPM with a culture medium containing a higher phosphate concentration in the feedstock. Equivalent: NPM feed medium with high phosphate concentration shows precipitation.

[0141] NPM feed medium was prepared using standard concentrations and glycerol as the carbon source. Increasing concentrations of phosphate were added to the salt solution. After diluting all the salts, the carbon source (e.g., glycerol) was added, and the solution was autoclaved at 121°C for 30 minutes. Precipitation events were monitored during preparation and after autoclaving.

[0142] The phosphate concentrations tested were 0 mM, 3 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, and 70 mM. KH2PO4 was used as the phosphate source (Table 4). [Table 4]

[0143] It is concluded that such NPM feed medium compositions containing moderate concentrations of calcium and magnesium salts, as well as phosphates less than 10 mM, may be used.

[0144] In equivalent feed media containing lower concentrations of calcium and magnesium salts, e.g., about 1 mM calcium salt and about 3 mM magnesium salt, higher phosphate concentrations, e.g., up to 10⁸, 10⁵, or up to 100 mM phosphates, may be used. In any case, the feed medium may contain less than 90% of the amount contained in the batch medium, preferably less than 27–10⁸ mM, compared to 30–120 mM in the batch medium. Comparative example: The culture media of the prior art described by Hardiman et al. (J. Biotechnol. 2007, 132:359~374) and European Patent No. 1584680 show precipitation.

[0145] Batch and feed media from prior art were prepared as described in Hardiman et al. (J. Biotechnol. 2007, 132:359-374) and European Patent No. 1584680 (illustrated in Example 1). Batch media were prepared, autoclaved, and the pH was set to 7.0. Feed media were prepared and autoclaved. Precipitation events were monitored during the different steps of preparation and completion (Table 5). [Table 5] [Table 6]

Claims

1. A method for reducing precipitate in a bacterial fermentation process for producing fermentation products, wherein the fermentation process is a) A step of culturing host cells for bacterial growth in a batch phase using batch medium, and b) The step of culturing the host cells using feed medium to produce the fermentation product in a fed batch phase, The feed medium and batch medium are both non-precipitated medium solutions. Here, i) The batch medium comprises a certain amount of calcium salt, magnesium salt, phosphate, and at least one chelating agent which is at least 5 mM of a certain amount of citrate and / or citric acid and / or at least 1 mM of a certain amount of EDTA, ii) The feed medium contains at least 1 mM and at least five times higher than the batch medium, a certain amount of calcium salt (M / M), iii) The feed medium is at least 3 mM and contains a certain amount of magnesium salt (M / M) that is at least 10 times higher than that of the batch medium, iv) The method wherein the feed medium contains a certain amount of phosphate (M / M) that is less than 90% of the amount contained in the batch medium.

2. a) The amount of calcium salt is a maximum of 1 mM in the batch medium, a maximum of 5 mM or a maximum of 4 mM in the feed medium, and / or b) The amount of magnesium salt is 1 to 3 mM in the batch medium, 3 to 100 mM, or 30 to 60 mM in the feed medium, and / or c) The method according to claim 1, wherein the amount of phosphate in the batch medium is 30 to 120 mM, and in the feed medium, the amount in the feed medium is less than 90% of the amount contained in the batch medium (M / M).

3. The method according to claim 1 or 2, wherein both the batch medium and the feed medium further comprise an organic carbon source and trace elements.

4. The method according to any one of claims 1 to 3, wherein the batch medium further comprises an ammonium salt.

5. The method according to any one of claims 1 to 4, wherein the fermentation process is carried out at a pH in the range of 6.7 to 7.

3.

6. The method according to any one of claims 1 to 5, wherein the host cells are grown in the batch phase to a density of at least 10 g of cell dry weight / liter, and the host cells are cultured in the fed batch phase under growth-restricted conditions.

7. The method according to any one of claims 1 to 6, wherein the bacterial host cell is a production host cell of a species selected from the group consisting of Escherichia, Pseudomonas, Bacillus, Lactococcus, Corynebacterium, Clostridium, Micrococcus, and Streptomyces.

8. The method according to any one of claims 1 to 7, wherein the fermentation product is one of the following: a protein of interest (POI), a metabolic pathway, RNA, or a recombinant DNA molecule such as plasmid DNA or a plasmid vector.

9. The method according to any one of claims 1 to 8, wherein the bacterial host cell comprises a gene construct that produces and / or expresses the fermentation product comprising an inducible promoter, and the expression of the gene construct is induced by restricting the cell culture components or by adding an inducing substance.

10. A culture medium system for use in a bacterial fermentation process, comprising batch medium and feed medium for culturing bacterial cells, wherein each medium is a non-precipitating medium solution. i) The batch medium comprises a certain amount of calcium salt, magnesium salt, phosphate, and at least one chelating agent which is at least 5 mM of a certain amount of citrate and / or citric acid and / or at least 1 mM of a certain amount of EDTA, ii) The feed medium contains at least 1 mM and at least five times higher than the batch medium, a certain amount of calcium salt (M / M), iii) The feed medium is at least 3 mM and contains a certain amount of magnesium salt (M / M) that is at least 10 times higher than that of the batch medium, iv) A culture medium system wherein the feed medium contains a certain amount of phosphate (M / M) that is less than 90% of the amount contained in the batch medium.

11. The following features, Call, a) The amount of calcium salt is a maximum of 1 mM in the batch medium, a maximum of 5 mM in the feed medium, or a maximum of 4 mM. b) The amount of magnesium salt is 1 to 3 mM in the batch medium, and 3 to 100 mM, or 30 to 60 mM in the feed medium. c) The amount of phosphate is 30 to 120 mM in the batch medium, and is less than 90% (M / M) of the amount contained in the batch medium. d) The amount of the chelating agent in the batch medium is a maximum of 100 mM citrate and / or citric acid, and / or a maximum of 30 mM EDTA. e) The batch medium and feed medium further contain an organic carbon source and trace elements, f) The culture medium system according to claim 10, characterized by comprising one or more of the following: the batch medium further comprises a nitrogen source and a phosphate.

12. a) The batch medium is (i) Preferably CaCl ２ Assuming a calcium salt of 0-1 mM, (ii) Preferably MgSO ４ Assuming a magnesium salt of 1-3 mM, (iii) 10-30 g / L of glucose and / or glycerol, (iv) For example, about 90 mM, preferably (NH ４ ) ２ SO ４ , NH ４ Cl, NH ４ H ２ PO ４ , (NH ４ ) ２ HPO ４ , (NH ４ ) ２ -H-citrate or NH ３ one or more of, 50-125 mM ammonium, (v) Preferably (NH ４ ) ２ SO ４ MgSO ４ , or K ２ SO ４ One or more of the following, 5-50 mM sulfate, (vi) Preferably KH ２ PO ４ NH ４ H ２ PO ４ , or (NH ４ ) ２ HPO ４ Or H ３ PO ４ , or NaH ２ PO ４ Na ２ HPO ４ Or K ２ HPO ４ One or more of the following, 30-120 mM phosphate, (vii) 5-100 mM citrate and / or citric acid, and / or 1-30 mM EDTA, (viiii) Trace elements such as one or more of copper, manganese, sodium, boron, zinc, and iron salts, (ix) and, if applicable, an antifoaming agent, or comprising the same Also, b) The feed medium is (i) A calcium salt, preferably CaCl2, 1 to 5 mM, (ii) Preferably MgSO ４ Magnesium salts of 3-100 mM or 30-60 mM, (iii) Preferably KH ２ PO ４ NH ４ H ２ PO ４ , or (NH ４ ) ２ HPO ４ Or H ３ PO ４ , or NaH ２ PO ４ Na ２ HPO ４ Or K ２ HPO ４ One or more of the following, phosphates in amounts less than 90% (M / M) of the batch medium, (iv) 500-800 g / L of glucose and / or glycerol, (v) Trace elements such as one or more of copper, manganese, sodium, boron, zinc, and iron salts, (vi) and, if more preferably, a chelating agent which is citrate and / or citric acid in an amount up to 20 mM, or EDTA in an amount up to 10 mM, the culture medium system according to claim 10 or 11.

13. Use of the culture medium system according to any one of claims 10 to 12 in a method for culturing bacterial host cells in a fed-batch fermentation process.

14. A method for producing fermentation products in a bacterial fermentation process, a) A batch phase for growing the host cells, followed by, b) A fed batch phase for producing fermentation products from the host cells, and c) Including isolation of fermentation products, A method using the culture medium system according to any one of claims 10 to 12.

15. The method according to claim 14, wherein the fermentation product is one of the following: a protein of interest (POI), RNA, or a recombinant DNA molecule such as plasmid DNA or a plasmid vector.

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