Method for producing extrachromosomal nucleic acid

Abstract

The present invention relates to a method for increasing the production of nucleic acids, comprising the step of limiting the total sulfur amount in the medium. The present invention further relates to the use of a medium containing a limited amount of sulfur for increasing the production of nucleic acids.

Description

Technical Field

[0001] The present invention is in the field of recombinant biotechnology, particularly in the field of nucleic acid production. The present invention relates to a method for increasing the production of nucleic acids, comprising the step of limiting the total amount of sulfur in the medium. The present invention further relates to the use of a medium containing a limited amount of sulfur for increasing the production of nucleic acids.

Background Art

[0002] The production of a protein of interest (POI) or a nucleic acid molecule of interest has been achieved using many prokaryotic hosts. The most prominent examples are bacteria such as Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces griseus, or Corynebacterium glutamicum. In recent years, a large number of biological pharmaceuticals (e.g., antibodies or their functional fragments, or nucleic acid molecules) have been produced and an increasing number are seeking approval for human use, but their efficient production remains a difficult challenge. An ever-increasing number of people require therapeutically effective doses, and thus a significant amount of such proteins or nucleic acid molecules are needed. Also, nucleic acid molecules are required, for example, for the production of proteins in production cells, plasmids for vaccination, rAAV vectors, etc., so a significant amount of such nucleic acid molecules are needed. Therefore, a considerable amount of molecules are required as active ingredients or templates for protein production, and efficient and cost-effective production becomes valuable.

[0003] Bacterial cell expression systems have long been and still are one of the main tools for producing this type of molecule. The main objective of process optimization is to achieve a high yield of the product with the required quality at the lowest possible cost, which is often determined by the characteristics of a specific expression construct or system. In very many cases, for example, in the production of nucleic acids or in the production of plasmids for vaccination such as AAV vectors, batch processes are used. However, batch processes require a large amount of labor as the bioreactor needs to be constantly monitored and thoroughly cleaned after each production. While the bioreactor is being cleaned, production cannot be carried out, making the batch process inefficient.

[0004] By applying continuous expression systems and / or fed-batch expression systems instead of batch processes, these drawbacks associated with batch processes can be overcome. However, even in continuous processes, high levels of recombinant nucleic acid molecule synthesis can sometimes overwhelm the metabolic capacity of the host cells, resulting in plasmid loss, reduced oxygen transfer, generation of toxic by-products, inclusion body formation, and / or induction of stress responses, thereby often impairing efficient nucleic acid molecule synthesis.

[0005] Towards this end, various approaches have been taken by scientists to address these problems when using batch or continuous processes. Nevertheless, there remains a need to effectively produce the target protein or target nucleic acid molecule in microbial host cells.

[0006] It is necessary to overcome the described drawbacks and further drawbacks. Accordingly, the present invention addresses these needs and technical objectives and provides the solutions described herein and defined in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0007]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0008] The present invention relates to a method for increasing the production of nucleic acids (preferably extrachromosomal nucleic acids) contained in host cells in cell culture, comprising the step of restricting the total sulfur amount in the medium of the cell culture over the course of culturing the cells, wherein the total sulfur amount is preferably approximately equal to or less than the amount of sulfur contained per gram dry cell weight of a reference host cell grown without restricting the total sulfur amount, per gram dry cell weight of the host cell for which it is desired to obtain in the cell culture, thereby increasing the production of nucleic acids.

[0009] As used herein, the term "restricting the total sulfur amount (alternatively, the total sulfur requirement; thus, for the purpose of expressing the same thing, the 'amount' and the'requirement' can be used interchangeably) in the medium over the course of cell culture" means that the total sulfur amount, i.e., 100% as preset as described herein, decreases over the course of cell culture in the medium of the host cells. For clarity, 100% of the total sulfur amount is the amount of sulfur content required to achieve the specific preset (or predefined) dry cell weight or cell concentration of the host cells described herein. How the total sulfur amount can be preset (or predefined) is described herein.

[0010] The decrease in the total sulfur amount over the course of cell culture in the cell medium is considered to be mainly due to the biomass production of the host cells, such as growth, cell division, etc.

[0011] The decrease in the total sulfur amount over the course of cell culture in the cell medium can be a decrease from 100% to 0%.

[0012] For example, for 1 g of dry cell mass of E. coli as an exemplary host cell, 8.5 mg of sulfur is required. Therefore, if 50 g of dry cell mass of E. coli is desired as an exemplary host cell, 425 mg of sulfur is required. Therefore, in that case, 425 mg of sulfur is regarded as 100% of the total sulfur amount according to the teachings of the present invention. Naturally, if 100 g of dry cell mass of E. coli is desired as an exemplary host cell, 850 mg of sulfur is required. Thus, in that case, 850 mg of sulfur is regarded as 100% of the total sulfur amount according to the teachings of the present invention.

[0013] As demonstrated in the examples and shown in the figures, but not limited thereto, the method of the present invention provides for decoupling the metabolism of the host cell from the production of a desired nucleic acid, such as a plasmid. As soon as sulfur is limited, the host cell, preferably a bacterial cell, can no longer synthesize biomass but will produce the desired nucleic acid, such as a plasmid. See also Figure 1.

[0014] Surprisingly, when implementing the method of the present invention, it was observed that when sulfur limitation is performed, the quality, i.e., the proportion, of the covalently closed circular (ccc) nucleic acid, such as plasmid DNA of the desired nucleic acid, preferably pDNA, produced by culturing under S limitation increases. See Figure 2.

[0015] Similarly, when implementing the method of the present invention, it was observed that when sulfur limitation is performed, the productivity of the desired nucleic acid, preferably ccc (covalently closed, circular) DNA, produced by culturing under S limitation increases. See Figure 3.

[0016] Also, when implementing the method of the present invention, it was observed that when sulfur limitation is performed, the yield of the desired nucleic acid, preferably cccDNA, produced by culturing under S limitation increases. See Figure 4.

[0017] Accordingly, the method of the present invention provides advantageous effects, in particular, an increase in the yield of the desired nucleic acid, a high quality of the desired nucleic acid, and / or a beneficial ratio of the desired nucleic acid to the cell dry mass. Each of these advantageous effects reduces the nucleic acid production cost and / or improves the effectiveness of downstream processes such as the purification of the desired nucleic acid.

[0018] As used herein, "increasing the production of a nucleic acid" means that, as described herein, the production of a nucleic acid by a host cell in a cell culture subject to a limitation of the total sulfur amount in the culture medium over the course of culturing the cells results in a higher yield of said nucleic acid as compared to the same host cell not subject to a limitation of the total sulfur amount in the culture medium over the course of culturing the cells. In other words, preferably the same host cell is not limited with respect to the total sulfur amount over the course of culturing the cells as compared to the host cell whose total sulfur amount is limited over the course of culturing the cells as described herein. "Higher yield" includes the yield of the desired nucleic acid, the volumetric yield, or the quality of the nucleic acid, for example, the amount of cccDNA in the case of a plasmid. Such a yield can, relatively speaking, be at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30% higher when applied by the method described herein and compared to the yield of the host cell subject to sulfur limitation as compared to the reference host cell described herein.

[0019] The total sulfur amount referred to herein may also be referred to herein as, for example, "(required) preset total sulfur amount" or "preset (required) total sulfur amount". In this specification, the terms "preset" or "predetermined" can be used interchangeably in relation to the total sulfur amount.

[0020] Means and methods for determining the amount of dry cell matter and the elemental composition of the cells contained therein, such as the host cells referred to herein, are well known in the art. See, for example, Heldal et al., Applied and Environmental Microbiology (1985), 50(5): 1251-1257 or Neidhardt, F. C. (2006), Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, Part I, Chapter 2 ”Chemical Composition of Escherichia coli” (ISBN 0-914826-89-1).

[0021] Alternatively, the host cells can be grown with different, e.g., three different amounts of sulfur until they reach the maximum OD600, the biomass can be measured by weighing, the cell dry mass can be plotted against the added sulfur content, and then a linear regression can be performed to enable the calculation of the amount of sulfur per OD unit or per dry weight of the host cells. See Examples 5 and Figure 5 for an explanation. The maximum OD600 is reached when the OD600 changes within the range of +0.5 OD600 to -0.5 OD600 units from the maximum over time.

[0022] To do so, the person skilled in the art understands the amount of C source, such as glucose, in the medium. In the case of conditions where sulfur is not limiting, the person skilled in the art is in a position to understand or easily determine the yield coefficient, for example, in a chemostat bioreactor in a steady state or fed-batch process. Thus, the person skilled in the art can determine the (expected) biomass. Therefore, the person skilled in the art can also determine the maximum OD600.

[0023] In parallel, one of ordinary skill in the art sets two additional conditions with less sulfur than the sulfur used under non-limiting sulfur conditions, and accordingly, the amount of C source, such as glucose, is made the same as the amount of glucose used under non-limiting sulfur conditions. As described above, when the maximum OD600 is reached, a subsequent linear regression is performed, which enables the calculation of the amount of sulfur per OD unit or per dry weight of the host cell.

[0024] Using these means and methods, one of ordinary skill in the art can preset (or pre-specify) the total sulfur amount (or required amount) described and referred to herein.

[0025] In connection with the present invention, surprisingly, it has been found that culturing host cells while limiting the available amount of sulfur in the medium leads to a significant increase in nucleic acid production. See, for example, Figure 3. In particular, as found in connection with the present invention, the amount of sulfur for growing a desired amount of host cells containing the nucleic acid molecule to be produced is the same as that contained in the same amount of reference host cells (preferably not containing said nucleic acid molecule) grown in a medium except that there was no sulfur limitation. Limiting the amount of sulfur in the medium for culturing the host cells containing the nucleic acid molecule to an amount of about 10 g or less results in a significant increase in nucleic acid production. For example, according to the present invention, an amount of X grams (dry weight) of reference host cells (grown without sulfur limitation) is produced, and 10 g of sulfur is contained in the X grams of reference host cells. If it is desired to produce the same amount of X grams of host cells containing the nucleic acid molecule to be produced according to the methods provided and described herein, the amount of sulfur in the medium for culturing the host cells containing the nucleic acid molecule is limited to about 10 g or less.

[0026] Accordingly, the present invention provides an efficient and simple method that increases the production of the desired nucleic acid and thereby even reduces the amount of resources required for nucleic acid production. Without being bound by theory, according to the present invention, by limiting the amount of sulfur in the medium, the metabolism of the host cell is decreased so as to enable an increase in the production of nucleic acid molecules by the host cell, thereby presumably decoupling the metabolism from nucleic acid production.

[0027] Preferably, in the method described and provided in connection with the present invention, the limiting step may include: (a1) presetting the amount of dry cell weight of the host cell that is desired to be obtained in the cell culture; and (b1) at the start of the cell culture, adding to the cell culture a total amount of sulfur per preset gram dry weight (i.e., the desired dry weight to be obtained) of the host cell, wherein the total amount of sulfur is approximately equal to or less than the amount of sulfur contained per gram dry weight of the reference host cell.

[0028] As used herein, "at the start" includes, for example, "prior to the start" such that sulfur is added prior to the start of the cell culture.

[0029] The above method, although not limited thereto, can be implemented as follows. That is, at the start of the cell culture, a total amount of sulfur per preset gram dry weight of the host cell is added, such that sulfur is restricted when the host cell reaches the preset dry cell weight. In practice, those skilled in the art, in accordance with the teachings of this application, preset the dry weight of the host cell that they want to make available for producing the desired nucleic acid when metabolism is decoupled from the production of the desired nucleic acid by restricting sulfur in the host cell. For this purpose, the host cell is grown without sulfur restriction until it reaches the preset dry cell weight. Those skilled in the art can determine the dry weight of the host cell grown in this way, and since those skilled in the art can also determine the amount of sulfur contained in the host cell grown in this way, they know the total amount of sulfur that must be added to the host cell at the start of the cell culture, such that sulfur is ideally restricted when the host cell reaches the preset dry cell weight. If so, metabolism will be decoupled from the production of the desired nucleic acid as described and taught herein.

[0030] Alternatively, but preferably also, in the method described and provided in connection with the present invention, the limiting step is (a2) presetting the amount of dry cell weight of the host cell that is desired to be obtained in the cell culture; and (b2) reducing, over the course of the cell culture, the amount of total sulfur available per gram dry weight of the host cell in the cell culture to be less than or equal to the amount of sulfur contained per gram dry weight of the reference host cell, which may include.

[0031] When performing alternative items (a2, b2), those skilled in the art preset the cell dry weight and the amount of sulfur as described above, as in the above alternative items (a1, b1). However, at the start of the cell culture, instead of adding the total sulfur amount per preset gram dry weight of the host cell, sufficient sulfur is added so that sulfur is not restricted, that is, sulfur is not a limiting factor. Optionally, the addition of sulfur is reduced over the course of the cell culture. For example, if a total of 10 g of sulfur is required for the preset dry cell weight of the host cell, this amount is not added all at once at the start of the cell culture but is added over a certain period of time, and the ratio of available sulfur to the dry weight of the host cell decreases over time, thereby making sulfur a limiting factor.

[0032] As a further alternative, but preferably, in the method described and provided in connection with the present invention, the limiting step is (a3) presetting the amount of dry cell weight of the host cell that is desired to be obtained in the cell culture; and (b3) adding to the cell culture, during the cell culture (from the start until the desired amount of the host cell containing the nucleic acid is obtained), the total sulfur amount per preset gram dry weight of the host cell (i.e., the dry weight that is desired to be obtained), the total sulfur amount being approximately equal to or less than the amount of sulfur contained per gram dry weight of the reference host cell, which may include adding.

[0033] When performing the alternative items (a3, b3), a person skilled in the art presets the dry cell weight and the amount of sulfur as described above, as in the above alternative items (a1, b1) or (a2, b2). However, sulfur is added until it reaches the desired amount of host cells from the start. For example, if 10 g of sulfur is required, during cell culture, these 10 g are added until the desired amount of host cells is achieved, and then no more sulfur is added.

[0034] The first alternative items, namely, (a1), (b1) are more preferred. See also the examples.

[0035] Preferably, in the methods described and provided in connection with the present invention, the amount of sulfur contained per gram dry weight of the reference host cell is approximately equal to or less than the amount of sulfur contained in the elemental composition per gram dry weight of the reference host cell. In this connection, the term "elemental composition" as used herein includes the composition of elements in the host cell, including sulfur and optionally additional elements such as, for example, sodium, magnesium, phosphorus, chloride, potassium, calcium, and / or other elements.

[0036] Generally, in connection with the present invention, sulfur can be added to the medium or cell culture in all suitable forms that enable cell culture, preferably in a form that enables the uptake of sulfur into the cells. For example, sulfur can be added in the form of (hydrated) sulfates, such as MgSO4, MnSO4, FeSO4, CuSO4, ZnSO4, (NH4)2SO4, or in the form of their respective hydrates, or in the form of dextran sulfate.

[0037] Preferably, in the methods described and provided in connection with the present invention, feeding nutrients (other than sulfur) to the cell culture can be done linearly while restricting the total sulfur amount. On the other hand, feeding nutrients (other than sulfur) to the cell culture can be done exponentially while restricting the total sulfur amount. As already explained herein (e.g., see (b1), (b2), and (b3) above), restricting the amount of sulfur is achieved by controlling the amount of sulfur added to the cell culture at the start of the cell culture and / or during the cell culture such that the total sulfur amount is approximately equal to or less than the amount of sulfur contained per gram dry weight of the reference host cell described herein.

[0038] In some cases, it may be necessary to add sulfur in the form of a sulfur salt, e.g., MgSO4, stepwise to the medium over a specific time range due to the solubility of such sulfur salts. Thus, as used herein, "start of the cell culture" encompasses a time range from 0 to 24 hours, e.g., 23 hours, preferably from 0 to 20 hours, more preferably from 0 to 16 hours, after or at the start of the cell culture, during which the total sulfur amount per pre-set gram dry weight (i.e., the dry weight desired to be obtained) of the host cell as described herein is added to the cell culture.

[0039] As shown in FIGS. 6A to 6D, when the preset total sulfur amount has not decreased to 0%, if sulfur is further added, for example, if 50% or 25% of the preset total sulfur amount is added, productivity (FIG. 6A) and volumetric yield (FIG. 6B) still show an increase, but the quality of the desired nucleic acid (FIG. 6C) and the cell dry mass (FIG. 6D) remain constant compared to the control. This means that the surprising discovery of the present invention can still be observed even when the total sulfur amount is not maintained at 0% but is preferably maintained at 50% or less, more preferably 25% or less of the preset total sulfur amount. Therefore, this surprising effect occurs when the preset total sulfur amount decreases to 0%, preferably within the range of 50% to 0% of the preset total sulfur amount.

[0040] Therefore, in the method of the present invention, preferably, when the preset total sulfur amount has decreased to 0% of the preset total sulfur amount in the medium, further sulfur is added to the medium. Such an amount is preferably added in the form of a feed.

[0041] When the total sulfur amount reaches 0%, those skilled in the art may add further sulfur to the medium so that an amount of, for example, 50% or less, for example, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the preset total sulfur amount is added, as described herein. Such an amount is preferably added in the form of a feed.

[0042] Further sulfur addition may be carried out at once, or may be repeated, for example, once, twice, three times, four times or more, or, as described herein, an amount of, for example, 50% or less, for example 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the preset total sulfur amount may be kept constant over the course of cell culture, or may be carried out at (desired) times such that it may decrease if no further sulfur is added. Such amounts are preferably added in the form of a feed.

[0043] Thus, when the total sulfur amount is 0%, one of ordinary skill in the art may preferably add 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less of the total amount of the preset total sulfur amount (described herein) to the medium. Such amounts are preferably added in the form of a feed.

[0044] Thus, when the total sulfur amount is 0%, one of ordinary skill in the art may more preferably add 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the total amount of the preset total sulfur amount (described herein) to the medium. Such amounts are preferably added in the form of a feed.

[0045] One of ordinary skill in the art can preset the total sulfur amount required per gram of cell dry weight as described herein. One of ordinary skill in the art also understands the time, i.e., the point in time, when the total sulfur amount will decrease to 0% over the course of cell culture.

[0046] The reason is that mainly due to the biomass production of the host cell, such as growth, cell division, etc., the total sulfur amount decreases over the course of cell culture, which is considered to change at a certain point.

[0047] As described above, a person skilled in the art can preset the total sulfur amount required per (desired) gram of cell dry weight as described herein. A person skilled in the art also understands the time, i.e., the point in time, when the total sulfur amount will decrease to 0% over the course of cell culture. Therefore, a person skilled in the art can also preset the time when the total sulfur amount can decrease, for example, to 50%.

[0048] In other words, the additional sulfur addition may be carried out when the preset total sulfur amount reaches 0%, or may be carried out when the preset total sulfur amount has decreased to preferably 50% or less, more preferably 25% or less of the preset total sulfur amount.

[0049] Therefore, instead of performing the additional sulfur addition described above when the total sulfur amount is 0%, additional sulfur may be added before the total sulfur amount decreases to 0% over the course of cell culture.

[0050] Therefore, in the method of the present invention, preferably, additional sulfur is added to the medium when the preset total sulfur amount has decreased to 50% or 25% or less of the preset total sulfur amount in the medium.

[0051] At that amount, a person skilled in the art may add sulfur to the medium so that the preset total sulfur amount reaches an amount of, for example, 50% or less, for example, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less as described herein.

[0052] Further addition of sulfur may be carried out at once, or may be repeated, for example, once, twice, three times, four times or more, or as described herein, an amount of, for example, 50% or less, for example, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the preset total sulfur amount may be kept constant over the course of cell culture as described herein, or may be carried out at (desired) times such that it may decrease when no more sulfur is added.

[0053] Thus, the decrease in the total sulfur amount over the course of cell culture in the cell culture medium may preferably be a decrease from 100% to 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less.

[0054] More preferably, the decrease in the total sulfur amount over the course of cell culture in the cell culture medium may preferably be a decrease from 100% to 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, for example, down to 0%.

[0055] In fact, as explained above, it is clear that the surprising effect of the invention described herein occurs in the range of preferably a decrease from 50% to 0% of the preset total sulfur amount, more preferably a decrease from 25% to 0%. Thus, before the surprising effect occurs, it is not necessary for the total sulfur amount to necessarily decrease to 0%, rather, when the preset total sulfur amount is a decrease of preferably from 50% to 0% of the preset total sulfur amount, more preferably from 25% to 0%, the surprising effect is considered to have already occurred.

[0056] As used herein, particularly in connection with the methods of the present application, a "reference host cell" is a host cell that is the same as the host applied in the methods described herein for increasing the production of a desired nucleic acid. The term "same" preferably refers to another cell of the same type, species and / or strain as the host cell containing the nucleic acid to be produced. However, the reference host cell is not affected by sulfur limitation, particularly when applied to the methods of the present application.

[0057] Preferably, the reference host cell can be grown in the same medium as the host cell applied in the methods described herein for increasing the production of a desired nucleic acid. The term "same medium" preferably relates to a cell culture medium having the same composition of components as the cell culture medium used for growing the host cell containing the nucleic acid to be produced, except for the sulfur content.

[0058] Preferably, the reference host cell can be grown under the same conditions as the host cell applied in the methods described herein for increasing the production of a desired nucleic acid, for example, with respect to temperature, pressure, gas environment, and / or mixing rate.

[0059] Preferably, the reference host cell does not contain the desired nucleic acid, particularly when applied to the methods of the present application. However, alternatively, the reference host cell can preferably contain the desired nucleic acid, particularly when applied to the methods of the present application. The latter alternative is more preferred than the first alternative.

[0060] Thus, a preferred reference host cell is not subject to sulfur limitation and does not contain the desired nucleic acid, particularly when applied to the methods of the present application. However, alternatively, a preferred reference host cell is not subject to sulfur limitation and contains the desired nucleic acid, particularly when applied to the methods of the present application.

[0061] The latter-mentioned alternative of the reference host cell is more preferred than the first-mentioned alternative of the reference host cell.

[0062] Therefore, a more preferred reference host cell is (i) As described herein, it is the same as the host applied to the method described herein for increasing the production of a desired nucleic acid, (ii) When particularly applied to the method of the present application, it is not subject to sulfur limitation, (iii) When particularly applied to the method of the present application, it contains the desired nucleic acid.

[0063] Furthermore, the more preferred reference host cell as described above, (iv) may be grown in the same medium as the host cell applied to the method described herein for increasing the production of the desired nucleic acid, and / or (v) For example, with respect to temperature, pressure, pH, gas environment, and / or mixing rate, it can be grown under the same conditions as the host cell applied in the method described herein for increasing the production of the desired nucleic acid.

[0064] Such a more preferred reference host cell may also be referred to herein as a "control", for example, a "control host cell", a "control culture", or in an example, a "control bacterial culture".

[0065] Preferably, in the methods described and provided in connection with the present invention, presetting the dry cell weight of the host cell can be achieved by obtaining a specified amount of a sample of the cell culture medium, harvesting the host cell, drying the host cell, and weighing the dried host cell. Means and methods for determining the amount of dry cell material and the elemental content of the cells contained therein are well known in the art. See, for example, Heldal et al., Applied and Environmental Microbiology (1985), 50(5): 1251-1257.

[0066] Preferably, in the methods described and provided in connection with the present invention, restricting the total sulfur content in the medium of the cell culture can be carried out when the host cells are in the logarithmic phase. Means and methods for determining whether a cell culture is in the logarithmic phase are known in the art, including measurement of the optical density (OD) of the cell culture. See, for example, https: / / www.implen.de / od600-diluphotometer / od600 / .

[0067] Preferably, in the methods described and provided in connection with the present invention, restricting the total sulfur content in the medium of the cell culture can be carried out when the dry weight of the host cells is at least about 5 g per liter of culture medium, preferably at least about 10 g, or at least about 20 g per liter of culture medium, such as 25 g per liter of culture medium, 30 g per liter of culture medium, 35 g per liter of culture medium, 40 g per liter of culture medium, 45 g per liter of culture medium, or 50 g per liter of culture medium.

[0068] Preferably, in the methods described and provided in connection with the present invention, the episomal nucleic acid may be a bacmid, cosmid, plasmid, or minicircle. The plasmid can be circular or linear. When circular, the plasmid is preferably a covalently closed circular (ccc). As described herein, "nucleic acid" also includes RNA molecules. Thus, the episomal nucleic acid can also be, for example, an RNA molecule in linear or circular form.

[0069] Preferably, the plasmid can be a low-copy number, medium-copy number, or high-copy number plasmid, and a high-copy number plasmid is preferred. Examples of high-copy number plasmids are plasmids having an origin of replication (ori) of a pMB1 derivative, ColE1 ori, pUC, or F1 ori. A high-copy number plasmid having a pUC ori is preferred.

[0070] Preferably, in the methods described and provided in connection with the present invention, the host cell can be a eukaryotic (e.g., fungal) or prokaryotic (e.g., bacterial) host cell, with prokaryotic cells being preferred. Preferred prokaryotic cells are E. coli cells.

[0071] In connection with the present invention, examples of eukaryotic host cells include fungal cells such as yeast, as well as microalgae (e.g., Chlorella) and protozoa.

[0072] As used herein, the term "host cell" preferably refers to a cell into which (or which can be introduced in the context of a reference host cell) a nucleic acid molecule that is not naturally contained by the host cell has been introduced, i.e., a genetically engineered (or which can be genetically engineered in the context of a reference host cell) cell. Preferred examples of prokaryotic host cells are E. coli. However, Pseudomonas species, Salmonella species, Bacillus species, Lactobacillus species, Corynebacterium species, Microbacterium species, or Actinomycetes species can also be envisioned in connection with the present invention. It should be understood that such terms are intended to refer not only to the particular subject cells but also to the progeny of such cells. Such progeny may not actually be identical to the parent cell due to either mutation or environmental influences that may occur in subsequent generations, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells can preferably be isolated cells grown in culture. In a preferred embodiment of the present invention, the bacterial host cell is E. coli. In a more preferred embodiment, the bacterial host cell is an E. coli K strain such as E. coli DH5α, or more preferably E. coli JM108. E. coli can also be derived from a B strain such as E. coli BL21, REL606, or a W strain, or any other derivative of E. coli. The bacterial host cells described herein can be produced by means and methods generally known in the art.

[0073] Preferably, the prokaryotic host cell may have an inactive recA and / or endA gene. Inactivation of recA and / or endA may be due to a complete or partial deletion or may be due to a mutation. Those skilled in the art recognize that, for example, by using the recA or endA of E. coli as a query sequence in a database or as a probe in a wet-lab experiment, the respective recA and / or endA genes can be found in the prokaryotic host.

[0074] Preferably, in the methods described and provided in connection with the present invention, the host cell may be a recombinant host cell.

[0075] Those skilled in the art recognize genetic engineering techniques known in the art for generating bacterial host cells for use in the systems or processes of the present invention. For example, for genetic engineering of bacterial host cells, various kits are available for randomly or targeted integration of nucleic acids containing nucleotide sequences into the bacterial genome. See, for example, Zhang et al. (1998), Nature Genetics 20, 123-128 or Sharan et al. (2009), Nature Protocols 4(2), 206-223. Those skilled in the art also recognize techniques for transformation of bacterial host cells and any other cloning techniques that can be used for the generation of extrachromosomal elements such as plasmids, cosmids, bacteriophages, mini-circles, etc.

[0076] In one embodiment of the present invention, the methods described and provided in connection with the present invention may be a method of fed-batch culture or continuous culture of the host cell in a fermenter, with fed-batch culture being preferred.

[0077] In the fed-batch culture or continuous culture of the host cells described herein, the feed can be linear or exponential. Thus, the fed-batch culture described herein can include a linear feed or an exponential feed. Similarly, the continuous culture described herein can include a linear feed.

[0078] The methods described and provided in connection with the present invention are based on a two-step process of decoupling host cell growth from the production of the nucleic acid of interest ("growth decoupling"). In the systems and processes of the present invention, growth, i.e., host cell propagation, can be spatially separated from the production of the nucleic acid of interest. In one bioreactor, i.e., the seed bioreactor, the host cells can be maintained under conditions ideal for their growth. Production of the nucleic acid of interest is either not induced at all or only slightly induced. Host cells from the seed reactor can be continuously transferred to a production bioreactor, which is the second bioreactor. Subsequently, the growth of the host cells can be inhibited, and production of the nucleic acid of interest can be induced in the production bioreactor. This concept is referred to herein as "growth decoupling". This enables continuous production. A portion of the host cells or a portion of the supernatant / media of the production reaction may be continuously removed from the production bioreactor, and the nucleic acid of interest can be harvested from the host cells. The fraction removed from the production bioreactor can preferably be continuously replaced by fresh host cells from the seed bioreactor. Thus, this technique enables continuous production of the nucleic acid of interest while maintaining high yield / productivity even with relatively small bioreactors, improving the S-T-Y (space-time yield) of the nucleic acid of interest.

[0079] The collection and isolation of nucleic acids such as plasmid DNA are well known to those skilled in the art. Generally, the production of the nucleic acids of the present invention involves growing a microbial / bacterial culture using the system or process of the present invention, followed optionally by collection and lysis of the microbial / bacterial host cells and optionally by purification of the nucleic acid molecule, such as plasmid DNA. Usually, the first step of the process is to lyse the microbial / bacterial host cells, preferably by alkaline lysis. The lysis reaction is carried out according to methods known in the art using an alkaline lysis solution containing a surfactant in a preferred embodiment of the present invention. A typical lysis solution consists of NaOH (0.2 M) and SOS (1%), but other alkaline solutions and other surfactants can also be used (see, for example, WO97 / 29190). After lysis, neutralization of the alkaline solution follows. Also, this step can in principle be carried out according to methods known per se, preferably in a gentle, continuous and automated mode. In a preferred embodiment, in the neutralization step, the lysed cell solution is mixed with the neutralization solution. Usually, a buffer of acidic pH and high salt concentration is used for neutralization. Preferably, this solution consists of potassium acetate (KAc) at pH 5.5 and 3 M. However, other neutralization salts can also be used or added. Thereafter, the nucleic acid molecule, such as plasmid DNA, can be precipitated by adding an aqueous solution containing about 70% ethanol. The nucleic acid can be pelleted by centrifugation, the supernatant aspirated and washed, and the nucleic acid containing the pellet resuspended in an aqueous solution containing about 70% ethanol and then pelleted again by centrifugation. After washing, the pelleted nucleic acid can be dried.

[0080] Alternatively, or in addition to this process, nucleic acid binding resins in the form of columns can also be used. Prior to capture / purification by the resin, it may be necessary to adjust the solution parameters (salt composition, conductivity, pH value, etc.) so that the desired biomolecule binds to the chromatography support (usually a resin) (this step is referred to as the "conditioning step" in the context of the present invention). The simplest conditioning procedure is, in particular, when the chromatography resin in the subsequent capture step is achieved by anion exchange chromatography, to dilute the clarified lysate with water or a low-salt buffer (WO97 / 29190). Further, especially when hydrophobic interaction chromatography is used as the first purification step, a high-concentration salt solution may be added and any resulting precipitate (present when the specific salt concentration in the solution is exceeded) may be separated by filtration or centrifugation (WO02 / 04027). When ammonium sulfate is used at a high concentration, this treatment reduces the RNA content (WO98 / 11208). For capture and purification, several steps are applied to obtain a highly purified biomolecule that meets the requirements as a pharmaceutical. As in the previous steps, enzymes, surfactants, and organic solvents should be avoided. Isolation and purification are carried out according to methods known in the art, in particular, by a combination of different chromatography techniques (anion exchange chromatography (AIEC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), ultrafiltration (dialysis), filtration or precipitation and extraction). A method that can be advantageously used to obtain plasmid DNA (pDNA) for therapeutic use in particular involves a combination of two steps based on different chromatography principles, where either of the two steps is selected from hydrophobic interaction chromatography (HIC), polar interaction chromatography (PIC), and anion exchange chromatography (AIEC), and in at least one, preferably both, of the two steps, the chromatography support is a rigid methacrylate-based monolith in the form of a porous monolithic bed, preferably a monolithic column.Suitable monolithic columns are commercially available under the trademark CIMmultus® from BIASeparations / Sartorius. This purification process can be advantageously carried out using a chromatographic support in the form of a single monolithic bed including a tube-in-tube system, where the outer tube and the inner tube carry different functional groups. In such a system, one of the monolithic tubes represents the support for the chromatographic principle of one step, and the other tube represents the support for the chromatographic principle of another step. Preferably, the capture / purification step can operate in batch mode, or in quasi-continuous or continuous mode, using techniques such as annular chromatography, carousel chromatography, or simulated moving.

[0081] As used herein, "bioreactor" or "reactor", including "seed bioreactor" or "production bioreactor", refers to a reaction vessel for fermentation to produce cells and biosynthetic products, and its size can range from a bench-top fermenter to an industrial tank. The bioreactor is preferably one that can automatically adjust the flow of oxygen, medium, and other nutrients, maintain the temperature and pH, minimize the possibility of contamination, and produce a higher density of cells than can be produced by conventional culture.

[0082] As used herein in the processes and systems of the present invention, "seed (bio)reactor" relates to the bioreactor used for the growth of host cells. In the seed bioreactor, the host cells of microorganisms or bacteria are in a non-induced / non-limiting state with respect to sulfur limitation that inhibits the growth of said host cells. Otherwise, growth in the seed bioreactor does not occur.

[0083] As used herein in the processes and systems of the present invention, "production (bio)reactor" relates to the bioreactor used for the production of the nucleic acid molecule of interest.

[0084] Bioreactors often include various sensors for monitoring important parameters such as pH, dissolved oxygen, or temperature, and means for controlling these parameters. Thus, the seed bioreactor preferably includes means for adjusting the pH. At least one production bioreactor preferably includes means for adjusting the pH. The means for adjusting the pH may include a pH probe, a device for recording the signal of the pH probe, and / or a device for adding an acid or a base to the medium, preferably a device for adding a base. The seed bioreactor preferably includes means for adjusting the dissolved oxygen. At least one production bioreactor preferably includes means for adjusting the dissolved oxygen. The means for adjusting the dissolved oxygen may include a pO2 probe, a control system for adjusting pO2 by means of pressure, ventilation rate, oxygen addition, agitation control, etc. The seed bioreactor preferably includes means for adjusting the temperature. At least one production bioreactor preferably includes means for adjusting the temperature. The means for adjusting the temperature may include a thermometer, a heating device, and a cooling device, and the temperature control by the heating device and / or the cooling device is preferably carried out by heating and / or cooling the mantle of the bioreactor. The seed bioreactor preferably comprises a gas inlet and a gas outlet, and means for regulating the gas flow. At least one production bioreactor preferably comprises a gas inlet and a gas outlet, and means for regulating the gas flow. At least one production bioreactor preferably includes a biomass sensor. The biomass concentration in the seed bioreactor can be adjusted by the feed inflow rate and / or the biomass outflow rate. The biomass concentration can be adjusted in at least one production bioreactor by the inflow of the feed, the inflow of the biomass, and / or the outflow of the biomass. The gas flow can be adjusted in at least one production bioreactor. The gas flow can be adjusted in at least one seed bioreactor.The seed and production bioreactor(s) are connected or coupled to enable the transfer of microorganisms or bacterial host cells from the seed bioreactor(s) to the production bioreactor(s). The flow of the medium containing the host cells from the seed bioreactor to the production bioreactor is preferably controlled. This control can be achieved, for example, by applying a chemostat or a turbidostat. Thus, the system of the present invention preferably further comprises means for operating the seed and production bioreactors as (c) connected chemostats or turbidostats. Preferably, the outflow of the seed bioreactor functions as the inflow to the production bioreactor.

[0085] The bioreactor of the present invention, i.e., the seed and / or production bioreactor, can be any bioreactor suitable for the purposes of the present invention. In one embodiment, the seed bioreactor is a stirred-tank bioreactor. In one embodiment, the seed bioreactor is a plug-flow bioreactor. In one embodiment, the production bioreactor is a stirred-tank bioreactor. In one embodiment, the production bioreactor is a plug-flow bioreactor.

[0086] Unless otherwise specified herein, as used herein in accordance with the present invention, the "growth" of a (host) cell used herein generally means an increase in the number of cells by cell division.

[0087] As used herein, "continuous fermentation" may relate to operating conditions in which a culture broth or nutrient medium, or a liquid medium such as cell broth, is added to a fermenter while the culture broth is discharged from the fermenter. The inflow of the liquid medium is preferably constant or intermittent at a certain rate. The discharge of the culture broth is preferably at the same rate so that the amount of liquid in the fermenter remains essentially constant. During continuous fermentation, the biomass preferably remains essentially unchanged. In the steady state, the fermentation conditions can be maintained, as necessary, by nutrient concentration, product concentration, constant pH, biomass, etc. The bioreactor used for continuous fermentation can be a stirred tank bioreactor or a tubular bioreactor. The "continuous fermenter" used herein is preferably a fermenter suitable for continuous fermentation.

[0088] For example, the (initial) startup of the continuous process according to the present invention may be carried out as follows. In both the seed and production bioreactors, it may be started as a batch culture, and subsequently, fed-batch culture may be continued until the required biomass concentration is achieved. Thus, during the startup of the process of the present invention, or in other words, during the start, the host cells are preferably in a state not induced with respect to the limitation of sulfur in the culture medium (in both the seed bioreactor and the production bioreactor). When the required cell density (e.g., logarithmic phase) is reached, production in the production bioreactor can be induced by limiting the amount of sulfur in the medium. In other words, the production bioreactor is in an induced state with respect to the amount of sulfur at this point. Furthermore, the continuous process can be started by transferring the host cells from the seed bioreactor to the production bioreactor, for example, by flowing the medium into the seed bioreactor and creating a flow from the outlet of the seed bioreactor to the inlet of the production bioreactor. Thus, in the continuous process of the present invention, cell growth is preferably inhibited in the production bioreactor.

[0089] Similarly, as contemplated in connection with the present invention, a fed-batch culture approach may be applied. Fed-batch culture processes are generally known in the art and are exemplified herein, and generally include processes in which different steps, such as feeding and culturing, are performed within the same bioreactor and the product (e.g., a nucleic acid molecule) remains within the bioreactor until the end of the culture. Fed-batch fermentation may be preferred for culture processes where it is necessary to control the specific amount or concentration of specific nutrients or culture components. In one embodiment of the present invention, fed-batch fermentation is applied in the methods described and provided herein.

[0090] Preferably, in the methods described and provided in connection with the present invention, the host cell may contain a recombinant extrachromosomal nucleic acid (e.g., a plasmid, bacteriophage, cosmid or minicircle, preferably a plasmid).

[0091] In the present invention, the nucleic acid molecule produced may be a recombinant extrachromosomal nucleic acid (e.g., a plasmid, bacteriophage, cosmid, or minicircle, preferably a plasmid). In one embodiment of the present invention, the nucleic acid (e.g., a recombinant extrachromosomal nucleic acid such as a plasmid, bacteriophage, cosmid, or minicircle, preferably a plasmid) contains a promoter sequence.

[0092] As used herein, a "promoter sequence" is a non-coding expression control sequence that is preferably inserted near the start of the coding sequence of an expression cassette and regulates its expression. Stated simply but basically correctly, it is the interaction between various special proteins called transcription factors and the promoter that determines whether a given coding sequence can be transcribed and ultimately translated into the actual protein encoded by that gene. Those skilled in the art will recognize that any suitable promoter can be used for recombinant expression within a host cell. The promoter itself may be preceded by upstream activation sequences, enhancer sequences, or combinations thereof. These sequences are known in the art as any DNA sequence derived from a gene encoding an extracellular or intracellular protein that exhibits strong transcriptional activity in cells. It will also be understood by those skilled in the art that termination sequences and polyadenylation sequences can be appropriately derived from the same source as the promoter.

[0093] As used herein, the term "inducible promoter" refers to a promoter that controls the expression of an operably linked gene or functional RNA in response to the presence or absence of an endogenous or exogenous stimulus. Such stimuli can be, but are not limited to, environmental signals such as compounds or temperature shifts. Examples of inducible promoters include, but are not limited to, pBAD, OR2-0R1-PR, pltetO, pllacO, PesR, plac, lacUV, tac promoter, pPrpB, pTetO, FixK2, pltet0-1, or PcpcG2.

[0094] The nucleic acid molecules (e.g., recombinant extrachromosomal nucleic acids such as plasmids, bacteriophages, cosmids, or minicircles, preferably plasmids) described, provided, and generated according to the present invention herein can be autonomously replicable within a host cell (e.g., the vector has an origin of replication that functions within the host cell). The nucleic acid molecule may have a linear, circular, or supercoiled configuration and may be complexed with other nucleic acids or other substances for a particular purpose.

[0095] Nucleic acid molecules produced according to the present invention described and provided herein (e.g., recombinant extrachromosomal nucleic acids such as plasmids, bacmids, cosmids, or minicircles, preferably plasmids) may also be suitable as vectors. Vectors typically contain, as elements of the expression cassette, transcriptional control elements suitable for promoting transcription, such as promoters, enhancers, polyadenylation signals, transcriptional pause, or transcription termination signals. For proper expression of a polypeptide, the vector preferably contains a preferably optimized 5' untranslated region leading to the ribosome binding site (RBS), e.g., a Kozak sequence for protein translation initiation suitable for recruiting ribosomes, and appropriate translation control elements such as a stop codon for terminating the translation process. In particular, the nucleotide sequence functioning as a selectable marker gene, as well as the nucleotide sequence encoding the protein of interest, can be transcribed under the control of transcriptional elements present in an appropriate promoter. The resulting transcripts of the selectable marker gene and the protein of interest carry functional translation elements that promote substantial levels of protein expression (i.e., translation) and appropriate translation termination. In one embodiment of the invention, the selectable marker used is the auxotrophic marker gene in a host strain containing a knockout or deletion of the auxotrophic marker gene. The vector may contain a polylinker (multiple cloning site, MCS), i.e., a short segment of DNA containing multiple restriction enzyme recognition sites, which is a standard feature of many plasmids used in molecular cloning. The multiple cloning sites typically contain 5, 10, 15, 20, 25, or more than 25 restriction sites. The restriction enzyme recognition sites within the MCS are usually unique (i.e., they occur only once within that particular plasmid). The MCS is generally used in procedures involving molecular cloning or subcloning.

[0096] In the context of the invention described and provided herein, one type of vector can be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments can be introduced by ligation or cloning without restriction. Other nucleic acid molecules (e.g., recombinant extrachromosomal nucleic acids such as plasmids, bacteriophages, cosmids, or minicircles, preferably plasmids) produced according to the present invention and described and provided herein that may be suitable as vectors include cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or minichromosomes. Another type of vector is a viral vector into which additional DNA fragments can be ligated to the viral genome. An expression cassette can be inserted into an expression vector as a DNA construct. This DNA construct can be recombinantly produced from synthetic DNA molecules, genomic DNA molecules, cDNA molecules, or combinations thereof. The DNA construct is preferably produced by ligating different fragments together according to standard techniques known in the art. The expression cassette can be part of an expression vector. Preferably, the expression vector is a DNA vector. The vector can conveniently contain sequences that facilitate the proper expression of the gene of interest and the antibiotic resistance gene. These sequences typically include, but are not limited to, the promoter sequences, transcription start sites, transcription termination sites, and polyadenylation functions described herein. The expression cassette can contain enhancers and / or introns. Usually, the intron is located at the 5' end of the open reading frame. Thus, an expression cassette for expressing a polypeptide of interest can contain an intron to increase the expression rate. The intron can be located between the promoter and / or promoter / enhancer element and the 5' end of the open reading frame of the polypeptide to be expressed. Several suitable introns that can be used in conjunction with the present invention are known in the art. The expression cassette or vector according to the present invention present in the host may be integrated into the host's genome or maintained extrachromosomally in some form. Furthermore, the expression cassette may contain an appropriate transcription termination site.This is a phenomenon known as promoter occlusion or transcriptional interference, where continuous transcription from an upstream promoter through a second transcription unit can inhibit the function of a downstream promoter. This event has been described in both prokaryotes and eukaryotes. Appropriate placement of transcription termination signals between two transcription units can prevent promoter occlusion. Transcription termination sites are well-characterized, and their incorporation into expression vectors has been shown to have various beneficial effects on gene expression.

[0097] As used herein, the terms "5'" and "3'" refer to the convention used to describe features of a nucleotide sequence in relation to either the position of a genetic element and / or the direction (5' to 3') of an event. For example, transcription by RNA polymerase or translation by ribosomes proceeding in the 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). Conventionally, nucleotide sequences, gene maps, vector maps, and RNA sequences are depicted in the 5' to 3' direction from left to right or the 5' to 3' direction is indicated by an arrow with the tip of the arrow pointing in the 3' direction. Thus, following this convention, 5' (upstream) indicates a genetic element located towards the left, and 3' (downstream) indicates a genetic element located towards the right.

[0098] As used herein, the term "expression" can preferably mean transcription of a nucleotide sequence. The nucleotide sequence preferably encodes a protein. Thus, the term also includes the production of mRNA (as a transcription product from the nucleotide sequence) and the translation of this mRNA to produce the corresponding gene product such as a polypeptide or protein.

[0099] In another embodiment of the methods described and provided in connection with the present invention, the method can be a method for generating a recombinant extrachromosomal nucleic acid (e.g., a plasmid, bacteriophage, cosmid, or minicircle, preferably a plasmid).

[0100] The present invention further relates to the use of a medium comprising a pre-set total sulfur amount per gram dry cell weight of a host cell that is desirably obtained by cell culture, wherein the total sulfur amount is preferably substantially equal to or less than the sulfur amount contained per gram dry cell weight of a reference host cell when grown without restricting the total sulfur amount, in order to increase the production of nucleic acids (preferably extrachromosomal nucleic acids) contained by the host cell in cell culture.

[0101] As used herein, the terms "nucleic acid" or "nucleic acid molecule" are used synonymously and include, inter alia, "oligonucleotide", "nucleic acid strand", etc., and can mean a polymer comprising one, two, or more nucleotides arranged in a single-stranded or double-stranded nucleotide strand, unless otherwise specifically defined.

[0102] In general, the nucleic acid molecules used in accordance with the present invention include, inter alia, DNA molecules, cDNA molecules, RNA molecules, oligonucleotide phosphorothioates, substituted ribooligonucleotides, or PNA molecules, and may preferably include DNA molecules or cDNA molecules. Further, the term "nucleic acid molecule" may refer to DNA, or RNA, or hybrids thereof, or any modifications thereof known in the art (for examples of modifications, see, for example, US5525711, US4711955, US5792608, or EP302175). The polynucleotide sequence can be single-stranded or double-stranded, linear or circular, natural or synthetic, and there is no size limitation. For example, the polynucleotide sequence can be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozyme RNA, or DNA encoding such RNA or chimeric plastids (Gamper, Nucleic Acids Research, 2000, 28, 4332-4339). The above polynucleotide sequence can be in the form of DNA or RNA of a vector, plasmid, or virus. Also described herein are nucleic acid molecules complementary to the above nucleic acid molecules and nucleic acid molecules capable of hybridizing to the nucleic acid molecules described herein. The nucleic acid molecules described herein may also be fragments of nucleic acid molecules in the context of the present invention. In particular, such fragments are functional fragments. An example of such a functional fragment is a nucleic acid molecule that can function as a primer.

[0103] According to the present invention, the nucleic acid molecule may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases, which will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution with analogs of one or more natural nucleotides, uncharged linkages (methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (phosphorothioates, phosphorodithioates, etc.), pendant moieties (polypeptides, etc.), intercalators (acridines, psoralens, etc.), chelating agents, alkylating agents, and internucleotide modifications such as modified linkages (alpha-anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a specified sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide bonds replace phosphate bonds within the molecular backbone. The nucleic acid can have any topological structure. For example, the nucleic acid may have a single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin, circular, or padlock-like conformation.

[0104] Embodiments characterizing the present invention are described herein, shown in the figures, illustrated in the examples, and reflected in the claims.

[0105] As used herein, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that can modify or replace the methods described herein.

[0106] Unless otherwise specified, the term "at least" preceding a series of elements is understood to refer to all of the elements in that series. One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0107] As used herein, the term "and / or" always includes the meanings of "and", "or", and "all or any other combination of the elements connected by said term".

[0108] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, more preferably within 5% or 2% of a given value or range, and also includes each exact numerical value.

[0109] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. As used herein, the term "comprising" can be replaced by the term "containing" or "including", and in some cases, when used herein, may also be replaced by the term "having".

[0110] As used herein, "consisting of" excludes elements, steps, or components not specified in the claims. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel features of the claims.

[0111] In each example of this specification, any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced by any of the other two terms.

[0112] It should be understood that the present invention is not limited to the specific procedures, customs, reagents, etc. described herein and can therefore vary. The terms used herein are for the purpose of describing only specific embodiments and do not limit the scope of the present invention, which is defined only by the claims.

[0113] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art. Generally, the nomenclature used in connection with the biochemistry, enzymology, molecular biology and cell biology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization techniques described herein are well known and commonly used in the art.

[0114] The methods and techniques of the present invention, unless otherwise indicated, are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), Ausubel et al., Current Protocols in Molecular Biology, J, Greene Publishing Associates (1992, and Supplements to 2002), Handbook of Biochemistry: Section A Proteins, Vol 1 1976 CRC Press, Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press. The nomenclature used in connection with the molecular biology and cell biology, protein biochemistry, enzymology, medicinal chemistry and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and generally used in the art.

[0115] All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) cited throughout the text of this specification are hereby incorporated by reference in their entirety, regardless of whether above or below. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. In case of conflict or inconsistency between the incorporated material and this specification, this specification shall prevail over any such material.

[0116] The present invention also relates to the following clauses. 1. A method for increasing the production of nucleic acids (preferably extrachromosomal nucleic acids) contained in host cells in cell culture, comprising, over the course of culturing the cells, the step of restricting the total sulfur content in the medium of the cell culture, wherein the total sulfur content is preferably per gram dry weight of the host cells desired to be obtained in the cell culture, or is approximately equal to or less than the amount of sulfur contained per gram dry weight of reference host cells when grown without restricting the total sulfur content, thereby increasing the production of the nucleic acids, said method comprising said step of restricting.

[0117] 2. The step of restricting comprises (a1) presetting the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and (b1) at the start of the cell culture, adding to the cell culture the total sulfur content per gram dry weight of the preset host cells, wherein the total sulfur content is approximately equal to or less than the amount of sulfur contained per gram dry weight of the reference host cells; said method according to clause 1, comprising said adding. The method according to clause 1, comprising.

[0118] 3. The step of restricting comprises (a2) presetting the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and (b2) over the course of the cell culture, reducing the available total sulfur content per gram dry weight of the host cells in the cell culture to at least not more than the amount of sulfur contained per gram dry weight of the reference host cells; said method according to clause 1, comprising. The method according to clause 1, comprising.

[0119] 4. The step of restricting comprises (a3) presetting the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and (b3) During the cell culture (from the start until the desired amount of the host cell containing the nucleic acid is obtained), adding to the cell culture a preset total sulfur amount per gram dry weight of the host cell, wherein the total sulfur amount is approximately equal to or less than the amount of sulfur contained per gram dry weight of the reference host cell, and the adding, The method according to item 1, comprising

[0120] 5. The method according to any one of items 1 to 4, wherein when the preset total sulfur amount is reduced to 0% of the preset total sulfur amount in the medium, additional sulfur is added to the medium.

[0121] 6. The method according to any one of items 1 to 4, wherein when the preset total sulfur amount is reduced to 50% or 25% or less of the preset total sulfur amount in the medium, additional sulfur is added to the medium.

[0122] 7. The method according to any one of the preceding items, wherein the amount of sulfur contained per gram dry weight of the reference host cell is approximately equal to or less than the amount of sulfur contained in the elemental composition per gram dry weight of the reference host cell.

[0123] 8. The method according to any one of the preceding items, wherein the reference host cell is identical to the host except that it does not contain the nucleic acid.

[0124] 9. The method according to any one of the preceding items, wherein the reference host cell is grown in the same medium as the host cell except that the total sulfur amount is restricted.

[0125] 10. The method according to any one of the preceding items, wherein presetting the dry cell weight of the host cell is achieved by obtaining a specified amount of a sample of the cell culture, collecting the host cell, and drying the host cell.

[0126] 11. The method according to any one of the preceding clauses, wherein restricting the total sulfur amount in the medium for the cell culture is carried out when the host cell is in the logarithmic phase.

[0127] 12. The method according to any one of the preceding clauses, wherein restricting the total sulfur amount in the medium for the cell culture is carried out when the dry weight of the host cell is at least about 5 g, preferably at least about 10 g, or at least about 20 g per liter of the culture medium.

[0128] 13. The method according to any one of the preceding clauses, wherein the extrachromosomal nucleic acid is a bacmid, cosmid, plasmid, or minicircle.

[0129] 14. The method according to any one of the preceding clauses, wherein the plasmid is a high-copy number plasmid having an origin of replication (ori) of a pMB1 derivative, ColE1 ori, pUC, or F1 ori.

[0130] 15. The method according to any one of the preceding clauses, wherein the host cell is a fungal host cell or a prokaryotic host cell.

[0131] 16. The method according to any one of the preceding clauses, wherein the host cell is a recombinant host cell.

[0132] 17. The method according to any one of the preceding clauses, which is a method for continuously culturing the host cell in a fermenter.

[0133] 18. The method according to any one of the preceding clauses, wherein the host cell contains a recombinant extrachromosomal nucleic acid (e.g., a plasmid).

[0134] 19. The method according to any one of the preceding clauses, which is a method for producing a recombinant extrachromosomal nucleic acid (e.g., a plasmid).

[0135] Use of a medium containing a pre-set total sulfur amount per gram dry cell weight of a host cell, which is desirably obtained by cell culture, wherein the total sulfur amount is preferably substantially equal to or less than the sulfur amount contained per gram dry cell weight of a reference host cell when grown without restricting the total sulfur amount, in order to increase the production of nucleic acids (preferably extrachromosomal nucleic acids) contained by the host cell in cell culture.

[0136] The present invention will be further described by the following examples. However, the examples and specific embodiments described therein should not be construed as limiting the present invention to such specific embodiments.

Examples

[0137] Materials and methods Fed-batch fermentation In fed-batch fermentation, E. coli cells, preferably E. coli JM108, were grown in a 2.1 L (net volume 1.0 L, batch volume 0.5 L) computer-controlled bioreactor (DASGIP parallel bioreactor system, Eppendorf AG, Germany). The bioreactor was equipped with a pH probe (Hamilton Bonaduz AG, Switzerland) and an optical DO probe (Hamilton Bonaduz AG). The pH was maintained at 7.0 ± 0.1 by adding a 12.5% ammonia solution (Carl Roth, Germany). The temperature was maintained at 37 ± 0.5 °C. The dissolved oxygen (O2) level was stabilized above 30% saturation by controlling the stirring speed, aeration rate, and gas composition. Foaming was suppressed by adding 2 mL of a 1:10 diluted Struktol J673A antifoam suspension (Scilla + Silacher, Germany) to the batch medium and by automatically adding a 1:10 diluted Struktol J673A controlled by a conductivity operating level sensor. A seed culture was used for inoculation of the bioreactor (25 mL of batch medium seeded with 250 μL of MCB in a 250 mL glass flask with baffles, shaken at 37 °C and 180 rpm). The seed culture was incubated until the final OD600 reached 2 - 4 and the specified amount was aseptically transferred to the bioreactor, reaching an initial ODini of 0.015.

[0138] The fermentation process was designed to have a final amount of 50 g CDM, of which 1.51 g was obtained in a 500 mL batch volume, and an additional 48.5 g was obtained in the feed stage by adding 500 mL of feed medium. The amount of glucose for a specific medium was calculated based on a yield coefficient (Yx / s) of 0.303 g / g and added as C6H12O6·H2O. For the preparation of the medium, all chemicals were purchased from Carl Roth GmbH (Germany) unless otherwise stated.

[0139] Regarding specific pDNA productivity, two different medium compositions were compared: one with a restricted sulfur source (S-restricted) and the other with an unrestricted sulfur source (control).

[0140] Medium for S-restriction According to the number of grams of CDM formed during the batch stage, the following components were added and then sterile filtered. In batch medium preparation, the following components were added and then sterile filtered. Yeast Extract (Bacto Yeast Extract) 0.05 g, NH4Cl 1.00 g, MgSO4·7H2O 0.12 g, KH2PO4 2.72 g, Na2HPO4·2H2O 3.69 g, glucose monohydrate 5 g, L-proline 0.1 g, L-isoleucine 0.1 g, 1% thiamine-HCl solution 0.05 mL, citric acid (anhydrous) 1.0 g, trace element solution 8.34 mL. The sterile filtered feed medium was composed of the following components according to the number of grams of CDM formed during the feed stage. 2.72 g KH2PO4, 3.69 g Na2HOP4·2H2O, 169.94 g glucose monohydrate, 3.0 g L-proline, 3.0 g L-isoleucine, 1.0 g citric acid (anhydrous), 1.50 mL of 1% thiamine-HCl stock solution, 25.0 mL trace element solution, 0.42 g MgCl2·7H2O. A total of 9.6 mL of 200 g / L MgSO4·7H2O stock solution was pulse-injected into the bioreactor (3.2 mL at the start of feeding, 3.2 mL 7 hours after the start of feeding, and 3.2 mL 15 hours after the start of feeding).

[0141] Control medium: According to the number of grams of CDM formed during the batch stage, the following components were added and then sterile filtered. In batch medium preparation, the following components were added and then sterile filtered. Yeast extract (Merck) 0.05 g, NH4Cl 1.00 g, MgSO4·7H2O 0.12 g, KH2PO4 2.72 g, Na2HOP4·2H2O 3.69 g, glucose monohydrate 5 g, L-proline 0.1 g, L-isoleucine 0.1 g, 1% thiamine-HCl solution 0.05 mL, citric acid (anhydrous) 1.0 g, trace element solution 8.34 mL. The sterile filtered feed medium was composed of the following components according to the number of grams of CDM formed during the feed stage. 3.60 g MgSO4·7H2O, 2.72 g KH2PO4, 3.69 g Na2HOP4·2H2O, 164.94 g glucose monohydrate, 3.0 g L-proline, 3.0 g L-isoleucine, 1.0 g citric acid (anhydrous), 1% thiamine-HCl stock solution 1.50 mL, trace element solution 25.0 mL. The trace element solution was prepared in 5 M HCl and contained as follows (g / L). 4.41 g CaCl2·2H2O, 3.34 g FeSO4·7H2O, 1.43 g CoCl2·6H2O, 1.03 g MnSO4·H2O, 0.15 g CuSO4·5H2O, 0.17 g ZnSO4·7H2O.

[0142] Feeding was started when the culture in the batch medium entered the stationary phase. A fed-batch culture regimen with a linear substrate feed (0.26 g / min, 13.91 mL / h each) was used for 35 hours (5 generations). During S-limited fermentation, the sulfur provided was consumed 23 hours after the start of feeding.

[0143] All cultures were repeated three times except for those shown in Figure 6. Average values were used for all figures and tables.

[0144] Offline analysis The bioreactor for offline analysis (OD600, CDM, product) was sampled during the fed-batch culture stage. OD600 was measured using an Ultrospec 500pro spectrophotometer (Amersham Biosciences, UK) after diluting the sample with phosphate-buffered saline to achieve a linear measurement range. For the determination of CDM, 1 mL of the cell suspension was transferred to a pre-weighed 2.0 mL reaction tube and centrifuged at 16,100 rcf and 4 °C for 10 minutes using an Eppendorf 5415R centrifuge. The supernatant was transferred to another reaction tube and may be stored at -20 °C for further analysis. As a washing step, the cell pellet was resuspended in 1.8 mL of RO-H2O, centrifuged, and the supernatant was discarded. Subsequently, the pellet was resuspended in 1.8 mL of RO-H2O and finally dried at 105 °C for 24 hours. The reaction tubes were cooled to room temperature in a desiccator before weighing them again.

[0145] For the analysis of the pDNA product, the sampling volume of the cell suspension corresponding to 20 mg of CDM was estimated by direct measurement of OD600. The calculated amount was transferred to a 2.0 mL reaction tube and centrifuged at 16,100 rcf and 4 °C for 10 minutes. The supernatant was discarded and the cell pellet was stored at -20 °C.

[0146] pDNA analysis The content of ccc-structured pDNA was determined using AIEX-HPLC (CIMac™ pDNA-0.3 analytical column, 1.4 μm; BIA Separations d.o.o., Slovenia). In the HPLC analysis, cell lysis was performed by the alkaline lysis method (Birnboim and Doly, 1979). The obtained lysate was directly analyzed by HPLC (Agilent 1100 with a quaternary pump, diode array detector (DAD)). The values derived from three biological replicates had a coefficient of variation of less than 10%.

[0147] Example 1 - Cell Dry Mass (CDM) In Figure 1, it can be confirmed that while the CDM of the control culture (without S limitation) continuously increases, the CDM of the culture under S limitation, i.e., the culture according to the method of the present invention, decreases at about 23 hours after the start of the culture. At this stage, S becomes limiting, and the bacterial cells no longer increase biomass but produce the desired nucleic acid, such as a plasmid. Therefore, immediately when S becomes limiting, metabolism is disconnected, in other words, from the production of the desired nucleic acid, such as a plasmid.

[0148] Example 2 - Quality of Plasmid DNA (pDNA) In Figure 2, it can be confirmed that the quality of the plasmid DNA (pDNA) generated by the culture under S limitation, i.e., the culture according to the method of the present invention, significantly improves at about 25.5 hours, while the quality of the pDNA produced by the control culture continuously decreases.

[0149] Example 3 - Productivity of Covalently Closed Circular (ccc) DNA When the productivity of covalently closed circular (ccc) DNA by bacterial culture according to the method of the present invention was directly compared with a control bacterial culture not subject to S limitation, it was significantly increased when S became limiting (see Figure 3). In contrast, in the control culture not subject to S limitation, the productivity of cccDNA decreased.

[0150] Example 4 - Volume Yield of cccDNA The volume yield of cccDNA achieved by bacterial culture according to the method of the present invention increases compared to a direct comparison with a control bacterial culture not subject to S limitation, while it decreases in the control culture. See Figure 4.

[0151] Example 5 - Method for Determining Sulfur Required for Growth - Plate the cells and grow them overnight on an agar plate - Prepare, for example, 120 ml of sulfur-free medium in a shaker flask. For example, the "medium for S limitation" described herein - Add different amounts of sulfur stock solution, e.g., MgSO4 or (NH4)2SO4, to the shaker flask. For example, 0, 25 μl, 50 μl, 100 μl of sulfur stock solution, etc. - Inoculate the plated cells (take care to minimize sulfur uptake from the pre - culture). - Grow the cells for at least 24 hours or until the maximum OD600 is reached. The maximum OD600 is achieved when the OD600 changes over time within the range of maximum +0.5 OD600 to -0.5 OD600 units. - Weigh the biomass content of each shaker.

[0152] The sulfur amount / required amount can be calculated as follows: - Plot the cell dry mass (CDM) against the calculated / added sulfur content of the shaker flask. - Perform a linear regression. - Calculate the grams of sulfur per gram of biomass. - The sulfur requirement is, for example, 0.0085 gS / gCDM as shown in the specific example of sulfur requirement calculation in Figure 5.

Claims

1. A method for increasing the production of nucleic acids (preferably extrachromosomal nucleic acids) contained in host cells in cell culture, comprising the step of restricting the total sulfur content in the medium of said cell culture over the course of culturing the cells, wherein the total sulfur content is preferably per gram dry weight of the host cells desired to be obtained in said cell culture, per gram dry weight of the reference host cells grown without restricting the total sulfur content The method comprising the step of restricting such that the amount of sulfur contained is approximately equal to or less than the amount of sulfur contained, thereby increasing the production of said nucleic acid.

2. The step of restricting comprises (a1) presetting the amount of dry cell weight of the host cells desired to be obtained in said cell culture; and (b1) adding to said cell culture at the start of said cell culture a total sulfur content per gram dry weight of said preset host cells, said total sulfur content being approximately equal to or less than the amount of sulfur contained per gram dry weight of said reference host cells; said adding. The method according to claim 1, comprising

3. The step of restricting comprises (a2) presetting the amount of dry cell weight of the host cells desired to be obtained in said cell culture; and (b2) reducing over the course of said cell culture the available total sulfur content per gram dry weight of said host cells in said cell culture to at least less than the amount of sulfur contained per gram dry weight of said reference host cells; said reducing. The method according to claim 1, comprising

4. The step of restricting comprises (a3) presetting the amount of dry cell weight of the host cells desired to be obtained in said cell culture; and (b3) adding to said cell culture during said cell culture (from the start until the desired amount of host cells containing said nucleic acid is obtained) a total sulfur content per gram dry weight of said preset host cells, said total sulfur content being approximately equal to or less than the amount of sulfur contained per gram dry weight of said reference host cells; said adding. The method according to claim 1, comprising

5. The method according to any one of claims 1 to 4, wherein when the preset total sulfur amount has decreased to 0% of the preset total sulfur amount in the medium, further sulfur is added to the medium.

6. The method according to any one of claims 1 to 4, wherein when the preset total sulfur amount has decreased to 50% or 25% or less of the preset total sulfur amount in the medium, further sulfur is added to the medium.

7. The method according to any one of the preceding claims, wherein the amount of sulfur contained per gram dry weight of the reference host cell is approximately equal to or less than the amount of sulfur contained in the elemental composition per gram dry weight of the reference host cell.

8. The method according to any one of the preceding claims, wherein the reference host cell is identical to the host, except that it does not contain the nucleic acid.

9. The method according to any one of the preceding claims, wherein the reference host cell is grown in the same medium as the host cell, except that the total sulfur amount is restricted.

10. The method according to any one of the preceding claims, wherein presetting the dry cell weight of the host cell is achieved by obtaining a specified amount of a sample of the cell culture medium, harvesting the host cell, and drying the host cell.

11. The method according to any one of the preceding claims, wherein restricting the total sulfur amount in the medium of the cell culture is carried out when the host cell is in the logarithmic phase.

12. The method according to any one of the preceding claims, wherein restricting the total sulfur amount in the medium of the cell culture is carried out when the dry weight of the host cell is at least about 5 g, preferably at least about 10 g, or at least about 20 g per liter of culture medium.

13. The method according to any one of the preceding claims, wherein the extrachromosomal nucleic acid is a bacmid, cosmid, plasmid, or minicircle.

14. The method according to any one of the preceding claims, wherein the host cell is a fungal host cell or a prokaryotic host cell.

15. The method according to any one of the preceding claims, which is a method for continuously culturing the host cell in a fermenter.

16. The method according to any one of the preceding claims, wherein the host cell contains a recombinant extrachromosomal nucleic acid (e.g., a plasmid).

17. Use of a medium containing a pre-set total sulfur amount per gram dry cell weight of a host cell, which is desirably obtained by cell culture, wherein the total sulfur amount is preferably substantially equal to or less than the sulfur amount contained per gram dry cell weight of a reference host cell when grown without restricting the total sulfur amount, in order to increase the production of nucleic acids (preferably extrachromosomal nucleic acids) contained by the host cell in cell culture.

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