Culture medium for plasmid production

Abstract

The present invention pertains to the field of culture media designed for plasmid production. Specifically, it relates to a specialized culture medium for bacterial growth that is optimized to achieve high plasmid yields.

Description

[0001] P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0002] “CULTURE MEDIUM FOR PLASMID PRODUCTION”

[0003] FIELD OF THE INVENTION

[0004] The present invention pertains to the field of culture media designed for plasmid production. Specifically, it relates to a specialized culture medium for bacterial growth that is optimized to achieve high plasmid yields.

[0005] STATE OF THE ART

[0006] Plasmid production is a critical process in biotechnology, particularly for applications in gene therapy, vaccine development, and the production of recombinant proteins. The culture medium plays a vital role in optimizing plasmid yield and quality. Traditionally, plasmid production has relied on complex media, which contain a variety of organic compounds, including peptones, yeast extract, and other undefined ingredients, providing a rich environment for bacterial growth. However, these media come with several challenges.

[0007] One of the main issues with complex media is the variability in composition, which can lead to batch-to-batch inconsistencies. This variability can affect the reproducibility of plasmid production, posing challenges for large-scale manufacturing and regulatory compliance. Furthermore, the undefined nature of the components makes it difficult to pinpoint the exact nutrients or factors that influence plasmid yield and quality, hindering process optimization.

[0008] In contrast, chemically defined (CD) media, which contain precisely known components, have emerged as an alternative. These media offer several advantages, such as reproducibility, scalability, and easier regulatory approval, as their composition is fully characterized. CD media typically include sources of carbon, nitrogen, salts, vitamins, and trace elements, all of which are carefully selected to support optimal bacterial growth and plasmid production. However, the use of CD media for plasmid production is not without its challenges. One of the main drawbacks is that CD media often do not support the same high yields of plasmid DNA (pDNA) as complex media. This is because the precise nutrient P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0009] balance required for optimal bacterial growth and plasmid replication is not always easy to achieve in a chemically defined medium. As a result, the efficiency of plasmid production may be suboptimal, particularly in large-scale fermentations. Another challenge with CD media is the optimization of carbon and nitrogen sources to support both bacterial growth and plasmid replication. The presence of specific nutrients, such as amino acids or growth factors, can influence the stability of the plasmid and the efficiency of its replication within the bacterial host. Additionally, the use of CD media can increase the cost of production, as the individual components are often more expensive than the bulk ingredients found in complex media.

[0010] Recent research has focused on optimizing CD media for plasmid production by carefully adjusting the composition to improve both plasmid yield and quality. Efforts have been made to identify key factors that enhance plasmid replication and minimize plasmid loss during fermentation. Innovations in this area include the development of tailored media formulations, which may include specific supplements or inducer molecules to maximize plasmid production. Moreover, advancements in fermentation technology, such as controlled feeding strategies and the use of fed-batch or continuous culture systems, have helped mitigate some of the limitations associated with CD media.

[0011] Despite these advancements, achieving a balance between high plasmid yield, quality, and cost-effectiveness remains a major challenge in plasmid production. The development of a reliable, high-performance chemically defined medium that can match or surpass the results obtained with complex media is still an ongoing area of research. This includes exploring alternative strategies for optimizing bacterial growth, plasmid replication, and overall fermentation efficiency.

[0012] Ultimately, the goal is to create a medium that not only supports efficient plasmid production but also meets the regulatory and scalability requirements necessary for industrial-scale applications.

[0013] SUMMARY OF THE INVENTION

[0014] The objective of the present invention is to develop a new chemically defined (CD) P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0015] medium for plasmid production through bacterial fermentation, ensuring that the plasmid yield and quality would be comparable to or exceed those achieved using traditional methods. This new medium was designed to optimize the production process while maintaining high standards of plasmid quantity and quality. To evaluate its effectiveness, the results of all experiments were compared against two reference media: a complex medium, which is the current gold standard used by the company, and a commercially available chemically defined medium. These comparisons allowed for a thorough assessment of the new medium's performance in plasmid production.

[0016] The present invention discloses a medium for plasmid production comprising: - Glycerol,

[0017] - (NH4)2SO4,

[0018] - MgSO4,

[0019] - KH2PO4,

[0020] - K2HPO4,

[0021] - NaCI,

[0022] - a vitamin mixture comprising: Thiamine, Pyridoxine hydrochloride and Nicotinic acid;

[0023] - a trace element mixture comprising: NaCI, ZnSO4*7 H2O, CuSO4*5H2O, MnCI2*4 H2O, FeCI3*6H2O, CaCI2*2 H2O, Na2MoO4*2 H2O, H2SO497%; and

[0024] - water.

[0025] Water for injection (WFI) or sterile water can be used for the dilution of all the components of the medium.

[0026] In a further aspect the use of the medium according to the invention for growing bacteria is described.

[0027] In the medium of the present invention, the following components can be defined as the fermentation medium:

[0028] - Glycerol,

[0029] - (NH4)2SO4,

[0030] - MgSO4, P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0031] - KH2PO4,

[0032] - K2HPO4, and

[0033] - NaCL

[0034] NaCI is present both in the fermentation medium and in the trace element mixture, and is added to each of the two independently for achieving the objective of the invention.

[0035] In a still further aspect, the invention relates to a method for plasmid production by bacterial fermentation, wherein the method comprises the step of using the medium herein disclosed.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The characteristics and advantages of the present invention will be apparent from the detailed description reported below, from the Experimental part given for illustrative and non-limiting purposes, and from the annexed Figures 1 -2.

[0038] Figure 1: comparison of Plasmid Yields. A) Volumetric yield; B) Specific yield.

[0039] Figure 2: restriction analysis and 1% agarose gel electrophoresis. On the left, base pairs are reported. Plasmid was linearized with Not1 restriction enzyme and digested with Not1 and Nco1 restriction enzymes. Three isoforms can be observed: open circular (OC), linearized (LIN) and supercoiled (SC). In the reference media, an extra isoform is highlighted by the black arrows.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] Plasmid production is a key process in biotechnology, especially for applications such as gene therapy, vaccine development, and the production of recombinant proteins. The present invention pertains to a culture medium, that has been formulated in order to maximize both the yield and quality of plasmids.

[0042] The present invention thus concerns a medium for plasmid production comprising: - Glycerol,

[0043] - (NH4)2SO4,

[0044] - MgSO4, P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0045] - KH2PO4,

[0046] - K2HPO4,

[0047] - NaCI,

[0048] - a vitamin mixture comprising: Thiamine, Pyridoxine hydrochloride and Nicotinic acid;

[0049] - a trace element mixture comprising: NaCI, ZnSO4*7 H2O, CuSO4*5H2O, MnCl2*4 H2O, FeCI3*6H2O, CaCI2*2 H2O, Na2MoO4*2 H2O, H2SO497%; and

[0050] - water.

[0051] The medium according to the invention provides a balanced concentration of salts, required to maintain the osmotic pressure of the medium and to provide essential ions for enzyme activity, stabilizing the plasmid DNA, assisting in the function of enzymes involved in DNA replication and other cellular processes.

[0052] In a preferred embodiment of the invention the medium comprises:

[0053] - Glycerol in the range from 25 to 35 g / L,

[0054] - (NH4)2SO4 in the range from 5 to 15 g / L,

[0055] - MgSO4 in the range from 1 to 5 mM,

[0056] - KH2PO4 in the range from 2 to 10 g / L,

[0057] - K2HPO4 in the range from 10 to 20 g / L, and

[0058] - NaCI in the range from 2 to 10 g / L.

[0059] The medium composition of the present invention was carefully designed to provide an optimal environment for bacterial growth and plasmid replication. The specific components of a plasmid culture medium play a crucial role in maximizing the yield, quality, and stability of plasmids.

[0060] In a more preferred embodiment, the medium according to the invention comprises the following components, wherein:

[0061] - Glycerol is in an amount of 30 g / L,

[0062] - (NH4)2SO4is in an amount of 10 g / L,

[0063] - MgSO4 is in an amount of 2 mM,

[0064] - KH2PO4 is in an amount of 6 g / L,

[0065] - K2HPO4 is in an amount of 14 g / L, and P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0066] - NaCI is in an amount of 5 g / L.

[0067] The vitamin mixture that was designed for the chemically defined medium of the invention supports bacterial metabolism and ensures the optimal growth of the culture.

[0068] These components are particularly important to provide just enough nutrients for growth, minimizing excess nutrients that could interfere with plasmid production. Still more preferred is a medium, wherein the vitamin mixture consists of: Thiamine, Pyridoxine hydrochloride and Nicotinic acid, each in the range from 1 to 5 mg / L, preferably 2 mg / L.

[0069] Accurately quantified trace elements are added to further reduce oxidative stress, to improve plasmid yield and stability of both the bacterial cells and the plasmid. In a more preferred embodiment, the medium according to the invention comprises the following trace element mixture wherein:

[0070] - NaCI is in the range from 0.01 to 0.1 g / L,

[0071] - ZnSO4*7 H2O is in the range from 0.001 to 0.01 g / L,

[0072] - CUSO4*5H2O is in the range from 0.0010 to 0.01 g / L,

[0073] - MnCl2*4 H2O is in the range from 0.010 to 0.1 g / L,

[0074] - FeCl3*6H2O is in the range from 0.010 to 0.1 g / L,

[0075] - CaCl2*2 H2O is in the range from 0.001 to 0.01 g / L,

[0076] - Na2MoO4*2 H2O is in the range from 0.0005 to 0.01 g / L, and

[0077] - H2SO497% is in the range from 0.01 to 0.1 mL / L.

[0078] More preferred is a medium wherein in the trace element mixture:

[0079] - NaCI is in an amount of 0.02 g / L,

[0080] - ZnSO4*7 H2O is in an amount of 0.004 g / L,

[0081] - CUSO4*5H2O is in an amount of 0.0016 g / L,

[0082] - MnCh*4 H2O is in an amount of 0.016 g / L,

[0083] - FeCl3*6H2O is in an amount of 0.019 g / L,

[0084] - CaCl2*2 H2O is in an amount of 0.006 g / L,

[0085] - Na2MoO4*2 H2O is in an amount of 0.002 g / L, and

[0086] - H2SO497% is in an amount of 0.0792 mL / L. P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0087] A still more preferred medium consists of:

[0088] - Glycerol in the range from 25 to 35 g / L,

[0089] - (NH4)2SO4 in the range from 5 to 15 g / L,

[0090] - MgSO4 in the range from 1 to 5 mM,

[0091] - KH2PO4 in the range from 2 to 10 g / L,

[0092] - K2HPO4 in the range from 10 to 20 g / L,

[0093] - NaCI in the range from 2 to 10 g / L,

[0094] - a vitamin mixture consisting of: Thiamine, Pyridoxine hydrochloride and Nicotinic acid, each in the range from 1 to 5 mg / L, and

[0095] - a trace element mixture consisting of:

[0096] - NaCI in the range from 0.01 to 0.1 g / L,

[0097] - ZnSO4*7 H2O in the range from 0.001 to 0.01 g / L,

[0098] - CUSO4*5H2O in the range from 0.0010 to 0.01 g / L,

[0099] - MnCl2*4 H2O in the range from 0.010 to 0.1 g / L,

[0100] - FeCl3*6H2O in the range from 0.010 to 0.1 g / L,

[0101] - CaCl2*2 H2O in the range from 0.001 to 0.01 g / L,

[0102] - Na2MoO4*2 H2O in the range from 0.0005 to 0.01 g / L, and

[0103] - H2SO497% in the range from 0.01 to 0.1 mL / L;

[0104] and

[0105] - water.

[0106] A further preferred medium consists of:

[0107] - Glycerol in an amount of 30 g / L,

[0108] - (NH4)2SO4in an amount of 10 g / L,

[0109] - MgSO4 in an amount of 2 mM,

[0110] - KH2PO4 in an amount of 6 g / L,

[0111] - K2HPO4 in an amount of 14 g / L,

[0112] - NaCI in an amount of 5 g / L,

[0113] - a vitamin mixture consisting of: Thiamine, Pyridoxine hydrochloride and Nicotinic acid, each in an amount of 2 mg / L, and

[0114] - a trace element mixture consisting of: P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0115] - NaCI in an amount of 0.02 g / L,

[0116] - ZnSO4*7 H2O in an amount of 0.004 g / L,

[0117] - CUSO4*5H2O in an amount of 0.0016 g / L,

[0118] - MnCl2*4 H2O in an amount of 0.016 g / L,

[0119] - FeCl3*6H2O in an amount of 0.019 g / L,

[0120] - CaCl2*2 H2O in an amount of 0.006 g / L,

[0121] - Na2MoO4*2 H2O in an amount of 0.002 g / L, and

[0122] - H2SO497% in an amount of 0.0792 mL / L;

[0123] and

[0124] - water.

[0125] Table 1 reports Chemical Defined (CD) medium final composition.

[0126] Ingredient Final Concentration Glycerol 30 g / L

[0127] (NH4)2SO410 g / L

[0128] MgSO42 mM

[0129] KH2PO46 g / L

[0130] K2HPO414 g / L

[0131] NaCI 5 g / L

[0132] Thiamine 2 mg / L

[0133] Pyridoxine hydrochloride 2 mg / L

[0134] Nicotinic acid 2 mg / L

[0135] NaCI 0.02 g / L

[0136] ZnSO4*7 H2O 0.004 g / L CUSO4*5H2O 0.0016 g / L

[0137] MnCI2*4 H2O 0.016 g / L

[0138]

[0139] P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0140] FeCI3*6H2O 0.019 g / L

[0141] CaCI2*2 H2O 0.006 g / L

[0142] Na2MoO4*2 H2O 0.002 g / L

[0143] H2SO497% 0.0792 mL / L

[0144]

[0145] Table 1. Chemical Defined (CD) medium final composition

[0146] Starting from the CD medium, a feed chemical defined medium composition was developed.

[0147] Table 2 reports Feed Chemical Defined medium final composition

[0148] Ingredient Final Concentration

[0149] Glycerol 600 g / L

[0150] (NH4)2SO45 g / L

[0151] MgSO4 50 mM

[0152]

[0153] Table 2. Feed Chemical Defined medium final composition

[0154] To avoid possible precipitation, phosphate buffer (KH2PO4 and K2HPO4) was added as last component. Once assembled, the CD medium was sterilized by 0,22 pm membrane filtration.

[0155] The CD medium can also be sterilized by autoclaving the whole medium apart from phosphate buffer and glycerol. Phosphate buffer and glycerol, as well as (NH4)2SO4, MgSO4, NaCI and vitamin mixture can be autoclaved separately in stock solution. It is preferable to avoid autoclaving trace elements in stock solution.

[0156] In a further aspect the use of the medium according to the invention for growing bacteria is described.

[0157] In the use according to the invention, the bacteria are E. Coli.

[0158] In a still further aspect, the invention relates to a method for plasmid production by bacterial fermentation, wherein the method comprises the step of using the medium herein disclosed.

[0159] In a preferred aspect of the method according to the invention the bacterial P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0160] fermentation produces a plasmid DNA having a volumetric plasmid yield of at least 45 mg / L and a specific plasmid yield of at least 0.95 mg / g, preferably the bacterial fermentation produces a plasmid DNA having a volumetric plasmid yield of at least 50 mg / L and a specific plasmid yield of at least 1 mg / g.

[0161] As used herein, the term “volumetric plasmid yield” expressed in mg / L refers to milligrams of plasmid obtained from a Liter of fermentation culture; and the term “specific plasmid yield”, expressed in mg / g, refers to milligrams of plasmid obtained from one gram of wet biomass.

[0162] EXPERIMENTAL PART

[0163] Reference is now made to the following experimental part, which together with the above descriptions illustrate some embodiments of the invention.

[0164] Results and discussion

[0165] In the first phases of the development of the CD medium, precipitation tests were performed in 50 mL tubes.

[0166] Various compositions of CD medium were tested varying the ingredients and their amounts in order to find the best CD medium (Table 1), to optimize the plasmid production process while maintaining high standards of plasmid quantity and quality. Once a stable composition was found (no visible precipitation), fermentation in flask (volume of 25 mL) were performed changing single components, with the goal to obtain a first selection. Candidate formulations were discarded in case of visible precipitation or no cell growth.

[0167] When a general composition was found, experiments were scaled-up with fermentations in higher volumes (100 mL). In this phase, experiments were characterized by upstream and downstream processes.

[0168] Upstream was based on a bacterial fermentation in Dasgip® parallel bioreactor system (Eppendorf®) with controlled pH, stirring, temperature and air influx. Downstream was based on alkaline lysis and FPLC purification with automated AKTA Pure system (GE Healthcare / Cytiva) and CIMmultus® DEAE 1 mL Monolithic Column (2 pm). P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0169] Finally, the eluted samples were submitted to restriction analyses and 1% agarose gel electrophoresis.

[0170] Parameters taken in consideration for the analysis were wet biomass (g / L), volumetric plasmid yield (mg / L), specific plasmid yield (mg / g).

[0171] At this point, a recipe for a CD medium was identified, obtaining surprisingly results as clearly shown in the figures.

[0172] In particular, the volumetric Plasmid Yields of the chemical defined medium of the invention (CD AMC) showed an increase by 26% and 25% compared to complex medium and commercial CD medium, respectively (Figure 1A) and the specific Plasmid Yields an increase by 36,4% and 18% compared to complex medium and commercial CD medium, respectively (Figure 1B).

[0173] Furthermore, in Figure 2 a comparison between control pmRVac4.2-EGFP, plasmid produced with the chemical defined medium of the invention (CD AMC) and the plasmid produced with the reference media (Complex medium and commercial CD Medium) surprisingly demonstrates that the chemical defined medium of the invention (CD AMC) allows to produce the three isoforms: open circular (OC), linearized (LIN) and the preferred supercoiled (SC) in a more abundant amount, avoiding the formation of the extra isoform, as occurs for the reference media (see the highlight by the black arrows in Figure 2).

[0174] Material and Methods

[0175] Fermentations were performed using NEB® 5-alpha Competent E. coli (High Efficiency), derivative of the DH5-Alpha K12 strain, T1 phage resistant (fhuA2) and endA deficient for high-quality plasmid preparation. Bacteria were transformed by thermic shock with pDNAs encoding Emerald Green Fluorescent Protein (pmEGFP, pmRVac4.2-EGFP).

[0176] Chemical Defined Medium: the chemical defined medium was obtained starting from concentrated stock solutions.

[0177] Stock solutions were prepared as follows in WFI (Water for Injection):

[0178] Glycerol: 100 g / L

[0179] (NH4)2SO4: 150 g / L P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0180] • MgSO4: 1 M

[0181] • NaCI: 100 g / L

[0182] • Phosphate buffer concentrated solution was prepared as follows in WFI: o KH2PO4: 60 g / L

[0183] o K2HPO4: 140 g / L

[0184] • Vitamin mixture concentrated solution was prepared as follows in WFI: o Thiamine: 5 g / L

[0185] o Pyridoxine hydrochloride: 5 g / L

[0186] o Nicotinic acid: 5 g / L

[0187] • Trace Elements concentrated solution was prepared as follows in WFI: o NaCI: 5 g / L

[0188] o ZnSO4* 7H2O: 1 g / L

[0189] o CuSO4* 5 H2O: 0,4 g / L

[0190] o MnCI2* 4 H2O: 4 g / L

[0191] o FeCI3* 6 H2O: 4,75 g / L

[0192] o CaCI2* 2 H2O: 1,5 g / L

[0193] o Na2MoO4* 2 H2O: 0,5 g / L

[0194] o H2SO497%: 19,8 mL / L

[0195] Cell transformation: E. coli DH5-Alpha cells were transformed with the plasmid pmEGFP. Plasmid was diluted with TE buffer or WFI (Water for Injection) and filtrated, reaching the final concentration of 0,2 pg / mL in a volume of 200 pL (the reaction was performed on 1 ng of plasmid).

[0196] Under laminar flow hood, 5 pL of plasmid were added to a sterile cryovial containing NEB® 5-alpha Competent E. coli (High Efficiency) and then incubated in ice for 30 minutes. The vial was transferred into a thermostatic bath at 42 °C for exactly 30 seconds, and then transferred back in ice. 250 pL S. O. C. (Super Optimal Broth with catabolite repression medium) without antibiotics were added to the cryovial under laminar hood and incubated in shaker (250 rpm, one hour, 37 °C).

[0197] Cell culture was plated at increasing volumes (20 pL, 50 pL, 100 pL) in 3 Petri dishes previously filled with 25 mL of LB - Agar broth and kanamycin 50 pg / mL. Dishes P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0198] were incubated overnight at 37 °C with the lid down.

[0199] 5 colonies were chosen from the 3 Petri dishes and inoculated into 5 different sterile tubes filled with 10 mL of LB + kanamycin 50 pg / mL. These pre-inoculums were incubated overnight at 30 °C.

[0200] Intermediates creation: 16-17 hours after incubation, OD600nm(optic density at 600 nm) of pre-inoculums were measured. Part of the cell culture was used for the creation of 5 intermediates - one for each colony - in cryovials and stored at -80 °C. These intermediates were composed by 50% freezing solution (50% LB + 50% glycerol) and 50% cell culture. Plasmid purification by QIAprep® Miniprep (Qiagen) was performed on the remanence of every pre-inoculum.

[0201] Colony selection: colony selection was based on both quantitative and qualitative data; the former by quantification with spectrophotometer, the latter by restriction analysis and 1% agarose gel.

[0202] Restriction analysis: enzymatic digestion was performed on 1 pg of pDNA, with reaction final volume of 50 pL. Vials were incubated for 1 hour at 37 °C and reactions were stopped placing vials in ice. In all cases, analysis was performed also on positive control (original pmEGFP). Samples were linearized with Not1 and double digested with Not1 + Nco1.

[0203] After restriction reactions, mixes for the electrophoretic run were prepared as described: pDNA, NEB® Gel Loading Purple Dye, WFL

[0204] Samples used corresponded to Ladder (Invitrogen™ High DNA Mass Ladder), nondigested samples, linearized samples, and double digested samples, with a final volume of 12 pL each. Electrophoresis on 1 % agarose gel was performed and bands were visualized with ChemiDoc System (Bio-Rad Laboratories, Inc.).

[0205] Master Cell Bank: a pre-inoculum was generated in a 50 mL tube (10 mL Chemical Defined Medium, kanamycin 50 pg / mL, 100 pL intermediate corresponding to the chosen colony) and was incubated at 30 °C and 250 rpm.

[0206] After 16 / 17 hours of incubation, OD600nmwas measured; this was necessary to generate a 100 mL culture in flask with starting OD600nmof 0.1.

[0207] Cell growth was monitored until the cell culture reached OD600nm= 0.8; cryovials P024299WO-01 Notarbartolo & Gervasi S.p. A.

[0208] were prepared with 500 pL sterile freezing solution (50% Chemical Defined Medium, 50% glycerol) and 500 pL cell culture. MCB cryovials were stored at -80 °C.

[0209] Fermentation Parameters: Bacterial fermentation was carried out in Dasgip® parallel bioreactor system (Eppendorf®), monitoring pH, Dissolved Oxygen and temperature using DASware® Control software.

[0210] 100 ml of the tested medium was inoculated for a starting OD600nmof 0.1, vessel temperature was set at 37°C, stirring and air influx was controlled programmatically in order to maintain a constant Dissolved oxygen of 30%. Growth culture was monitored offline, at the beginning of exponential growth phase a constant feed was applied.

[0211] The fermentation was stopped after 20h of feeding and the biomass was collected by centrifugation.

[0212] Fermentation process: all fermentations were performed on 3 working days. The chemical defined medium according to the invention was specifically designed for bacterial growth in a culture broth. On the first day, a pre-inoculum (Volume: 100 mL) in flask was incubated at 37 °C and 250 rpm overnight. On the second day, a new flask was inoculated with a starting OD600nmof 0,2. Bacterial growth was monitored until OD600nmreached the value of 0,8; cell culture was inoculated in Dasgip® parallel bioreactor system (Eppendorf®) with a starting OD600nmof 0,2. On the third day OD600nmwas measured and bacterial fermentation was stopped. Cell broth was collected and wet pellet was frozen at -20 °C.

[0213] From the above description and the above-noted examples, the advantage attained by the product described and obtained according to the present invention are apparent.

Claims

P024299WO-01 Notarbartolo & Gervasi S.p. A.CLAIMS1. A medium for plasmid production comprising:- Glycerol,- (NH4)2SO4,- MgSO4,- KH2PO4,- K2HPO4,- NaCI,- a vitamin mixture comprising: Thiamine, Pyridoxine hydrochloride and Nicotinic acid;- a trace element mixture comprising: NaCI, ZnSO4*7 H2O, CuSO4*5H2O, MnCI2*4 H2O, FeCI3*6H2O, CaCI2*2 H2O, Na2MoO4*2 H2O, H2SO497%; and- water.

2. The medium according to claim 1, wherein:- Glycerol is in the range from 25 to 35 g / L,- (NH4)2SO4is in the range from 5 to 15 g / L,- MgSO4is in the range from 1 to 5 mM,- KH2PO4is in the range from 2 to 10 g / L,- K2HPO4is in the range from 10 to 20 g / L, and- NaCI is in the range from 2 to 10 g / L.

3. The medium according to any one of claims 1 or 2, wherein:- Glycerol is in an amount of 30 g / L,- (NH4)2SO4is in an amount of 10 g / L,- MgSO4is in an amount of 2 mM,- KH2PO4is in an amount of 6 g / L,- K2HPO4is in an amount of 14 g / L, and- NaCI is in an amount of 5 g / L.P024299WO-01 Notarbartolo & Gervasi S.p. A.

4. The medium according to any one of claims 1 -3, wherein said vitamin mixture consists of: Thiamine, Pyridoxine hydrochloride and Nicotinic acid, each in the range from 1 to 5 mg / L, preferably 2 mg / L.

5. The medium according to any one of claims 1 -4, wherein in said trace element mixture:- NaCI is in the range from 0.01 to 0.1 g / L,- ZnSO4*7 H2O is in the range from 0.001 to 0.01 g / L,- CUSO4*5H2O is in the range from 0.0010 to 0.01 g / L,- MnCl2*4 H2O is in the range from 0.010 to 0.1 g / L,- FeCl3*6H2O is in the range from 0.010 to 0.1 g / L,- CaCl2*2 H2O is in the range from 0.001 to 0.01 g / L,- Na2MoO4*2 H2O is in the range from 0.0005 to 0.01 g / L, and- H2SO497% is in the range from 0.01 to 0.1 mL / L.

6. The medium according to any one of claims 1 -5, wherein in said trace element mixture:- NaCI is in an amount of 0.02 g / L,- ZnSO4*7 H2O is in an amount of 0.004 g / L,- CUSO4*5H2O is in an amount of 0.0016 g / L,- MnCl2*4 H2O is in an amount of 0.016 g / L,- FeCl3*6H2O is in an amount of 0.019 g / L,- CaCl2*2 H2O is in an amount of 0.006 g / L,- Na2MoO4*2 H2O is in an amount of 0.002 g / L, and- H2SO497% is in an amount of 0.0792 mL / L.

7. The medium according to any one of claims 1-6, wherein said medium consists of:- Glycerol in the range from 25 to 35 g / L,- (NH4)2SO4 in the range from 5 to 15 g / L,P024299WO-01 Notarbartolo & Gervasi S.p. A.- MgSO4 in the range from 1 to 5 mM,- KH2PO4 in the range from 2 to 10 g / L,- K2HPO4 in the range from 10 to 20 g / L,- NaCI in the range from 2 to 10 g / L,- a vitamin mixture consisting of: Thiamine, Pyridoxine hydrochloride and Nicotinic acid, each in the range from 1 to 5 mg / L, and- a trace element mixture consisting of:- NaCI in the range from 0.01 to 0.1 g / L,- ZnSO4*7 H2O in the range from 0.001 to 0.01 g / L,- CUSO4*5H2O in the range from 0.0010 to 0.01 g / L,- MnCl2*4 H2O in the range from 0.010 to 0.1 g / L,- FeCl3*6H2O in the range from 0.010 to 0.1 g / L,- CaCl2*2 H2O in the range from 0.001 to 0.01 g / L,- Na2MoO4*2 H2O in the range from 0.0005 to 0.01 g / L, and- H2SO497% in the range from 0.01 to 0.1 mL / L;and- water.

8. The medium according to any one of claims 1-7, wherein said medium consists of:- Glycerol in an amount of 30 g / L,- (NH4)2SO4in an amount of 10 g / L,- MgSO4 in an amount of 2 mM,- KH2PO4 in an amount of 6 g / L,- K2HPO4 in an amount of 14 g / L,- NaCI in an amount of 5 g / L,- a vitamin mixture consisting of: Thiamine, Pyridoxine hydrochloride and Nicotinic acid, each in an amount of 2 mg / L, and- a trace element mixture consisting of:- NaCI in an amount of 0.02 g / L,P024299WO-01 Notarbartolo & Gervasi S.p. A.- ZnSO4*7 H2O in an amount of 0.004 g / L,- CUSO4*5H2O in an amount of 0.0016 g / L,- MnCl2*4 H2O in an amount of 0.016 g / L,- FeCl3*6H2O in an amount of 0.019 g / L,- CaCl2*2 H2O in an amount of 0.006 g / L,- Na2MoO4*2 H2O in an amount of 0.002 g / L, and- H2SO497% in an amount of 0.0792 mL / L;and- water.

9. Use of the medium according to any one of claims 1 -8 for growing bacteria.

10. The use according to claim 9, wherein said bacteria are E. Coli.

11. A method for plasmid production by bacterial fermentation, said method comprising the step of using the medium according to any one of claims 1 -8.

12. The method according to claim 11, wherein said bacterial fermentation produces a plasmid DNA having a volumetric plasmid yield of at least 45 mg / L and a specific plasmid yield of at least 0.95 mg / g.

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