Culture medium for plasmid and recombinant protein production

Abstract

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

Description

[0001] P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0002] “CULTURE MEDIUM FOR PLASMID AND RECOMBINANT PROTEIN

[0003] PRODUCTION”

[0004] FIELD OF THE INVENTION

[0005] The present invention pertains to the field of culture media designed for plasmid and recombinant protein production. Specifically, it relates to a specialized culture medium for bacterial growth that is optimized to achieve high plasmid and recombinant protein yields.

[0006] STATE OF THE ART

[0007] Plasmid and recombinant protein production are key processes in biotechnology, especially for applications such as gene therapy, vaccine development, targeted drugs and diagnostics

[0008] The culture medium plays a vital role in optimizing plasmid or recombinant protein yield and quality. Traditionally, plasmid or recombinant protein 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.

[0009] 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 and recombinant protein 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 or recombinant protein yield and quality, hindering process optimization.

[0010] 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 P024299WO-02 Notarbartolo & Gervasi S.p.A. 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 or recombinant protein 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) or recombinant protein as complex media. This is because the precise nutrient balance required for optimal bacterial growth and plasmid replication or protein synthesis is not always easy to achieve in a chemically defined medium. As a result, the efficiency of plasmid or recombinant protein production may be suboptimal, particularly in large-scale fermentations.

[0011] Another challenge with CD media is the optimization of carbon and nitrogen sources to support both bacterial growth and plasmid replication or protein synthesis. 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.

[0012] Recent research has focused on optimizing CD media for plasmid or recombinant protein production by carefully adjusting the composition to improve both plasmid or recombinant protein yield and quality. Efforts have been made to identify key factors that enhance plasmid replication and minimize plasmid or recombinant protein 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 or recombinant protein 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.

[0013] Despite these advancements, achieving a balance between high plasmid or recombinant protein yield, quality, and cost-effectiveness remains a major challenge in plasmid or recombinant protein production. The development of a reliable, high- performance chemically defined medium that can match or surpass the results P024299WO-02 Notarbartolo & Gervasi S.p.A. obtained with complex media is still an ongoing area of research. This includes exploring alternative strategies for optimizing bacterial growth, plasmid replication, protein synthesis and overall fermentation efficiency.

[0014] Ultimately, the goal is to create a medium that not only supports efficient plasmid and recombinant protein production but also meets the regulatory and scalability requirements necessary for industrial-scale applications.

[0015] SUMMARY OF THE INVENTION

[0016] The object of the present invention is to develop a new chemically defined (CD or CD AMC) medium for plasmid and / or recombinant protein production through bacterial fermentation, ensuring that the plasmid and recombinant protein 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 and recombinant protein 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 (Commercial CD medium). These comparisons allowed for a thorough assessment of the new medium's performance in plasmid and recombinant protein production.

[0017] The present invention discloses a medium for plasmid and / or recombinant protein production comprising:

[0018] - Glycerol,

[0019] - (NH4)2SO4,

[0020] - MgSO4,

[0021] - KH2PO4,

[0022] - K2HPO4,

[0023] - NaCI,

[0024] - a vitamin mixture comprising: Thiamine, Pyridoxine hydrochloride and Nicotinic acid;

[0025] - a trace element mixture comprising: NaCI, ZnSO4*7 H2O, CuSO4*5H2O, MnCI2*4 P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0026] H2O, FeCl3*6H2O, CaCl2*2 H2O, Na2MoO4*2 H2O, H2SO4 97%; and

[0027] - water.

[0028] Water for injection (WFI) or sterile water can be used for the dilution of all the components of the medium.

[0029] In a further aspect the use of the medium according to the invention for growing bacteria is described.

[0030] In the medium of the present invention, the following components can be defined as the fermentation medium:

[0031] - Glycerol,

[0032] - (NH4)2SO4,

[0033] - MgSO4,

[0034] - KH2PO4,

[0035] - K2HPO4, and

[0036] - NaCI.

[0037] 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.

[0038] In a still further aspect, the invention relates to a method for plasmid and recombinant protein production by bacterial fermentation, wherein the method comprises the step of using the medium herein disclosed.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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 -6.

[0041] Figure 1 : comparison of Plasmid Yields. A) Volumetric yield; B) Specific yield.

[0042] 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, P024299WO-02 Notarbartolo & Gervasi S.p.A. an extra isoform is highlighted by the black arrows.

[0043] Figure 3: comparison Protein Yield of the DARPin protein. A) Volumetric yield of wet biomass (g / L): the bar graph shows the comparison of the total wet biomass of fermentation (scaled to 1 L) between the CD AMC medium and the Commercial CD medium. The reference medium resulted in a slightly higher biomass yield in comparison to CD AMC medium. B) Specific recombinant protein yield (mg / g): the bar graph illustrates the specific recombinant protein yield, expressed in milligrams per gram of biomass (mg / g), comparing the CD AMC medium and the Commercial CD medium. The CD AMC medium outperforms the Commercial CD Medium resulting in a 2-fold higher recombinant protein production. In detail the average protein specific yield resulted in 19,8 mg / g for CD AMC medium and 8,0 mg / g for Commercial CD Medium. C) Volumetric recombinant protein yield (mg / L): the bar graph displays the volumetric recombinant protein yield, measured in milligrams per liter (mg / L), comparing the CD AMC medium and the Commercial CD medium. The CD AMC medium outperforms the Commercial CD Medium resulting in a 2-fold higher volumetric yield (748 mg / L for CD AMC medium and 329 mg / L for the reference).

[0044] Figure 4: comparison Protein Yield of the EGFP protein. A) Volumetric yield of wet biomass (g / L): the bar graph shows the comparison of the total wet biomass of fermentation (scaled to 1 L) between the CD AMC medium and the Commercial CD medium. The reference medium resulted in a slightly higher biomass yield in comparison to CD AMC medium. B) Specific recombinant protein yield (mg / g): the bar graph illustrates the specific recombinant protein yield, expressed in milligrams per gram of biomass (mg / g), comparing the CD AMC medium and the Commercial CD medium. The CD AMC medium achieved a mean yield of approximately 2.5 mg / g, slightly superior of the Commercial CD Medium, which showed a mean yield of roughly 1.9 mg / g. C) Volumetric recombinant protein yield (mg / L): the bar graph displays the volumetric recombinant protein yield, measured in milligrams per liter (mg / L), comparing the CD AMC medium and the Commercial CD medium. The data indicates that the CD AMC medium resulted in a slightly higher volumetric yield of P024299WO-02 Notarbartolo & Gervasi S.p.A. the recombinant protein compared to the commercial medium.

[0045] Figure 5: SDS-PAGE of expression and purification samples of DARPin protein resulted from fermentation in Commercial CD Medium (A) and CD AMC (B). 1. Ladder, 2. Not-induced Cells, 3. Induced Cells, 4. Cell Debris after Lysis, 5. Supernatant after cell lysis and clarification. 6. Flow through of affinity purification, 7. Normalized purified recombinant protein, 8. Purified protein (20 pg), 9. Purified Protein (50 pg), 10. Purified Protein (100 pg).

[0046] Figure 6: SDS-PAGE of Expression and purification samples of EGFP protein resulted from fermentation in Commercial CD Medium (A) and CD AMC (B). 1. Ladder, 2. Not-induced Cells, 3. induced Cells, 4. Cell debris after lysis, 5. Supernatant after cell lysis and clarification. 6. Flow through of affinity purification, 7. Normalized purified recombinant protein, 8. Purified protein (20 pg), 9. Purified Protein (50 pg), 10. Purified Protein (100 pg). The recombinant protein in the eluted samples is highlighted with a square.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] Plasmid and recombinant protein production are key processes in biotechnology, especially for applications such as gene therapy, vaccine development, targeted drugs and diagnostics. The present invention pertains to a culture medium, that has been formulated in order to maximize both the yield and quality of plasmids and recombinant proteins.

[0049] A recombinant protein is a protein produced in a lab by genetically engineering cells (like bacteria or yeast) to express a specific gene, encoding a protein. The protein can be identical or similar to a natural one, but in large, pure quantities for research, diagnostics and therapeutic uses.

[0050] The present invention thus concerns a medium for plasmid and / or recombinant protein production comprising:

[0051] - Glycerol,

[0052] - (NH4)2SO4,

[0053] - MgSO4, P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0054] - KH2PO4,

[0055] - K2HPO4,

[0056] - NaCI,

[0057] - a vitamin mixture comprising: Thiamine, Pyridoxine hydrochloride and Nicotinic acid;

[0058] - a trace element mixture comprising: NaCI, ZnSO4*7 H2O, CuSO4*5H2O, MnCL*4 H2O, FeCl3*6H2O, CaCI2*2 H2O, Na2MoO4*2 H2O, H2SO4 97%; and

[0059] - water.

[0060] 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 protein synthesis.

[0061] In a preferred embodiment of the invention the medium comprises:

[0062] - Glycerol in the range from 25 to 35 g / L,

[0063] - (NH4)2SO4 in the range from 5 to 15 g / L,

[0064] - MgSO4 in the range from 1 to 5 mM,

[0065] - KH2PO4 in the range from 2 to 10 g / L,

[0066] - K2HPO4 in the range from 10 to 20 g / L, and

[0067] - NaCI in the range from 2 to 10 g / L.

[0068] The medium composition of the present invention was carefully designed to provide an optimal environment for bacterial growth and plasmid and / or recombinant protein production. The specific components of a culture medium play a crucial role in maximizing the yield, quality, and stability of plasmids and recombinant proteins.

[0069] In a more preferred embodiment, the medium according to the invention comprises the following components, wherein:

[0070] - Glycerol is in an amount of 30 g / L,

[0071] - (NH4)2SO4 is in an amount of 10 g / L,

[0072] - MgSO4 is in an amount of 2 mM,

[0073] - KH2PO4 is in an amount of 6 g / L,

[0074] - K2HPO4 is in an amount of 14 g / L, and P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0075] - NaCI is in an amount of 5 g / L.

[0076] 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.

[0077] These components are particularly important to provide just enough nutrients for growth, minimizing excess nutrients that could interfere with plasmid production and / or recombinant protein synthesis.

[0078] 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.

[0079] Accurately quantified trace elements are added to further reduce oxidative stress, to improve both plasmid and recombinant protein yield and stability of the bacterial cells.

[0080] In a more preferred embodiment, the medium according to the invention comprises the following trace element mixture wherein:

[0081] - NaCI is in the range from 0.01 to 0.1 g / L,

[0082] - ZnSO4*7 H2O is in the range from 0.001 to 0.01 g / L,

[0083] - CUSO4*5H2O is in the range from 0.0010 to 0.01 g / L,

[0084] - MnCl2*4 H2O is in the range from 0.010 to 0.1 g / L,

[0085] - FeCl3*6H2O is in the range from 0.010 to 0.1 g / L,

[0086] - CaCl2*2 H2O is in the range from 0.001 to 0.01 g / L,

[0087] - Na2MoO4*2 H2O is in the range from 0.0005 to 0.01 g / L, and

[0088] - H2SO4 97% is in the range from 0.01 to 0.1 mL / L.

[0089] More preferred is a medium wherein in the trace element mixture:

[0090] - NaCI is in an amount of 0.02 g / L,

[0091] - ZnSO4*7 H2O is in an amount of 0.004 g / L,

[0092] - CUSO4*5H2O is in an amount of 0.0016 g / L,

[0093] - MnCl2*4 H2O is in an amount of 0.016 g / L,

[0094] - FeCl3*6H2O is in an amount of 0.019 g / L,

[0095] - CaCl2*2 H2O is in an amount of 0.006 g / L, P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0096] - Na2MoO4*2 H2O is in an amount of 0.002 g / L, and

[0097] - H2SO4 97% is in an amount of 0.0792 mL / L.

[0098] A still more preferred medium consists of:

[0099] - Glycerol in the range from 25 to 35 g / L,

[0100] - (NH4)2SO4 in the range from 5 to 15 g / L,

[0101] - MgSO4 in the range from 1 to 5 mM,

[0102] - KH2PO4 in the range from 2 to 10 g / L,

[0103] - K2HPO4 in the range from 10 to 20 g / L,

[0104] - NaCI in the range from 2 to 10 g / L,

[0105] - a vitamin mixture consisting of: Thiamine, Pyridoxine hydrochloride and Nicotinic acid, each in the range from 1 to 5 mg / L, and

[0106] - a trace element mixture consisting of:

[0107] - NaCI in the range from 0.01 to 0.1 g / L,

[0108] - ZnSO4*7 H2O in the range from 0.001 to 0.01 g / L,

[0109] - CUSO4*5H2O in the range from 0.0010 to 0.01 g / L,

[0110] - MnCL*4 H2O in the range from 0.010 to 0.1 g / L,

[0111] - FeCl3*6H2O in the range from 0.010 to 0.1 g / L,

[0112] - CaCl2*2 H2O in the range from 0.001 to 0.01 g / L,

[0113] - Na2MoO4*2 H2O in the range from 0.0005 to 0.01 g / L, and

[0114] - H2SO4 97% in the range from 0.01 to 0.1 mL / L; and

[0115] - water.

[0116] A further preferred medium consists of:

[0117] - Glycerol in an amount of 30 g / L,

[0118] - (NH4)2SO4 in an amount of 10 g / L,

[0119] - MgSO4 in an amount of 2 mM,

[0120] - KH2PO4 in an amount of 6 g / L,

[0121] - K2HPO4 in an amount of 14 g / L,

[0122] - NaCI in an amount of 5 g / L,

[0123] - a vitamin mixture consisting of: Thiamine, Pyridoxine hydrochloride and Nicotinic P024299WO-02 Notarbartolo & Gervasi S.p.A. acid, each in an amount of 2 mg / L, and

[0124] - a trace element mixture consisting of:

[0125] - NaCI in an amount of 0.02 g / L,

[0126] - ZnSO4*7 H2O in an amount of 0.004 g / L, - CUSO4*5H2O in an amount of 0.0016 g / L,

[0127] - MnCl2*4 H2O in an amount of 0.016 g / L,

[0128] - FeCl3*6H2O in an amount of 0.019 g / L,

[0129] - CaCl2*2 H2O in an amount of 0.006 g / L,

[0130] - Na2MoO4*2 H2O in an amount of 0.002 g / L, and - H2SO4 97% in an amount of 0.0792 mL / L; and

[0131] - water.

[0132] Table 1 reports Chemical Defined (CD) medium final composition. P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0133] Table 1. Chemical Defined (CD) medium final composition

[0134] Starting from the CD medium, a feed chemical defined medium composition was developed.

[0135] Table 2 reports Feed Chemical Defined medium final composition Table 2. Feed Chemical Defined medium final composition

[0136] 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.

[0137] 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.

[0138] In a further aspect the use of the medium according to the invention for growing bacteria is described. In the use according to the invention, the bacteria are E. Coli.

[0139] In a still further aspect, the invention relates to a method for plasmid and / or P024299WO-02 Notarbartolo & Gervasi S.p.A. recombinant protein production by bacterial fermentation, wherein the method comprises the step of using the medium herein disclosed.

[0140] In a preferred aspect of the plasmid production method according to the invention the 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, 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.

[0141] In a preferred aspect of the recombinant protein production method according to the invention the bacterial fermentation produces a recombinant protein having a volumetric plasmid yield of at least 90 mg / L and a specific protein yield of at least 2.50 mg / g, preferably the bacterial fermentation produces a recombinant protein having a volumetric protein yield of at least 500 mg / L and a specific protein yield of at least 15 mg / g, more preferably a volumetric protein yield of at least 700 mg / L and a specific protein yield of at least 18 mg / g.

[0142] As used herein, the term “volumetric plasmid yield” expressed in mg / L refers to milligrams of plasmid obtained from a Liter of fermentation culture; the term “volumetric protein yield” expressed in mg / L refers to milligram of recombinant protein 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; the term “specific protein yield”, expressed in mg / g, refers to milligrams of recombinant protein obtained from 1 g of wet biomass.

[0143] EXPERIMENTAL PART

[0144] Reference is now made to the following experimental part, which together with the above descriptions illustrate some embodiments of the invention.

[0145] Results and discussion

[0146] In the first phases of the development of the CD medium, precipitation tests were performed in 50 mL tubes.

[0147] 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 P024299WO-02 Notarbartolo & Gervasi S.p.A. 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.

[0148] 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.

[0149] 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).

[0150] Finally, the eluted samples were submitted to restriction analyses and 1 % agarose gel electrophoresis.

[0151] Parameters taken in consideration for the analysis were wet biomass (g / L), volumetric plasmid yield (mg / L), specific plasmid yield (mg / g).

[0152] At this point, a recipe for a CD medium was identified, obtaining surprisingly results as clearly shown in the figures.

[0153] 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 1 B).

[0154] 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, P024299WO-02 Notarbartolo & Gervasi S.p.A. avoiding the formation of the extra isoform, as occurs for the reference media (see the highlight by the black arrows in Figure 2).

[0155] It was then verified whether the chemically defined (CD) medium of the invention was efficient for recombinant protein production via bacterial fermentation. The requirement for the medium formulation was to guarantee a recombinant protein production comparable to a reference medium in both quality and quantity.The results of all experiments were compared with the commercial chemically defined medium used as the reference culture medium.

[0156] Experiments were performed by fermentation and recombinant protein induction with IPTG (Isopropyl [3-D-1 -thiogalactopyranoside). In this phase, experiments were characterized by upstream and downstream processes.

[0157] Upstream was based on a bacterial fermentation (100 mL) carried out in Dasgip® parallel bioreactor system (Eppendorf®) with controlled pH, stirring, temperature and air influx. The Downstream process involved a mechanical disruption of bacterial cell by sonication (high-frequency sound waves to create shock waves that break the membrane cells) followed by FPLC purification with automated AKTA Pure system (Cytiva) and HisTrap™ HP (Cytiva) His tag protein purification columns (1 mL). Finally, the purified proteins were analysed by SDS-Page and quantified spectrophotometrically. The parameters considered for yield evaluation were: wet biomass (g / L), volumetric recombinant protein yield (mg / L) and specific recombinant protein yield (mg / g).

[0158] Evaluation of the CD AMC minimal medium reveals performance characteristics that match or exceed those of the commercial standard.

[0159] The results obtained using CD AMC medium in EGFP protein production (Figure 4) reveal similar or slightly better performance than the commercial reference, while in DARPin protein manufactuting (Figure 3) CD AMC medium significantly enhanced specific recombinant protein yields, which achieved a two-fold improvement over the reference medium.

[0160] Moreover, the purified DARPin and EGFP proteins, obtained using CD AMC, showed less impurity, respect to the protein purified obtained using commercial P024299WO-02 Notarbartolo & Gervasi S.p.A. media (lane 10 Figure 5a versus lane 10 Figure 5b and lane 10 Figure 6a versus lane 10 Figure 6b).

[0161] Even if biomass accumulation was marginally lower using CD AMC medium (Figure 3A and 4A), the concomitant increase in specific protein yield (Figure 3B and 4B) suggests a favorable reallocation of metabolic flux. This indicates that the medium composition promotes high-level of heterologous protein production instead of improving microbial cells growth.

[0162] Material and Methods

[0163] Fermentations for plasmid production 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 Enhanced Green Fluorescent Protein (pmEGFP, pmRVac4.2-EGFP).

[0164] Fermentations for protein expression were carried out using One Shot™ BL21 Star™ (DE3) (Invitrogen TM) Chemically Competent E. coli, a derivative of the E. coli B strain that does not contain the Ion protease and is also deficient in the outer membrane protease OmpT. One Shot™ BL21 Star™ (DE3) (Invitrogen TM) has been engineered to carry the ADE3 lysogen that contains the T7 RNA polymerase gene under the control of the lacllV5 promoter. IPTG is required to induce expression of the T7 RNA polymerase cascade system that in turn transcribes the T7-promoter-regulated target gene. One Shot™ BL21 Star™ (DE3) (Invitrogen ™) were transformed by thermic shock with two different pDNAs:

[0165] - (pET-6xHis / EGFP) encoding Enhanced Green Fluorescent protein;

[0166] - (pET-6xHis / { DARpin E_01 }) encoding DARpin protein.

[0167] Chemical Defined Medium: the chemical defined medium was obtained starting from concentrated stock solutions.

[0168] Stock solutions were prepared as follows in WFI (Water for Injection):

[0169] • Glycerol: 100 g / L

[0170] • (NH4)2SO4: 150 g / L

[0171] • MgSO4: 1 M P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0172] • NaCI: 100 g / L

[0173] • Phosphate buffer concentrated solution was prepared as follows in WFI: o KH2PO4: 60 g / L o K2HPO4: 140 g / L

[0174] • Vitamin mixture concentrated solution was prepared as follows in WFI: o Thiamine: 5 g / L o Pyridoxine hydrochloride: 5 g / L o Nicotinic acid: 5 g / L

[0175] • Trace Elements concentrated solution was prepared as follows in WFI: o NaCI: 5 g / L o ZnSO4* 7H2O: 1 g / L o CuSO4* 5 H2O: 0,4 g / L o MnCI2* 4 H2O: 4 g / L o FeCI3* 6 H2O: 4,75 g / L o CaCh * 2 H2O: 1 ,5 g / L o Na2MoO4* 2 H2O: 0,5 g / L o H2SO4 97%: 19,8 mL / L

[0176] Cell transformation: NEB® 5-alpha Competent E. coli (High Efficiency) or One Shot™ BL21 Star™ (DE3) (Invitrogen ™) cells were transformed with the plasmid (pmEGFP, pmRVac4.2-EGFP or pET-6xHis / EGFP or pET-6xHis / { DARpin E_01 }). 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).

[0177] Under laminar flow hood, 5 pL of plasmid were added to a sterile cryovial containing NEB® 5-alpha Competent E. coli (High Efficiency) or One Shot™ BL21 Star™ (DE3) (Invitrogen™) 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). P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0178] 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 or Ampicillin 100 pg / mL. Dishes were incubated overnight at 37 °C with the lid down.

[0179] 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 or Ampicillin 100 pg / mL. These pre-inoculums were incubated overnight at 30 °C.

[0180] Intermediates creation: 16-17 hours after incubation, ODeoonm (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.

[0181] Colony selection for plasmid production: 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.

[0182] Restriction analysis for plasmid production: 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.

[0183] After restriction reactions, mixes for the electrophoretic run were prepared as described: pDNA, NEB® Gel Loading Purple Dye, WFI.

[0184] 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.).

[0185] Master Cell Bank: a pre-inoculum was generated in a 50 mL tube (10 or 5 mL Chemical Defined Medium, kanamycin 50 pg / mL or Ampicillin 100 pg / mL and 100 pL intermediate corresponding to the chosen colony) and was incubated at 30 °C and 250 rpm. P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0186] After 16 / 17 hours of incubation, ODeoonm was measured; this was necessary to generate a 100 mL culture in flask with starting ODeoonm of 0.1.

[0187] Cell growth was monitored until the cell culture reached ODeoonm = 0.8; cryovials 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.

[0188] Fermentation Parameters: Bacterial fermentation was carried out in Dasgip® parallel bioreactor system (Eppendorf®), monitoring pH, Dissolved Oxygen and temperature using DASware® Control software.

[0189] 100 ml of the tested medium was inoculated for a starting ODeoonm of 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.

[0190] The fermentation was stopped after 20h of feeding and the biomass was collected by centrifugation.

[0191] Fermentation process: For plasmid production 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 preinoculum (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 ODeoonm of 0,2. Bacterial growth was monitored until ODeoonm reached the value of 0,8; cell culture was inoculated in Dasgip® parallel bioreactor system (Eppendorf®) with a starting ODeoonm of 0,2, stirring and air influx was controlled programmatically to maintain a constant dissolved oxygen of 30%.

[0192] On the third day ODeoonm was measured and bacterial fermentation was stopped. Cell broth was collected and wet pellet was frozen at -20 °C.

[0193] 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. P024299WO-02 Notarbartolo & Gervasi S.p.A.

[0194] For protein production all fermentation were performed on 4 working days. 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 100 mL of the tested medium was inoculated to a starting OD600nm of 0,1 , vessel temperature was set at 30°C, stirring and air influx was controlled programmatically to maintain a constant dissolved oxygen of 30%.

[0195] On the third day, at the end of the batch phase of the fermentation (approximately 17h after inoculation) an IPTG solution was added to the culture medium to reach a final concentration of 1 mM to induce protein expression. Simultaneously with induction, a constant feed was applied and the vessel temperature was set at 25°C. The fermentation was halted on the fourth day no later than 20 h after the start of feeding. Cell broth was collected and wet pellet was frozen at -20 °C.

[0196] Cell Lysis: Bacterial cells were lysed via sonication. Bacterial pellet was resuspended in 20mL of the same buffer that was used for column equilibration in next step of purification (150 mM Sodium Chloride, 50mM Trizma Base, 30 mM Imidazole)

[0197] 30 pL of Halt™ Protease Inhibitor single-use Cocktail (100X) (Thermofisher Scientific) was added to reduce the degradation of the recombinant protein by proteases.

[0198] The solution was exposed to high-frequency sound waves for 8 cycles of 30 seconds with a 2 minutes break between each cycle. The samples were kept on ice to minimize protein heat exposure. Post-sonication samples were clarified by centrifugation at 12000 ref for 30 minutes.

[0199] Purification step: Protein purification was performed by using FPLC with automated AKTA Purifier system (Cytiva) and HisTrap™ HP (Cytiva) His tag protein purification columns 1 mL. Column equilibration was performed by using 150 mM Sodium Chloride, 50mM Trizma Base, 30 mM Imidazole (equilibration buffer). After loading the sample, the column was washed with a wash buffer to reduce the nonspecific binding of contaminant proteins. P024299WQ-02 Notarbartolo & Gervasi S.p.A.

[0200] The target protein was eluted using a gradient of the equilibration buffer versus the elution buffer (150 mM Sodium Chloride, 50mM Trizma Base 500 mM Imidazole). The increasing concentration of Imidazole causes the target protein (containing the His tag) to dissociate from the column resin.

[0201] Qualitative analyses (SDS-Page): The quality of the target protein was evaluated by SDS-Page in denaturation condition, using a Mini-PROTEAN® TGX Stain- Free™ precast Gel (Bio-Rad Laboratories, Inc). The samples were prepared by using a protein sample buffer for SDS-PAGE (4x Laemmli Sample Buffer Bio-Rad Laboratories, Inc.) and heated at 90°C for 5 minutes prior to loading. Precision Plus Protein™ Dual Color (Bio-Rad Laboratories, Inc.) was used as a molecular weight ladder. The protein bands in the polyacrylamide gel were visualized using a Chemidoc System (Bio-Rad Laboratories, Inc.).

[0202] Protein quantification: Quantification of target protein was carried out by spectrophotometric analysis using a Nanophotometer (Implen GmbH).

[0203] The Molar Extinction Coefficient (E) was theoretically determined for each target protein sequence using the ProtParam online bioinformatics tool.

[0204] Quantification was performed by measuring absorbance at 280 nm and applying the theoretically determined s for each protein.

Claims

P024299WO-02 Notarbartolo & Gervasi S.p.A.CLAIMS1. A medium for plasmid and recombinant protein 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, MnCL*4 H2O, FeCl3*6H2O, CaCI2*2 H2O, Na2MoO4*2 H2O, H2SO4 97%; and- water.

2. The medium according to claim 1 , wherein:- Glycerol is in the range from 25 to 35 g / L,- (NH4)2SO4 is in the range from 5 to 15 g / L,- MgSO4 is in the range from 1 to 5 mM,- KH2PO4 is in the range from 2 to 10 g / L,- K2HPO4 is 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)2SO4 is in an amount of 10 g / L,- MgSO4 is in an amount of 2 mM,- KH2PO4 is in an amount of 6 g / L,- K2HPO4 is in an amount of 14 g / L, and- NaCI is in an amount of 5 g / L.P024299WO-02 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- H2SO4 97% 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- H2SO4 97% 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-02 Notarbartolo & Gervasi S.p.A.- MgSCM 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,- MnCL*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- H2SO4 97% 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)2SO4 in 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-02 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- H2SO4 97% 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 and recombinant protein 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.

13. The method according to claim 11 , wherein said bacterial fermentation produces a recombinant protein having a volumetric plasmid yield of at least 90 mg / L and a specific protein yield of at least 2.50 mg / g.

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