Large scale plasmid DNA production process

Abstract

The invention relates to a method for isolating a covalently closed circular (ccc) DNA molecule from a microbial cell comprising a ccc DNA molecule, comprising the steps of: contacting the microbial cell with a lysis agent and passing the composition through a tubing system with a flow having a Reynolds number of at least 3000 to obtain a lysed composition; incubating the lysed composition to obtain a lysate, contacting the lysate with a neutralization solution to obtain a neutralized lysate; and further processing the neutralized lysate to obtain the ccc DNA molecule.

Description

Technical Field

[0001] This invention relates to a method for large-scale isolation of cccDNA molecules from microbial cells containing covalently closed circular (ccc) DNA molecules, comprising the following steps: Microbial cells are contacted with a lysis agent, and the composition is passed through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; the lysis composition is incubated to obtain a lysate, the lysate is contacted with a neutralization solution to obtain a neutralized lysate; and the neutralized lysate is further processed to obtain ccc DNA molecules. Background Technology

[0002] ccc DNA molecules, particularly plasmids, are essential for the production of therapeutic molecules, especially antibodies or therapeutic nucleotide sequences. For example, large quantities of ccc DNA molecules are required to produce therapeutic antibodies. Methods for the production and purification of plasmid DNA are provided, for example, in WO 2004 / 060277, WO 01 / 79486, or WO 2005 / 026331. However, there is a need for improved methods for purifying plasmid DNA that achieve high purity and high yield of ccc DNA molecules. In particular, there is a need for improved large-scale plasmid DNA purification methods that achieve high purity and high yield of ccc DNA molecules.

[0003] Purpose and content of the invention

[0004] Therefore, there is a need for an improved large-scale ccc DNA production process, especially a large-scale production process capable of achieving high yields.

[0005] To meet this need, the present invention provides a method for isolating ccc DNA molecules from microbial cells containing covalently closed circular (ccc) DNA molecules, comprising the following steps: a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution; and d) Further process the neutralized lysis buffer to obtain ccc DNA molecules. Step b) is carried out in two parallel tubes.

[0006] The inventors have discovered that, in large-scale processing, using two parallel tubes for step b) is beneficial for achieving turbulence, a suitable Reynolds number (>2300), and a suitable pyrolysis time of 3 to 6 minutes.

[0007] A method for producing ccc DNA molecules in microbial cells has also been envisioned, comprising the following steps: (i) Fermentation of microbial cells containing ccc DNA molecules; (ii) Harvesting microbial cells; a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution; and d) Further process the neutralized lysate to obtain ccc DNA molecules.

[0008] The inventors have discovered that when microbial cells come into contact with a lysis agent, i.e., in a mixing tube prior to lysis step c), and the flow has a Reynolds number of at least 3000, the yield of ccc DNA molecules can be increased.

[0009] In particular, the inventors have discovered that applying a Reynolds number of at least 3000 in step a) can increase the yield of ccc DNA molecules.

[0010] Typically, the pyrolysis step b) lasts for 6 minutes or less, preferably 2 to 6 minutes, more preferably 3 to 6 minutes.

[0011] In one implementation, the total duration of steps a) and b) is 6 minutes or less, preferably 2 to 6 minutes, more preferably 3 to 6 minutes.

[0012] Step d) includes filtering the neutralized lysate through at least one filtration step.

[0013] In some embodiments, diatomaceous earth is preferably used to perform alluvial filtration in step d). Additionally, ultrafiltration / percolation can be performed, preferably using a hollow fiber filter. Other alkaline earth metal (e.g., CaCl2) salts can be added to the neutralized lysis buffer. After this precipitation, tangential flow filtration can be performed. Anion exchange (AEX) chromatography can be performed after tangential flow filtration. The sample can be further processed by adding potassium phosphate, such as dipotassium hydrogen phosphate or ammonium sulfate, to the neutralized lysis buffer. Furthermore, hydrophobic interaction chromatography or thiophilic interaction chromatography can be performed. Additional percolation steps can be performed. In one embodiment, step d) does not include AEX chromatography.

[0014] Typically, the diameter of the tubing system in step a) can range from 2 to 50 mm, preferably 4 to 40 mm. In step (b), the pyrolysis composition can be passed through a tubing system with a diameter ranging from 2 to 50 mm, preferably 5 to 30 mm. For fermentation scales of 500 L–1000 L, the diameter can be increased, preferably to 30 to 40 mm. In step c), the pyrolysis liquid is passed through a tubing system ranging from 2 to 60 mm, preferably 4 to 52 mm.

[0015] Typically, the flow rate in step a) ranges from 0.3 to 1.5 m / s, preferably from 0.5 to 1.5 m / s. In step b), the pyrolysis composition can flow through the pipe system at a flow rate ranging from 0.05 to 1 m / s, preferably from 0.1 to 0.5 m / s. In step c), the pyrolysis liquid can flow through the pipe system at a flow rate ranging from 0.3 to 1.5 m / s, preferably from 0.5 to 1.5 m / s.

[0016] In another embodiment with a scale of 500L–1000L, the flow rate in step a) ranges from 0.05 to 1.5 m / s, preferably from 0.1 to 1.5 m / s. In step b), the pyrolysis composition may flow through the piping system at a flow rate ranging from 0.01 to 1 m / s, preferably from 0.05 to 0.5 m / s. In step c), the pyrolysis fluid may flow through the piping system at a flow rate ranging from 0.05 to 1.5 m / s, preferably from 0.1 to 1.5 m / s.

[0017] Using the method of the present invention, a yield of at least 65% of ccc DNA molecules can be obtained after step c). The homogeneity of the ccc DNA molecules obtained by step c) is at least 70% ccc, preferably 80%, more preferably at least 90% ccc, and even more preferably at least 93%, for example 95% ccc.

[0018] Using the method of the present invention, a yield of at least 65% ccc DNA molecules can be obtained after step d). The homogeneity of the ccc DNA molecules obtained by step d) is at least 70% ccc, preferably 80%, more preferably at least 90% ccc, and even more preferably at least 93%, for example 95% ccc. Homogeneity can be evaluated by capillary gel electrophoresis (CGE).

[0019] The final pDNA may contain residual HCP (% w / w pDNA) of less than 0.05, less than 0.03, or less than 0.01, for example, less than 0.001 or less than 0.003. Residual HCP can be assessed by ELISA.

[0020] The final pDNA may contain less than 1%, less than 0.5%, or less than 0.1% residual RNA (% w / w pDNA). The residual RNA can be assessed by qPCR.

[0021] The final pDNA may contain less than 2%, preferably less than 1%, of residual gDNA (% w / w pDNA). Residual genomic DNA can be assessed by qPCR.

[0022] The final pDNA may contain endotoxin levels below 5 EU / mg, below 1 EU / mg, or below 0.5 EU / mg. Endotoxin levels can be assessed using the LAL test. The method is particularly suitable for large-scale implementation.

[0023] DNA molecules obtained by the methods described herein were also considered.

[0024] Graphic Legend

[0025] Figure 1: Fermentation and Cell Harvesting Process

[0026] Figure 2 Exemplary pyrolysis device Detailed Implementation

[0027] Before describing the invention in detail in conjunction with several preferred embodiments, the following general definitions are provided.

[0028] The invention described below can be suitably practiced without any elements or limitations not specifically disclosed herein.

[0029] The present invention will be described below with reference to specific embodiments and certain accompanying drawings; however, the present invention is not limited thereto, but is defined only by the claims.

[0030] When the term "comprising" is used in this specification and claims, it does not exclude other elements. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this is also understood to disclose a group preferably consisting only of those embodiments.

[0031] Unless otherwise specified, when referring to singular nouns, the use of indefinite or definite articles, such as "a," "an," or "the," includes the plural form of the noun. In this invention, the term "about" or "approximately" indicates a range of accuracy that, as those skilled in the art will understand, still ensures the technical effectiveness of the stated feature. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0032] Technical terms are used according to their common sense. If a specific meaning is conveyed to certain terms, the definition of the term will be given below in the context in which the term is used.

[0033] This invention relates to a method for isolating ccc DNA molecules from microbial cells containing covalently closed circular (ccc) DNA molecules, comprising the following steps: a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution; and d) Further process the neutralized lysis buffer to obtain ccc DNA molecules. Step b) is carried out in two parallel tubes.

[0034] Furthermore, this invention relates to a method for producing ccc DNA molecules in microbial cells, comprising the following steps: (i) Fermentation of microbial cells containing ccc DNA molecules; (ii) Harvesting microbial cells; a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution; and d) Further process the neutralized lysis buffer to obtain ccc DNA molecules. Step b) is carried out in two parallel tubes.

[0035] Contacting microbial cells with a lysis agent means adding the lysis agent to the microbial cells. Typically, this means that a tube containing microbial cells and an optional buffer solution and a tube containing the lysis agent are joined at a valve leading to a mixing tube.

[0036] In the large-scale method, step b) is performed in two parallel tubes. The lysis tubes are connected at the front and rear ends with Y-joints. In other words, a single tube containing the mixed biomass slurry in the dissolution buffer is connected to two parallel tubes using Y-joints. The composition is passed through the two parallel tubes. After this lysis step, the two tubes are reconnected via the Y-joints before the lysed cells are mixed with the neutralization buffer.

[0037] The inventors have discovered that if a composition containing a lysing agent and microbial cells passes through the system at a Reynolds number of at least 3000, then after step c), i.e. in the neutralized lysate, the yield of ccc DNA is high, i.e., more than 60%, such as 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

[0038] Incubating the lysis composition to obtain a lysis buffer means incubating the composition for a certain period of time after mixing the microbial cells with the lysis agent. Therefore, the lysis composition typically flows in a lysis tube for a certain period of time. The lysis tube is usually directly connected to the mixing tube.

[0039] A covalently closed circular DNA molecule refers to a complete circular double-stranded DNA molecule, meaning that neither strand has been cleaved. The method of this invention particularly relates to a method for isolating and generating covalently closed circular recombinant DNA molecules.

[0040] Circular (ccc) DNA molecules can be plasmids, granules, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors, or hybrids thereof. Preferably, the ccc DNA molecule is a plasmid. Typically, the size of a ccc DNA molecule ranges from 2 kbp to 20 kbp.

[0041] Microbial cells can be, for example, bacterial cells, fungal cells, algal cells, slime mold cells, or protozoan cells. Preferably, the microbial cell is a fungal cell, such as a yeast cell or a bacterial cell. More preferably, the microbial cell is a bacterial cell. In the context of this invention, microorganisms capable of replicating ccc DNA molecules, such as plasmids, are particularly preferred. Bacterial cells can be Gram-positive or Gram-negative cells. Preferably, the bacterial cell is a Gram-negative bacterial cell. More preferably, the bacterial cell is *Escherichia coli*.

[0042] In this context, Escherichia coli strains used for plasmid production, such as DH1, DH5α, DH10B, Stbl3, or JM109, are particularly preferred. In some embodiments, the DH5α strain is particularly preferred.

[0043] The term "fermentation" and its various parts of speech, or "fermentation production," refers to the bulk growth of microorganisms on a growth medium under aerobic or anaerobic conditions. Preferably, the fermentation reported herein refers to bacteria under anaerobic conditions. In the context of this invention, the term specifically refers to a fermentation process in which a covalently closed circular recombinant DNA molecule, such as a replication origin of a plasmid, is replicated by microorganisms.

[0044] Step a) Contact with and mix with the pyrolysis agent

[0045] Microbial cells can be contacted with lysing agents after fermentation, or microbial cells can be frozen and thawed after fermentation.

[0046] In some embodiments, the initial concentration of microbial cells used in step a) is 30 to 250 g / L, preferably 50 to 150 g / L (referring to the concentration of microbial cells before contact with the lysis agent). The initial concentration can be adjusted by a buffer solution, typically a physiological buffer solution with a pH of about 8.0, containing substances capable of chelating divalent metal cations such as Mg2+ and Ca2+ required for the function of DNA-degrading enzymes (e.g., via a buffer solution referred to herein as LY1).

[0047] Step a) begins by contacting the microbial cells with the lysing agent. The lysing agent is a strongly alkaline solution, including a strong base such as NaOH and a detergent such as sodium dodecyl sulfate (SDS).

[0048] The viscosity range of the composition of microbial cells and lysis agents is typically 1.2–1.3 mm. 2 / s (according to Ph.Eu. (European Pharmacopoeia) 9.8, Chapter 2.2.9, capillary viscometer method).

[0049] The Reynolds number is used to describe different fluid flow conditions. At low Reynolds numbers, viscous forces dominate, resulting in laminar flow, characterized by smooth and constant fluid motion. At high Reynolds numbers, inertial forces dominate, resulting in turbulent flow.

[0050] For flow in a circular pipe, the Reynolds number is usually defined as...

[0051] Where v m The average velocity of fluid through the pipe diameter

[0052] d is the inner diameter of the pipe

[0053] v is the viscosity of the fluid.

[0054] Critical Reynolds number ≈ 2040 ± 10.

[0055] The critical Reynolds number marks the transition between laminar and turbulent flow.

[0056] The inventors have surprisingly discovered that Reynolds numbers far above the critical number, i.e. at least 3000, at least 3300, preferably 3500 to 6000, and more preferably 4000 to 5500, lead to high yields of ccc DNA molecules.

[0057] Therefore, in a specific implementation, the Reynolds number in step a) is at least 3000, at least 3300, preferably 3500 to 6000, more preferably 4000 to 5500, for example 5000 to 5500.

[0058] Therefore, after cleavage, i.e. after neutralization step c), the yield of ccc DNA molecules can reach at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% (relative to the total amount of recombinant DNA molecules produced).

[0059] In some embodiments, the flow is turbulent throughout the pyrolysis process, including neutralization. In other words, in steps a), b), and c), the composition flows through the pipe system in a turbulent state. In other words, the Reynolds number is at least 2100 in steps a), b), and c).

[0060] Therefore, in the specific implementation, the Reynolds number in step a) is at least 3000, and the Reynolds number in steps b) and c) is at least 2100.

[0061] Therefore, some implementations involve a method for isolating ccc DNA molecules from microbial cells containing covalently closed circular (ccc) DNA molecules, which includes the following steps: a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution, and allow the composition to flow through a pipe system at a Reynolds number of at least 2100; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution, and allow the composition to flow through the pipe system at a Reynolds number of at least 2100; d) Further process the neutralized lysis buffer to obtain ccc DNA molecules. Step b) is carried out in two parallel tubes.

[0062] The specific implementation scheme relates to a method for isolating ccc DNA molecules from microbial cells containing covalently closed circular (ccc) DNA molecules, which includes the following steps: a) Contact the microbial cells with the lysis agent and pass the composition through the tubular system at a Reynolds number of 4000-5500 to obtain the lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution, and allow the composition to flow through a pipe system at a Reynolds number of at least 2500-5400; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution, and allow the composition to flow through the pipe system at a Reynolds number of at least 4500 to 6500; d) Further process the neutralized lysis buffer to obtain ccc DNA molecules. Step b) is carried out in two parallel tubes.

[0063] The composition is passed through a piping system via conventional means (i.e., via a controllable pump), the piping system typically comprising a mixing tube.

[0064] In a specific implementation, the diameter of the pipe system (i.e., the mixing pipe) in step a) ranges from 2 to 50 mm, preferably from 4 to 40 mm. Typically, the flow velocity in step a) ranges from 0.3 to 1.5 m / s, preferably from 0.5 to 1.5 m / s.

[0065] Step b): Pyrolysis

[0066] The lysis step is typically an alkaline lysis step, in which the detergent contained in the lysis solvent disrupts the cell membrane. Furthermore, the strong base contained in the lysis solvent denatures the chromosome and ccc DNA. In the subsequent neutralization step, a neutralizing solution, i.e., an acidic solution, is added. This solution renatures the ccc DNA but not the chromosomal DNA of the microorganism.

[0067] Typically, in step b), the pyrolysis composition passes through a tubular system, i.e., a pyrolysis tube. The movement of the pyrolysis composition is controlled by conventional means (i.e., a controlled pump). Step b) begins when the pyrolysis composition enters the pyrolysis tube and ends before the pyrolysis solution comes into contact with the neutralization solution (i.e., step c).

[0068] Preferably, step b) lasts for 2 to 6 minutes, more preferably 3 to 6 minutes.

[0069] The pyrolysis composition flows through the pipe system at a flow rate ranging from 0.05 to 1 m / s, preferably from 0.1 to 0.5 m / s.

[0070] Typically, the diameter of the pipe system in step b) ranges from 2 to 50 mm, preferably from 5 to 30 mm.

[0071] In some implementations, the Reynolds number in step b) is at least 2100, preferably 2100 to 6000, and more preferably 2500 to 5500.

[0072] To achieve high Reynolds numbers in a large-scale system, step b) is performed in two parallel tubes. The lysis tubes are connected at the front and rear ends with Y-joints. In other words, a single tube containing the mixed biomass slurry in the dissolution buffer is connected to two parallel tubes using Y-joints. The composition is passed through the two parallel tubes. After this lysis step b), the two tubes are reconnected via the Y-joints before the lysed cells are mixed with the neutralization buffer (i.e., b)).

[0073] In one embodiment, at the beginning of step b), each tube is pre-filled with buffer. In another embodiment, at the beginning of step b), each tube is pre-filled with lysis buffer LY1.

[0074] In another embodiment, the pyrolysis tube is mounted at an angle. This prevents air bubbles from forming within the pyrolysis tube. This angle can be achieved, for example, by winding the pyrolysis tube in a gradually increasing manner. In other words, the pyrolysis tube is fixed to the drum at a certain slope to prevent air bubbles.

[0075] Step c): Neutralization

[0076] In step c), the lysis solution is contacted with the neutralization solution to neutralize it.

[0077] The addition of the neutralization solution allows ccc DNA to renature, but the chromosomal DNA of microbial organisms cannot renature.

[0078] The neutralizing solution is an acidic solution. Preferably, the neutralizing solution contains an acetate buffer, such as potassium acetate or ammonium acetate. As other examples, a combination of potassium acetate or ammonium acetate may also be used. More preferably, the neutralizing solution is potassium acetate (e.g., 1000-5000 mM, more preferably 2000 to 4000 mM, e.g., 3000 mM).

[0079] Typically, the pyrolysis solution and the neutralization solution come into contact in the piping system via a Y-connector.

[0080] In step c), the pyrolysis solution can be passed through a pipe system, i.e., a neutralization pipe. Typically, the diameter of the pipe system in step c) ranges from 2 to 60 mm, preferably from 4 to 52 mm.

[0081] In some embodiments, the pyrolysis fluid flows through the pipe system at a flow rate ranging from 0.3 to 1.5 m / s, preferably from 0.5 to 1.5 m / s.

[0082] After contacting the pyrolysis solution with the neutralization solution, the neutralized pyrolysis solution can be pumped into a collection tank.

[0083] In some implementations, the Reynolds number in step c) is at least 3000, preferably 3500 to 6500, and more preferably 4000 to 6100.

[0084] Step d) Further processing

[0085] In one embodiment, the neutralized pyrolysis solution is kept at below 15°C for 12 hours or less, such as 8 to 12 hours, preferably 10 to 12 hours, before proceeding with one or more further processing steps.

[0086] In a preferred embodiment, the neutralized pyrolyte is processed directly, i.e., one or more processing steps are performed immediately after neutralization, without maintaining it below 15°C. Direct processing of the neutralized pyrolyte is advantageous because it is time-efficient and avoids cooling.

[0087] Therefore, in a preferred embodiment, steps a) to d) are performed at room temperature.

[0088] Step d) may include one or more of the following processing steps in any order: - Filter aid filtration - Ultrafiltration / Percolation - Anion exchange chromatography - Precipitation - Hydrophobic interaction chromatography In one implementation, step d) may include one or more of the following steps: - Filter aid filtration - Ultrafiltration / Percolation - Deep filtration - Anion exchange chromatography For further processing steps, particularly filter aid filtration and ultrafiltration, the composition (i.e., the neutralized lysate) can be divided into sub-lots. The size of the sub-lots can be from 500L to 2000L, for example from 800L to 1500L, such as 800L or 1000L.

[0089] Alluvial filtration

[0090] It is recommended to use a combination of a highly permeable filter aid and a low-porosity filter plate. The filter aid material can be added to the pyrolysis solution in solid or suspension form. The size of the filtration equipment should be selected accordingly to achieve effective clarification of the pyrolysis solution. After pyrolysis, the pyrolysis solution can be pumped into a storage tank containing the filter aid material. The mixture of pyrolysis solution and filter aid material is clarified by filter aid filtration, for example, using a filter with a rejection ratio of 0.5 to 12 µm, preferably 3 to 12 µm. Typically, the filter aid material is diatomaceous earth.

[0091] In one embodiment, the filter aid material is added to the lysis buffer in solid form. The filter material can be added at a final concentration of 20 to 30 g / L, for example, about 20 g / L or 25 g / L. In a preferred embodiment, the filter material can be added at a final concentration of 25 g / L.

[0092] In this step, the neutralized pyrolysis solution can be divided into sub-lots of approximately 800 L to 1000 L, preferably 800 L, and a specific amount of filter aid material is added to each lot to a final concentration of approximately 20 to 30 g / L, preferably 25 g / L. During the addition of the filter aid, the solution can be stirred, for example at 100 to 500 rpm, preferably 200 to 500 rpm, more preferably 250 to 350 rpm, such as 300 rpm or 330 rpm. In this process step, the composition can be further stirred, for example for 20 to 120 minutes, 30 to 60 minutes, such as 45 minutes. The solution can be continuously stirred (e.g., at 300 rpm or 330 rpm) and pumped to the filter plates. Pumping can be carried out at a pumping rate of 1000 l / h to 2400 l / h, for example 1000 l / h to 2200 l / h, for example 1000 l / h to 2000 l / h, 1200 L / h to 1600 L / h, for example 1400 l / h, preferably 1600 l / h to 2400 l / h, and more preferably 1800 l / h to 2200 l / h, for example 2040 l / h. The size of the filtration equipment should be selected accordingly to achieve effective clarification of the pyrolysis liquid. In one embodiment, for each sub-batch of about 800 to 1000 L, a 4.52 m... 2 The total area.

[0093] In one embodiment, the duration of filter aid filtration is about 10 to 20 hours, preferably 11 to 22 hours, and more preferably 12 to 20 hours.

[0094] Ultrafiltration / Percolation

[0095] Typically, ultrafiltration and percolation, which can be performed directly after filter aid filtration, can be carried out as tangential flow filtration. In a specific embodiment, a hollow fiber filter assembly is used. Typically, an application time of 2000 seconds is applied. -1 up to 8000s -1 Optimal 4000s -1 Up to 6000 s -1 A constant shear rate. Filtration is divided into a first concentration stage, namely ultrafiltration. In the concentration step, the sample can be concentrated to ~5 to ~50 times, preferably ~6 to ~30 times, more preferably ~7 to ~20 times.

[0096] In the percolation step (5 to 6x), the buffer solution (20 mM Tris, 10 mM EDTA, 666 mM NaCl, pH 8.0) is exchanged. The retentate can be collected after percolation. The hollow fiber assembly can be washed, and the wash fraction can be combined with the retentate for further processing.

[0097] Optionally: Precipitation with alkaline earth metal salts

[0098] Preferably, the alkaline earth metal salt is a calcium salt.

[0099] Optional: TFF (percolation) (5x)

[0100] After precipitation with alkaline earth metal salts, preferably calcium salts, a further percolation step can be performed to exchange the buffer solution in preparation for subsequent AEX chromatography.

[0101] Depth filtration

[0102] A deep filtration step, also known as deep filtration, can be performed. Preferably, the deep filtration step is performed before the chromatography step. In particular, the deep filtration step can be performed before the anion exchange chromatography step.

[0103] The terms deep filtering and deep filtering are used synonymously in this document.

[0104] Optional: Anion exchange chromatography

[0105] Anion exchange chromatography can be performed in a binding mode, where ccc DNA molecules bind to the resin and are eluted after at least one washing step. For the washing step, an alkaline washing buffer can be used.

[0106] The anion exchange resin can be a weak anion exchange resin or a strong anion exchange resin, preferably a strong anion exchange resin, such as a quaternary ammonium or quaternized polyethyleneimine (e.g., Poros 50 HQ, Thermo Fisher).

[0107] ccc DNA molecules can be eluted by gradient elution or by stepwise gradient elution, preferably by stepwise gradient elution.

[0108] Hydrophobic interaction chromatography / sulfur-affinity interaction chromatography

[0109] Further hydrophobic interaction chromatography and / or thiophilic interaction chromatography can be performed. Hydrophobic interaction chromatography or thiophilic interaction chromatography can be performed after the addition of potassium phosphate or ammonium sulfate. K₂HPO₄ is preferred as the potassium phosphate, and further filtration is optional.

[0110] Preferably, aromatic resins, such as Capto Phenyl ImpRes (GE Healthcare), are used for hydrophobic interaction chromatography.

[0111] Optional: Ultrafiltration / Percolation

[0112] The final ultrafiltration / percolation step can be performed using tangential flow filtration. In some implementations, hollow fiber modules are employed.

[0113] After ultrafiltration / distillation, the material can be filtered (0.2 μm) and stored.

[0114] In one implementation, for large-scale purification, step d) sequentially includes the following steps: (i) Filter aid filtration (ii) Ultrafiltration / Percolation (iii) Deep filtration (iv) Optional anion exchange chromatography (v) Hydrophobic interaction chromatography.

[0115] In one implementation, for large-scale purification, step d) sequentially includes the following steps: (i) Filter aid filtration (ii) Ultrafiltration / Percolation (iii) Deep filtration (iv) Anion exchange chromatography (v) Hydrophobic interaction chromatography.

[0116] In a particularly preferred embodiment, a method for isolating ccc DNA molecules from microbial cells containing covalently closed circular (ccc) DNA molecules includes the following steps: a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution; and d) Further process the neutralized lysis buffer to obtain ccc DNA molecules. Step b) is performed in two parallel tubes, and in which Step d) includes the following steps in sequence: (i) Filter aid filtration (ii) Ultrafiltration / Percolation (iii) Deep filtration (iv) Anion exchange chromatography (v) Hydrophobic interaction chromatography.

[0117] In one implementation, step d) includes the following steps in sequence: (i) Filter aid filtration 1 (ii) Ultrafiltration 1 / Percolation 1 (iii) Deep filtration (iv) Optional anion exchange chromatography (v) Hydrophobic interaction chromatography (vi) Optionally, perform final processing on the product according to project-specific requirements. In one implementation, step d) includes the following steps in sequence: (i) Filter aid filtration 1 (ii) Ultrafiltration 1 / Percolation 1 (iii) Precipitation (iv) Filter aid filtration 2 / percolation 2 (v) Optional anion exchange chromatography (vi) Hydrophobic interaction chromatography (vii) Perform final processing on the product according to the specific requirements of the project.

[0118] More specifically, in the specific implementation plan, step d) includes the following steps: (i) Filtration using diatomaceous earth as a filter aid (ii) Ultrafiltration / percolation using hollow fiber tangential flow filtration modules (iii) Deep filtration (iv) Optional anion exchange chromatography, wherein during AEX chromatography, ccc DNA molecules bound to the AEX resin are washed with alkaline wash buffer. (v) Hydrophobic chromatography using aromatic resins In a specific implementation, after performing all processing steps (i) to (v) in step d), a yield of 10% to 50%, preferably 15% to 45%, and more preferably 20% to 40% can be achieved.

[0119] This method can be carried out on a large scale. The term "large scale" refers to fermentation scales of 10 l and larger, such as 100 l, 200 l, 500 l, 800 l, 1000 l or larger.

[0120] It also includes ccc DNA molecules obtained by the methods described herein.

[0121] experiment

[0122] result: The yield after the pyrolysis step was measured using the workflow described in detail below and by applying different Reynolds numbers.

[0123]

[0124] Table 1: Comparison of different Reynolds numbers applied to the pyrolysis tube.

[0125] method: As described in Section 2, the entire process flow is divided into two parts: Upstream processes include seed fermentation, primary fermentation, and fermentation harvesting; while downstream processes include cell lysis, lysis buffer clarification and conditioning, and chromatographic purification steps. And the final TFF used for stock solution conditioning. All unit operations are described in detail in the following sections.

[0126] Example 1

[0127] Upstream processes

[0128] For example, batch fermentation and fed-batch fermentation strategies have been applied to pDNA production. This fermentation protocol follows a general process flow ( Figure 1 Escherichia coli (such as DH5α or DH10B) is the preferred cell source. Depending on the process requirements, a fed-batch fermentation strategy can be selected for pDNA production.

[0129] Batch fermentation

[0130] Shake flask / seed fermentation tank seed fermentation

[0131] The components of the seed fermentation medium “Batch” are described in Table 2. Kanamycin solution, thiamine hydrochloride and magnesium sulfate solution were prepared separately and added to the basal medium after sterilization and before fermentation began. The basal seed medium “Batch” was sterilized by autoclaving, while the thiamine-HCl / MgSO4 and antibiotics were aseptically filtered.

[0132]

[0133] Table 2. Composition of seed fermentation medium

[0134] The key process parameters and related set values ​​for shake-flask seed fermentation are shown in Table 3.

[0135]

[0136] Table 3: Key process parameters for seed fermentation in "Batch" shake flasks

[0137] The key process parameters and related set values ​​for seed fermentation in the seed fermentation tank are shown in Table 3b.

[0138]

[0139] Table 3b: Key process parameters of the "Batch" seed fermentation tank

[0140] Primary fermentation "Batch"

[0141] The composition of the primary fermentation medium is listed in Table 4. The basal primary fermentation medium “Batch” was prepared and autoclaved. Kanamycin solution and thiamine-HCl / MgSO4 solution were prepared separately, aseptically filtered, and added to the sterile basal medium before the start of primary fermentation.

[0142]

[0143] Table 4: Composition of the main fermentation medium

[0144] The key process parameters and related settings for the primary fermentation "Batch" are shown in Table 5.

[0145]

[0146] Table 5: Key process parameters for the primary fermentation "Batch"

[0147] Based on the cultured *E. coli* strain, the primary fermentation was conducted at 30–39 °C (7.5 L / min aeration, pO2 ≥ 30%, initial stirring speed 300 rpm). Throughout the primary fermentation, the pH was maintained at 7.0 ± 0.2 using 1 M phosphoric acid and 25% (NH4)OH. PPG was used as an antifoaming agent. A control cascade was implemented, initially affecting the stirrer rate and subsequently influencing oxygen enrichment in the influent gas flow. The fermentation endpoint was indicated by a sharp increase in dissolved oxygen (“pO2 peak”). The fermentation endpoint was reached when the pO2 peak was detected and the substrate was depleted, and cell harvesting was initiated.

[0148] Feeding and batch fermentation

[0149] Shake flask / seed fermentation tank seed fermentation

[0150] The components of the seed fermentation medium "Fed-Batch" are shown in Table 6. Thiamine-HCl and magnesium sulfate solutions were prepared separately and added to the basal medium after sterilization and before fermentation began. The basal seed medium "Fed-Batch" was sterilized by autoclaving, while the thiamine-HCl and MgSO4 were aseptically filtered.

[0151]

[0152] Table 6. Composition of seed fermentation medium

[0153] The key process parameters and related set values ​​for shake-flask seed fermentation are shown in Table 7.

[0154]

[0155] Table 7: Key process parameters for "Fed-Batch" shake flask seed fermentation.

[0156] The key process parameters and related set values ​​for seed fermentation in the seed fermentation tank are shown in Table 7b.

[0157]

[0158] Table 7b: Key process parameters for “Fed-Batch” seed fermentation tanks.

[0159] Primary fermentation "Fed-Batch"

[0160] The composition of the primary fermentation medium is listed in Table 8. The basal primary fermentation medium, “Fed-Batch,” was prepared and autoclaved. Thiamine-HCl and MgSO4 solutions were prepared separately, aseptically filtered, and added to the sterile basal medium before the start of primary fermentation.

[0161]

[0162] Table 8: Composition of the main fermentation medium

[0163] The composition of the fed culture medium is listed in Table 9. Prepare the fed culture medium and filter (0.2 μm).

[0164]

[0165] Table 9: Composition of fed culture medium

[0166] The key process parameters and related settings for the primary fermentation "Fed-Batch" are shown in Table 10.

[0167]

[0168] Table 10: Key process parameters for primary fermentation "Fed-Batch"

[0169] Based on the cultured *E. coli* strain, the primary fermentation was conducted at 30–39 °C (7.5 L / min aeration, pO2 ≥ 30%, initial stirring speed 300 rpm). Throughout the primary fermentation, the pH was maintained at 7.0 ± 0.2 using 1 M phosphoric acid and 25% (NH4)OH. PPG was used as an antifoaming agent. A control cascade was implemented, initially affecting the stirring rate, then increasing the inlet gas flow, followed by increasing the tank pressure. The end of the batch stage was indicated by a sharp rise in dissolved oxygen (“pO2 peak”) – at which point the predetermined indexed feed program was immediately initiated (see Equation 1). The fermentation endpoint was reached when the cell growth plateaued, or when pO2 < 5% due to the pO2 cascade reaching its maximum, and cell harvesting was initiated.

[0170]

[0171] Equation 1: Calculation of the predetermined index replenishment procedure

[0172] F = Feed flow rate [mL / h]

[0173] µ w =Growth rate setpoint [h] -1 ]

[0174] V L0 = Reactor liquid volume at the start of feeding [mL]

[0175] c XL = Cell dry weight at the start of feeding [g / mL]

[0176] Y X / S = Substrate / biomass yield coefficient [g / g]

[0177] c S = Carbon source concentration in the feed solution [g / mL]

[0178] t = Processing time [h]

[0179] t0 = Feeding start time [h]

[0180] Fermentation Harvest

[0181] Cells are harvested using classical centrifugation or tangential flow filtration (TFF). The harvested cells are stored as solid precipitate or biomass. Key process parameters for fermentation and TFF harvesting are shown in Tables 11 and 11b.

[0182]

[0183] Table 11: Centrifugation Harvest

[0184] Table 11b: TFF

[0185] TFF was then subjected to three percolation cycles with resuspension buffer LY1.

[0186] Downstream processes

[0187] The RH / pDNA downstream process consists of eleven main unit operations.

[0188] Cell lysis and plasma release are based on alkaline lysis. The lysate is clarified and further purified by adding CaCl2 followed by TFF adjustment to enable AEX capture. After AEX capture chromatography, potassium phosphate or ammonium sulfate is added to the AEX library to allow HIC binding. The clarified feed stream is loaded onto a HIC purification column. The pDNA in the HIC eluent can be ultimately buffer-exchanged via TFF or microfiltered, depending on project-specific requirements. Stock solutions are typically stored at -70°C.

[0189] Pyrolysis

[0190] Figure 2 The lysis assembly is shown. Microbial cells in buffer (25 or 50 mM Tris-HCl, 10 mM EDTA; pH 8.0) were contacted with lysis buffer (0.8% (w / v) NaOH, 1% (w / v) SDS) in a mixing tube. The lysis composition was incubated in the lysis tube. The lysis buffer was then contacted with neutralization solution (3M KAc, 3M KAc, pH 5.5 or 1 M KAc + 3 M (NH4)Ac, or 7M CH3COONH4, 1 M KAc) in a neutralization tube. Buffer solutions LY1 (25 or 50 mM Tris-HCl, 10 mM EDTA; pH 8.0), LY2 (0.8% (w / v) NaOH, 1% (w / v) SDS), and LY3 (3 M KAc, pH 5.5 or 3 M CH3COONH4, 1 M KAc, or 7 M CH3COONH4, 1 M KAc) were used in the alkaline lysis step. Key process parameters for lysis are shown in Table 12.

[0191]

[0192] Table 12: Key Process Parameters for Pyrolysis

[0193] Depending on the scale of the main fermentation, different lysis devices are used for cell lysis (see Table 13 for details on pipe dimensions).

[0194] Table 13

[0195] The cell lysis step begins with the thawing of the EoF biomass / EoF biomass slurry. The thawed EoF biomass / EoF biomass slurry is adjusted to a biomass concentration of 100 g / L using lysis buffer LY1. The actual lysis step is initiated by pumping the homogenized biomass slurry (LY1) and lysis buffer LY2 to the T-connector, where the biomass slurry is mixed with the LY2 buffer. Turbulent flow with a calculated Reynolds number of 4000-6000 is achieved in the mixing tube. Figure 2 ).

[0196] The lysis tube is connected directly downstream of the mixing tube. The lysis time is determined by the flow rate and the size of the lysis tube, keeping it within the range of 3-6 minutes. At the end of the lysis tube, neutralization buffer LY3 is mixed with the lysis buffer via a Y-shaped connector. The neutralized lysis buffer is then transferred to the collection tank through the neutralization tube.

[0197] lysis buffer clarification: Using standard techniques described in the prior art, clarification of the lysis buffer is preferably achieved through a filter aid filtration step (using diatomaceous earth as the filter aid material). Suitable clarification steps are described, for example, in Eibl, R., Eibl, D. (eds.), “Single-Use Technology in Biopharmaceutical Manufacture,” 2nd ed., 2019, JohnWiley & Sons, Inc., pp. 271-77. It is recommended to use a combination of a highly permeable filter aid and a low-porosity filter plate. The filter aid material can be added to the lysis buffer in solid or suspension form. The size of the filtration equipment should be selected accordingly to achieve effective lysis buffer clarification.

[0198] TFF 1 lysis buffer conditioning

[0199] The lysis buffer matrix was exchanged to DF1 buffer (20 mM Tris, 10 mM EDTA, 666 mM NaCl; pH 8.0±0.1) via TFF to allow pDNA to bind to the AEX capture resin. Key process parameters for percolation 1 harvest are shown in Table 14.

[0200]

[0201] Table 14: Key Process Parameters for Ultrafiltration / Percolation 1

[0202] Considering the scale of the main fermentation, different TFF components were used for lysis buffer conditioning.

[0203] Table 15: TFF Components for Ultrafiltration / Diffusion 1

[0204] TFF (Thin-fiber ion exchange) for pyrolysis fluid conditioning was performed using a hollow fiber assembly. The TFF was applied at 4000-6000 s. - The process was performed at a constant shear rate and approximately 0.5 bar TMP. TFF was divided into a first concentration stage and a subsequent percolation stage. TFF was performed in two sub-batch stages: the clarified lysate was divided into two batches, each concentrated 20-fold. The retentates were combined, and the combined lysate matrix was buffer-exchanged with 5 volumes of DF1 buffer in a continuous percolation. After percolation, the retentate was collected and the hollow fiber assembly was rinsed in a washing step. The retentate and wash were combined for further processing.

[0205] CaCl2-precipitate

[0206] Adjust the DF-combination to 800 mM CaCl2 using a 5M CaCl2 stock solution and stir for 10 minutes. Then, store the solution overnight at 2–8°C.

[0207] TFF 2 lysis buffer conditioning: percolation 2

[0208] The clarified CaCl2 precipitate was exchanged 5-fold with TFF to DF1 buffer (20 mM Tris, 10 mM EDTA, 666 mM NaCl; pH 8.0 ± 0.1) to allow pDNA to bind to the AEX capture resin. The key process parameters for percolation 2 (hollow fiber, buffer) were the same as for TFF1. After percolation, the sample adjusted accordingly was used directly as the capture load for AEX chromatography.

[0209] AEX capture tomography

[0210] The adjusted lysis buffer was directly loaded onto an AEX capture chromatography column (resin: Poros 50 HQ, column height: 15-25 cm; maximum pDNA loading 6-7 g / L). cv ).

[0211]

[0212] Table 16: AEX Capture Bars

[0213] Before loading the feed stream, equilibrate the AEX column with AXA (20 mM Tris-HCl, 10 mM EDTA, 666 mM NaCl; pH 8.0 ± 0.1) at 150 cm / h. Load the sample at a feed stream rate of 60 cm / h. After loading, wash the AEX column with buffer AXA at ​​150 cm / h to a volume of five (CV). Perform a five-volume step elution at 60 cm / h with elution buffer containing 800 mM NaCl (buffer AXA / AXB: 20 ​​mM Tris-HCl, 10 mM EDTA, 1 M NaCl; pH 8.0 ± 0.1) to elute the product. Collect the eluted product and combine the appropriate fractions.

[0214] HIC purification chromatography

[0215] The AEX consumables were mixed with a potassium phosphate stock solution (4 M K₂HPO₄, 10 mm Tris-HCl, pH 8.0 ± 0.1) to adjust the K₂HPO₄ concentration to 2.0 M. Alternatively, the salinity of the AEX consumables could be increased to a final concentration of ≥2.0 M ammonium sulfate using 3.0 M ammonium sulfate. The feed stream was filtered again before loading into the HIC (Sartopore 2 0.45 / 0.2 µm, Sartorius). Key process parameters for HIC purification are shown in Table 17.

[0216]

[0217] Table 17: Key Process Parameters for HIC Purification Chromatography

[0218] Based on the scale of the primary fermentation, HIC

[0219] Chromatography uses different column specifications:

[0220] Table 18: HIC Purification Columns

[0221] HIC purification columns (resin: Capto Phenyl ImpRes, BH: 15-25 cm) were equilibrated with HIA buffer (2.0 mM MK2HPO4, 10 mM Tris-HCl, pH 8.0) or an alternative equilibration buffer (2160 mM (NH4)2SO4, 20 mM Tris-HCl, 10 mM EDTA, pH 7.0). The feed stream was loaded at a linear flow rate of 150 cm / h. After loading, the HIC column was washed with 5 column volumes of HIA buffer at 150 cm / h. Elution of the product can be achieved by linear gradient elution over 7.5 CV to 1.5 M K₂HPO₄, or elution to 1.62 M ammonium sulfate (buffer HIA / HIB: 10 mM Tris-HCl, pH 8.0, or 20 mM Tris-HCl, 10 mM EDTA, pH 7.0), and maintained at 6 CV under 1.5 M K₂HPO₄ or 1.62 M ammonium sulfate conditions before 100% B. Alternatively, step elution can be applied. The entire chromatography process is completed at 150 cm⁻¹ h.

[0222] Alternatively, this method can be performed without the initial AEX step. The clarified lysis buffer is then mixed with 3M ammonium sulfate, 10 mM EDTA, 20 mM Tris, pH 7.0 (adjusted with 25% HCl) to a final concentration of 2.16M for adjustment, and filtered using a filter capsule (e.g., PALL P700 and Supracap 100) or through a filter aid before HIC loading.

[0223] The key process parameters for HIC are shown in Table 18.

[0224]

[0225] Table 18: Key process parameters for HIC purification chromatography without pre-AEX process

[0226] The HIC column (resin: Capto Phenyl ImpRes, BH: 20 cm) was equilibrated with 5 column volumes of HIA buffer (Phase A: 2.16 M ammonium sulfate, 20 mM Tris, 10 mM EDTA, pH 7.0 (adjusted with 25% HCl), flow rate 150 cm / h). Feed loading was performed at a linear flow rate of 75 cm / h. After loading, the HIC column was washed with 5 column volumes of HIA buffer at 150 cm / h. Product elution was performed using a linear gradient of 0–20% Phase B (buffer HIA / HIB: Phase B: 20 ​​mM Tris, 10 mM EDTA, pH 7.0). Linear gradient elution was performed at 38 cm / h over a 7.5 column volume, followed by 6 column volumes at 38 cm / h under 20% B conditions, and then a step flow rate of 150 cm / h to 100% B was maintained for 2 column volumes.

[0227] The final process step may include ultrafiltration dialysis filtration 3: Ultrafiltration / Percolation 3 In the final TFF step, the HIC elution matrix can be exchanged for project-specific DF2 buffer. Hollow fiber modules (membrane: PES, NMWCO: 100 kDa, fiber ID: 0.5 mm, fiber length: 41.5 cm) can be specified for TFF conditioning (shear rate 4000–6000 s⁻¹; TMP ≈ 0.5 bar).

[0228] Depending on the scale of the primary fermentation, different TFF (Thyrate-Free Fermentation) components are used for lysate conditioning:

[0229] Table 19

[0230] TFF can be performed at a constant shear rate of 4000–6000 s⁻¹. The HIC eluent is first concentrated to a predetermined target concentration. Subsequently, the lysis buffer matrix is ​​exchanged for DF2 buffer in a continuous percolation process. The retentate is collected after percolation and the hollow fiber assembly is rinsed in a single washing step. The retentate and wash fractions are combined.

[0231] Optional step: Filtration of the concentrate

[0232] The stock solution after UF / DF 3 was filtered through a 0.2 µm filter and then stored.

[0233] buffer solution

[0234] Table 20: Composition of lysis buffer LY1

[0235] Table 21: Composition of lysis buffer LY2

[0236] Table 22: Composition of lysing buffer LY3

[0237] Table 23: Composition of Alternative Lysis Buffer LY3

[0238] Table 23.1: Composition of the alternative lysis buffer LY3

[0239] Table 24: Composition of Percolation Buffer DF1

[0240] Table 25: Composition of AEX electrophoresis buffer AXA

[0241] Table 26: Composition of AEX electrophoresis buffer AXB

[0242] Table 27: Composition of AEX CIP buffer AXCIP1

[0243] Table 28: Composition of AEX CIP buffer AXCIP2

[0244] Table 29: Composition of HIC Adjustment Buffer

[0245] Table 30: Composition of HIC Electrophoresis Buffer HIA (Equilibration Buffer)

[0246] Table 31: Composition of HIC electrophoresis buffer HIB

[0247] Table 32: Composition of Alternative HIC Adjustment Buffer

[0248] Table 33: Composition of Alternative HIC Running Buffer HIA (Equilibrium Buffer)

[0249] Table 34: Composition of Alternative HIC Running Buffer (HIB)

[0250] Table 35: Composition of HIC adjustment buffer for AEX-free HIC chromatography

[0251] Table 36: Composition of the corresponding HIC running buffer HIA (equilibration buffer)

[0252] Table 37: Composition of the corresponding HIC running buffer HIB (elution buffer)

[0253] In the following example data, unless otherwise stated, the parameters are the same as described above:

[0254] Table 38: Substitute plasmid size and corresponding USP yield when using E. coli DH10B

[0255] Table 39: Pyrolysis Units Depending on the Scale of the Primary Fermentation

[0256] Table 40: Quality after DSP processing, including AEX

[0257] Table 41: Quality after DSP without AEX

[0258] Large-scale production of pDNA

[0259] In large-scale applications (≥1000L), a pipe size was developed for the cell lysis step to achieve turbulence and a Reynolds number >2300. The lysis tube has an inner diameter of 19.1 mm and a length of approximately 30 m. Lysis is performed in two independent lysis tubes connected by a Y-joint, the aforementioned inner diameter and length ensuring turbulence and a lysis time of 3 to 6 minutes.

[0260] After lysis and before mixing the lysed cells with neutralization buffer LY3, reconnect the two tubing into one and mix the neutralization buffer LY3 with the lysis buffer using the Y-shaped connector.

[0261] The neutralized pyrolysis solution is pumped through a neutralization pipe and collected in a collection tank.

[0262] For the tubing dimensions used for cell lysis at a scale of 1000 L, see Table 42.

[0263]

[0264] Table 42

[0265] Before clarifying the pyrolysis solution by filter aid filtration (using diatomaceous earth as the filter aid material), the neutralized pyrolysis solution can be kept at below 15°C and stirred at 100 rpm for up to 12 hours. It is preferable to use a highly permeable filter aid combined with a low-porosity filter plate. The filter aid material is added to the pyrolysis solution in solid form at a concentration of approximately 20 g / L. In this step, the neutralized pyrolysis solution is divided into 1000 L sub-batches, to which 20 kg of filter aid material is added. During the addition of the filter aid, the solution is stirred at 300 rpm, and stirred for another 45 min after complete addition. The solution is continuously stirred at 300 rpm and pumped through the filter plate at a pumping rate of 1400 L / h. The size of the filtration equipment should be selected accordingly to achieve effective pyrolysis solution clarification. For each 1000 L sub-batch, two filter assemblies (total area 4.52 m²) are used. 2 The two processes are operated in parallel. After filtration, the clarified lysis buffer is collected in a 1000 L bag for subsequent UF / DF treatment as described above (see TFF 1 lysis buffer conditions). The target volume after UF should be 250 L, followed by 5 percolation cycles as described above. A final filtration step is performed before AEX capture chromatography (Zeta-Plus 60 SP, 4.5–0.2 µm or PDH4 15–0.5 µm). AEX and HIC are performed as described above.

[0266] The quality of pDNA (drug stock solution) after DSP is listed in Table 43.

[0267]

[0268] Table 43

[0269] Homogeneity was measured by capillary gel electrophoresis (CGE); residual HCP was measured by ELISA; residual RNA was measured by qPCR; residual genomic DNA was measured by qPCR; and residual endotoxins were detected using the LAL assay. Example 2: Example 2 is performed as in Example 1, except for the following changes: The lysis buffer was clarified and (without the addition of CaCl2) directly applied to TFF conditioning, followed by AEX capture.

[0270] The neutralized pyrolysis solution was clarified by a filter aid filtration step (using diatomaceous earth as the filter aid material) and continuously treated at 20°C for 12-20 hours. (The pyrolysis solution was not kept below 15°C).

[0271] A combination of a highly permeable filter aid and a low-porosity filter plate is preferred. The filter aid material is added to the lysis buffer in solid form at a concentration of approximately 20-30 g / L. In this step, the neutralized lysis buffer is divided into 800 L sub-batches, to which 20-30 kg of filter aid material is added. During the addition of the filter aid, the solution is stirred at 330 rpm, and stirred for another 45 min after complete addition. The solution is continuously stirred at 330 rpm and pumped through the filter plate at a pumping rate of 2040 L / h. The size of the filtration equipment should be selected accordingly to achieve effective lysis buffer clarification. For each 800 L sub-batch, two filter assemblies (total area 4.52 m²) are used. 2 The two processes operate in parallel. After filtration, the clarified lysis buffer is collected in a 1000L bag container for subsequent UF / DF treatment as described above (see TFF 1 lysis buffer conditions).

[0272] TFF (Thin-fiber ion exchange) for pyrolysis fluid conditioning was performed using a hollow fiber assembly. The TFF was applied at 4000-6000 s. - The process was performed at a constant shear rate and approximately 0.5 bar TMP. TFF was divided into a first concentration stage and a subsequent percolation stage. TFF was performed in two sub-batch stages: the clarified lysate was divided into two batches, each concentrated 20-fold. The retentates were combined, and the combined lysate matrix was buffer-exchanged with 5 to 6 volumes of DF1 buffer in a continuous percolation. After percolation, the retentate was collected and the hollow fiber assembly was rinsed in a washing step. The retentate and wash were combined for further processing.

[0273] The target volume after UF should be 220 L, followed by 5 to 6 percolation cycles as described above. A final filtration step (Zeta-Plus 60 SP, 4.5–0.2 µm or PDH4 15–0.5 µm) should be performed before AEX capture chromatography. AEX and HIC are performed as described above.

[0274] The quality of the DSP-treated batches of pDNA (drug stock solution) according to Example 2 is listed in Table 44.

[0275]

[0276] Table 44

[0277] This application also includes the following: Item 1. A method for isolating ccc DNA molecules from microbial cells containing covalently closed circular (ccc) DNA molecules, comprising the following steps: a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution; and d) Further process the neutralized lysis buffer to obtain ccc DNA molecules. Step b) can be performed either in two parallel tubes.

[0278] Item 2. A method for producing ccc DNA molecules in microbial cells, comprising the following steps: (i) Fermentation of microbial cells containing ccc DNA molecules; (ii) Harvesting microbial cells; a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution; and d) Further process the neutralized lysate to obtain ccc DNA molecules.

[0279] Item 3. The method according to item 1 or 2, wherein in step a), the composition flows through the pipe system at a Reynolds number of at least 3300, preferably 3500 to 6000, more preferably 4000 to 5500.

[0280] Item 4. The method according to any one of items 1 to 3, wherein step b) lasts for 6 minutes or less.

[0281] Item 5. The method according to any one of the preceding items, wherein step b) lasts for 2 to 6 minutes, preferably 3 to 6 minutes.

[0282] Item 6. The method according to any one of the preceding items, wherein in steps b) and c), the Reynolds number is at least 2100.

[0283] Item 7. The method according to any one of the preceding items, wherein in step b), the Reynolds number is at least 2100, preferably 2100 to 6000, more preferably 2500 to 5500.

[0284] Item 8. The method according to any one of the preceding items, wherein in step c), the Reynolds number is at least 3000, preferably 3500 to 6500, more preferably 4000 to 6100.

[0285] Item 9. The method according to any one of the preceding items, wherein step d) comprises filtering the neutralized pyrolysis solution by at least one filtration step.

[0286] Item 10. The method according to Item 9, wherein in step d), diatomaceous earth is preferably used to perform the filter aid filtration step.

[0287] Item 11. The method according to Item 10, wherein an ultrafiltration / percolation step is performed in step d), wherein preferably, the ultrafiltration / percolation filter is a hollow fiber filter.

[0288] Item 12a. The method according to any one of the preceding items, wherein step d) comprises adding an alkaline earth metal salt to the neutralized pyrolysis solution.

[0289] Item 12b. The method according to any one of the preceding items, wherein step d) does not include adding an alkaline earth metal salt to the neutralized pyrolysis solution.

[0290] Item 13. The method according to Item 12a, wherein the alkaline earth metal salt is a calcium salt, preferably calcium chloride.

[0291] Item 14. The method according to Item 13, wherein a tangential flow filtration step is performed after the addition of the alkaline earth metal salt.

[0292] Item 15. The method according to any one of the preceding items, wherein step d) comprises performing anion exchange (AEX) chromatography.

[0293] Item 16. The method according to Item 15, wherein AEX chromatography is performed in a combined mode.

[0294] Item 17. The method according to Item 15 or 16, wherein during AEX chromatography, ccc DNA molecules bound to AEX resin are washed with an alkaline washing buffer.

[0295] Item 18. The method according to any one of items 15 to 17, wherein during AEX chromatography, stepwise gradient elution of ccc DNA molecules is used.

[0296] Item 19. The method according to any one of the preceding items, wherein step d) comprises adding a potassium salt or an ammonium salt to the neutralized pyrolysis solution.

[0297] Item 20. The method according to Item 16, wherein the potassium salt is potassium phosphate and the ammonium salt is ammonium sulfate.

[0298] Item 21. The method according to any one of items 1 to 14, 19 and 20, wherein the method does not include performing anion exchange (AEX) chromatography.

[0299] Item 22. The method according to any one of items 1 to 14, 19 to 21, wherein step d) does not include performing anion exchange (AEX) chromatography.

[0300] Item 23. The method according to any one of the preceding items, wherein step d) comprises performing hydrophobic interaction chromatography or thiophilic interaction chromatography.

[0301] Item 24. The method according to Item 23, wherein an aromatic resin is used for hydrophobic interaction chromatography.

[0302] Item 25. The method according to Item 24, wherein a further ultrafiltration / percolation step is performed after hydrophobic interaction chromatography.

[0303] Item 26. The method according to any one of the preceding items, wherein the diameter of the pipe system in step a) is in the range of 2 to 50 mm, preferably in the range of 4 to 40 mm.

[0304] Item 27. The method according to any one of the preceding items, wherein in step (b), the pyrolysis composition is passed through a tubular system.

[0305] Item 28. The method according to Item 27, wherein the diameter of the pipe system in step b) ranges from 2 to 50 mm, preferably from 5 to 30 mm.

[0306] Item 29. The method according to any one of the preceding items, wherein in step c), the lysis solution is passed through a pipe system.

[0307] Item 30. The method according to Item 29, wherein the diameter of the pipe system in step c) ranges from 2 to 60 mm, preferably from 4 to...

[0308] 52 mm.

[0309] Item 31. The method according to any one of the preceding items, wherein the flow velocity in step a) is in the range of 0.3 to 1.5 m / s, preferably in the range of 0.5 to 1.5 m / s.

[0310] Item 32. The method according to any one of items 26 to 30, wherein the pyrolysis composition described in step b) flows through the pipe system at a flow rate ranging from 0.05 to 1 m / s, preferably from 0.1 to 0.5 m / s.

[0311] Item 33. The method according to any one of items 29 to 32, wherein in step c), the pyrolysis fluid flows through the pipe system at a flow rate ranging from 0.3 to 1.5 m / s, preferably from 0.5 to 1.5 m / s.

[0312] Item 34. The method according to any one of the preceding items, wherein in step a), the viscosity of the composition is in the range of 1.2-1.3 mm. 2 / s (according to Ph.Eu. (European Pharmacopoeia) 9.8, Chapter 2.2.9, capillary viscometer method).

[0313] Item 35. The method according to any of the preceding items, wherein the ccc DNA molecule is selected from plasmids, granules, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors, or hybrids thereof.

[0314] Item 36. The method according to Item 30, wherein the ccc DNA molecule is a plasmid.

[0315] Item 37. The method according to any one of the preceding items, wherein the size of the ccc DNA molecule ranges from 2 kbp to 20 kbp.

[0316] Item 38. The method according to any one of the preceding items, wherein the microbial cell is a bacterium, preferably a Gram-negative bacterial cell.

[0317] Item 39. The method according to any one of the preceding items, wherein in step (b), the microbial cell is Escherichia coli.

[0318] Item 40. The method according to any one of the preceding items, wherein the pyrolysis agent comprises NaOH and SDS.

[0319] Item 41. The method according to any one of the preceding items, wherein the neutralizing solution comprises an acetate buffer, preferably potassium acetate, or ammonium acetate and potassium acetate.

[0320] Item 42. The method according to any one of the preceding items, wherein the yield of ccc DNA molecules after step c) is at least 65%.

[0321] Item 43. The method according to any one of the preceding items, wherein the homogeneity of the ccc DNA molecules obtained by step c) is at least 70% ccc, preferably at least 80%, more preferably at least 90% ccc, for example at least 92%, at least 93%, or at least 95% ccc.

[0322] Item 44. The method according to any one of the preceding items, wherein the method is performed on a large scale.

[0323] Item 45. The method according to any one of the preceding items, wherein microbial cells with a fermentation volume of 500 l or more, preferably 800 l or more, more preferably 1000 l or more are used.

[0324] Item 46. The method according to any one of the preceding items, wherein in step b), the length of each pyrolysis tube is 30 m.

[0325] Item 47. The method according to any one of the preceding items, wherein the lysis solution from the two parallel tubes is combined into one tube after step b) and before step c).

[0326] Item 48. The method according to any one of the preceding items, wherein the pyrolysis composition in step b) is passed through the piping system at a pumping rate ranging from 100 to 200 L / h, preferably from 123 to 160 L / h, and more preferably from 140 to 160 L / h.

[0327] Item 49. The method according to any one of the preceding items, wherein the pyrolysis composition in step a) is passed through the piping system at a pumping rate ranging from 100 to 200 L / h, preferably from 123 to 160 L / h, and more preferably from 140 to 160 L / h.

[0328] Item 50. The method according to any one of the preceding items, wherein the pyrolysis composition in step c) is passed through the piping system at a pumping rate ranging from 100 to 200 L / h, preferably from 120 to 160 L / h, and more preferably from 140 to 150 L / h.

[0329] Item 51. ccc DNA molecules, which are obtained by the method described in any one of items 1 to 43.

Claims

1. A method for isolating ccc DNA molecules from microbial cells containing covalently closed circular (ccc) DNA molecules, comprising the following steps: a) Contacting microbial cells with a lysis agent and passing the composition through a tubular system at a Reynolds number of at least 3000 to obtain a lysis composition; b) Incubate the pyrolysis composition to obtain a pyrolysis solution; c) Contact the pyrolysis solution with a neutralization solution to obtain a neutralized pyrolysis solution; as well as d) Further process the neutralized lysis buffer to obtain ccc DNA molecules. Step b) is carried out in two parallel tubes.

2. The method according to claim 1, wherein microbial cells with a fermentation volume of 500 l or more, preferably 800 l or more, more preferably 1000 l or more are used.

3. The method according to claim 1 or 2, wherein in step b), the length of each pyrolysis tube is 30 m.

4. The method according to any one of the preceding claims, wherein the lysis solutions from the two parallel tubes are combined into one tube after step b) and before step c).

5. The method according to any one of the preceding claims, wherein in step a), the composition flows through the pipe system at a Reynolds number of at least 3300, preferably 3500 to 6000, more preferably 4000 to 5500, and even more preferably 5000 to 5500.

6. The method according to any one of the preceding claims, wherein step b) comprises passing the pyrolysis composition through a pipe system at a Reynolds number of at least 2100, and wherein step c) comprises passing the neutralized pyrolysis solution through a pipe system at a Reynolds number of at least 2100.

7. The method according to claim 6, wherein in step b), the Reynolds number is at least 2100, preferably 2100 to 6000, more preferably 2500 to 5500, and even more preferably 2500 to 3000.

8. The method according to claim 6 or 7, wherein in step c), the Reynolds number is at least 3000, preferably 3500 to 6500, more preferably 4000 to 6100.

9. The method according to any one of the preceding claims, wherein step b) lasts for 6 minutes or less, preferably 2 to 6 minutes, more preferably 3 to 6 minutes.

10. The method according to any one of the preceding claims, wherein the diameter of the pipe system in step a) is in the range of 2 to 50 mm, preferably in the range of 4 to 40 mm, 10 to 30 mm, or 15 to 25 mm.

11. The method according to any one of the preceding claims, wherein in step b), the pyrolysis composition is passed through a tubing system, preferably wherein the diameter of the tubing system in step b) is in the range of 2 to 50 mm, preferably in the range of 5 to 30 mm, 10 to 30 mm, or 15 to 25 mm.

12. The method according to any one of the preceding claims, wherein in step c), the lysis solution is passed through a pipe system.

13. The method according to claim 12, wherein the diameter of the pipe system in step c) ranges from 2 to 60 mm, preferably from 4 to... 52 mm, preferably 20 to 30 mm.

14. The method according to any one of claims 10 to 13, wherein the pyrolysis composition in step b) is passed through the piping system at a pumping rate ranging from 100 to 200 L / h, preferably from 123 to 160 L / h, and more preferably from 140 to 160 L / h.

15. The method according to any one of the preceding claims, wherein the yield of ccc DNA molecules after step c) is at least 65% and / or wherein the homogeneity of the ccc DNA molecules obtained by step c) is at least 70% ccc, preferably 80%, more preferably 90% ccc.

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