WO2025027059A1

WO2025027059A1 - Method for purification of plasmid dna

Info

Publication number: WO2025027059A1
Application number: PCT/EP2024/071645
Authority: WO
Inventors: Alisa STROBEL; Oliver RAMMO; Eric VOLK; Duc Do
Original assignee: Merck Patent Gmbh
Priority date: 2023-08-02
Filing date: 2024-07-31
Publication date: 2025-02-06

Abstract

The present invention relates to a method for the chromatographic purification of plasmid DNA. For this phosphate is added to the wash and/or elution buffer in anion exchange chromatographic purification of plasmid DNA (pDNA).

Description

Method for purification of plasmid DNA

The present invention relates to a method for the chromatographic purification of plasmid DNA. For this phosphate is added to the wash and/or to the elution buffer in anion exchange chromatographic purification of plasmid DNA (pDNA).

The demand for rapid and efficient methods for obtaining high-purity nucleic acids like plasmid DNA from biological sources is constantly increasing, owing to the increasing importance of recombinant DNA for exogenous expression or therapeutic applications. In particular, the demand for purification methods which can also be carried out on a larger scale is also increasing. The use of highly pure plasmid DNA is crucial in various applications like polymerase chain reaction (PCR) amplification, DNA sequencing, in vitro mRNA synthesis, and subcloning of transgenes. Therefore, protocols for generating plasmid DNA with high yield and quality have earned serious attention.

Many known methods for the purification of, in particular, relatively large amounts of nucleic acids like plasmid DNA include a chromatographic purification step. The efficiency of this step generally also determines the efficiency and effectiveness of the manufacturing process.

The major challenge in the production of pDNA is the separation of the target supercoiled (scDNA) isoform from other impurities to achieve the target purity without significantly reducing the process yield. The isolation of scDNA from impurities such as RNA, proteins, genomic DNA, and inactive isoforms (linear and open circular pDNA) is difficult due to their similar physiochemical characteristics such as negative charge and molecular mass.

The removal of these impurities usually requires a plurality of subsequent purification steps, anion-exchange chromatography being one possibility. In WO20174085 plasmid DNA is purified by contacting the sample comprising pDNA with an anion exchange material in the presence of a kosmotropic salt and eluting the pDNA with a chaotropic salt.

Although it has been tried to optimize anion-exchange chromatographic purification of plasmid DNA, there is still a need for a process combining enhanced performance and high yield with high purity.

Downstream processes in the biopharmaceutical and biotechnological industries often rely on chromatographic steps with bead-based resins in a packed-bed column as the stationary phase. The resin beads typically have diameters between 30 and 500 pm and generally provide an efficient chromatographic technique with high binding capacity. In addition, several other stationary phases, including monoliths and membranes, have been developed in the last few decades as possible alternatives to classical chromatographic supports. The main advantage of using membranes or monoliths is attributed to short diffusion times, as the interactions between molecules and active sites in the membrane or monolith occur in convective through-pores rather than in stagnant fluid inside the resin pores. Therefore, membrane and monolith chromatography have the potential to operate at high flow rates and low pressure drops.

But as described above membrane or monolith-based chromatography, among others due to the absence of pore diffusion and the higher flow rates, might show different chromatographic behavior and thus different separation properties.

It has been found that when using certain types of wash and/or elution buffers, plasmid DNA purification by anion exchange chromatography can be improved either on bead based matrices as well as on a membrane or monolith as chromatographic matrix When using a wash and/or elution buffer comprising phosphate, impurities can be more efficiently separated and removed from the target pDNA material, allowing both for high yields and high purity in the final product.

The present invention is therefore directed to a method for purifying plasmid DNA comprising a) Providing a sample comprising said plasmid DNA b) Loading the sample onto a chromatography matrix comprising anion exchange groups c) Washing the chromatography matrix with a wash buffer d) Eluting plasmid DNA bound to the chromatography matrix with an elution buffer whereby the wash buffer or the elution buffer or the wash buffer and the elution buffer comprise at least 50 mM phosphate (PO4 ).

In another preferred embodiment the wash buffer comprises 50 to 2500 mM, preferably 150 to 2500 mM, 250 to 2000 mM phosphate (PO4 )■

In another preferred embodiment the wash buffer comprises 50 to 1500 mM, preferably 150 to 1000 mM, chloride (Cl’).

In another preferred embodiment the elution buffer comprises 50 to 2500 mM, preferably 150 to 2500 mM, 250 to 2000 mM phosphate (PCU’ )■

In a preferred embodiment the elution buffer additionally comprises 50 to 2000 mM, preferably 150 to 2000 mM, chloride (Cl’).

In a preferred embodiment, in step b), the sample that is loaded onto the chromatography matrix comprises chloride (Cl’) in a concentration between 50 mM and 400 mM, preferably between 100 and 200 mM. In another preferred embodiment the wash and the elution buffer do not comprise any detergent.

In a preferred embodiment the chromatography matrix is a membrane, most preferred a hydrogel membrane.

In one embodiment the plasmid DNA is supercoiled plasmid DNA (sc pDNA).

In another embodiment, the removal of RNA in the purified plasmid DNA obtained in step d) compared to the sample provided in step a) is at least 90%, preferably at least 96%, most preferred at least 98%. That means at least 90%, preferably at least 96%, most preferred at least 98% (w/w) of the RNA impurities which are present in the sample provided in step a) are removed by the method of the present invention.

In another embodiment the yield of the purified plasmid DNA in step d) is at least 70%, preferably at least 85%, most preferred at least 90%.

In a preferred embodiment the anion exchange groups comprise trimethylammonium (-CH2N(CH3)3⁺) or triethylammonium (-CH₂CH₂N(CH₂CH3)3⁺.

Figures:

Figure 1 shows the chromatogram of a chromatographic purification process according to the present invention. The pDNA only eluted during the higher second peak in the elution. Further details can be found in the Examples.

Figure 2 shows the HPLC analysis of the fractions obtained in the chromatographic purification process of Figure 1. Further details can be found in the Examples. Figure 3 shows a chromatogram of a gradient elution according to the state of the art without phosphate present in the wash 2 and elution buffers. The pDNA and the impurities elute in one peak. Further details can be found in the Examples.

Figure 4 shows the chromatogram of a chromatographic purification process according to the present invention. Further details can be found in the Examples under Process 2.

Figures 5 to 9 show the HPLC analysis of the fractions obtained in the chromatographic purification process of Figure 4. Further details can be found in the Examples under Process 2.

Figure 10 shows the HPLC analysis of elution with different phosphate concentrations. Further details can be found in the Examples under Process 4.

Definitions

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such may vary. It must be noted that, as used in this specification and the appended claims, the singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a ligand" includes a plurality of ligands and reference to "an antibody" includes a plurality of antibodies and the like.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. The following terms are defined for purposes of the invention as described herein. The term "plasmid DNA" refers to any distinct nucleic acid entity that is not part of or a fragment of a host cell's primary genome. As used herein, the term "plasmid DNA" may refer to either circular or linear molecules composed of DNA or DNA derivatives. The term "plasmid DNA" may refer to either single stranded or double stranded molecules. Plasmid DNA includes naturally occurring plasmids as well as recombinant plasmids encoding a gene of interest including, e.g., marker genes or therapeutic genes.

Plasmids are typically epigenomic circular DNA molecules having a length of between 4 and 20 kB, which corresponds to a molecular weight of between 2.6x10⁶ and 13.2x10⁶ Daltons often capable of autonomous replication in a producing cell. Even in their compact form (super coil), plasmid DNA molecules normally have a size of several hundred nm.

Plasmid DNA often exists in different conformations: apart from the "native" super-coiled (sc) conformation, plasmid DNA may also be present in open circular (oc), or even in linear form. Since super-coiled plasmid DNA (sc pDNA) typically represents the desired (and commercially relevant) conformation for plasmid DNA, the biomolecule of interest in some embodiments of this aspect of the present invention is sc pDNA. The plasmid DNA to be purified/isolated may typically comprise mammalian DNA, bacterial DNA, non-coding DNA, or viral DNA. In some instances, the plasmid DNA will comprise DNA capable of expressing a polypeptide of interest. The purification method will generally not depend on the size of the plasmid DNA, i.e. minivectors with only 350 bp as well as large plasmid vectors comprising up to 20 genes (35 kbp), and anything in-between may be effectively purified by the method described herein.

As used herein, and unless stated otherwise, the term “sample” refers to any composition or mixture that contains plasmid DNA. Samples may be derived from biological or other sources. Biological sources include eukaryotic and prokaryotic sources, such as plant and animal cells, tissues and organs. The sample may also include diluents, buffers, detergents, and contaminating species, debris and the like that are found mixed with the target molecule, in this case plasmid DNA. The sample may be "partially purified" (i.e., having been subjected to one or more purification steps, such as filtration steps) or may be obtained directly from a host cell or organism producing the nucleic acid (e.g., the sample may comprise harvested cell culture fluid).

The term "impurity" or “contaminant” as used herein, refers to any foreign or objectionable molecule, including one or more host cell proteins, endotoxins, lipids, nucleic acids and one or more additives which may be present in a sample containing the plasmid DNA that is being separated from one or more of the foreign or objectionable molecules using the process of the present invention.

The terms "purifying," "separating," or "isolating," as used interchangeably herein, refer to increasing the degree of purity of the target plasmid DNA from a composition or sample comprising the target plasmid DNA and one or more impurities. Typically, the degree of purity of the target nucleic acid is increased by removing (completely or partially) at least one impurity from the composition.

The term "chromatography" refers to any kind of technique which separates an analyte of interest (e.g. a target plasmid DNA) from other molecules present in a sample like impurities. Usually, the target plasmid DNA is separated from other molecules as a result of differences in rates at which the individual molecules of the mixture bind to and/or migrate through a chromatography matrix under the influence of a moving phase.

The term "matrix" or "chromatography matrix" are used interchangeably herein and refers to a solid phase though which the sample migrates in the course of a chromatographic separation. The matrix typically comprises a base material and ligands covalently bound to the base material. The matrix of the present invention comprises or consists of particles, a membrane or a monolith, preferably the base material is a membrane or monolith, most preferred a membrane.

A “ligand” is a functional group that is part of the chromatography matrix, typically it is attached to the base material of the matrix, and that determines the binding properties and interaction properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interactions groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the aforementioned). It is also possible that one ligand has more than one binding I interaction property. The matrix of the present invention comprises at least anion exchange groups. Anion exchange groups can be subdivided into strong anion exchange groups, such as quaternary ammonium groups like trimethylammonium (-CH2N(CH3)3⁺) or triethylammonium (- CH₂CH₂N(CH₂CH3)3⁺ as well as weak anion exchange groups, such as ammonium (NHs⁺), N,N diethylamino or diethyl-aminoethyl DEAE. The matrix may additionally comprise further other types of ligands so that the matrix is a mixed mode matrix. Such ligands may e.g., have hydrophobic interaction groups, such as phenyl, butyl, propyl, hexyl. The groups may be part of the base material, they may also be part of a ligand. One ligand may comprise one or several different anion exchange groups. According to the present invention, the term anion exchange chromatography matrix covers matrices which only comprise one or more types of anion exchange groups as well as matrices comprising one or more types of anion exchange groups in combination with other types of functional groups like hydrophobic interaction groups, i.e. , mixed mode matrices.

The ligands can be attached to the base material of the matrix by any type of covalent attachment. Covalent attachment can for example be performed by directly bonding the functional groups to suitable residues on the base material like OH, NH2, carboxyl, phenol, anhydride, aldehyde, epoxide or thiol etc. It is also possible to attach the ligands via suitable linkers. It is also possible to generate the matrix by polymerizing monomers comprising the ligands and a polymerizable moiety. Examples of matrices generated by polymerization of suitable monomers are polystyrene, polymethacrylamide or polyacrylamide-based matrices generated by polymerizing suitable styrole or acryloyl monomers.

In another embodiment the stationary phase can be generated by grafting the ligands onto the base material or from the base material. For grafting from processes with controlled free-radical polymerisation, such as, for example, the method of atom -transfer free-radical polymerisation (ATRP), are suitable. A very preferred one-step grafting from polymerisation reaction of acrylamides, methacrylates, acrylates, methacrylates etc. which are functionalized e.g. with ionic, hydrophilic or hydrophobic groups can be initiated by cerium(IV) on a hydroxyl-containing support, without the support having to be activated.

When the chromatography matrix is used in a chromatographic separation it is typically used in a separation device, also called housing, as a means for holding the matrix.

In one embodiment, the device comprises a housing with an inlet and an outlet and a fluid path between the inlet and the outlet. In a preferred embodiment the device is a chromatography column. Chromatography columns are known to a person skilled in the art. They typically comprise cylindrical tubes or cartridges filled with the stationary phase as well as filters and/or means for fixing the stationary phase in the tube or cartridge and optionally connections for solvent delivery to and from the tube or cartridge. The size of the chromatography column varies depending on the application, e.g. analytical or preparative. In one embodiment the column or generally the separation device is a single use device. The term "anion exchange matrix" is thus used herein to refer to a chromatography matrix which carries at least anion exchange groups. That means it typically has one or more types of ligands that are positively charged under the chromatographic conditions used, such as quaternary amino groups.

A "buffer" is a solution that resists changes in pH by the action of its acidbase conjugate components. Various buffers which can be employed depending, for example, on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non- limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations of these.

According to the present invention the term “buffer” or “solvent” is used for any liquid composition that is used to load, wash, elute, re-equilibrate, strip and/or sanitize the chromatography matrix.

Detergents are defined as “surface active molecules”. The term "detergent" as used herein refers to compounds that lower the surface tension between two liquids or between a liquid and a solid. Detergents may act as wetting agents, emulsifiers, foaming agents, and dispersants. Examples of detergents are non-ionic, ionic or zwitterionic detergents.

When “loading” a chromatography column in bind and elute mode, the sample or composition comprising the target molecule and one or more impurities is loaded onto a chromatography column. In preparative chromatography, the sample can be loaded directly without the addition of a loading buffer. If a loading buffer is used, the buffer has a composition, a conductivity and/or pH such that the target nucleic acid is bound to the stationary phase while ideally all the impurities are not bound and flow through the column. Typically, the loading buffer, if used, has the same or similar composition as the equilibration buffer used to prepare the column for loading.

The final composition of the sample loaded on the column is called feed. The feed may comprise the sample obtained from clarification and the loading buffer.

By “wash” or "washing" a chromatography matrix is meant passing an appropriate liquid, e.g. a buffer through or over the matrix. Typically washing is used after loading to remove weakly bound contaminants from the matrix in bind/elute mode prior to eluting the target molecule. Additionally, wash steps can be used to reduce levels of residual detergents, enhance viral clearance and/or alter the conductivity carryover during elution.

To "elute" a molecule (e.g. the target plasmid DNA) from a matrix means that the molecule is removed therefrom. Elution may take place by altering the solution conditions such that a buffer different from the loading and/or washing buffer competes with the molecule of interest for the ligand sites on the matrix or alters the equilibrium of the target molecule between stationary and mobile phase such that it favors that the target molecule is preferentially present in elution buffer.

A non-limiting example is to elute a molecule from an ion exchange resin by altering the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material.

Elution can be performed by using a buffer gradient. A gradient typically starts with an elution buffer similar to the equilibration buffer or wash buffer and is then, in the course of the gradient, changed so that some of its properties, like pH, conductivity or composition, differ more and more from the equilibration buffer. This causes elution conditions which at the beginning provide adequate compound retention so that not all compounds will immediately elute from a chromatography column. Compounds that are retained less elute first. By further changing the elution buffer in the course of the gradient compounds that are retained stronger on the matrix will then also elute from the matrix. An elution gradient thus ideally separates the target molecule from its impurities by causing elution at different elution buffer composition.

There are two main gradient designs:

-Linear gradient - a linear change of a least one property like pH or conductivity in the elution buffer

-Step gradient - a stepwise change in least one property like pH or conductivity in the elution buffer

Elution can also be performed by isocratic elution.

The term “conductivity" as used herein, refers to an inherent property of most materials, that quantifies how strongly it resists or conducts electric current. In aqueous solutions, such as buffers, the electrical current is carried by charged ions. The conductivity is determined by the number of charged ions, the amount of charge they carry and how fast they move. Hence, for most aqueous solutions, the higher the concentration of dissolved salts, the higher the conductivity. Raising the temperature enables the ions to move faster, hence increasing the conductivity. Typically, the conductivity is defined in room temperature, if not otherwise indicated. The basic unit of conductance is Siemens (S). It is defined as the reciprocal of the resistance in Ohms, measured between the opposing faces of a 1 cm cube of liquid. Therefore, the values are estimated in mS/cm. A membrane as chromatographic matrix can be distinguished from particle-based chromatography by the fact that the interaction between a solute, e.g. the target nucleic acids or contaminants, and the matrix does not take place in the dead-ended pores of a particle, but mainly in the throughpores of the membrane. Exemplary types of membranes are flat sheet systems, stacks of membranes, microporous polymer sheets with incorporated cellulose, polystyrene or silica-based membranes as well as radial flow cartridges, hollow fiber modules and hydrogel membranes. Preferred are hydrogel membranes. Such membranes comprise a membrane support and a hydrogel formed within the pores of said support. The membrane support provides mechanical strength to the hydrogel. The hydrogel determines the properties of the final product, like pore size and binding chemistry.

The membrane support can consist of any porous membrane like polymeric membranes, ceramic based membranes and woven or nonwoven fibrous material. Suitable polymeric materials for membrane supports are cellulose or cellulose derivatives as well as other preferably inert polymers like polyethylene, polypropylene, polybutylenterephthalate or polyvinylidene-difluoride.

The hydrogels can be formed through in-situ reaction of one or more polymerizable monomers with one or more crosslinkers and/or one or more cross-linkable polymers to form a cross-linked gel that has preferably macropores. Suitable polymerizable monomers include monomers containing vinyl or acryl groups. Preferred are monomers comprising an additional functional group that either directly forms the ligand of the matrix or is suitable for attaching the ligands. Suitable crosslinkers are compounds containing at least two vinyl or acryl groups. Further details about suitable membrane supports, monomers, crosslinkers etc. as well as suitable production conditions can be found in WO04073843 and WO2010/027955. Especially preferred are membranes made of an inert, flexible fiber web support comprising assembly within and around the fiber web support a porous polyacrylamide hydrogel with quaternary ammonium groups (strong anion exchange groups), like Natrix® Q Chromatography membrane, Merck KGaA, Germany.

Depending on the membrane device used, the respective processes are conducted by different operating principles like dead-end operation, crossflow operation and radial flow operation systems. Dead-end operation is preferred.

Examples of suitable membranes to be used in the method of the present invention are

- Membranes with a polyethersulfone (PES)-based support and a crosslinked polymeric coating, functionalized with quaternary ammonium groups (strong anion exchange groups), like Mustang® Q, Pall.

- Membranes made of stabilized reinforced cellulose, functionalized with quaternary ammonium groups (strong anion exchange groups) or with DEAE groups (diethylaminoethyl, weak ion exchange groups), like Sartobind® membranes, Sartorius.

- Membranes made of stabilized reinforced cellulose, comprising a hydrogel with quaternary ammonium groups (strong anion exchange groups), like Sartobind® Jumbo Membranes made of stabilized reinforced cellulose, functionalized with quaternary ammonium groups (strong anion exchange groups), like Sartobind® Jumbo membranes, Sartorius.

- Membranes made of a fine fiber non-woven scaffold comprising a hydrogel with quaternary ammonium groups (strong anion exchange groups), like 3M^TM Emphaze™ AEX Hybrid Purifier, 3M.

- Membranes made of an inert, flexible fiber web support comprising within and around the fiber web support a porous polyacrylamide hydrogel with quaternary ammonium groups (strong anion exchange groups), like Natrix® Q Chromatography membrane, Merck KGaA, Germany. A monolith or a monolithic sorbent, similar to a membrane, has throughpores, like interconnected channels, so that liquid can flow from one side of the monolith, through the monolith, to the other side of the monolith.

Since the mobile phase is flowing through these throughpores, molecules to be separated are transported by convection rather than by diffusion. Due to their structure monolithic sorbents show flow rate independent separation efficiency and dynamic capacity.

The monolith is typically formed in situ from reactant solutions and can have any shape or confined geometry, typically with frit-free construction, which guarantees convenience of operation. Preferably, monolithic materials have a binary porous structure, mesopores and macropores. The micron-sized macropores are the throughpores and ensure fast dynamic transport and low backpressure in applications; mesopores contribute to sufficient surface area and thus high loading capacity.

The monoliths can be made of organic, inorganic or organic/inorganic hybrid materials. Preferred are organic polymer-based monoliths.

The synthesis of organic polymer monoliths is typically done by a one- step polymerization providing a tunable porous structure with tailored functional groups. Generally, a pre-polymerization mixture consisting of the monomers, crosslinkers, porogenic solvents, and initiators in an appropriate ratio is polymerized in a suitable container, also called mould, determining the format of the monolith. Polymerization is typically initiated by heating, use of UV radiation, microwave or y-ray radiation in the presence of initiators. After reaction for the prescribed time at an appropriate temperature, the resulting material is typically washed with solvents to remove unreacted components and porogenic solvents.

Suitable organic polymers are polymethacrylates, polyacrylamides, polystyrenes, polyurethanes, etc., like Poly(methacrylic acid-ethylene dimethacrylate), Poly(glycidyl methacrylate-ethylene dimethacrylate) or Poly(acrylamide-vinylpyridine-N,N'-methylene bisacrylamide).

Inorganic monoliths can be made of silica or other inorganic oxides. Preferably they are made of silica. Silica monoliths are normally prepared via a sol-gel method with phase separation. This mainly includes hydrolysis, condensation, and polycondensation of silica precursors. Typically, tetraethoxysilane (TEOS) or tetramethylorthosilicate (TMOS) is distributed in a suitable solvent in the presence of a porogen (e.g. poly(ethylene glycol) (PEG)), followed by the addition of a catalyst, acid or base, or a binary catalyst, acid and base in sequence. After reaction for a prescribed time, the resulting gel-like product is washed with solvents to remove unreacted precursor, porogen, and catalyst, followed by the proper post treatment, typically a heat treatment.

The monoliths can be modified with suitable functional groups, preferably at least ion exchange groups, to generate the targeted interaction with the sample comprising the target molecule and thus the targeted separation.

Membranes and monoliths can also be produced by 3D printing processes.

Typically, the monoliths are contained in a housing like a column.

Particle-based resins intended for liquid chromatography are normally comprised of particles that are packed together in a tubular cylinder called column to form a bed. The packed bed shows a distinct space between the particles, so called void volume, which mainly defines the liquid fluid permeability and hydrodynamic properties of the packed bed.

The particles typically consist of a cross-linked polymer matrix in spherical, bead-like or granular shape with relatively uniform size for improved chromatographic and hydrodynamic characteristics of the packed bed. They can have a dense structure with discrete or very small pores but usually exhibit a porous multichannel or reticular structure forming an inner pore volume and additional surface area inside the particle. The particle surface area can be modified with a variety of functional groups suitable for chromatography applications either by using functional monomers for the backbone-polymer structure, coupling of functional groups to the particle surface directly of via ligands or short polymers structures (grafts).

Particulate base materials can be prepared, for example, from organic polymers. Organic polymers of this type can be polysaccharides, such as agarose, dextranes, starch, cellulose, etc., or synthetic polymers, such as poly(acrylamides), poly(methacrylamides), poly(acrylates), poly(methacrylates), hydrophilic substituted poly(alkyl allyl ethers), hydrophilic substituted poly(alkyl vinyl ethers), poly(vinyl alcohols), poly(styrenes) and copolymers of the corresponding monomers. These organic polymers can preferably also be employed in the form of a crosslinked hydrophilic network. This also includes polymers made from styrene and divinylbenzene, which can preferably be employed, like other hydrophobic polymers, in a hydrophilized form.

Alternatively, inorganic materials, such as silica, zirconium oxide, titanium dioxide, aluminium oxide, etc., can be employed as particulate base materials. It is equally possible to employ composite materials, i.e., for example, particles which can themselves be magnetised by copolymerisation of magnetisable particles or of a magnetisable core. It is also possible to use core shell materials whereby the shell, i.e. at least the surface or a coating, has OH groups.

However, preference is given to the use of hydrophilic base materials which are stable to hydrolysis or can only be hydrolysed with difficulty since the materials according to the invention should preferably withstand alkaline cleaning or regeneration at e.g., basic pH over an extended use duration.

The base matrix may consist of irregularly shaped or spherical particles, whose particle size can be between 2 and 1000 pm. Preference is given to average particle sizes between 3 and 300 pm, in a most preferred embodiment the average particle size is between 20 - 63 pm.

The particulate base material may be in the form of non-porous or preferably porous particles. The average pore sizes can be between 2 and 300 nm. Preference is given to pore sizes between 5 and 200 nm, most preferred average pore size is between 40 - 110 nm.

In a very preferred embodiment, the particulate base material is formed by copolymerisation of a hydrophilic substituted alkyl vinyl ether selected from the group of 1 ,4-butanediol monovinyl ether, 1 ,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclo-'hexane-'dimethanol monovinyl ether and divinylethyleneurea (1 ,3- divinylimidazolin-2-one) as crosslinking agent.

An example of a suitable commercially available vinylether based base material is Eshmuno®, Merck KGaA, Germany.

In a preferred embodiment the polymer to be used as a particulate matrix in the method of the present invention is derivatised by graft polymerisation with tentacle-like structures, which can in turn carry the corresponding ligands or be functionalised by means of the latter. The grafting is preferably carried out in accordance with EP 0 337 144 page 12 example 8 or US 5453186 page 9 example 8 using N-(2- Trimethylammoniumethyl)-acrylamide and/or another monomer carrying suitable functional groups. The polymerisation catalyst employed is cerium(IV) ions, since this catalyst forms free-radical sites on the surface of the base material, from which the graft polymerisation of the monomers is initiated. The polymerisation is terminated by termination reactions involving the cerium salts. For this reason, the (average) chain length can be influenced by the concentration ratios of the base material, the initiator and the monomers. Furthermore, uniform monomers or also mixtures of different monomers can be employed; in the latter case, grafted copolymers are formed.

Suitable monomers for the preparation of the graft polymers and further details about the grafting procedure are e.g., disclosed in WO 2007/014591 , EP 0337 144, especially page 12, example 8 and US 5453186 page 9, example 8.

Preferably the matrix is derivatised with cationic groups by graft polymerisation whereby the resulting chains that are grafted onto the base material have a length of between 2 and 100, preferably 5 and 60, in particular between 10 and 30 monomer units, each unit typically carrying one cationic group.

The matrix might carry additional other functional groups like hydrophobic or hydrophilic groups in addition to the anion exchange groups but in any case, it has anion exchange groups.

Preferred particulate matrices are matrices with weak anion exchange and/or strong anion exchange groups, e.g., with trimethylammoniumethyl (TMAE) groups, like Eshmuno® Q or Fractogel® TMAE, Merck KGaA, Germany, or with dimethylethanolamine (DMAE) groups, like Fractogel® DMAE, Merck KGaA, Germany, or with diethylaminoethyl (DEAE) groups like Fractogel® DEAE, Merck KGaA, Germany

The base material may equally also be in the form of fibres, hollow fibres or coatings.

Detailed Description The plasmid DNA to be purified according to the method of the present invention may originate from any natural, genetic-engineering or biotechnological source, such as, for example, prokaryotic cell cultures. If plasmid DNA from cell preparations is to be purified, the cells are firstly digested by known methods, such as, for example, lysis. If the sample to be purified has already been pre-treated in another way, lytic digestion is unnecessary. For example, the sample can be obtained from biological material by removal of the cell debris and a precipitate of RNA, from nucleic acid samples which have already been pre-purified and, for example, are present in buffer, or alternatively from nucleic acid solutions which have been formed after amplification. Filtration, precipitation or centrifugation steps may be necessary. The person skilled in the art is able to select a suitable digestion method depending on the source of the plasmid DNA to be purified. The sample is preferably a lysate obtained from cells, such as, for example, a clarified lysate.

Clarification can be a centrifugation and/or microfiltration process in which relatively larger components such as lysed cells and/or impurities are removed from a solution. Clarification filters include depth filtration, charged depth filtration and similar microfiltration techniques.

Centrifugation can for example be a low-speed centrifugation to remove larger particles like cellular debris. This can be for example done at 10000 to 12000 g for 10 to 30 minutes. The target plasmid DNA can be found in the supernatant.

The resulting sample is a clarified lysate, also called clarified sample.

For the purification of plasmid DNA from E. coli, the cells are, for example, firstly lysed by alkaline lysis with NaOH/SDS solution. Addition of an acidic potassium-containing neutralization buffer then causes the formation of a precipitate, which can be removed by centrifugation or filtration. The clear supernatant remaining, the clarified lysate, can be employed as starting material, i.e. as sample, for the method according to the invention. It is also possible firstly to concentrate or pre-purify the clarified lysate by known methods, such as dialysis or precipitation.

The sample comprising the plasmid DNA and potentially other impurities from which the plasmid DNA shall be purified, e.g. RNA, is then subjected to a chromatographic separation on a chromatography matrix comprising anion exchange groups. For this the sample is loaded onto the chromatography matrix. The final composition of the sample loaded onto the matrix is called the feed. In one embodiment of the present invention the feed does not comprise any detergent. In another embodiment the feed comprises chloride (Ch) in a concentration between 50 mM and 400 mM, preferably between 100 and 200 mM.

Typically, the concentration of the chloride in the feed, if present, is adjusted by adding a suitable amount of a chloride salt, such as potassium chloride or preferably sodium chloride. The optimal concentration of chloride can be determined using a batch assay in microtiter plate format or by screening chromatography studies, measuring plasmid binding capacity at increasing chloride salt concentrations. The optimal chloride concentration in the sample to be loaded is molecule specific. The goal is to maximize the yield, i.e. to maximize the loading of the target plasmid DNA, and to minimize the impurities. This requires evaluation of the dynamic binding capacity for the specific target material and the chromatography device.

Preferably, the feed does not comprise any significant amounts of phosphate or any other kosmotropic ions, with the exception of ions added during the cell lysis process, e.g 1.0 M acetate. That means the sample that is loaded on the chromatography matrix does not comprise phosphate or any other kosmotropic salt in a concentration above 200 mM, preferably it does not comprise phosphate or any other kosmotropic salt beside acetate in a concentration above 50 mM. Typically, the feed preparation and also the chromatographic separation are performed at or around room temperature. But it is also possible to work at other temperatures, e.g. between 5 and 40 °C.

The feed preferably is adjusted to an electrolytic conductivity between 40 to 90 mS/cm, most preferably to 75 and 85 mS/cm.

Conductivity adjustment is done by addition of salt, salt concentrate solutions, or, respectively, dilution with a low conductivity buffer or neat water. For feed conductivity adjustment by salt supplementation preferably sodium or potassium chloride are used, but any other salt commonly used in purification applications such as e.g. salts from sulfate, acetate, carbonate/bicarbonate, phosphate or citrate might be considered as well depending on the adjustment of the concentration of chloride, the adjustment of the conductivity, and effect on the binding capacity of the matrix.

The feed typically shows pH values between 4.5 to 5.5 but the method might also be conducted to feeds showing pH values ranging from 4.0 up to 9.0.

Column equilibration buffer are typically buffers matching the pH and conductivity of the feed loaded onto the chromatography material. Typically buffers with pH below 6.0 and conductivity between 40 to 90 mS/cm are selected but buffers out of that range are applicable as well.

Suitable, exemplary equilibration buffers comprise between 500 and 1500 mM, preferably around 1 M, acetate and chloride concentration between 100 and 200 mM. The equilibration buffer pH is for example around 5.0 and conductivity between 60 and 90 mS/cm.

After loading the matrix is washed with at least one wash buffer. The wash buffer might be identical to the equilibration buffer or different from the equilibration buffer. The matrix might also be washed with 2, 3 or 4 different wash buffers. Preferably, one of the wash buffers comprises 50 to 2500 mM, preferably 150 to 2500 mM, more preferred 250 to 2000 mM phosphate (PO4 ), and 50 to 1500 mM, preferably 150 to 1000 mM, chloride (Ch). The content of phosphate and chloride need to be adjusted so that the target plasmid DNA is not eluted. The skilled person can choose a suitable combination of phosphate and chloride based on a few experiments.

It is known that the addition of detergents to the feed, the wash buffer and/or the elution buffer can especially improve the removal of endotoxins from plasmid DNA. But the removal of detergents from the purified sample might cause problems. With the method of the present invention, typically, endotoxin removal can be performed without the need to add detergents or with less detergents.

Preferably, none of the one or more wash buffers or the elution buffer comprises a detergent. Alternatively, wash buffers and/or elution buffers used in the method of the present invention provide equivalent removal of endotoxins with less detergent compared to a process using the same method and buffers but without more than 50 mM phosphate.

Preferably the pH and the ionic strength of the first wash buffer is identical or similar to the pH and the ionic strength of the equilibration buffer and the load feed.

Preferably, the pH of the wash buffer comprising more than 50 mM phosphate is between pH 7 and 10, preferably between pH 8 and 9. The conductivity and ionic strength of the wash buffer comprising phosphate is typically different than that of the equilibration buffer and the load feed. In a preferred embodiment, at least two wash buffers are used whereby the pH and the ionic strength of the first wash buffer is identical or similar to the pH and the ionic strength of the equilibration buffer and the load feed and the first wash buffer does not comprise more than 50 mM phosphate and one of the following wash buffers, preferably the second wash buffer comprises more than 50 mM phosphate.

Elution of the target plasmid DNA is then done by using an elution buffer. If one of the wash buffers comprises above 50 mM, preferably between 250 and 2500 mM, phosphate, in one embodiment, the elution buffer does not comprise phosphate.

In another embodiment, independent of the composition of the wash buffer, the elution buffer comprises between 50 and 2500 mM phosphate, most preferred between 250 and 2500 mM. The elution buffer has a different pH and/or different ionic strength than the equilibration/loading buffer.

Phosphate can e.g. be added to the wash and/or elution buffer by the addition of sodium and/or potassium phosphates like the mono- sodium/potassium phosphates, the di- sodium/potassium phosphates or the tris-odium/potassium phosphates, most preferred dipotassium hydrogenphosphate (K2HPO4).

In one embodiment the elution buffer has a higher pH and/or a higher ionic strength than the equilibration/loading buffer. In one embodiment the pH of the elution buffer is above pH 7, preferably between pH 8.0 and 9.5. In one embodiment the elution buffer comprises between 500 and 1500 mM chloride, typically sodium chloride.

Elution can be performed isocratically, in one step, with an elution buffer that is not changed during elution. Elution can also be performed by gradient elution with linear or stepwise change of the elution buffer.

The term “column volume” (CV) refers to the volume inside of a packed column or generally packed housing. This volume includes the chromatography matrix, the interstitial volume (volume outside of the matrix), and the own internal porosity (pore volume) of the matrix. If the matrix is a membrane the column volume is typically called the membrane volume (MV).

In a preferred embodiment, for elution, the applied linear or step gradient lasts around 50 to 500 column/membrane volumes (CV/MV) plus an optional additional hold step at target elution buffer for at least 10 CV/MV.

In a very preferred embodiment, elution is done with a buffer comprising phosphate and chloride. In case of gradient elution the phosphate content of the elution buffer is preferably kept constant while the chloride content is increased, e.g. from 0 to 100 mM to 500 to 2000 mM.

In another embodiment, the applied single step wash and elution steps in which the buffer is not changed last around 3 to 150 CV/MV. In the case of single step wash and elution, the phosphate and chloride concentrations are kept constant in the respective buffers and steps.

Preferably, in the method of the present invention a sample comprising a clarified lysate and 50 to 400 mM chloride is loaded on a chromatography matrix comprising anion exchange groups. The matrix is washed with at least one wash buffer comprising 50 to 2000 mM phosphate and 50 mM to 1500 mM chloride. Elution is performed with an elution buffer comprising between 0 and 2000 mM phosphate and 50 to 2000 mM chloride.

In a preferred embodiment, the chromatography matrix is a membrane. It has been found that the method of the present invention even works when using membranes at flow rates between 5 and 20 MV/min.

By performing the method of the present invention, the target plasmid DNA can be obtained with high yields and high purity. The inventors have found that, unexpectedly, the addition of phosphate to the wash buffer and/or the elution buffer has an influence on the elution of the target plasmid DNA and impurities like RNA and open circular plasmid DNA that also bind to the chromatography matrix. The presence of phosphate in the wash and/or elution buffers seems to provide for stabilization of the binding of the target plasmid DNA to the chromatography matrix so that the impurities are eluted prior to the target plasmid DNA.

This finding provides more flexibility in the purification of plasmid DNA, since depending on the phosphate concentration in the buffer, the ionic strength, usually relating to the chloride concentration, necessary for pDNA elution, varies. Up to now, there was the need to find a balance between loading efficiency of the target plasmid DNA and binding of impurities. If too many impurities were bound to the chromatography matrix they could not be removed by anion exchange chromatography so that often reduction in yield had to be accepted to gain high purity. The method of the present invention offers the possibility to gain both, high yield and high purity as the improved removal of bound impurities allows for optimal loading of plasmid DNA despite potential additional binding of impurities. The impurities can now be removed by using an elution and/or a wash buffer comprising phosphate. Based on the method described above it is possible to remove at least 90%, preferably at least 96%, most preferred at least 98% of the RNA impurities.

The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

The entire disclosure of all applications, patents, and publications cited above and below are hereby incorporated by reference. Examples

The following examples represent practical applications of the invention.

Protocols for Plasmid DNA Capture

For the small volume membrane screening devices where the system holdups are disproportionately large, very high volumes were used for wash 1 , wash 2, elution, cleaning in place (CIP) and equilibration. At larger scale these values can be reduced and flow direction reversed for enhancing individual steps. This is a standard practice and common knowledge for any one proficient in art.

Chromatography Materials

Buffers

Buffer composition of Equilibration buffer. Adjusted to pH 5.0 with acetic acid.

Chemical M (g/mol) C (mol/L)

Sodium chloride 58.44 0.110 - 0.190

Potassium acetate 98.14 1.000

Buffer composition of Wash 2 buffer. Adjusted to pH 9.0 with phosphoric acid.

M

Chemical C (mol/L)

(g/mol)

Tris(hydroxymethyl)aminomethane 121.14 0.100 Dipotassium hydrogenphosphate 174.18 2.000

Buffer composition of Elution A buffer. Adjusted to pH 9.0 with phosphoric acid.

Chemical M (g/mol) C (mol/L)

Tris(hydroxymethyl)aminomethane 121.14 0.100

Dipotassium hydrogenphosphate 174.18 1.000

Buffer composition of Elution B buffer. Adjusted to pH 9.0 with phosphoric acid.

Chemical M (g/mol) C (mol/L)

Tris(hydroxymethyl)aminomethane 121.14 0.100

Dipotassium hydrogenphosphate 174.18 1.000

Sodium Chloride 58.44 1.000

Process 1

Method description for AKTA systems

Step volume / Flow rate /

Step Buffer

MVor mL mL/min

Equilibration 50 MV / 10 mL 2 mL/min Equilibration buffer

Load 2 mL/min Adjusted cell lysate material

Wash 1 50 MV / 10 mL 2 mL/min Equilibration buffer

Wash 2 50 MV / 10 mL 1 mL/min Wash 2 buffer

Gradient 500 MV / 1 mL/min Elution A buffer

Elution* 100 mL Elution B buffer

Strip 1 25 MV / 5 mL 1 mL/min Strip buffer

CIP 50 MV / 10 mL 1 mL/min CIP solution

Strip 2 25 MV / 5 mL 2 mL/min Strip buffer

Re- 50 MV / 10 mL 2 mL/min Equilibration buffer equilibration * Gradient elution mixes Elution A and Elution B buffers. The mixture starts with 100% Elution A buffer and 0% Elution B buffer changing over the 500 MV of the step duration to 0% Elution A buffer and 100% Elution B buffer.

Plasmid Feed

8 kb plasmid DNA from E. coli clarified cell lysate. The sample was supplemented with NaCI in the range between 100 and 190 mM required for optimal binding of pDNA to the chromatography matrix.

Process

The clarified harvest is adjusted to the target salt concentration with 1 .0 M potassium acetate, 3.0 M sodium chloride (NaCI), pH 5.0 buffer.

This buffer minimizes changes in the cell lysate content and minimizes the dilution. After the load step, a wash step with equilibration buffer is performed to complete the loading and flush out any leftover load material. This is followed by a second wash with 0.100 M TRIS, 2.0 M dipotassium phosphate (K2HPO4), pH 9.0 for partial removal of any bound RNA. For this chromatography process a gradient elution is used to collect the bound material. The gradient increases the NaCI concentration from 0.0 M to 1 .0 M at a rate of 0.01 M per 1 mL process volume. At the same time the remaining components and conditions are kept constant at 0.100 M TRIS, 1.0 M K2HPO4, pH 9.0.

Results

Figure 1 shows the chromatogram from the run performed with the conditions described above. The UV detection is absorbance at 256 nm. Peaks can be observed during the Wash 2 step and the gradient elution. Through HPLC analysis it was observed that there was no pDNA present in the wash peak and in the first peak during the elution. The pDNA only eluted during the higher second peak in the elution. Figure 2 shows the HPLC analysis of the wash 2 fraction, and the two elution peaks using UV absorbance at 260 nm. The HPLC analysis of the collected fractions showed that there is no pDNA eluting during the wash step and the first peak in the elution. Those peaks primarily contain impurities. pDNA was only present in the second peak during the gradient elution.

Figure 3 shows a chromatogram of a gradient elution without phosphate present in the wash 2 and elution buffers. The process steps and loading amount are the same as the process in Figure 1. During the gradient elution, the pDNA and the RNA eluted together resulting in a single peak. HPLC analysis was performed to confirm this. This shows that in absence of phosphate in the elution buffer the impurities cannot be separated.

Process 2

The general information on materials and buffers given above also applies for this process unless defined otherwise below.

Results

The results are shown in Figures 4 to 9. Overall, one can see that wash 2 with a wash buffer comprising phosphate but no sodium chloride some impurities can be removed. But wash 3 with a wash buffer comprising phosphate and sodium chloride the removal of impurities is more efficient.

Figure 4 shows the process chromatogram of application of step wash and elution with phosphate for purification of pDNA.

Figure 5 shows the HPLC chromatogram of purified pDNA material, which is used as standard to identify pDNA isoform peaks. Peak at approximately 6.2 min corresponds to open circular pDNA, while peak at 7.5 min corresponds to covalently closed circular pDNA.

Figure 6 shows the HPLC chromatogram of initial cell lysate material used to load the membrane for chromatography. The larger peaks correspond to RNA and genomic DNA impurities, while the small peaks close to 6.2 min and 7.5 min correspond to the pDNA material. Figure 7 shows the HPLC chromatogram of Wash 2 fraction. No pDNA was detected in the collected fraction, only genomic DNA, which is considered an impurity.

Figure 8 shows the HPLC chromatogram of Wash 3 fraction. No pDNA was detected in the collected fraction, only genomic DNA, which is considered an impurity.

Figure 9 shows the HPLC chromatogram of the Elution fraction. Only pDNA in different isoforms was detected in the sample.

Process 3

Buffers

Buffer composition of Wash 2 buffer. Adjusted to pH 9.0 with phosphoric acid.

Buffer composition of Wash 3 buffer. Adjusted to pH 9.0 with phosphoric acid.

Buffer composition of Elution buffer. Adjusted to pH 9.0 with phosphoric acid.

Method description for AKTA systems

the start as 100%, flushing out the previous step buffer.

Plasmid feed

8 kb plasmid DNA from E.coli clarified cell lysate. The sample was supplemented with NaCI in the range between 100 and 190 mM required for optimal binding of pDNA to the chromatography matrix.

Process

The clarified harvest is adjusted to the target salt concentration with 1 .0 M potassium acetate, 3.0 M sodium chloride (NaCI), pH 5.0 buffer. This buffer minimizes changes in the cell lysate content and minimizes the dilution. After the load step, a wash step with equilibration buffer is performed to complete the loading and flush out any leftover load material. This is followed by a second wash with 0.100 M TRIS, 1 .0 M dipotassium phosphate (K2HPO4), pH 9.0 for partial removal of any bound RNA and genomic DNA. For this chromatography process a third wash step is utilized to further remove bound impurities. The buffer used in the third wash is composed of 0.1 OO M TRIS, 1.0 M dipotassium phosphate (K2HPO4), 0.700 M sodium chloride (NaCI), adjusted to pH 9.0. This is followed by a step elution to collect the bound pDNA material. The elution buffer is applied immediately at the start of the step. The elution buffer is composed of 0.1 OO M TRIS, 1.0 M dipotassium phosphate (K2HPO4), 1.5 M sodium chloride (NaCI), adjusted to pH 9.0.

This shows that also a step elution with a buffer comprising phosphate and sodium chloride is efficient.

Process 4

Figure 10 shows the results of gradient elution using elution buffers with different phosphate concentrations. For all the runs shown, the loadings were performed to the same target and at the same conditions. The NaCI concentration is linearly increasing from 0.0 to 1.5 M. Phosphate concentration is kept constant during the elution. This shows that the concentration of phosphate in the elution buffer has an influence on target elution.

Claims

1 . A method for purifying plasmid DNA comprising a) Providing a sample comprising said plasmid DNA b) Loading the sample onto a chromatography matrix comprising anion exchange groups c) Washing the chromatography matrix with a wash buffer d) Eluting plasmid DNA bound to the chromatography matrix with an elution buffer whereby the wash buffer and/or the elution buffer comprise at least 50 mM phosphate (PO ).

2. Method according to claim 1 , characterized in that the elution buffer and/or the wash buffer additionally comprises chloride.

3. Method according to claims 1 or 2, characterized in that the wash buffer comprises 50 to 2500 mM phosphate (PO ).

4. Method according to one or more of claims 1 to 3, characterized in that the elution buffer comprises 50 to 2500 mM phosphate (PO )

5. Method according to one or more of claims 1 to 4, characterized in that, in step b), the sample that is loaded onto the chromatography matrix comprises chloride (Ch) in a concentration between 50 mM and 400 mM.

6. Method according to one or more of claims 1 to 5, characterized in that the wash and the elution buffer do not comprise any detergent.

7. Method according to one or more of claims 1 to 6, characterized in that the chromatography matrix is a membrane.

8. Method according to one or more of claims 1 to 7, characterized in that the plasmid DNA is super-coiled plasmid DNA (sc pDNA).

9. Method according to one or more of claims 1 to 8, characterized in that at least 90% of the RNA comprised in the sample provided in step a) is removed in the plasmid DNA obtained in step d).

10. Method according to one or more of claims 1 to 9, characterized in that the yield of the recovered purified plasmid DNA obtained in step d) is at least 70%.

11. Method according to one or more of claims 1 to 10, characterized in that the anion exchange groups comprise trimethylammonium (- CH₂N(CH₃)3⁺) and/or triethylammonium (-CH2CH2N(CH2CH3)3⁺and/or diethyl-aminoethyl and/or dimethylethanolamine.

12. Method according to one or more of claims 1 to 11 , characterized in that the sample comprises a clarified lysate.

13. Method according to one or more of claims 1 to 12, characterized in that step d) is performed by gradient elution with an elution buffer comprising a constant concentration of phosphate (PO4 ) between 50 to 2500 mM and an increasing amount of chloride (Ch) starting from a concentration between 0 and 100 mM to a concentration between 500 to 2000 mM.

14. Method according to one or more of claims 1 to 13, characterized in that step c) is performed with a wash buffer comprising a concentration of phosphate (PO4 ) between 50 to 2500 mM and a concentration of chloride (Ch) between 0 mM to 1500 mM.