Method for the continuous separation of nucleic acids from a liquid medium

Abstract

The present invention is directed to a method for the continuous separation of nucleic acid molecules from a liquid medium in a separation cell (1), wherein the cell (1) comprises a separation chamber (2), comprising an inlet (3) and an outlet (4), which define therebetween a fluid path (5), and at least one first electrode (6) and at least one second electrode (7) arranged along, preferably parallel to said fluid path, the electrodes being arranged and configured to generate an electric field (E) therebetween, preferably proximal to the fluid path, wherein the method comprises at least one adsorption phase followed by at least one desorption phase, both based on the principle of electrosorption, and to the separation cell (1) applicable in this method.

Description

[0001] CO12408 / WI GR

[0002] METHOD FOR THE CONTINUOUS SEPARATION OF NUCLEIC ACIDS FROM A LIQUID MEDIUM

[0003] The present invention is directed to a method for the continuous separation of nucleic acid molecules from a liquid medium in a separation cell (1 ) , wherein the cell (1 ) comprises a separation chamber (2 ) , comprising an inlet (3) and an outlet (4 ) , which define therebetween a fluid path (5) , and at least one first electrode ( 6) and at least one second electrode (7 ) arranged along, preferably parallel to said fluid path, the electrodes being arranged and configured to generate an electric field (E) therebetween, preferably proximal to the fluid path, wherein the method comprises at least one adsorption phase followed by at least one desorption phase, both based on the principle of electrosorption, and to the separation cell (1 ) applicable in this method.

[0004] Nucleic acid therapeutics are rapidly advancing for treating genetic diseases, cancer, and viral infections, and the production process of the extrachromosomal plasmid DNA (pDNA) in microorganisms such as Escheri chia coli is inexpensive and well-established. Thus, nucleic acid separation is a fundamental technique in molecular biology, essential for isolating DNA and RNA from a complex mix of cellular materials . Traditionally, this process is performed using batch techniques such as centrifugation, chromatographic methods such as anion exchange chromatography or hydrophobic interaction chromatography, or electrophoresis, each with specific requirements and limitations . These methods often involve labor-intensive steps, are time-consuming, and are limited in their ability to handle large volumes of material, which restricts their scalability and throughput .CO12408 / WI GR

[0005] 2

[0006] US 2001 / 0008762 Al is directed to an electrostatic separation approach and discloses nucleic acid captured from a mixture of cellular debris produced by cell lysis by exposing the mixture to an electrode and applying a voltage to the electrode which is then removed from the mixture carrying said nucleic acid. This application is solely limited to batchwise separation, which is associated with severe limitations .

[0007] With the increasing demand for high-throughput nucleic acid analysis in fields like diagnostics, research, and therapeutic development, there is an urgent need for improved methods for separating, isolating or purifying nucleic acids, especially for continuous separation processes .

[0008] A continuous method offers several advantages : it reduces processing time, allows for automation and scalability, ensures consistent quality of the separated nucleic acids, and improves overall efficiency. By transitioning from batch to continuous processes, laboratories can achieve higher throughput, reduced operational costs, and better integration into streamlined workflows . Despite these clear benefits, achieving effective nucleic acid separation continuously remains a significant challenge, primarily due to the delicate nature of nucleic acids and the complex interactions involved in their isolation .

[0009] This application addresses the problem of efficiently separating nucleic acid molecules from complex liquid mixtures in a continuous manner .

[0010] The application provides solutions for the challenges associated with traditional nucleic acid separation methods, which often involve batch processing, lengthy processing times, and difficulties in maintaining the integrity of nucleic acids during separation . The need for a continuous separation process that allows for effective adsorption and desorption of nucleicCO12408 / WI GR

[0011] 3

[0012] acids while minimizing contamination and loss of target molecules is critical in various applications, including molecular biology, diagnostics, and therapeutic development . The described method aims to enhance the efficiency and reliability of nucleic acid purification by utilizing an electric field in a separation cell, thereby addressing the limitations of existing techniques .

[0013] In contrast to US 2001 / 0008762 Al, the present invention facilitates the continuous separation of nucleic acids by utilizing a separation cell with electrodes that generate an electric field, allowing for adsorption and subsequent desorption phases, offering higher efficiency and adaptability for various nucleic acid types compared to the batch process described. While US 2001 / 0008762 Al provides a foundational approach to nucleic acid separation, it lacks the continuous operation, precise control, and versatility offered by the current invention ' s claimed method.

[0014] The problem is solved by a first aspect of the present invention directed to a method for the continuous separation of nucleic acid molecules from a liquid medium in a separation cell (1 ) ,

[0015] wherein the cell (1 ) comprises a separation chamber (2 ) , comprising an inlet (3) and an outlet (4 ) , which define therebetween a fluid path (5) , and at least one first electrode ( 6) and at least one second electrode (7 ) arranged along, preferably parallel to said fluid path, the electrodes being arranged and configured to generate an electric field (E) therebetween, preferably proximal to the fluid path,

[0016] wherein the method comprises at least one adsorption phase followed by at least one desorption phase,

[0017] the adsorption phase comprising:CO12408 / WI GR

[0018] 4

[0019] (a) Continuously providing a separation mixture (M) of at least one nucleic acid molecule in a liquid medium along the fluid path (5) ,

[0020] (b) Generating said electric field (E) by applying voltage to the at least one first and at least one second electrode, wherein the at least one nucleic acid molecule adsorbs to the at least one first electrode,

[0021] (c) Maintaining said electric field (E) over a certain adsorption time period ta,

[0022] (d) Optionally, washing the at least one first electrode and the at least one second electrode by replacing the flow of the separation mixture (M) along the fluid path (5) with a flow of a washing liquid,

[0023] and the desorption step comprising:

[0024] (e) Replacing the flow of the separation mixture (M) , or optionally the flow of the washing liquid (if a washing step (d) has been carried out in the adsorption step) , along the fluid path (5) with a flow of a desorption medium (D) along the fluid path (5) ,

[0025] (f ) Desorbing the at least one nucleic acid molecule from the at least one first electrode into the desorption medium (D) by modifying the electrical field (E) and / or by convection of the at least one first electrode within the chamber (2 ) ,

[0026] (g) Optionally, collecting the desorption medium (D) comprising the at least one nucleic acid molecule when leaving the chamber (2 ) via the outlet (4 ) .CO12408 / WI GR

[0027] 5

[0028] The continuous provision of a separation mixture along the fluid path ensures a steady-state operation, which can lead to higher throughput and efficiency in nucleic acid separation compared to batch processes .

[0029] The use of an electric field to adsorb nucleic acid molecules to electrodes provides a selective and controllable means of separation, potentially resulting in higher purity of the isolated nucleic acids .

[0030] The optional washing step allows for the removal of contaminants and impurities, which can further increase the purity of the nucleic acids collected during the desorption phase .

[0031] The separation mixture (M) according to the invention is to be understood as a mixture comprising the liquid medium and the at least one nucleic acid molecule . The at least one nucleic acid molecule is to be separated by applying the method according to the invention .

[0032] The adsorption time period ta preferably ranges 1 to 120 min .

[0033] Preferably, desorbing according to step (f ) is carried out for a desorption time period tn ranging from 1 to 120 min .

[0034] The separation mixture (M) preferably enters the separation chamber (2 ) via the inlet (3) and leaves the separation chamber (2 ) via the outlet (4 ) .

[0035] Alternatively, the separation mixture (M) can be cycled through the cell in step (a) by fluidly connecting the outlet (4 ) with the inlet (3) . In particular, cycling is achieved by a transportation means, such as a pump, transferring the separation mixture (M) leaving the chamber (2 ) via the outlet (4 ) to the inlet (3) into the chamber (2 ) .CO12408 / WI GR

[0036] 6

[0037] During the adsorption phase, the mixture (M) is depleted from the at least one nucleic acid molecule when passing the cell along the fluid path (5) , preferably amounting to at least 70 % compared to the original content of the at least one nucleic acid molecule, more preferably at least 80 %, even more preferably at least 90 % . In particular, the mixture (M) is essentially depleted from the at least one nucleic acid molecule when passing the cell along the fluid path (5) . The at least one nucleic acid adsorbs onto the at least one first electrode .

[0038] The washing liquid preferably enters the separation chamber (2 ) via the inlet (3) and leaves the separation chamber (2 ) via the outlet ( 4 ) .

[0039] The desorption medium (D) preferably enters the separation chamber (2 ) via the inlet (3) and leaves the separation chamber (2 ) via the outlet (4 ) .

[0040] During the desorption phase, the desorption medium (D) is enriched with the at least one nucleic acid molecule when passing the cell along the fluid path (5) . The at least one nucleic acid desorbs from the at least one first electrode .

[0041] Replacing different liquids (separation mixture (M) , washing liquid, desorption medium (D) ) with each other can be realized by a valve in liquid connection to the inlet (3) .

[0042] In a preferred method, volumetric flow rates of the desorption medium (D) through the chamber (2 ) during the desorption phase are higher than volumetric flow rates of the separation mixture (M) through the chamber (2 ) during the adsorption phase .

[0043] This ensures that adsorbed nucleic acid molecules are desorbed into the desorption medium (D) quantitatively.CO12408 / WI GR

[0044] 7

[0045] Employing higher volumetric flow rates during the desorption phase can enhance the efficiency of nucleic acid elution, reducing the time required for the desorption step .

[0046] The increased flow rates during desorption can help to prevent clogging and fouling of the electrodes, maintaining the performance of the separation cell over time .

[0047] Faster desorption flow rates can lead to a more concentrated nucleic acid product, as the desorbed molecules spend less time in the separation chamber, reducing dilution .

[0048] During the adsorption phase, preferred volumetric flow rates of the separation mixture (M) through the chamber (2 ) range from 0.1 to 10 cv / min, more preferably 0.1 to 1 cv / min .

[0049] "cv" according to the invention represents column volumes, i . e . the volume of the chamber (2 ) without the volume of the at least one first and at least one second electrodes . For instance, suitable column volumes are in a range of 0.1 mL to 40000 mL, preferably 0.1 mL to 1000 mL, even more preferably 0.2 to 10 mL, in particular 0.5 to 2 mL .

[0050] High cv may require more than at least one first and at least one second electrode to ensure sufficient adsorption .

[0051] During the desorption phase, preferred volumetric flow rates of the desorption medium (D) through the chamber (2 ) range from 0.1 to 10 cv / min, preferably 1 to 5 cv / min .

[0052] The washing liquid can pass the chamber at volumetric flow rates ranging from 0.1 to 1 mL / min .CO12408 / WI GR

[0053] 8

[0054] By adjusting the flow rate, the amount of adsorbed nucleic acid - and consequently the amount desorbed - can be precisely controlled. A low flow rate during the adsorption step and a high flow rate during the desorption step significantly increase the desorbed amount .

[0055] Preferably, the total amount of the at least one nucleic acid molecule in the separation mixture (M) is lower in the separation mixture (M) leaving the chamber (2 ) via the outlet (4 ) compared to the separation mixture (M) entering the chamber (2 ) via the inlet ( 3 ) .

[0056] The reduction of nucleic acid concentration in the separation mixture after passing through the chamber indicates effective adsorption, which can be correlated with high capture efficiency of the target molecules .

[0057] The amount of the at least one nucleic acid can be determined by known means in the art, such as UV260, fluorescence spectroscopy, probing, antibody detection .

[0058] Measuring the concentration of the at least one nucleic acid molecule within the chamber (2 ) at sequential time intervals allows for the monitoring of the kinetic behavior of the at least one nucleic acid molecule over time, thereby providing a time-resolved profile of the at least one nucleic acid concentration changes within the chamber (2 ) . Measuring can be achieved for example by applying a UV electrode prior and after the chamber (2 ) .

[0059] Monitoring the decrease in nucleic acid concentration can serve as an in-process control, providing real-time feedback on the separation process and allowing for adjustments to optimize performance .CO12408 / WI GR

[0060] 9

[0061] The ability to quantify the reduction in nucleic acid concentration can be used to calculate the binding capacity of the electrodes, aiding in the design and scaling of the separation system.

[0062] If more than one adsorption phase is carried out, washing of the electrodes can be carried out between at least two adsorption phases or between each of the absorption phases, wherein the washing comprises replacing the flow of the separation mixture (M) along the fluid path (5) with a flow of a washing liquid.

[0063] Preferably, the washing according to step (d) lasts for at least 2 to 10 cv, more preferably 2 to 5 cv.

[0064] In a preferred embodiment, modifying the electrical field (E) is achieved by deactivating or reversing the electrical field (E) , more preferably by reversing the electrical field (E) .

[0065] Deactivating or reversing the electrical field provides a simple and rapid means of inducing desorption, which can be easily integrated into automated systems for high-throughput applications .

[0066] It has surprisingly been shown that a combination of high flow rates during the desorption phase and reversing the electrical field (E) results in a more complete desorption of nucleic acids from the electrodes, potentially increasing the yield and reducing the risk of carry-over contamination in subsequent separation cycles .

[0067] The ability to modify the electrical field allows for the fine-tuning of the separation process, enabling the selective desorption of different nucleic acid molecules under different electrical conditions .CO12408 / WI GR

[0068] 10

[0069] Convection of the at least one first electrode within the chamber can be achieved by moving the at least one first electrode relatively to the flow of the desorption medium, preferably by vibrating the at least one first electrode, the chamber (2 ) and / or the cell (1 ) .

[0070] In a preferred embodiment, step (f ) is carried out by a combination of modifying the electrical field (E) and by convection of the at least one first electrode within the chamber, more preferably by a combination of reversing the electrical field (E) and by convection of the at least one first electrode within the chamber .

[0071] In an alternative embodiment, step (f ) is carried out by solely modifying the electrical field (E) .

[0072] Preferably, the adsorption phase is repeated at least once before conducting the desorption phase .

[0073] In this case, the flow of the first mixture (M) , which is depleted from the at least one nucleic acid molecule after performing the first adsorption phase is replaced by a flow of another identical separation mixture (M) of at least one nucleic acid molecule (identical to the nucleic acid molecule as before) in a liquid medium or another batch of the separation mixture (M) in step (a) of the repeated adsorption phase .

[0074] Preferably, the desorption medium is enriched with the least one nucleic acid when leaving the cell (2 ) via the outlet (4 ) by the means of step (f ) .

[0075] The liquid medium preferably comprises water, in particular DI water, and optionally further constituents selected from buffer systems, preferably biological buffer systems, such as Tris (Tris (hydroxymethyl ) aminomethane ) , HEPES (4- (2-hydroxyethyl) -1-CO12408 / WI GR

[0076] 11

[0077] piperazineethanesulf onio acid) , MOPS (3- (N-morpholino) propanesulf onio acid) , MES (2- (N-morpholino) ethanesulf onio acid) , Naphosphate buffer, citrate buffer, acetate buffer, salts, such as NaCl, KOI, NH4CI, Na2SO4, (NH4) 2SO4, CaCl2, Z1C12, LiCl2, MgCl2, CH3COOK, CHsCOONa, CH3COONH4 EDTA, PCR fragments and cell lysates, such as proteins, endotoxins, unwanted nucleic acids, NTPs and dNTPs and other biomolecules different from the at least one nucleic acid molecule to be separated.

[0078] The concentration of the salts preferably ranges from 1 to 2500 m , preferably 5 to 1000 mM, such as 10 mM.

[0079] The option to add various constituents to the liquid medium provides versatility in the method, allowing it to be tailored to separate specific nucleic acids or to handle complex samples such as cell lysates or biological fluids .

[0080] The washing liquid can be water, in particular DI water .

[0081] In this case, it is advisable to maintain the electrical field (E) during washing.

[0082] The desorption medium (D) can comprise, water, in particular DI water, and optionally further stabilizers for the at least one nucleic acid molecule to be separated.

[0083] The optional presence of stabilizers in the desorption medium provides versatility to the method, allowing for the customization of the medium to stabilize specific nucleic acid molecules and enhance the efficiency of their recovery.

[0084] Said stabilizers can be selected from Tris (Tris (hydroxymethyl ) aminomethane ) , HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulf onio acid) , MOPS (3- (N-CO12408 / WI GR

[0085] 12

[0086] morpholino) propanesulf onic acid) , MES (2- (N-morpholino) ethanesulf onio acid) , phosphate buffer, citrate buffer, acetate buffer salts, such as NaCl, KC1, NH4CI, Na2SO4, (NH4) 2SO4, CaCl2, Z1C12, L1C12, MgCl2, CH3COOK, CHsCOONa, CH3COONH4, EDTA.

[0087] The selection of stabilizers from a broad range of buffering agents and salts enables the fine-tuning of the separation medium to maintain the structural integrity and functionality of the nucleic acids during the separation process .

[0088] The use of common laboratory buffers and salts as stabilizers allows for easy replication of the method in different laboratory settings, facilitating standardization and consistency in nucleic acid separation protocols across various research and diagnostic applications .

[0089] Using a desorption medium based on water has several advantages :

[0090] • Dl-Water in the desorption medium passing the chamber (2 ) significantly weakens the electrical field (E) , thereby accelerating the desorption process of the at least one nucleic acid molecules, which have been adsorbed onto the at least one first electrode .

[0091] • According to the known means in the art, nucleic acid molecules are separated using anion exchange chromatography, which uses excessive amounts of salts during the desorption process from the exchange column . High salt loads present in the medium containing the separated nucleic acid molecules make subsequent use more difficult . The method according to the invention does not require such large salt loads .CO12408 / WI GR

[0092] 13

[0093] The liquid medium and / or the desorption medium may exhibit a pH value ranging from 4 to 9, preferably from 4 to 8 .

[0094] The control of the pH value within the range of 4 to 9 ensures that the separation process occurs under mild conditions, which preserves the biological activity of the nucleic acids and prevents hydrolytic damage .

[0095] The preferred pH range of 4 to 8 provides an optimal balance between the stability of the nucleic acids and the efficiency of their interaction with the separation matrix, leading to improved selectivity and yield in the separation process .

[0096] Deactivating of the electrical field (E) is carried out by generating a short-circuit on the electrodes, preferably for a time period ranging from 0.5 to 10 min .

[0097] In a preferred embodiment, the electrodes are carbon-based electrodes, more preferably consisting of carbon .

[0098] Carbon electrodes, especially those consisting entirely of carbon, are robust and resistant to corrosion, thereby increasing the durability of the separation apparatus and reducing the frequency of electrode replacement .

[0099] The carbon-based electrodes can be mechanically treated prior usage, such as by sandblasting.

[0100] The mechanical treatment of carbon-based electrodes, such as sandblasting, can increase the surface area, which in turn enhances the electrode ' s ability to establish more effective electrical contact with the at least one nucleic acid molecule to be separated.CO12408 / WI GR

[0101] 14

[0102] Such treatment can also remove surface contaminants and impurities, leading to improved electrode performance and longevity .

[0103] Preferably, the electrodes are porous electrodes exhibiting pore sizes ranging from 1 to 1000 nm and / or modal pore diameters ranging from 30 to 70 nm, measured by known means in the art, such as mercury infiltration .

[0104] Modal pore diameter is the diameter of the most frequently occurring pores . This value results as a peak in the distribution function .

[0105] In a preferred embodiment, the at least one first electrode represents the anode and the at least one second electrode represents the cathode .

[0106] The at least one nucleic acid molecules are in particular negatively charged and, thus, can adsorb to the positively charged anode .

[0107] The electrodes can be cylindrical electrodes, preferably exhibiting a length of 6 to 18 cm.

[0108] The electrodes can exhibit an accumulated specific pore surface area ranging from 15 to 30 m2g-1, preferably 18 to 25 m2g-1, determined by mercury inf iltration / porosimetry (e . g. conform to ISO 15901-1 ) .

[0109] The preferred range for specific pore surface area ensures that the electrodes have sufficient active sites for electrosorption, which can lead to higher capacitance and energy storage capabilities .CO12408 / WI GR

[0110] 15

[0111] Preferably, the electric field (E) exhibits a field strength ranging from 0 to 1400 V m-1(simulated via COMSOL Multiphysics software) .

[0112] The voltage applied to the electrodes can range from -1600 to 1600 mV.

[0113] Preferably, the at least one first electrode and the at least one second electrode are spaced from each other in a range of 0.1 to 3 cm.

[0114] The surface of the electrodes can be irregular, preferably characterized by multiline surface analysis revealing profile height deviations from the mean line Ra of (0. 95 ± 0.25) pm (arithmetic mean) and a maximum peak-to-valley height of the profile Rz of (5.7 1 1.3) pm, as determined by VK-X3000 3D laser scanning microscope .

[0115] The electrodes can exhibit a pore volume ranging from 150 to 260 cm3g-1, as determined by mercury inf iltration / porosimetry .

[0116] The electrodes can exhibit a total pore surface area ranging from 18 to 25 m2g-1] , as determined by mercury inf iltration / porosimetry .

[0117] The specified pore surface area of the electrodes ensures a high degree of accessibility for the at least one nucleic acid molecule to be separated, which can lead to improved separation performances .

[0118] Preferably, the at least one nucleic acid is a ribonucleic acid (RNA) , such as a mRNA, or desoxyribonucleic acid (DNA) , preferably a desoxyribonucleic acid with a length of 2 to 25 kbp, preferably a circular desoxyribonucleic acid (= plasmid DNA) , more preferably plasmid DNA of open circular (oc) andCO12408 / WI GR

[0119] 16

[0120] supercoiled (ccc) form, even more preferably of supercoiled form.

[0121] In a preferred embodiment, the mixture (M) contains at least one ribonucleic acid and at least one desoxyribonucleic acid and allows for selective separation of the at least one ribonucleic acid from the at least one desoxyribonucleic acid (i . e . the at least one desoxyribonucleic acid remains in the mixture (M) during the method according to the invention) or for selective separation of the at least one desoxyribonucleic acid from the at least one ribonucleic acid (i . e . the at least one ribonucleic acid remains in the mixture (M) during the method according to the invention) . Possible applications are the purification of ccc plasmid DNA or the separation of RNA from in vitro transcriptions .

[0122] It has surprisingly been shown that plasmid DNA as the at least one nucleic acid molecule to be separated can be selectively desorbed from the electrodes in the oc form if salts are present in the desorption medium (D) , preferably in amounts ranging from 5 to 10 mM.

[0123] In a preferred embodiment, the voltage applied to the electrodes is higher than -1600 and lower than 1600 mV, more preferably ranges from -1000 to 1000 mV.

[0124] Applying above voltage values, it has been surprisingly shown that a simultaneous separation of oc and ccc forms can be achieved .

[0125] Thus, the method according to the invention surprisingly allows for a selective separation of the at least one nucleic acid to be separated, such as for a selective separation of oc and ccc forms of plasmid DNA as the at least one nucleic acid molecule to be separated.CO12408 / WI GR

[0126] 17

[0127] Alternatively, the method according to the invention for the separation of nucleic acids from a liquid medium can also be used to enrich (concentrate) nucleic acids in a medium, by ensuring that the total volume of the desorption medium (D) is minimized. Preferably, the mixture (M) represents a mixture of at least one pre-purified nucleic acid molecules in a liquid medium.

[0128] Alternatively, the method according to the invention can be used to separate unwanted nucleic acid molecules (contaminants) , such as RNA or oc DNA, from the separation mixture (M) . In this case, the liquid medium leaving the cell (2 ) via the outlet (4 ) can be collected without these unwanted contaminants . The at least one desorption phase facilitates in a second process step the cleaning of the electrodes to which the unwanted nucleic acid molecules have been bound during the at least one adsorption phase .

[0129] A second aspect of the present invention is directed to a separation cell (1 ) for separating at least one nucleic acid molecule from a liquid medium, wherein the cell comprises : a separation chamber (2 ) , comprising an inlet (3) and an outlet (4 ) , which define therebetween a fluid path (5) for the liquid medium, a volume for containing the liquid medium provided along said fluid path, and at least one first electrode ( 6) and at least one second electrode (7 ) arranged along said fluid path, the electrodes being arranged and configured to generate an electric field (E) therebetween, preferably proximal to the fluid path, wherein the cell is a dynamic flow cell .

[0130] Figure 1 depicts a schematic flow cell according to the invention and shows the separation cell (1 ) containing a chamber (2 ) with an inlet (3) and an outlet (4 ) , defining a fluid path (5) . Inside the chamber (2 ) , a first electrode ( 6) and one second electrodeCO12408 / WI GR

[0131] 18

[0132] (7 ) are arranged along, in particular parallel to the fluid path (5) . An electric field (E) is generated between the electrodes, influencing the flow of the separation mixture . The design facilitates the adsorption and desorption phases for nucleic acid separation .

[0133] The configuration of the cell facilitates easy integration into larger systems, such as continuous flow reactors or analytical instruments, providing versatility in various industrial and laboratory settings .

[0134] In a preferred embodiment, the electrodes are carbon-based electrodes, preferably consisting of carbon .

[0135] The use of carbon-based electrodes, especially those consisting entirely of carbon, offers high electrical conductivity and chemical stability, which can result in longer-lasting and more reliable electrochemical devices .

[0136] In a preferred embodiment, the separation cell comprises one first and one second electrode .

[0137] The simplicity of the cell design with only two electrodes can facilitate ease of manufacturing and maintenance, as well as scalability for both small-scale laboratory use and large-scale industrial processes .

[0138] The cell can comprise at least two first electrodes and at least two second electrodes, preferably wherein two of the first electrodes are arranged side-by-side and two of the second electrodes are arranged side-by-side .

[0139] The first electrode can have a tubular structure (that is one electrode or several electrodes arranged in a circle) and eachCO12408 / WI GR

[0140] 19

[0141] of the at least one second electrode has a cylindrical structure and is arranged concentrically within the first electrode .

[0142] The tubular structure of the first electrode, combined with the concentric arrangement of the cylindrical second electrodes, maximizes the contact surface area between the electrodes and the at least one nucleic acid molecule to be separated, which can lead to increased separation performance .

[0143] The electrodes can be connected to a power supply via two small holes that allow for an electrical disconnect device . Two stainless steel wires can be inserted into these holes and sealed with silicone, two-component adhesive or similar to prevent liquid leakage . The external ends of the wires can connect to the power supply using crocodile clips .

[0144] The electrodes can be replaced within the cell and are preferably contacted from below through the wires .

[0145] The cell can be sealed using a sealing ring and a tri-clamp closure . This tri-clamp design facilitates easy replacement of the electrodes .

[0146] To ensure a secure and sealed connection within the cell, a special press-fit adapter can be attached to both ends of the flow cell, which uses a locking mechanism with counter plates, M6 screws, and nuts to maintain the necessary locking pressure without interfering with the connector to the cell .

[0147] Preferably, the cell (1 ) does not comprise another stationary phase, such as an ion exchange membrane .

[0148] Preferably, the at least one first electrode and the at least one second electrode is not based on carbon nano tubes .CO12408 / WI GR

[0149] 20

[0150] Figure 1 depicts a schematic flow cell according to the invention .

[0151] Figure 2 shows an exemplary flow cell for EMS experiments . Figure 3 shows an UV absorbance profile for an exemplary EMS run .

[0152] Figure 4 shows an UV absorbance profile for an exemplary EMS run .

[0153] Figure 5 shows an Agarose gel electrophoresis analysis of eluted pVAXl plasmid DNA.

[0154] Figure 6 shows an UV absorbance profile for an exemplary EMS run .

[0155] Figure 7 shows an Agarose gel electrophoresis analysis of Flue mRNA.CO12408 / WI GR

[0156] 21

[0157] Examples

[0158] The following examples are included to further describe embodiments of the invention . However, the present invention is not way limited to the following examples .

[0159] Preparation of pencil graphite electrodes (PGE)

[0160] Two types of cylindrical graphite pencil leads (Farber Castell, Germany) , 6B and 8B, were used as electrodes for electromodulated separation (EMS) experiments, each undergoing different preparation steps .

[0161] For the 6B pencil leads, which contain manufacturing additives such as wax, a burning process was conducted to remove these impurities . This burning process was carried out using a Fireboy Plus Bunsen burner (Integra Biosciences, Switzerland) with CV360 butane gas (Campingaz GmbH, Germany) under a fume hood. To prevent damage and cracking of the pencil leads during burning, the air and gas supplies were kept at low levels to ensure a controlled flame . The burning process was maintained until melted wax was no longer visible, and the pencil leads started to glow faintly. After cooling to room temperature, the 6B leads were cut into 60 mm lengths . The cut 6B leads were then immersed in 70% ethanol for 30 minutes to remove any wax residues . Afterward, they were wiped with KIMTECH tissue, placed in ddH2O for another 30 minutes, and wiped again . A second immersion in ddH2O was performed to ensure that all ethanol was removed. Following this step, the leads were equilibrated and stored in either ddH2O or in the respective buffer system used for the experiment . This equilibration step prevents the release of trapped air from the porous structure of the leads, which could otherwise form air bubbles during experiments and affect nucleic acids motion . Additionally, if water or electrolyte molecules enter the pores of the leads, it could increase the measuredCO12408 / WI GR

[0162] 22

[0163] concentration of the nucleic acids in solution, potentially altering the experimental outcomes . The 8B pencil leads did not contain additives or wax . Therefore, the burning step was not required. These 8B leads were cut into 60 mm, gently wiped with KIMTECH tissue to remove any dust, and stored in either ddH2O or in the respective buffer system used for the experiment until they were used in the experiments .

[0164] Design and set up of EMS flow cell

[0165] A continuous flow cell was designed in Inventor 2025 (Autodesk, Inc . , USA) and 3D-printed. The parts were printed in Clear Resin V4 (Formlabs, USA) , with a layer height of 25 pm, using a Formlabs 3B+ SLA printer (Formlabs, USA) . The flow cell can accommodate four pencil electrode mines (PGEs) , two as anodes and two as cathodes, in a small volume chamber . To ensure a tight fit and a constant distance of 1.5 mm between the two pairs of electrodes, four cylindrical cutouts and a removable spacer were implemented. For regular replacement of the PGEs, a Tri-clamp construction was implemented at the top part of the cell . This consists of two angled cylinders with a rubber seal in the middle that can be pressed together from the outside by a clamp . This allows the chamber to be opened and closed with a reduced risk of leakage . At the bottom part, two 1 mm channels allowed the connection with stainless steel wires between an external power supply and the PGEs inside the cell . The wire contacting the electrodes was fixed with 2-part epoxy adhesive (Loctite® HY 4070, Henkel AG & Co . KGaA, Germany) .

[0166] An AKTA pure™ chromatography system (Cytiva, USA) was used to transport liquid samples through the flow cell . To connect the system to a module the standard M6 male thread adapters (Cytiva, Sweden) with embedded capillary were used. Further, a special press-fit adapter system was designed and implemented at the inlet and outlet of the cell . The design incorporated and securedCO12408 / WI GR

[0167] 23

[0168] nylon M6 nuts for the mounting of the AKTA adapters . To seal this assembly, matching silicone seals were cutted. This allowed the nuts to be pressed on tightly, reducing the risk of a leakage .

[0169] Figure 2 shows an exemplary flow cell for EMS experiments . The design allows the removable mounting of two pairs of PGEs (2 anodes, 2 cathodes) with fixed spacing within the flow cell . The PEGs are connected to a power supply through two small holes to form the electric separation setup . With the Tri-Clamp design implemented, the PGEs can be removed easily. To ensure a sealed connection of the cell to an AKTA system, a special press-fit adapter can be implemented at both ends of the design .

[0170] The exemplary flow cell was designed to accommodate varying lengths, volumes, and electrode configurations . For the experiments, the flow cell according to Figure 2 was equipped with four 8B Faber-Castell pencil leads (0 2.8 mm) without wax mixture impregnation, each cut to a length of 60 mm. For the flow cell according to the experiment, the volume has been determined to be around 1 mL . The module was filled with DI-H2O to ensure that the sealing was free of air bubbles . Before the start of the experiment, a fixed potential was applied and a multimeter (Tacklife DM01M, Shenzhen Temie Technology Co . Ltd. , China) was used to check for proper contact between the electrode clamp, wire and electrode at all four electrodes in the open but filled module .

[0171] Electro modulated separation (EMS) with nucleic acids in flow cell

[0172] To evaluate the EMS of nucleic acids, preferably pDNA or mRNA, e . g. pVAXl (Thermo Fisher Scientific) or Flue mRNA (firefly luciferase mRNA, Promega) , an experimental setup was developed to enable the parallel operation of the AKTA system and aCO12408 / WI GR

[0173] 24

[0174] potentiostat (Gamry G750, Gamry Instruments Inc . , USA) , which applies external potential via the wires . The AKTA system' s UV monitor, located downstream of the column, was set to 260 nm to measure the absorbance of DNA (A260) or RNA, providing real-time information about its concentration eluting from the module .

[0175] During the EMS experiments, the potentiostat was operated in a two-electrode setup . Potentials were applied using chronoamperometry (CA) at - 1.0 V or -1. 6 V versus the open circuit potential (EOC) . For experiments conducted without an applied potential, the system was maintained at open circuit potential (OCP) . CA involves applying a step potential to the electrochemical cell and measuring the resulting current response over time . The CA method was initiated simultaneously with the corresponding AKTA method. Analysis of the CA scan curve provides insights into the charge transfer caused by the current response to each applied potential step .

[0176] The following steps give an overview of the method applied: Step AKTA System Potentiostat Gamry 1. Module washed with DI- Initial OCP applied; Equilibration H2O to establish a step potential constant UV baseline applied via CA to signal . fully form the electric field before Step 2 .

[0177] 2. Load & 50 pL of pDNA / mRNA Potential maintained Wash solution injected into at the same level to the AKTA loop and promote pDNA / mRNA carried through the adsorption onto the module; system reverts PGEs inside the to DI-H2O. module .CO12408 / WI GR

[0178] 25

[0179] 3. Elution 1 System wash with DI-H2O Potential adjusted to to remove unbound induce desorption of pDNA / mRNA. adsorbed pDNA / mRNA. 4. Elution 2 System wash with 1 M Potential switched NaCl or DI-H2O to off during this step , remove residual

[0180] pDNA / mRNA; optionally

[0181] combined with a

[0182] switching potential .

[0183] 5. Re- Module washed with DI- No potential applied, equilibration H2O to remove any

[0184] residual buffer or

[0185] pDNA / mRNA before the

[0186] next experiment .

[0187] During each step 5 times the column volume of the used flow cell is flowing through the flow cell . Using these defined steps, a control experiment as well as experimental runs were performed. The control experiment triplicate was conducted with the flow cell, whereby no potential (OCP) was applied during the whole experiment . Subsequently, experimental runs were performed in triplicate under varying conditions (e . g. potential magnitude and flow rate) to determine the optimal process parameters . A base experiment was conducted with a flow rate of 1 mL min-1and an applied potential of 1.0 V or 1. 6 V in Step 1 and Step 2 and switched to - 1.0 V or - 1. 6 V in Step 3. Later, the flow rates were adjusted for optimizing the method. For all experiments, 0.3 mg mL- 1pVAXl / Fluc mRNA in DI-H2O was injected. In selected experiments, fractions were collected for subsequent analysis .

[0188] For quantifying the amount of eluted pDNA / mRNA from the flow cell, quantitative measures for determining favorable experimental methods were defined. Therefore, a triplicate bypass injection of 0.3 g L-1pDNA / or a single bypass injection of 0.3 g L-1mRNA with a volume of 50 pL was done and the UVCO12408 / WI GR

[0189] 26

[0190] signal was recorded. This is identical to the sample used during experiments . The recorded average breakthrough peak area is a control for how much sample should be inside the module for any given experiment . Different quantitative results can be derived using this value, now called bypass peak area . For the calculation of the quantitative values, the UV signal of Elution2 and Re-equilibration were not directly taken into account . A measure for the effectiveness of the desorption is the relative total desorption (RTD) given in formula (1 ) .

[0191] desorption peak area

[0192] RTD= bypass peak area w

[0193] This value resembles the yield value often used in bioseparation processes and is known to the skilled person . Higher RTD percentages reflect higher yields for different methods .

[0194] pVAXl Gelelectrophoresis and PATfix analysis

[0195] Agarose gel electrophoresis was employed to assess the degradative effects of EMS on pVAXl . A 0.8 % (w / w) agarose gel was prepared by dissolving agarose (Sigma Aldrich, MA, USA) in lx TAE buffer . The TAE buffer used for electrophoresis was prepared from a 50x stock solution (ROTIPHORESE® 50x TAE Buffer, Carl Roth GmbH + Co . KG, Karlsruhe, Germany) . The stock solution was diluted to a lx working concentration (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.5) with deionized water before use . After cooling for 2 min, 3.5 pL of Midori Green Xtra staining dye (MG09 Sample, Nippon Genetics Europe, Germany) was added. pVAXl samples were mixed with TAE buffer and loading dye and then loaded into the gel with a 1 kb DNA ladder (N3232S; NEB, MA, USA) and / or Supercoiled DNA Ladder (New England Biolabs, USA) . Electrophoresis was conducted at 95 V (= 7 V cm- 1) for 55 min using a PowerEase Touch 120 W power supply (Invitrogen, CA, USA) . Band detection was performed using an Amersham Typhoon biomolecular imager (Cytiva, Sweden) at 488 nm, and data wereCO12408 / WI GR

[0196] 27

[0197] processed with ImageQuantTL to visualize and quantify the size distribution of the samples .

[0198] In addition to agarose gel electrophoresis, the PATfix® pDNA platform was employed for the quantitative analysis of supercoiled (sc) and open circular (oc) isoforms of pVAXl . This analysis was performed using PATfix® pDNA platform equipped with a 1.4 pm CIMac™ pDNA monolithic column (Sartorius BIA Separations d. o . o . , Slovenia) . Prior to analysis, samples were centrifuged to remove any solids, such as precipitates . After centrifugation, the samples were diluted with equilibration buffer to achieve a conductivity of less than 40 mS cm-1, ensuring optimal binding conditions . 50 pL of the prepared sample was injected into the PATfix® system at a flow rate of 1 mL min-1. The system operates with an equilibration buffer (100 mM Tris, 300 mM Gu-HCl, pH 8.0) and elution buffer (100 mM Tris, 300 mM Gu-HCl, 700 mM NaCl, pH 8.0) . The A260 and conductivity were continuously recorded during the analysis to monitor DNA elution profiles .

[0199] The system was calibrated using the pDNA Analytical Standard pFix5 sc / oc (Sartorius BIA Separations d. o . o . , Slovenia) . The results were analyzed using the PATfix® software .

[0200] Flue mRNA Gelelectrophoresis

[0201] Agarose gel electrophoresis was employed to assess the degradative effects of EMS on firefly luciferase mRNA (Flue mRNA, Promega) (1805 nt) . A 1.5 % agarose gel was prepared by dissolving agarose (Sigma Aldrich, MA, USA) in lx TBE buffer . The TBE buffer used for electrophoresis was prepared from a lOx stock solution (lOx TBE Buffer, Thermo Scientific, USA) . The stock solution was diluted to a lx working concentration (89 mM Tris, 89 mM boric acid, 20 mM EDTA) with deionized water before use . After cooling for 2 min, 2.5 pL of RNA loading dyeCO12408 / WI GR

[0202] 28

[0203] (10359119, Thermo Scientific, USA) was added. Flue mRNA samples were mixed with TBE buffer and loading dye and then heated up at 95°C for 2 min before loaded into the gel with RNA Millenium Marker (AM7150; Invitrogen, USA) . Electrophoresis was conducted at 80 V for 60 min using a PowerEase Touch 120 W power supply (Invitrogen, USA) . Band detection was performed using an Amersham Typhoon biomolecular imager (Cytiva, Sweden) at 488 nm, and data were processed with ImageQuantTL to visualize and quantify the size distribution of the samples .

[0204] EMS of pVAXl in the exemplary flow cell

[0205] A control experiment was performed in triplicate using the flowcell according to Figure 2. The flow rate was maintained at 1 mL min- 1, and no potential was applied (OCP) . Following this, the base experimental method was conducted under identical conditions with the addition of pVAXl plasmid DNA (pDNA) and an applied potential of -1. 6 V.

[0206] The UV spectroscopy results revealed similar UV signals across replicates, though with varying breakthrough peaks . The desorption peaks displayed a consistent profile during elution 1, but virtually no further desorption peak was observed during Elution 2 . This absence suggests that no pDNA was left on the graphite electrodes and the second elution process with 1 M NaCL may not be necessary.

[0207] In this experiment a sample volume of 50 pL pVAXl at a concentration of 0.3 mg / mL is first injected into the system and enters the flow cell chamber . The process then follows the steps outlined in Figure 3 :

[0208] Step "Load + Wash" : During this step, the injected sample flows through the flow cell chamber, where the nucleic acid molecules adsorb onto the stationary phase, which in this case is theCO12408 / WI GR

[0209] 29

[0210] electrode surface . This adsorption is driven by electrostatic interactions facilitated by the applied potential (1. 6 V) . After the adsorption phase, the system is washed with water over 5 chamber volumes (CV) to remove unbound and interfering components . This ensures that only the adsorbed nucleic acid molecules remain on the electrode surface . The transition from "Load" to "Wash" occurs automatically as the wash step follows immediately after the sample injection is completed.

[0211] The peak observed during this step represents the elution of unbound components from the system. Additionally, it may include a small fraction of sample molecules that failed to adsorb onto the electrode surface . The intensity of this peak reflects the breakthrough behavior and the adsorption efficiency of the electrode as the stationary phase .

[0212] The first elution peak during Elution 1 (-1. 6 V) occurs when a negative potential is applied to the electrode, triggering the electrochemical desorption of the adsorbed nucleic acid molecules, e . g. pDNA. This results in the release of the target molecules into the flow system, generating a distinct peak in the chromatogram.

[0213] The second elution peak during Elution 2 (1 M NaCl) is produced by introducing a high-salt buffer into the flow cell chamber . The ionic strength disrupts residual interactions between the nucleic acid molecules, e . g. pDNA and the electrode surface, desorbing any remaining adsorbed material . This step ensures that the surface is thoroughly cleaned for subsequent use .

[0214] Figure 3 shows this UV absorbance profile at 260 nm plotted against elution volume for 0.3 mg mL- 1pVAXl . The chromatogram captures Steps 2 through 4 : Load + Wash, Elution 1 and Elution 2, each performed for 5 column volumes (CV) , where 1 CV corresponds to 1.02 mL for the flow cell according to theCO12408 / WI GR

[0215] 30

[0216] experiment . A breakthrough UV signal is observed during Step 2 : Load + Wash, with a peak at 1 mL elution volume . A desorption peak is detected during Step 3 : Elution 1 (-1. 6 V) . Experiments were conducted at a flow rate of 1.0 mL min- 1. Triplicate runs are overlaid to demonstrate reproducibility, showing minimal variation in retention time and peak intensity. A control run at open circuit potential (OCP) is included for comparison .

[0217] Following the base experiment, a triplicate experiment using the previously determined optimized conditions (flow rate of 0.5 mL min-1 during Step 2 : Load + Wash and 2 mL min-1 during Step 3 : Elution 1 ) was conducted. The UV spectroscopy results revealed slight differences in breakthrough and desorption peak height as well as baseline level . Nevertheless, the three UV signals were relatively similar and proved a potential-based desorption . A low peak with a late co-eluting peak and an overall wavy baseline could be observed during Step 4 : Elution 2 . The elution peak was more than double that of the base experiment (RTD of 11.04 % and 25. 90 % respectively) , proving that adjusting the flow rates significantly improves pDNA adsorption and subsequent desorption behavior .

[0218] Figure 4 shows the UV absorbance profile at 260 nm plotted against the elution volume for 0.3 mg mL-1 pVAXl . The chromatogram encompasses Step 2 through Step 4 each performed for 5 CV, where 1 CV equals 1.02 mL for the flow cell according to Figure 2. A breakthrough UV signal is observed during Step 2 : Load + Wash, with a peak at 1 mL elution volume . The chromatogram displays wide and high desorption peaks during Step 3 : Elution 1, which was done at -1. 6 V. This results in a RTD of 25.21 % . During Step 4 : Elution 2 and Step 5 : Re-equilibration, additional peaks and an unstable UV signal baseline can be observed. The analysis was conducted at a flow rate of 1.0 mL min-1 except for Step 2 : Load + Wash, which was done at 0.5 mL min-1, and Step 3 : Elution 1, which was done at 2. 0 mL min-1. Triplicate runs areCO12408 / WI GR

[0219] 31

[0220] overlaid to illustrate reproducibility, showing minimal variation in retention time and peak intensity.

[0221] pVAXl Integrity via gel electrophoresis and PATfix system

[0222] An agarose gel was done with samples fractionated from optimized flow-cell experiments to have a higher intensity on the gel . For both used and new PGEs (1 ) , an OCP breakthrough sample (2, 5) , an applied potential breakthrough peak sample (3, 6) , and an applied potential desorption peak sample (4, 7 ) was fractionated. OCP breakthrough and potential breakthrough peak sample intensities are similar within the same PGE conditions, while the potential desorption peak sample intensity is lower for both . Interestingly, the sc band of the desorption peak sample is present when using new PGEs while a sample that has been desorbed from used PGEs shows only an oc but no sc band. The main difference between used and unused PGEs is that the used ones have meet 1 M NaCl buffer and may still contain salt residues inside the pores and on the surface .

[0223] Figure 5 shows an Agarose gel electrophoresis analysis of eluted pVAXl plasmid DNA from the same sample fractionated during two optimized experiments using the flow cell according to Figure 2, comparing results with used PGEs and new PGEs . The gel lanes (from left to right) include : (LI ) Generuler linear DNA ladder for size reference, (1 ) the unused original pVAXl sample (2, 5) OCP (control) breakthrough during Load + Wash (3, 6) collected fractions of eluted pVAXl during breakthrough during Load + Wash (4, 7 ) collected fractions of eluted pVAXl during Elution 1 (L2 ) Supercoiled DNA ladder for size reference . The gel was run on 0.8% agarose at 95 V (~7.3 V cm- 1) for 55 minutes .CO12408 / WI GR

[0224] 32

[0225] Quantitative Analysis of pVAXl Isoforms Using PATfix® Platform.

[0226] In addition to qualitative analysis through agarose gel electrophoresis, quantitative analysis of pVAXl isoforms was conducted using the PATfix® pDNA platform. The PATfix results for the samples of the gel quantitatively confirmed the shift of the ratio of sc to oc or a degrading effect of sc in the presence of salt .

[0227] Table 1 below shows the percentage distribution of supercoiled and open circular isoforms of pVAXl in the different collected fractions conditions, as determined by the PATfix® pDNA platform. The percentage values are calculated based on the peak area of each isoform in the PATfix chromatogram.

[0228] Label Sample / collected Electrode Potential oc sc in fraction area area Figure [%] [%] 5

[0229] (1 ) Original pVAXl - - 17 83 (2 ) Load + Wash used PGE OCP 25 75 (3) Load + Wash used PGE 1. 6 V 22 78 (4 ) Elution 1 used PGE -1. 6 V 67 33 (5) Load + Wash new PGE OCP 37 63 ( 6) Elution 1 (2 ) new PGE 1. 6 V 34 66 (7 ) Load + Wash (3) new PGE -1. 6 V 34 66

[0230] EMS of Flue mRNA in the exemplary flow cell

[0231] Building upon the optimized procedure established for pVAXl, a proof-of-concept study was conducted to assess the feasibility of applying the electrosorption process to Flue mRNA (1805 nt) within the exemplary flow cell . The objective was to evaluateCO12408 / WI GR

[0232] 33

[0233] whether the current and optimized process parameters are applicable to other nucleic acids .

[0234] An initial experiment was performed under optimized conditions, utilizing a flow rate of 0.5 mL min- 1during Step 2 (Load + Wash) and 3.0 mL min- 1during Step 3 (Elution 1 ) in deionized water (DI-H2O) . An open-circuit potential (OCP) control was included to validate the findings . Flue mRNA was adsorbed at 1.0 V and desorbed at -1.0 V, in accordance with preliminary results obtained from static setup experiments, which confirmed the feasibility of electrosorption without compromising mRNA integrity .

[0235] UV spectroscopic analysis in Figure 6 confirmed potentialdependent mRNA desorption during Elution 1, with the elution peak exhibiting a residence time distribution (RTD) value of 18 %, aligning with the positive impact of optimized flow rate adjustments observed for pDNA adsorption and desorption .

[0236] Figure 6 shows the UV absorbance at 260 nm plotted against elution volume for 0.3 mg mL- 1Flue mRNA. The chromatogram captures Steps 2 through 4 : Load + Wash, Elution 1 and Elution 2, each performed for 5 CV, where 1 CV corresponds to 1.02 mL for the flow cell according to the experiment . A breakthrough UV signal is observed during Step 2 : Load + Wash, with a peak at 1 mL elution volume . A desorption peak is detected during Step 3 : Elution 1 (-1.0 V) . The analysis was conducted at a flow rate of 1.0 mL min-1 except for Step 2 : Load + Wash, which was done at 0.5 mL min-1, and Step 3 : Elution 1, which was done at 3.0 mL min-1. A control run at open circuit potential (OCP) is included for comparison .

[0237] The experimental findings successfully demonstrated the desorption of Flue mRNA via the applied potential (Figure 6) ,CO12408 / WI GR

[0238] 34

[0239] thereby confirming the applicability of the electrosorption process to other nucleic acids . These results underscore the broader potential of the electrosorption approach for various nucleic acid types, paving the way for expanded biotechnological applications .

[0240] Integrity of Flue mRNA after electrosorption

[0241] A preliminary investigation was conducted to evaluate the feasibility of utilizing the smaller and more sensitive Flue mRNA nucleotide within an electrosorption potential framework, ensuring no observable degradation occurs . The experimental setup employed a static adsorption system, utilizing the same pencil graphite electrodes (PGEs) as in the dynamic setup to assess Flue mRNA structural integrity under controlled conditions (Figure 7) .

[0242] The static setup consists of a 3D-printed pencil holder, a semimicro cuvette, and two PGEs, which are fixed at the same distance as in the dynamic set up (1.5 mm) and immersed in 1.5 mL of sample solution . A Ruzizao DC power supply (Shenzhen Kuaiqu Electronics Co . Ltd. , China) applies 1.0 V for 60 minutes, followed by desorption through convection .

[0243] Figure 7 shows an agarose gel electrophoresis (AGE) analysis (1.5%, 80 V = 4 V cm- 1, 60 min) of Flue mRNA integrity during EMS in ddH2O, with Millennium™ RNA-Marker LI as a reference . Samples are collected before adsorption (2 ) and after washing / desorption (Lanes 4, 5, 7, and 8) for two 1.0 V replicates, along with an OCP control (Lanes 3 and 6) . The figure provides a comparative analysis of Flue mRNA stability in ddH2O, with the distinct bands indicating the preservation of the initial forms without visible degradation or conformational change during EMS in this aqueous environment .CO12408 / WI GR

[0244] 35

[0245] The present invention is also described by the following items :

[0246] 1 . Method for the continuous separation of nucleic acid molecules from a liquid medium in a separation cell (1 ) ,

[0247] wherein the cell (1 ) comprises a separation chamber (2 ) , comprising an inlet (3) and an outlet (4 ) , which define therebetween a fluid path (5) , and at least one first electrode ( 6) and at least one second electrode (7 ) arranged along, preferably parallel to said fluid path, the electrodes being arranged and configured to generate an electric field (E) therebetween, preferably proximal to the fluid path,

[0248] wherein the method comprises at least one adsorption phase followed by at least one desorption phase,

[0249] the adsorption phase comprising:

[0250] (a) Continuously providing a separation mixture (M) of at least one nucleic acid molecule in a liquid medium along the fluid path (5) ,

[0251] (b) Generating said electric field (E) by applying voltage to the at least one first and at least one second electrode, wherein the at least one nucleic acid molecule adsorbs to the at least one first electrode,

[0252] (c) Maintaining said electric field (E) over a certain adsorption time period ta,

[0253] (d) Optionally, washing the at least one first electrode and the at least one second electrode by replacing the flow of the separation mixture (M) along the fluid path (5) with a flow of a washing liquid,CO12408 / WI GR

[0254] 36

[0255] and the desorption step comprising:

[0256] (e) Replacing the flow of the separation mixture (M) , or optionally the flow of the washing liquid, along the fluid path (5) with a flow of a desorption medium (D) along the fluid path (5)

[0257] (f ) Desorbing the at least one nucleic acid molecule from the at least one first electrode into the desorption medium (D) by modifying the electrical field (E) and / or by convection of the at least one first electrode within the chamber (2 ) ,

[0258] (g) Optionally, collecting the desorption medium (D) comprising the at least one nucleic acid molecule when leaving the chamber (2 ) via the outlet (4 ) .

[0259] 2. Method according to item 1, wherein volumetric flow rates of the desorption medium (D) through the chamber (2 ) during the desorption phase are higher than volumetric flow rates of the separation mixture (M) through the chamber (2 ) during the adsorption phase .

[0260] 3. Method according to item 1 or 2, wherein the total amount of the at least one nucleic acid molecule in the separation mixture (M) is lower in the separation mixture (M) leaving the chamber (2 ) via the outlet (4 ) compared to the separation mixture (M) entering the chamber (2 ) via the inlet (3) .

[0261] 4. Method according to any of the preceding items, wherein modifying the electrical field (E) is preferably achieved by deactivating or reversing the electrical field (E) , preferably by reversing the electrical field (E) .CO12408 / WI GR

[0262] 37

[0263] 5. Method according to any of the preceding items, wherein the liquid medium comprises water, in particular DI water, and optionally further constituents selected from buffer systems, preferably biological buffer systems, such as Tris (Tris (hydroxymethyl) aminomethane) , HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulf onio acid) , MOPS (3- (N-morpholino) propanesulf onio acid) , MES (2- (N-morpholino) ethanesulf onio acid) , phosphate buffer, citrate buffer, acetate buffer, salts, such as NaCl, KOI, NH4CI, Na2SO4, (NH4) 2SO4, CaCl2, Z1C12, L1C12, MgCl2, CH3COOK, CHsCOONa, CH3COONH4, EDTA, PCR fragments, cell lysates, proteins, endotoxins, unwanted nucleic acids, NTPs and dNTPs and other biomolecules different from the at least one nucleic acid molecule to be separated .

[0264] 6. Method according to any of the preceding items, wherein the washing liquid is water, in particular DI water .

[0265] 7. Method according to any of the preceding items, wherein the desorption medium (D) comprises, water, in particular DI water, and optionally comprises further stabilizers for the at least one nucleic acid molecule to be separated.

[0266] 8. Method according to item 7, wherein said stabilizers can be selected from Tris (Tris (hydroxymethyl) aminomethane) , HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulf onio acid) , MOPS (3- (N-morpholino) propanesulf onio acid) , MES (2- (N-morpholino) ethanesulf onio acid) , phosphate buffer, citrate buffer, acetate buffer salts, such as NaCl, KC1, NH4CI, Na2SO4, (NH4) 2SO4, CaCl2, Z1C12, L1C12, MgCl2, CH3COOK, CHsCOONa, CH3COONH4, EDTA, .

[0267] 9. Method according to any of the preceding items, wherein the liquid medium and / or the desorption medium exhibits a pH value ranging from 4 to 9, preferably from 4 to 8 .CO12408 / WI GR

[0268] 38

[0269] 10. Method according to any of the preceding items, wherein the electrodes are carbon-based electrodes, preferably wherein the electrodes are consisting of carbon .

[0270] 11. Method according to item 10, wherein the carbon-based electrodes are mechanically treated prior usage, such as by sandblasting .

[0271] 12. Method according to any of the preceding items, wherein the electrodes are porous electrodes exhibiting pore sizes ranging from 1 to 1000 nm and / or modal pore diameters ranging from 30 to 70 nm, measured by known means in the art, such as mercury infiltration .

[0272] 13. Method according to any of the preceding items, wherein the at least one first electrode represents the anode and the at least one second electrode represents the cathode .

[0273] 14. Method according to any of the preceding items, wherein the electrodes are cylindrical electrodes, preferably exhibiting a length of 6 to 18 cm.

[0274] 15. Method according to any of the preceding items, wherein the electrodes exhibit an accumulated specific pore surface area ranging from 15 to 30 m2g-1, preferably 18 to 25 m2g-1, determined by mercury inf iltration / porosimetry (e . g. conform to ISO 15901-1 ) •

[0275] 16. Method according to any of the preceding items, wherein the electric field (E) exhibits a field strength ranging from 0 to 1400 V m-1(simulated via COMSOL Multiphysics software) .

[0276] 17. Method according to any of the preceding items, wherein the voltage applied to the electrodes ranges from -1600 to 1600 mVCO12408 / WI GR

[0277] 39

[0278] 18. Method according to any of the preceding items, wherein the at least one first electrode and the at least one second electrode are spaced from each other in a range of 0.1 to 3 cm.

[0279] 19. Method according to any of the preceding items, wherein the surface of the electrodes is irregular, preferably characterized by multiline surface analysis revealing profile height deviations from the mean line Ra of (0. 95 ± 0.25) pm (arithmetic mean) and a maximum peak-to-valley height of the profile Rz of (5.7 ± 1.3) pm, as determined by VK-X3000 3D laser scanning microscope .

[0280] 20. Method according to any of the preceding items, wherein the electrodes exhibit a pore volume ranging from 150 to 260 cm3g-1, as determined by mercury inf iltration / porosimetry .

[0281] 21. Method according to any of the preceding items, wherein the electrodes exhibit a total pore surface area ranging from 18 to 25 m2g-1, as determined by mercury inf iltration / porosimetry .

[0282] 22. Method according to any of the preceding items, wherein the at least one nucleic acid is a ribonucleic acid (RNA) , such as mRNA, or desoxyribonucleic acid (DNA, preferably a desoxyribonucleic acid with a length of 2 to 25 kbp, preferably a circular desoxyribonucleic acid (= plasmid DNA) , more preferably plasmid DNA of open circular (oc) and supercoiled (ccc) form, even more preferably of supercoiled form.

[0283] 23. Separation cell (1 ) for separating at least one nucleic acid molecule from a liquid medium, wherein the cell comprises : a separation chamber (2 ) , comprising an inlet (3) and an outlet (4 ) , which define therebetween a fluid path (5) for the liquid medium,CO12408 / WI GR

[0284] 40

[0285] a volume for containing the liquid medium provided along said fluid path, and

[0286] at least one first electrode ( 6) and at least one second electrode (7 ) arranged along said fluid path, the electrodes being arranged and configured to generate an electric field (E) therebetween, preferably proximal to the fluid path,

[0287] wherein the cell is a dynamic flow cell .

[0288] 24. Separation cell according to item 23, wherein the electrodes are carbon-based electrodes, preferably consisting of carbon .

[0289] 25. Separation cell according to item 23 or 24, wherein the separation cell comprises one first and one second electrode .

[0290] 26. Separation cell according to any of items 23 to 25, wherein the cell comprises at least two first electrodes and at least two second electrodes, preferably wherein two of the first electrodes are arranged side-by-side and two of the second electrodes are arranged side-by-side .

[0291] 27. Separation cell according to any of items 23 to 26, wherein the first electrode has a tubular structure (that is one electrode or several electrodes arranged in a circle) and each of the at least one second electrode has a cylindrical structure and is arranged concentrically within the first electrode .

Claims

CO12408 / WI GR41Claims1 . Method for the continuous separation of nucleic acid molecules from a liquid medium in a separation cell (1 ) ,wherein the cell (1 ) comprises a separation chamber (2 ) , comprising an inlet (3) and an outlet (4 ) , which define therebetween a fluid path (5) , and at least one first electrode ( 6) and at least one second electrode (7 ) arranged along said fluid path, the electrodes being arranged and configured to generate an electric field (E) therebetween,wherein the method comprises at least one adsorption phase followed by at least one desorption phase,the adsorption phase comprising:(a) Continuously providing a separation mixture (M) of at least one nucleic acid molecule in a liquid medium along the fluid path (5) ,(b) Generating said electric field (E) by applying voltage to the at least one first and at least one second electrode, wherein the at least one nucleic acid molecule adsorbs to the at least one first electrode,(c) Maintaining said electric field (E) over a certain adsorption time period ta,(d) Optionally, washing the at least one first electrode and the at least one second electrode by replacing the flow of the separation mixture (M) along the fluid path (5) with a flow of a washing liquid,and the desorption step comprising:CO12408 / WI GR42(e) Replacing the flow of the separation mixture (M) , or optionally the flow of the washing liquid, along the fluid path (5) with a flow of a desorption medium (D) along the fluid path (5)(f ) Desorbing the at least one nucleic acid molecule from the at least one first electrode into the desorption medium (D) by modifying the electrical field (E) and / or by convection of the at least one first electrode within the chamber (2 ) ,(g) Optionally, collecting the desorption medium (D) comprising the at least one nucleic acid molecule when leaving the chamber (2 ) via the outlet (4 ) .

2. Method according to claim 1, wherein volumetric flow rates of the desorption medium (D) through the chamber (2 ) during the desorption phase are higher than volumetric flow rates of the separation mixture (M) through the chamber (2 ) during the adsorption phase .

3. Method according to any of the preceding claims, wherein modifying the electrical field (E) is preferably achieved by deactivating or reversing the electrical field (E) .

4. Method according to any of the preceding claims, wherein the liquid medium comprises water and optionally further constituents selected from buffer systems, salts, PCR fragments, cell lysates, proteins, endotoxins, unwanted nucleic acids, NTPs and dNTPs and other biomolecules different from the at least one nucleic acid molecule to be separated.

5. Method according to any of the preceding claims, wherein the desorption medium (D) comprises water and optionallyCO12408 / WI GR43comprises further stabilizers for the at least one nucleic acid molecule to be separated.

6. Method according to any of the preceding claims, wherein the liquid medium and / or the desorption medium exhibits a pH value ranging from 4 to 9.

7. Method according to any of the preceding claims, wherein the electrodes are carbon-based electrodes .

8. Method according to claim 7, wherein the carbon-based electrodes are mechanically treated prior usage .

9. Method according to any of the preceding claims, wherein the voltage applied to the electrodes ranges from -1600 to 1600 mV.

10. Method according to any of the preceding claims, wherein the electrodes exhibit a pore volume ranging from 150 to 260 cm3g-1, as determined by mercury inf iltration / porosimetry .

11. Method according to any of the preceding claims, wherein the electrodes exhibit a total pore surface area ranging from 18 to 25 m2g-1, as determined by mercury inf iltration / porosimetry .

12. Method according to any of the preceding claims, wherein the at least one nucleic acid is a ribonucleic acid (RNA) or desoxyribonucleic acid (DNA) .

13. Separation cell (1 ) for separating at least one nucleic acid molecule from a liquid medium, wherein the cell comprises : a separation chamber (2 ) , comprising an inlet (3) and an outlet (4 ) , which define therebetween a fluid path (5) for the liquid medium,CO12408 / WI GR44a volume for containing the liquid medium provided along said fluid path, andat least one first electrode ( 6) and at least one second electrode (7 ) arranged along said fluid path, the electrodes being arranged and configured to generate an electric field (E) therebetween,wherein the cell is a dynamic flow cell .

14. Separation cell according to claim 13, wherein the electrodes are carbon-based electrodes .

15. Separation cell according to any of claims 13-14, wherein the first electrode has a tubular structure and each of the at least one second electrode has a cylindrical structure and is arranged concentrically within the first electrode .

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