Separation device and separation method

Abstract

The present invention relates to an easy-to-automate method for separating biomolecules, such as proteins or nucleic acids. More particularly, the present invention relates to a separation device comprising a column provided with a bottom liquid inlet / outlet and adapted to receive a separation medium. At least a portion of an elongated frit is arranged substantially parallel to the column side wall, separating the separation medium from the bottom liquid inlet / outlet. The elongated frit can be tubular and should have a porosity for retaining the separation medium within the column during sample processing. Furthermore, the present invention relates to a method for separating at least one target molecule from a liquid, the method comprising providing at least one chromatographic column having a bottom liquid inlet / outlet and optionally a top liquid inlet. The column retains a separation medium; and wherein at least a portion of an elongated frit is arranged substantially parallel to the column side wall, to separate the separation medium from the bottom liquid inlet / outlet. A liquid sample in the column is treated in a method comprising aspirating and dispensing fluid from the bottom liquid inlet / outlet; adding liquid at the top liquid inlet; or any combination thereof.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 087,996, filed October 6, 2020, and U.S. Provisional Application No. 63 / 166,431, filed March 26, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of target molecule separation, preferably through automated liquid handling processes. More specifically, this invention relates to separation apparatus capable of efficiently separating target molecules, for example, through parallel processing. The invention also includes methods for separating target molecules, such as automated methods for processing liquid samples containing target molecules, with sample volumes ranging from 100 mL to 5 L. Background Technology

[0004] A conventional chromatographic column can consist of a packed bed of media enclosed by top and bottom frits. Specific ranges of columns are... A pipette tip column (Phynexus Inc., phynexus.com) consists of a pipette tip column. Glass flakes are located at the top and bottom distal ends of the tip, with a column of media placed between the two glass flakes. Currently, the largest commercially available... The pipette tip column is 20 mL.

[0005] In chromatography, it is known that a larger bed volume limits the amount of space in the chamber above the upper glass buffer, thus limiting the volume of sample that can be processed. However, a potentially more serious problem is that as the bed volume increases, the column back pressure increases dramatically, thereby slowing the flow rate of sample and buffer through the column. Large sample volumes are required; however, the ability to process large sample volumes is affected by limitations in column volume and flow rate.

[0006] US 9,733,169 relates to extraction devices used in sample preparation processes for chemical analysis. More specifically, according to US 9,733,169, conventional pipette extraction products, such as disposable pipette extraction products, including those described in US Patent No. 6,566,145, use a loose adsorbent inside the pipette tip, wherein the adsorbent is contained by using a lower sieve or filter and an upper porous barrier. This type of pipette tip extraction device uses a dispersed SPE, where the term "dispersed" means that the solid-phase adsorbent is completely mixed with the liquid solution aspirated to the pipette tip.

[0007] However, according to US 9,733,169, this single-use pipette extraction is not suitable for narrow and low-volume tips. In fact, it is claimed that this single-use pipette extraction is not reproducible when incorporated into 1 mL pipette tips, and is typically used for 96-well plates and robotic techniques.

[0008] To overcome this problem, it has been proposed to introduce an intermediate porous barrier that ensures the adsorbent mixes with the solution during suction and distribution, and allows the liquid solution to pass through but prevents the adsorbent from passing through.

[0009] According to the '169 patent, the proposed intermediate barrier could lead to recovery losses because some liquid solution would inevitably be trapped by the porous membrane. Furthermore, the presence of this barrier would create a back pressure problem. Therefore, the aim of the '169 patent is to provide a dispersion-type SPE device for processing liquid sample solutions for reproducible chemical analysis with low back pressure.

[0010] To this end, '169 patent describes a pipette tip device comprising a pipette tip with a volume of approximately 1 mL or less, having a lower barrier at its narrow end (lower end) and a porous upper barrier at its wide opening (upper end), with loose adsorbent between the barriers. To provide turbulent mixing of the adsorbent and the liquid, '169 patent proposes a baffle system inside the pipette tip.

[0011] In addition, automated systems for large-scale purification can be purchased from Phynexus Inc. https: / / phynexus.com / wp-content / uploads / AutoPlasmid_MMG_Automated_Plasmid_ Purification_v52019.pdf PhyNexus AutoPlasmid MMG offers fully automated purification of plasmid DNA across a range of scales, from maxiprep to megaprep and gigaprep. It utilizes dual-flow chromatography and includes 200+ or ​​1000+ PhyTip columns with low dead volume glass frit at both the top and bottom.

[0012] WO 01 / 88185 relates to a filter element that can be incorporated into a device for separating nucleic acids from solid contaminants in a liquid sample. Specifically, the filter element may be formed of a material having a rigid porous structure with pore sizes of about 10 micrometers to 200 micrometers. The filter element has end walls and one or more side walls extending from the plane of the end walls, such that when a liquid sample containing both nucleic acids and solid contaminants is introduced into the element, the liquid containing nucleic acids is filtered through the side walls and / or end walls, while the solid contaminants are retained. In one embodiment, the filter element is tubular and has a closed end, such that when a sample containing both liquid and solid material comes into contact with the filter element, for example when it is drawn into a pipette tip or syringe with a filter, the liquid is filtered through the side walls and flows out from the open end of the tube, while the solid material initially accumulates and remains on the end walls.

[0013] EP 2 177 915 relates to a column tip treatment apparatus and method. Its objective is to enhance the contact between the packing material and the fluid to be treated, thereby achieving efficient reaction and precise treatment. By varying the ratio between the volume of the beads forming the packing material and the capacity of the tip-shaped container, particularly by selecting the packing volume such that the given ratio allows for gaps rather than a densely closed state within the tip-shaped container, suction or discharge is performed at a flow rate that allows for liquid turbulence at the column tip, ensuring sufficient contact between the packing material and the fluid, thus achieving efficient treatment. Furthermore, the appropriate suction or discharge flow rate can be determined by considering the pore size or the number of pores per unit area.

[0014] However, due to the increasing demand for faster and more efficient liquid processing of large volumes of samples, the field needs to improve the ability to process large volumes of liquid samples and increase the flow rate of samples and buffers through the column without increasing the column back pressure.

[0015] Samples suitable for the above-mentioned treatment types typically involve biomolecules such as proteins, nucleic acids, and related molecules. These molecules are used in many different applications, such as cell and gene therapy. Gene therapy based on adeno-associated virus (AAV) vectors has reported numerous successes. Currently, the FDA has approved two AAV-based gene therapies: Luxturna in 2017 for the treatment of a rare inherited retinal dystrophy, and Zolgensma last year for the treatment of spinal muscular atrophy. Furthermore, clinical trials based on AAV vectors have reported positive results in the treatment of early childhood blindness, hemophilia, and many other diseases.

[0016] AAV purification methods are currently primarily based on affinity chromatography targeting the unique properties of each AAV capsid. One example of an affinity resin is AVB agarose gel (AVB Sepharose). AVB agarose gel is based on single-domain antibody fragments from camelids. The ligands used in AVB agarose gel are isolated from llamas that have been naturally exposed to wild-type AAV. Therefore, many AAV serotypes bind to this resin. Other resins include POROS. TM Capture Select TM AAV resins (ThermoFisher): AAV8, AAV9, and AAVX.

[0017] Ion-exchange chromatography can be used for virus purification. For example, charge-difference-based methods can be used to separate empty AAV capsids from genomic vectors containing AAV capsids. Affinity and ion-exchange chromatography can be combined.

[0018] Purification of mammalian viruses, such as influenza virus, can be performed using hybrid-mode CHT (ceramic hydroxyapatite) media. CHT media are a set of hybrid-mode calcium affinity / cation exchange carriers obtained from BioRad. The high negative surface charge on many viruses allows them to bind tightly to CHT calcium sites, resulting in high-yield purification. Summary of the Invention

[0019] A first aspect of the invention is a separation device comprising at least one column having a bottom liquid inlet / outlet and adapted to receive a separation medium; wherein at least a portion of an elongated frit is disposed substantially parallel to the column sidewall to separate the separation medium from the bottom liquid inlet / outlet.

[0020] A second aspect of the invention is a method for separating at least one target molecule from a liquid, the method comprising providing at least one column having a bottom liquid inlet / outlet and optionally a top liquid inlet, the column retaining a separation medium; wherein at least a portion of an elongated glass frit is disposed substantially parallel to the column sidewalls and separates the separation medium from the bottom liquid inlet / outlet; and processing a liquid sample in the column by aspirating and dispensing liquid from the bottom liquid inlet / outlet; adding liquid at the top liquid inlet; or any combination thereof.

[0021] Other embodiments, details and advantages of the invention will be shown in the dependent claims and the overall description of the invention.

[0022] definition

[0023] The term "separation medium" is used herein to refer to any conventional solid-phase medium, such as chromatographic media in particulate form, where the particles are porous and include separating functional groups. Chromatographic media are generally classified into media containing organic or inorganic solid particles. Organic separation media are generally classified into natural polymers, such as agarose and glucose; and synthetic polymers, such as styrene and divinylbenzene (DVB); while inorganic media include solid phases, such as silica and diatomaceous earth. Separating functional groups can be ligands coupled to a surface; and / or chemical entities of a solid support that can be used and are capable of interacting with target molecules.

[0024] The term "bed" as used herein is used extensively to refer to a layer of separation medium that has not yet been dispersed by a liquid flow. Therefore, the "bed" as used herein does not require filling by applying external pressure—as those skilled in the art will recognize, it can be more or less densely filled by the liquid flowing downwards in the column described herein, or simply settled.

[0025] The term "dispersed medium" is used herein to refer to a medium dispersed by a fluid flow, such as a liquid. "Dispersed" medium particles move freely; and it does not exist as a packed bed.

[0026] The term "vertical" flow is used in this document to refer to flow that exits from the side of a slender glass flot and enters the separation medium more or less in the direction of the column side (column wall).

[0027] The term "double-flow chromatography," also known as "reciprocating flow," refers to moving a liquid into and out of the column two or more times, entering and leaving the distal end of the column.

[0028] The term "virus" is a submicroscopic infectious agent that replicates only within the living cells of other organisms. As obligate intracellular parasites, viruses must infect host cells to complete their life cycle; they reproduce by co-opting or "hijacking" the molecular mechanisms of the host cell. When not inside an infected cell or during the process of infecting a cell, it exists as an independent viral particle. Most viral particles have extremely simple structures and are very small, averaging only 1% the size of a typical bacterium. Viruses are present in almost every ecosystem on Earth and infect all types of life forms, from animals and plants to microorganisms such as bacteria and archaea. For the purposes of this invention, the term virus includes the terms viruses, virions, and viral particles.

[0029] The terms used in this article to describe sample sizes—“maxi-prep”, “mega-prep”, and “giga-prep”—are commonly used in the field of plasmid isolation. Therefore, “maxi-prep” typically includes an initial culture volume of 100 to 200 mL with an expected DNA yield of 500 to 850 μg; “mega-prep” typically includes an initial culture volume of 500 mL to 2.5 L with an expected DNA yield of 1.5 to 2.5 mg; and “giga-prep” typically includes an initial culture volume of 2.5 to 5 L with an expected DNA yield of 7.5 to 10 mg. The term “tera-prep” is 10 times larger than “giga-prep”.

[0030] The term "axial flow" in this text, as opposed to radial flow, refers to flow from the top to the bottom of a vertically positioned chromatographic column; or vice versa. Attached Figure Description

[0031] Figure 1 is provided to illustrate how the liquid flow is guided upward from the bottom liquid inlet 1a during the liquid suction phase in the column of the separation device according to the invention.

[0032] Figure 2 shows how liquid flows in opposite directions during liquid distribution in the same column as in Figure 1, i.e. from the top liquid inlet 1b to the bottom liquid outlet 1a.

[0033] Figure 3 is an enlarged image of the elongated glass flotation 6 arranged in the separation medium 7, which is also a tubular glass flotation. The glass flotation liquid channel 11 is perforated as shown in the figure (dimensions not shown in the image). An inclined column sidewall 2b is provided at the lower end of the column.

[0034] Figure 4 illustrates how an elongated glass flotation 6 is assembled by wrapping a mesh 10 around a tubular hollow support 9. In Figure 4a, the hollow support, as shown, has holes 8 that allow fluid to pass through. In Figure 4b, the tubular glass flotation—an example of an elongated glass flotation 6 according to the invention—is assembled from the tubular hollow support 9, which is wrapped with the porous mesh 10.

[0035] Figure 5 is a table summarizing the general performance of plasmid purification according to the present invention. Detailed Implementation

[0036] The invention will now be described with reference to images. As previously stated, in a first aspect, the invention relates to a separation apparatus comprising at least one column having a bottom liquid inlet / outlet 1a and adapted to receive a separation medium; wherein at least a portion of an elongated glass flotation 6 is disposed substantially parallel to the column sidewall 2a and separates the separation medium 7 from the bottom liquid inlet / outlet 1a.

[0037] Therefore, by appropriately selecting the size, especially the porosity, the slender glass frit 6 can prevent the separation medium 7 from leaving the column.

[0038] The bottom inlet / outlet 1a is adapted for liquid entry and exit from the column, allowing for liquid aspiration and distribution. Additionally, the column may include a top liquid inlet 1b, adapted for liquid entry into the column, i.e., flow from top to bottom.

[0039] In this document, the term "column sidewall" 2a should be understood to refer to the parallel (e.g., tubular) wall of the column, and not to any inclined top or end portion of the column, referred to as the column base wall 2b. Furthermore, in other words, at least a portion of the elongated glass block 6 according to the invention is located substantially vertically within the column. As those skilled in the art will know, during operation, the column will be vertically positioned.

[0040] The elongated glass frit 6 is arranged according to the invention to provide a direction of liquid flow substantially perpendicular to the column wall 2 as the liquid passes through the column during sample processing. In other words, the flow direction of the liquid is substantially horizontal when viewed from the perspective of a vertically positioned column as it passes through the elongated glass frit 6. Furthermore, the elongated glass frit is configured not to collect any solid contaminants, as shown in the more detailed disclosure below.

[0041] The liquid flow in the vertically arranged separation column according to the invention will be a combination of vertical and axial flow, as the flow direction will change as the liquid passes through the elongated glass frit 6, optionally flowing in a back-and-forth reciprocating pattern. In one embodiment, the liquid flow is axial in the lower part of the column and vertical in the upper part, with a transitional liquid flow region in between. Advantageously, at the outer edge of the column where the elongated glass frit operates, particularly at the inlet of the column chamber, there are no or very small dead flow regions.

[0042] When the liquid is drawn from the bottom, the flow provided according to the invention will disperse the separation medium 7, thereby providing effective mixing and capturing of target molecules through the separation medium 7.

[0043] As liquid is dispensed from the column to the bottom inlet / outlet 1a, the separation medium will settle. In this document, it should be understood that the bottom inlet / outlet 1a is located at the distal end of the column, as shown in the figure.

[0044] As those skilled in the art will understand, the separation medium 7 can be located in the column as a more or less dense packed bed, which can be advantageously dispersed by the upflow obtained as described above, and can be refilled or returned to a more precipitated form during liquid distribution. Advantageously, the packed bed volume can be a smaller size of about 20, 15, 10, 5, 3, 2, and 1 mL, or even as small as 50 and 100 μL; and a larger size of up to about 100 mL, 1 L, and 10 L, or even up to about 50, 100, or 200 L. Advantageous separation bed volumes are selected from 20, 10, and 5 mL. As discussed in more detail below with respect to the second aspect of the invention, the column according to the invention allows for repeated back-and-forth flow.

[0045] In this document, the term "substantially parallel to the column wall" should be understood as being arranged non-horizontally in a vertically (upright) column, for example, at an angle that provides the aforementioned flow direction. For example, assuming the column is arranged vertically, at least a portion of the slender glass block 6 can be arranged at an angle over a wide range, for example, at approximately 45 to 90° with respect to the horizontal plane.

[0046] In this document, the term “at least a portion” should be understood in a broad sense, referring to the liquid flow that constitutes a portion of the total flow rate as it passes through the column sidewall 2a during sample processing.

[0047] The upper glass frit 5 can be positioned in the column to facilitate the column receiving the lower separation medium 7. Therefore, the upper glass frit 5 is used conventionally to hold the separation medium 7 and prevent it from reaching the top of the column during liquid sample processing.

[0048] According to the present invention, the volume of the column chamber 3 can be positioned above the upper glass frit 5 in the column, as shown in Figures 1 and 2. Therefore, after the separation medium 7 is dispersed, the liquid flow is guided upward through the upper glass frit 5 and into the column chamber 3. The volume of the column chamber 3 can be, for example, about 40 mL, allowing the processing of 40 mL aliquots of sample liquid. However, the column according to the present invention can practically be configured to almost any size and column chamber size, including almost any volume of separation medium 7.

[0049] As understood by those skilled in the art, a fundamental factor for effective treatment is that the liquid flow into the column should be strong enough to at least partially disperse the separation medium. Those skilled in the art will also recognize that larger columns and lower suction back pressures will require faster liquid flow.

[0050] In the illustrative embodiments described herein, the liquid flow rate is 100 mL / min.

[0051] Furthermore, the position of the upper glass flotation 5 according to the present invention allows for free space between the separation medium 7 and the upper glass flotation 5, as shown in Figures 1 and 2. In other words, when the separation medium 7 is placed in the column, for example in the form of a packed bed, there will be free volume between the separation medium 7 and the upper glass flotation 5. During processing, this volume will be more or less occupied by the dispersed separation medium, as described above.

[0052] During sample processing, the dispersion of the separation medium 7 according to the invention will effectively mix the liquid sample with the separation medium. During this mixing, molecules from the sample, such as biomolecules, interact with the available surface of the medium, for example through ionic interactions; through hydrophobic interactions; or through "lock-key" type affinity interactions, depending on the type of separation medium selected. Thus, target molecules, such as biomolecules, can be bound, captured, or otherwise retained according to any known separation principle.

[0053] The column according to the invention can retain any separation medium 7 suitable for processing a particular sample, such as particulate chromatography media. As will be understood by those skilled in the art, the porosity of the upper glass frit 5 and the properties of the elongated glass frit 6 should be adjusted to hold the separation medium 7 in place during processing.

[0054] Therefore, the elongated glass frit 6 according to the invention may contain a mesh 10 having a size suitable for the intended separation.

[0055] The mesh 10 of the elongated glass frit 6 can be held in position by a hollow support 9 located in place, which includes one or more holes 8 located in place and of appropriate size to allow liquid to pass through the mesh 10.

[0056] For example, the mesh 10 can be positioned sufficiently close to the support 9 to maintain a location through which the aforementioned advantageous flow passes and whose flow is substantially perpendicular to the elongated glass flake 6. Thus, the mesh 10 can be attached to, coated on, or wrapped around the support 9. As those skilled in the art will understand, such a support 9 can be made of any suitable material (e.g., plastic) that is inert to the conditions of the intended treatment.

[0057] Specifically, the support member 9 should be hollow to allow liquid to enter at one end and flow out along the length of the supported net 10. The support member 9 may be defined by a circular, elliptical, triangular, or other angular cross-section, such as a basically circular cross-section forming a tubular support member.

[0058] Furthermore, in order to create the desired flow, the support 9 should include some kind of liquid channel, such as holes 8. As shown in FIG4, the elongated glass frit 6 according to the invention may include a tubular support 9, which includes uniformly positioned holes 8, and a mesh 10 has been wound thereon.

[0059] This invention encompasses various designs for elongated glass frits. For example, as described above, the elongated glass frit is tubular, or at least partially tubular. The elongated glass frit or a portion thereof may be designed to have a variable cross-section, for example, a tube with a smaller diameter at its lower end than its upper end; or a tube with a smaller diameter at its upper end than its lower end. The diameter in such a design may vary with a constant and resemble a cone, or an inverted cone. Alternatively, it may be designed as a sphere or any other shape, as long as it provides at least a partial flow of liquid through the aforementioned elongated glass frit.

[0060] As shown in Figures 1 to 3, the column of the device according to the invention may be provided with a tubular column end 4. Compared to a conventional column, the tubular column end 4 can be relatively long, and for example, can have a length that constitutes an appropriate portion of the column length, such as approximately one-third of the column length. Those skilled in the art can adjust the lengths of the column end 4 and the column bottom wall 2b for the desired process. Advantageously, as described elsewhere in the invention, the elongated tube is configured to have a length shorter than the actual column length, providing a portion of the column in which the liquid and dispersion medium are located above the upper end of the elongated glass stock, as shown. Thus, in a particular embodiment, the elongated glass stock has an end above which the separation medium is provided. Furthermore, this elongated glass stock will allow the liquid to flow substantially vertically, thereby avoiding the accumulation of solid contaminants within the glass stock.

[0061] As shown in Figures 1 to 3, the tubular column end 4 and the slender glass flotation 6 can be configured as an integral part (a single part, a tube, or a molten part) so that the liquid flows through both in either direction.

[0062] This chromatographic column is advantageously sized to fit liquid processing systems. These systems are commonly used in chromatography and other separation fields, such as biomolecular processing, and may include a computer that controls all or part of the process using appropriate software.

[0063] Therefore, the device according to the invention may include means for transferring liquid between the upper and lower ends of a column, which is capable of providing positive and negative pressure.

[0064] As understood by those skilled in the art, the device may be a pump, such as a piston pump.

[0065] Alternatively, or additionally, specific devices are provided to support top-down flow, i.e., for adding liquid to the top liquid inlet 1b of the column and allowing the liquid to descend by gravity or by pumping. As those skilled in the art will understand, the devices according to the invention can be used for up-and-down and / or suction / discharge, or any mixture or combination thereof. The invention can, for example, provide a method in which the sample and eluent are aspirated and dispensed at the bottom liquid inlet / outlet 1a, while the washing liquid can be added at the top liquid inlet 1b and, for example, allowed to pass through the column by gravity. In this washing step, the column can be removed from the liquid processing step for convenient washing using the top liquid inlet 1b, and reinserted after washing for, for example, steps to elute target molecules.

[0066] As described above, the separation medium 7 present in the column can be dispersed during liquid aspiration, i.e., during the upward stroke of the pump. This dispersion enhances the contact between the sample and the column medium, and the capture of target molecules is faster and more thorough than many existing techniques.

[0067] During the downstroke of the pump, the column bed is refilled and fluid flows through the packed bed. The liquid flow is redirected, moving perpendicular to the separation medium through the elongated glass frit and exiting the end of the column. This process can be repeated multiple times as needed. Therefore, the apparatus of the present invention can be advantageously used for “back-and-forth” flow, also known as dual-flow chromatography (DFC).

[0068] This invention has shown that it can reduce the back pressure in automated liquid sample processing, allowing it to flow rapidly into the chromatographic column, thereby effectively processing the sample.

[0069] Finally, this design increases the column volume of the separation device according to the invention, enabling up to 40 mL of sample to be processed in a single stroke.

[0070] The apparatus according to the invention may include two or more chromatographic columns for parallel processing (e.g., automated processing).

[0071] The apparatus according to the invention has been developed for capturing, cleaning, and recovering biomolecules. In other words, the apparatus can be used in one or more steps of a sample preparation protocol prior to the analysis of a target molecule. Alternatively, the invention can be used to separate target molecules from complex liquid contents, wherein the apparatus of the invention can be used in one or more purification steps, optionally in combination with conventional purification steps for biomolecules, such as centrifugation, chromatography, filtration, etc.

[0072] Therefore, in a second aspect, the present invention relates to a method for separating at least one target molecule from a liquid, the method comprising providing at least one column having a bottom liquid inlet / outlet 1a and optionally a top liquid inlet 1b, the column containing a separation medium 7; wherein at least a portion of an elongated glass frit 6 is arranged substantially parallel to the column wall 2 and spaced the separation medium 7 from the bottom liquid inlet / outlet 1a; and processing a liquid sample in the column by including drawing and dispensing liquid from the bottom liquid inlet / outlet 1a; adding liquid to the top liquid inlet 1b; or using any combination of the bottom liquid inlet / outlet 1a and the top liquid inlet 1b.

[0073] More specifically, the method of the present invention may include the following steps:

[0074] a) Provide at least one column having a bottom liquid inlet / outlet 1a and containing a separation medium 7; wherein at least a portion of an elongated glass flotation 6 is arranged substantially parallel to the column wall 2 and separates the separation medium 7 from the bottom liquid inlet / outlet 1a;

[0075] b) Provide a liquid comprising at least one target molecule that can be mixed with other molecules;

[0076] c) Place the bottom liquid inlet / outlet 1a of the column into the liquid;

[0077] d) During the suction phase, a negative pressure is applied to the column to allow liquid to pass through the liquid inlet / outlet 1a at the bottom of the column and through the elongated glass frit 6 in a flow direction substantially perpendicular to the column wall 2, thereby dispersing the separation medium 7; and optionally, time is allowed to allow the target molecules to be captured through the separation medium 7.

[0078] The method may also include the following steps:

[0079] e) During the distribution phase, a positive pressure is applied to the column to cause the liquid to flow through the elongated glass frit 6 in a direction substantially perpendicular to the column wall 2, toward the liquid inlet / outlet 1a at the bottom of the column; and

[0080] f) Collect the liquid flowing out from the bottom liquid inlet / outlet 1a of the column.

[0081] Optionally, the following steps may be performed after the above steps:

[0082] g) Repeat steps d) and f) using the eluent that released the target molecules from separation medium 7; and

[0083] h) Collect the eluent containing the released target molecules through the liquid inlet / outlet 1a at the bottom of the column, optionally followed by a step of recovering and / or analyzing the eluted target molecules.

[0084] The analysis of the target molecule may include any conventional or advantageous method, such as mass spectrometry (MS), optionally in combination with chromatography (e.g., HPLC).

[0085] Furthermore, this method can utilize the reciprocating flow of the sample, washing and / or eluent. Reciprocating flow, also known as two-flow chromatography, is now well-known to those skilled in the art, for example, see US 7,482,169, which describes in detail a process using reciprocating flow.

[0086] As mentioned above, since liquid can also be added from the liquid inlet 1b at the top of the column, technicians can flexibly change the direction of liquid flow according to their specific circumstances.

[0087] As understood by those skilled in the art, one or more washing steps may be included after sample aspiration. These washing steps can be performed in the same manner as the aspiration steps. One or more types of buffer solutions may be used in the washing, and these washings may be performed in successive steps.

[0088] As understood by those skilled in the art, washing can be carried out in a top-down flow of fluid, with the fluid introduced from the liquid inlet 1b at the top of the column. The fluid can flow through the column by gravity, or a pump can be used to force the fluid through the column.

[0089] Elution can also be carried out in a back-and-forth fluid flow or a top-down fluid flow.

[0090] The target molecule can be a biomolecule, such as a protein or nucleic acid, or a fragment thereof.

[0091] For example, the target molecule can be a protein selected from peptides and antibodies, such as a Fab fragment.

[0092] Alternatively, the target molecule can be a DNA or RNA molecule, such as a plasmid, such as a conjugating plasmid or a non-conjugating plasmid.

[0093] The liquid provided in step b) may be derived from cell culture or chemically synthesized.

[0094] In step d), the volume of liquid aspirated can range from about 20 mL to about 1 L. For example, a 40 mL liquid sample can be aspirated, or any larger volume, depending on the application.

[0095] The positive and negative pressures in steps d) and f) can be provided by a suitable pump, such as a piston pump.

[0096] In step a), at least two chromatographic columns may be provided and processed in parallel in subsequent steps.

[0097] The column may contain different separation media; and / or different liquids may be drawn into the column for separation. "Back-and-forth" flow, also known as dual-flow chromatography (DFC), can be used for sample aspiration and / or elution to increase the contact time between the liquid and the separation medium.

[0098] The separation device according to the invention can be used in this method. Therefore, all the details provided herein regarding the device of the first aspect can be applied to this method, and vice versa.

[0099] In one aspect of the invention, the aforementioned biomolecule may be a carrier of genetic material (e.g., DNA or RNA). The carrier may be a modified or unmodified virus.

[0100] In this paper, the term "unmodified virus" refers to a virus carrying at least one gene encapsulated by a protein coating and / or a lipid envelope, which is capable of multiplying within the cells of a living host.

[0101] The term "modified virus" in this document refers to any virus that has been modified compared to its natural state, such as functional modifications that result in reduced virulence, or structural modifications. The term "modified virus" also includes variant viral forms.

[0102] In particular, the target molecule isolated according to the present invention can be adeno-associated virus (AAV), which is a replication-defective non-enveloped virus with a linear single-stranded DNA (ssDNA) genome.

[0103] Because they cannot replicate, AAVs are often used as carriers for delivering genetic material, such as in gene therapy or vaccination.

[0104] Therefore, in one particular aspect, the present invention relates to a method for isolating genetic information vectors using the isolation device described herein, preferably in the form of DNA or RNA, and advantageously, the vector is a virus, such as adeno-associated virus (AAV).

[0105] This invention includes the isolation of viruses containing genetic information. On the other hand, this invention includes the isolation of viral envelopes, i.e., viruses that do not contain any genetic information. The latter "empty virus" can be used as a vector in vaccine or gene therapy applications, enabling its user to insert any relevant genetic material to achieve the desired results.

[0106] Since the separation device of the present invention is easily adaptable to automated processing, this aspect of the present invention can constitute a step in an automated process for manufacturing vaccines or gene therapy formulations.

[0107] Therefore, in this respect, the present invention relates to a method for isolating viruses, the method using at least one column equipped with a bottom liquid inlet / outlet and containing a separation medium; wherein at least a portion of an elongated glass frit is arranged substantially parallel to the sidewall of the column and separates the separation medium from the bottom liquid inlet / outlet.

[0108] This column can be part of the separation apparatus described in this invention. The illustrative volume of such a column can be in the range of 1 or 2 liter chamber sizes, wherein the size of the bed at the bottom end can be 10 or 20 mL, and the volume of the separation medium can reach 200 or 400 mL. However, larger columns are also useful, for example, chamber volumes of 3 or 4 liters or more. This aspect of the invention can also be applied to larger biomanufacturing sizes, such as columns with a diameter of 1 meter commonly used in the art.

[0109] The aforementioned vector can be a virus, such as adeno-associated virus (AAV) or a portion or fragment thereof. This AAV may contain genetic information; or it may not contain genetic information and may be a virus capable of inserting DNA or RNA as needed using techniques well known to those skilled in the art.

[0110] Ion exchange carriers can be applied to any virus. Other affinity carriers can also be applied to viruses. This involves developing affinity groups that can select for proteins on the surface of the virus and attaching these groups to the separation medium.

[0111] The separation media used in this regard may include natural polymers, such as agarose or cellulose. Such separation media are well known to those skilled in the art and can be obtained, for example, by using affinity chromatography, for the capture step of the carriers described herein, wherein a sample from a mammalian cell culture is applied to the column. Such samples may be pretreated to remove cells, which is preferred for mammalian cells into which the resulting carriers are secreted.

[0112] Therefore, the present invention includes an affinity chromatography method wherein the separation medium comprises ligands specifically designed to capture viruses.

[0113] An advantage of this aspect of the invention is the low pressure, which allows the use of plastics and column liners, a significant advantage over currently used stainless steel when separating fragile biomolecules such as viral vectors. Furthermore, supplying the separation medium to the column described herein is very simple, as uniform packing and pressure packing are not required.

[0114] Detailed description of the attached figures

[0115] Figure 1 is provided to illustrate how, in the liquid aspiration phase of the column of the separation apparatus according to the invention, the liquid flow is guided upward from the bottom liquid inlet 1a. It can be clearly seen how the liquid is drawn in and passes through the column end 4 and then into the elongated glass frit 6, shown here as a hollow tube integrated with the column end 4, the drawn-in liquid flowing at an angle substantially perpendicular to the upper glass frit 5; or at an angle substantially perpendicular to the surface of the separation medium 7.

[0116] A pump (not shown) can be installed at the top of the column to apply both negative and positive pressure. In dispersion mode, negative pressure is applied to the top of the column, and liquid flows into the column at the distal end. The liquid flows through the elongated glass frit 6 into the column and disperses the separation medium 7. It continues to flow into the column chamber 3. The dispersed separation medium 7 is contained within the column by the upper glass frit 5. A typical column has a total length of 206 mm, an end tube length of 67 mm, and a column diameter of 35 mm.

[0117] The liquid flows into the column to disperse the separation medium 7, enabling it to rapidly capture target molecules, such as plasmids.

[0118] Figure 2 illustrates how, in the same column as in Figure 1, the liquid flows in opposite directions during liquid distribution, i.e., from the liquid inlet at the top of the column to the liquid outlet at the bottom. It can be clearly seen how the liquid passes through the elongated glass frit 6 at an angle substantially perpendicular to the upper glass frit 5, so as to exit the column through the elongated glass frit 6 and the column end 4.

[0119] The liquid flowing out of the column will refill the separation medium, making it similar to its original state, such as a more or less filled bed.

[0120] Figure 3 is an enlarged image of the elongated glass frit 6 arranged in the separation medium 7, which is also formed as a tubular glass frit. The glass frit liquid channel 11 is perforated as shown (dimensions not shown in the image). In the embodiment shown herein, the bottom orifice 8 is larger, allowing for better discharge of the column in a counter-current mode (distributing liquid). The number, size, and density of orifices will vary depending on the specific column embodiment and the intensity of the pump's dispersion of the separation medium. As those skilled in the art will understand, as the orifice size decreases, the liquid flow velocity increases, correspondingly increasing the dispersion capacity of the elongated glass frit.

[0121] Figure 3 also shows an advantageous feature of the column of the device of the present invention, namely, the parallel column wall 2 at the ends, and the lower wall having a slope of about 45° with respect to the vertical direction. The slope of the lower wall can vary, but advantageously it is not 95° with respect to the vertical direction.

[0122] Figure 4 shows how to assemble the elongated glass flake 6 by wrapping the mesh 10 around the tubular hollow support 9.

[0123] In Figure 4a, the hollow support shown has a hole 8 that allows fluid to pass through.

[0124] In Figure 4b, the tubular glass flotation—an example of the elongated glass flotation 6 according to the invention—is assembled from a tubular hollow support 9, which is covered with a porous mesh 10. In particular, each hole 8 covered by the mesh 10 will form a glass flotation liquid channel 11. Therefore, due to the mesh 10, the holes 8 of the hollow support 9 will not allow the separation medium 7 to leave the column.

[0125] Figure 5 is a table summarizing the general performance of plasmid purification according to the present invention, with particular consideration given to three sizes of elongated glass frits designed as tubular glass frits.

[0126] Experimental Section

[0127] The embodiments of the present invention provided are for illustrative purposes only and should not be construed as limiting the invention as defined in the appended claims. All references provided below and elsewhere in this patent are incorporated herein by reference.

[0128] Example 1. Large-scale preparation of pure plasmids using a 5 mL bed column in an automated instrument for plasmid purification. Chemical process

[0129] Table 1: Buffers used for large-scale purification

[0130]

[0131] The buffer solution includes:

[0132] Resuspension buffer: 50 mM Tris-HCl, pH 8.0; 10 mM EDTA

[0133] Lysis buffer: 0.2M NaOH; 2.5% SDS

[0134] Precipitation buffer: 2.04M KOAc, pH 4.5

[0135] Endotoxin removal buffer: surfactant

[0136] Equilibration buffer: 50 mM Tris HCl, pH 7.0

[0137] Washing buffer: 50 mM Tris HCl, pH 7.0; 300 mM NaCl

[0138] Elution buffer: 50 mM Tris HCl, pH 8.5; 500 mM NaCl

[0139] Equal portions of buffer (manual)

[0140] Divide the buffer into equal portions according to the table below, making the volume sufficient for a single sample.

[0141] Add 29 μL of thymolphthalein to the resuspension buffer.

[0142] Before use, store the buffer solution overnight at the recommended temperature.

[0143] Preparation of cell lysates (manual)

[0144] Weigh 3g of moistened, fully thawed E. coli cell granules.

[0145] Add RNase (20 mg) to the resuspension buffer and mix thoroughly.

[0146] Add resuspension buffer and added RNase to the cell particles and shake vigorously to break up the particles.

[0147] Add lysis buffer to the cell mixture and gently shake the mixture until the solution turns completely blue (approximately 20 to 30 times).

[0148] Add precipitation buffer to the cell mixture and gently shake the mixture until the solution turns completely white and no longer shows any blue color (approximately 20 times).

[0149] Invert the cell flask until separation between cell lysates and cell debris is observed.

[0150] Gently pour the cell lysate sample into the filter of the instrument's sample cartridge to separate the cell lysate from cell debris.

[0151] The final collected cell lysate sample was approximately 60 to 80 mL.

[0152] Automated instrumentation: A fully automated instrument for plasmid purification was obtained from Phynexus (http / phynexus.com), suitable for the sample volume used in this paper, and equipped with an elongated glass buffer designed as a tubular glass buffer at its bottom. In particular, the tubular glass buffer used in this paper is designed according to the accompanying drawings.

[0153] Re-filtering:

[0154] Filtered sample volume: 60 to 80 mL

[0155] Draw 20 mL from the sample cartridge and expel 20 mL at a rate of 30 mL / min through the top of the filter.

[0156] Repeat this process 10 times.

[0157] nourish:

[0158] Transfer 15 mL of ERB endotoxin removal buffer to the sample cartridge.

[0159] Let it sit for 30 minutes

[0160] Balance column:

[0161] Joined tubular glass column

[0162] Inhale and expel 30 mL EQB (4 times).

[0163] capture:

[0164] Inhale 30 mL of the semi-clarified lysate and expel it to position 1, A1B1

[0165] Inhale and expel 25 mL, repeat 7 times.

[0166] Enter a pause after each cycle (to collect agarose gel samples).

[0167] washing:

[0168] Aspirate and expel 30 mL of washing buffer.

[0169] Draw 30 mL of wash buffer from the wash buffer container.

[0170] Discharge 30 mL of washing buffer into the waste disposal area.

[0171] Add 60 mL of washing buffer.

[0172] Enter pause to collect the washing sample from the agarose gel.

[0173] Elution: Large-scale preparation

[0174] Aspirate 30 mL of elution buffer.

[0175] Drain 30 mL of elution buffer into another conical tube.

[0176] Collect 20 mL, concentration 150 g / mL

[0177] Example 2: Buffer volume and particle size distribution for purifying plasmids used in large-scale and ultra-large-scale preparations. Particle weight

[0178] The instrument described in Example 1 is used in this Example 2.

[0179] Table 2a. Buffer volumes and particle weights used for purifying plasmids used in large-scale preparations

[0180]

[0181] Re-filter

[0182] Transfer 40 mL of the semi-clarified lysate from the sample container to position 1 (A1B1).

[0183] Inhale 20 mL of the semi-clarified lysate and expel 20 mL (30 mL / min) from the top of the filter.

[0184] Repeat 10 times

[0185] nourish

[0186] Add 25 mL of endotoxin removal buffer (ERB) to the sample container.

[0187] Incubate for 28 minutes

[0188] balance

[0189] Aspirate and expel 30 mL of equilibration buffer from the column, and perform 4 cycles.

[0190] capture

[0191] Transfer 30 mL of the lysate from the sample cassette to positions A1B1 to A4B4.

[0192] Inhale 25 mL (500 mL / min) and expel 25 mL (70 mL / min) per cycle.

[0193] The column will cycle 8, 4, 2 and 2 times in A1B1, A2B2, A3B3 and A4B4 respectively.

[0194]

[0195] Elution: Large-scale preparation

[0196] Draw 30 mL of elution buffer.

[0197] Displace 30 mL of elution buffer into another conical tube.

[0198] Repeat this process for the next sample, dividing the sample into equal parts.

[0199] Table 2b: Sample volume for large-scale preparation

[0200] Example 3. Buffer volume and particle weight for ultra-large-scale plasmid purification.

[0201] The instrument described in Example 1 is used in this Example 3.

[0202] Table 3: Buffer volume and particle weight for plasmid purification for ultra-large-scale preparation.

[0203]

[0204] Example 4. Purification of wtAd5AAV virus

[0205] Any solid-phase separation medium having functional groups or solid surfaces capable of binding or partially binding virus particles can be used in the column of this invention.

[0206] In this embodiment, serotype-specific capture-select resins AAV8 and AAV9 were used to purify wtAd5 AAV virus. AAV vectors were prepared via transient transfection. HEK293 cells were transfected with polyethyleneimine (PEI) and three plasmids in a 1:1:1 ratio (inverted terminal repeat (ITR) vector, AAV rep / cap, and Ad helper plasmid). Transfected cells were grown in the presence of puromycin, and AAV productivity was screened after infection with wtAd5 virus. Cell particles were collected by centrifugation. The cell particles were resuspended in 20 mM Tris [pH 7.5], 150 mM NaCl, and 10 mM MgCl2. Nuclease and 0.1% Triton X-100 were added to the suspended cells, and the mixture was incubated at 37°C for 15 minutes to lyse the cells. The lysed mixture was centrifuged, and the supernatant was removed. The supernatant was filtered to produce a purified cell lysate containing the AAV vector.

[0207] Two 50 mL chamber tubular glass columns were filled with 10 mL of AAV8 and AAV9 capture media, respectively. The columns were run in a reciprocating flow at a rate of 100 mL / min. The columns were equilibrated with phosphate-buffered saline (PBS) at a pH of 7 to 7.5.

[0208] In other embodiments, the column can be equilibrated with 10 to 50 mM sodium phosphate or Tris at pH 6 to 8. 0.1 to 0.2 M NaCl or KCl can be added to the conditioning buffer to prevent nonspecific adsorption due to protein / protein interactions.

[0209] 40 mL of purified cell lysate containing the rAAV vector was loaded into the column. The column was fully loaded. Additional aliquots of the sample virus were added as needed to fully load the column. After virus loading, unbound non-viral material was washed out of the column.

[0210] Typically, two 40 mL washes are used. Samples with high impurity content may require additional washing steps with fresh waste liquid. Using a double or intermediate wash can improve impurity removal rates and make impurity removal more predictable, especially when there is an interaction between the target protein and the impurity.

[0211] The second wash may optionally include a high-salt wash with up to 1 M NaCl. Tris buffer may be added to test the effect of pH. Other additives include surfactants, such as up to 0.05% (v / v) Tween 20. A ionizing salt, such as up to 0.2 M MgCl2, may be added. However, phosphate-containing buffers must be avoided to prevent interaction with Mg. 2+ Cation precipitation. If the lipid and capsid structure of the virus is not damaged, up to 20% ethanol can be added.

[0212] The binding and elution behaviors of target virus particles differ, and optimal elution conditions were determined experimentally. A starting point can be 50 to 100 mM citric acid at pH 3.0. This can be lowered to pH 2. Other available elution buffer components include phosphates, hydrochloric acid, glycine, acetate, or other components that buffer well at low pH values. Other additives may include up to 2 M MgCl2 at pH 7 and / or up to 50% propylene glycol. Combinations of these components can be used to optimize elution conditions.

[0213] Impurities were removed using a graded gradient elution to obtain a concentrated elution fraction. A fully loaded column was used. Otherwise, virus release may not be efficient due to recombination events during elution. The reconstituted virus was immediately neutralized to prevent denaturation of some molecules at low pH. The eluted virus solution was neutralized by adding 1M Tris-HCl at pH 8.7.

[0214] Example 5. Purification of influenza and dengue viruses

[0215] Influenza and dengue viruses were purified using a one-step method with CHT XT media (BioRad). A tubular glass column with a 50 mL chamber was filled with 20 mL of the media. Two samples were tested: ssRNA influenza virus with a diameter of 80–120 nm and ssRNA dengue virus with a diameter of 50 nm.

[0216] Influenza virus (strain A / Cifornia / 07 / 2009) was cultured on MOCK cells in 225 mL flasks in minimal essential medium (MEM; Gibco) containing 10% fetal bovine serum (FBS) and L-glutamine. When the cells were nearly confluent, the influenza virus was inoculated at a 1:3000 dilution onto a cell monolayer in 75 mL of D-MEM / F-12 containing trypsin, penicillin, and streptomycin (influenza medium). The supernatant was harvested on days 3, 4, and 11 and clarified through a 0.45 μm filter.

[0217] Preparation of dengue virus: Type 2 dengue virus strain ThNH7 / 93 was cultured on C6 / 36 cells. Cells were grown in 225 mL flasks pre-coated with 100 μg / mL poly-L-lysine phosphate-buffered saline (PBS). Cells were cultured for 1 week in modified EMEM minimal essential medium (MP Biomedicals) containing 10% FBS at 28°C. After cell confluence, dengue virus type 2 was inoculated at a 1:1000 dilution onto a cell monolayer in 75 mL of modified EMEM containing 0.5% FBS and MEM vitamin solution (Invitrogen Corporation), and then cultured at 28°C. The medium was changed on day 3, and the culture medium (approximately 75 mL) was collected on day 7. The culture medium was filtered through a 0.45 μm filter to remove cells and large cell debris.

[0218] Run the column back and forth repeatedly at a rate of 100 mL / min. Perform virus purification as shown in Table 4 for influenza virus and Table 5 for dengue virus.

[0219] Table 4: Influenza virus purification protocols

[0220]

[0221] Table 5: Dengue virus purification protocol

[0222]

[0223]

[0224] Washing and loading conditions can be altered to optimize virus yield and purity.

Claims

1. A separation apparatus comprising at least one column having a bottom liquid inlet / outlet (1a) and adapted to receive a separation medium; wherein at least a portion of an elongated glass frit (6) is disposed substantially parallel to the column sidewall (2a) to separate the separation medium (7) from the bottom liquid inlet / outlet (1a), The upper glass material (5) is positioned at a location on the column such that the column receives the separation medium (7) below the upper glass material (5). The elongated glass frit (6) includes a mesh (10), and the mesh (10) is held in place by a hollow support (9) containing at least one hole (8).

2. The apparatus according to claim 1, wherein when the column is vertically positioned, the elongated glass piece (6) is not horizontally positioned in the column.

3. The apparatus according to claim 2, wherein when the column is vertically positioned, at least a portion of the elongated glass block (6) is arranged at an angle ranging from 45 to 90° with respect to the horizontal plane of the column.

4. The apparatus according to any one of claims 1-3, wherein the elongated glass frit (6) is configured to allow liquid flow perpendicular to and parallel to the column sidewall (2a).

5. The apparatus according to any one of claims 1-3, wherein it is equipped with a top liquid inlet (1b).

6. The apparatus according to claim 1, wherein the upper glass flotation (5) is arranged at one position in the column, resulting in a volume consisting of the separation medium (7) and the free space below the upper glass flotation (5).

7. The device according to claim 1, wherein the hollow support (9) is defined as a circular, elliptical, triangular or other angled cross-section.

8. The apparatus according to any one of claims 1-3, wherein the elongated glass flotation (6) is composed of a tubular support (9) on which a mesh (10) is wound around the side facing the separation medium (7).

9. The device according to any one of claims 1-3, wherein a tubular column end (4) is disposed at the bottom of the column.

10. The device according to claim 9, wherein the length of the tubular column end (4) is one-third of the column length.

11. The device according to claim 9, wherein the tubular column end (4) and the elongated glass material (6) are arranged as a single integral component.

12. The device according to any one of claims 1-3, wherein the column is provided with an inclined column base wall (2b).

13. The apparatus according to any one of claims 1-3, wherein the column is arranged in the liquid processing system.

14. The apparatus according to any one of claims 1-3, further comprising means for transferring liquid between the upper end and the lower end of the column.

15. The apparatus of claim 14, wherein the apparatus provides positive or negative pressure.

16. The apparatus of claim 14, wherein the apparatus includes a pumping device.

17. The apparatus according to any one of claims 1-3, comprising two or more columns arranged for parallel processing.

18. A method for separating at least one target molecule from a liquid, the method comprising providing at least one column having a bottom liquid inlet / outlet (1a) and optionally a top liquid inlet (1b), said column containing a separation medium (7); wherein at least a portion of an elongated glass frit (6) is arranged substantially parallel to the column wall (2) and spaced the separation medium (7) from the bottom liquid inlet / outlet (1a); and treating a liquid sample in said column by aspirating and dispensing liquid from the bottom liquid inlet / outlet (1a); and / or adding liquid at the top liquid inlet (1b); or any combination thereof, The upper glass material (5) is positioned at a location on the column such that the column receives the separation medium (7) below the upper glass material (5). The elongated glass frit (6) includes a mesh (10), and the mesh (10) is held in place by a hollow support (9) containing at least one hole (8).

19. The method of claim 18, wherein the method comprises the following steps: a) Provide at least one column having a bottom liquid inlet / outlet (1a) and an optional top liquid inlet (1b), the column containing a separation medium (7); wherein elongated glass frits (6) are arranged substantially parallel to the column sidewalls (2a) and space the separation medium (7) from the bottom liquid inlet / outlet (1a); b) Provide a liquid comprising at least one target molecule that can be mixed with other molecules; c) Place the bottom liquid inlet / outlet (1a) of the column into the liquid; d) During the suction phase, a negative pressure is applied to the column to allow liquid to pass through the bottom liquid inlet / outlet (1a) of the column and flow along a direction substantially perpendicular to the column sidewall (2a) through the elongated glass frit (6). The separation medium (7) is dispersed by this liquid flow; and optionally, time is allowed to allow the target molecules to be captured by the separation medium (7).

20. The method of claim 19, further comprising the following steps: e) During the distribution phase, a positive pressure is applied to the column to cause the liquid to flow through the elongated glass frit (6) in a direction substantially perpendicular to the column sidewall (2a), toward the liquid inlet / outlet (1a) at the bottom of the column; and f) Collect the liquid flowing out from the bottom liquid inlet / outlet (1a) of the column; Optionally, the following steps may be performed after the above steps: g) Repeat steps d) and f) using the eluent that releases the target molecules from the separation medium; and h) Collect the eluent containing the released target molecules through the liquid inlet / outlet (1a) at the bottom of the column, optionally followed by a step of recovering and / or analyzing the eluted target molecules.

21. The method according to any one of claims 18 to 20, wherein the target molecule is a biomolecule.

22. The method of claim 21, wherein the target molecule is a protein or nucleic acid, or a fragment thereof.

23. The method of claim 21, wherein the target molecule is a protein selected from peptides and antibodies.

24. The method of claim 23, wherein the target molecule is a Fab fragment.

25. The method of claim 21, wherein the target molecule is a plasmid.

26. The method according to claim 19 or 20, wherein the liquid provided in step b) is derived from cell culture; or from a chemical synthesis method.

27. The method according to claim 19 or 20, wherein in step d), the volume of liquid aspirated is in the range of 20 mL to 5 L.

28. The method of claim 27, wherein the volume of the aspirated liquid is in the range of 100 mL to 200 mL.

29. The method of claim 27, wherein the volume of the aspirated liquid is in the range of 500 mL to 2.5 L.

30. The method of claim 27, wherein the volume of the aspirated liquid is in the range of 2.5L to 5L.

31. The method according to claim 19 or 20, wherein the positive and negative pressures in steps d) and f) are provided by a piston pump.

32. The method according to claim 19 or 20, wherein in step a), at least two pillars are provided and processed in parallel in subsequent steps.

33. The method of claim 32, wherein the at least two columns comprise separation media (7) having different functions from each other.

34. The method according to claim 33, wherein the separation medium (7) having different functions from each other is an affinity medium and an ion exchange medium.

35. The method of claim 32, wherein in step d), different liquids are drawn into at least two separate columns.

36. The method of claim 19 or 20, wherein step d) comprises applying at least one back-and-forth flow to the inhaled liquid.

37. The method according to any one of claims 18 to 20, wherein the separation device according to any one of claims 1 to 17 is used.

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