Methods for separating biomolecules

Abstract

To provide an improved method for separating biomolecules.SOLUTION: The present disclosure is directed to a method for separating a biomolecule from a cell culture or from a biological solution, the method comprising the steps of: (a) providing magnetic particles comprising ligands capable of binding the biomolecule; (b) contacting a cell culture or biological solution comprising the biomolecule with the magnetic particles to obtain magnetic particles comprising the bound biomolecule; (c) retaining the magnetic particles in a magnetic separator by means of a magnetic field; (d) optionally washing the magnetic particles with a washing liquid; (e1) providing a flow of an elution liquid through the magnetic separator to elute the bound biomolecule from the magnetic particles while retaining the magnetic particles in the magnetic separator by means of the magnetic field ; (f1) forwarding the biomolecule eluted from the magnetic separator to a membrane chromatography device; and (g1) separating the biomolecule from impurities and / or contaminants by membrane chromatography.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to the field of biomolecule separation, and more particularly to methods for separating biomolecules from cell cultures or from biological solutions within a magnetic separator.

Background Art

[0002] The use of magnetic adsorption beads to separate biomolecules is known in the art. For example, U.S. Patent No. 7,506,765 B2 describes a high-gradient magnetic separator for the selective separation of magnetic particles from a suspension, which is carried out through a matrix of plate-like separation structures of magnetic material disposed within a magnetic field. Further, U.S. Patent No. 6,602,422 B1, which is hereby incorporated by reference in its entirety, relates to a separation and release method for purifying biological substances on a microseparation column. The method includes the release of biological substances from a magnetic carrier and elution from a microseparation column, provided that the magnetic carrier remains magnetically retained within the microseparation column by a matrix of ferromagnetic particles. International Publication No. 2018 / 122246 describes a method for separating biomolecules from a cell culture, the method including binding the biomolecules to magnetic beads, separating the magnetic beads containing the bound biomolecules from the remaining cell culture by using a magnetic separator, transporting the magnetic beads containing the bound biomolecules as a slurry together with an added buffer to separate eluted cells, and eluting the biomolecules from the magnetic beads within the eluted cells. Also, International Publication No. 2018 / 122089 relates to magnetic beads comprising a porous matrix and one or more magnetic particles embedded within the matrix, and further comprising an immunoglobulin-binding ligand covalently coupled to the porous matrix.

[0003] Magnetic bead processing is an effective method for directly purifying target biomolecules from unpurified feed cell stocks, and this technique can replace clarification tools such as centrifugation, filtration, and capture chromatography. Magnetic beads are functionalized with ligands that have affinity for the target biomolecules. The magnetic beads are added and mixed with the unpurified feed for a predetermined time to specifically bind to all target biomolecules within the feed. After binding, the magnetic beads are trapped by a magnet, while cells and impurities are decanted. Wash buffer is added to the magnetic beads, the magnet is released, and the wash buffer and magnetic beads are mixed together. After mixing, the magnet is activated to trap the beads, and the wash buffer is decanted. After several washes, elution buffer is added to the magnetic beads, similar to the wash buffer, and the target biomolecules are released from the magnetic beads using several batch elution steps. Since all washing and elution steps are performed in batch mode, and several batch elution steps are necessary to achieve high elution yield, the target product is more diluted compared to column chromatography when using this technique. Furthermore, when using batch elution, optimizing elution conditions is more difficult because the amount of washing buffer needs to be set to achieve the appropriate elution conditions for releasing target biomolecules from magnetic beads, and different or large amounts of elution buffer are required for each elution step. One way to overcome this is to transfer the washed beads into a chromatography column for the elution step to reduce the elution buffer volume, and then collect the elution pool using fractionation with a single optimized elution buffer. However, this requires additional equipment and column packing, and is time-consuming. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent No. 7506765B2 [Patent Document 2] U.S. Patent No. 6602422B1 [Patent Document 3] International Publication No. 2018122246 [Patent Document 4] International Publication No. 2018122089 [Patent Document 5] U.S. Patent No. 20140296464A1 [Patent Document 6] U.S. Patent No. 20160288089A1 [Patent Document 7] International Publication No. 2018011600A1 [Patent Document 8] International Publication No. 2018037244A1 [Patent Document 9] International Publication No. 2013068741A1 [Patent Document 10] International Publication No. 2015052465A1 [Patent Document 11] U.S. Patent No. 7867784B2 [Patent Document 12] Indian Patent No. 201911019289 [Non-patent literature]

[0005] [Non-Patent Document 1] https: / / www.gelifesciences.com / en / us / solutions / bioprocessing / products-and-solutions / downstream-bioprocessing / fibro-chromatography [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, in this field, there is a need for improved methods for separating biomolecules, including efficient capture and purification of biomolecules from unpurified feeds, while achieving shorter process times and more cost-effective processes. [Means for solving the problem]

[0007] The objective of providing an improved method for separating biomolecules is achieved by this disclosure, relating to a method for separating biomolecules by simultaneously applying flow elution and stirring within a magnetic separator. As shown herein, this makes it possible to flow-elute target biomolecules directly from the magnetic separator with remarkably high yield and purity using a small elution volume. The precision of the method is equivalent to that of column chromatography, while the required process time is substantially shorter.

[0008] More specifically, the present disclosure relates to a method for separating biomolecules from cell cultures or biological solutions, (a) A step of providing magnetic particles containing ligands that can bind to biomolecules, (b) A step of bringing a cell culture or biological solution containing biomolecules into contact with magnetic particles to obtain magnetic particles containing bound biomolecules, (c) A step of holding magnetic particles in a magnetic separator using a magnetic field, (d) A step of optionally cleaning magnetic particles with a cleaning solution, (e) A step of stirring magnetic particles on at least one surface of the magnetic separator to form a fluid bed of magnetic particles within the magnetic separator, (f) A step of eluting bound biomolecules from magnetic particles by using a magnetic field to hold magnetic particles in a magnetic separator and providing a flow of eluent in a direction substantially perpendicular to at least one surface. This relates to methods that include this.

[0009] A further improvement achieved by the present inventors is that magnetic separation can be followed by membrane chromatography to further purify the biomolecules. Therefore, this disclosure relates to a method for separating biomolecules from cell cultures or biological solutions, (a) Providing magnetic particles comprising a ligand capable of binding to a biomolecule; (b) Contacting a cell culture or biological solution containing a biomolecule with the magnetic particles to obtain magnetic particles containing the bound biomolecule; (c) Retaining the magnetic particles in a magnetic separator using a magnetic field; (d) Optionally, washing the magnetic particles with a washing solution; (e1) While retaining the magnetic particles in a magnetic separator using a magnetic field, providing a flow of eluate passing through the magnetic separator to elute the bound biomolecule from the magnetic particles; (f1) Transporting the biomolecule eluted from the magnetic separator to a membrane chromatography device; (g1) Separating the biomolecule from impurities and / or contaminants by membrane chromatography; A method comprising the above is provided.

[0010] Preferred embodiments of the present disclosure are described below in the mode for carrying out the invention and the dependent claims.

Brief Description of the Drawings

[0011] [Figure 1] It is a figure showing a flowchart of a method for separating a biomolecule based on the present disclosure. [Figure 2] It is a figure schematically showing a system suitable for use in the method of the present disclosure. [Figure 3] It is a figure showing a schematic view of a non-limiting embodiment of a magnetic separator suitable for use in the method of the present disclosure. [Figure 4] It is a figure showing a graph representing an elution curve obtained from the polyclonal IgG experiment described in Example 1 below. [Figure 5A] It is a figure showing a graph representing an elution curve obtained from the monoclonal antibody (mAb) experiment described in Example 2 below. [Figure 5B]This figure shows a graph representing the elution curve obtained from the monoclonal antibody (mAb) experiment described in Example 2 below. [Figure 5C] This figure shows a graph representing the elution curve obtained from the monoclonal antibody (mAb) experiment described in Example 2 below. [Figure 6] This figure shows a flowchart of an alternative method for separating biomolecules based on this disclosure. [Figure 7] This figure shows a cross-section of a membrane chromatography device that may be used based on one embodiment of an alternative method for separating biomolecules according to this disclosure. [Figure 8A] This figure shows a graph representing the elution curve obtained from the monoclonal antibody (mAb) experiment described in Example 3 below. [Figure 8B] This figure shows a graph representing the elution curve obtained from the monoclonal antibody (mAb) experiment described in Example 3 below. [Figure 8C] This figure shows a graph representing the elution curve obtained from the monoclonal antibody (mAb) experiment described in Example 3 below. [Figure 8D] This figure shows a graph representing the elution curve obtained from the monoclonal antibody (mAb) experiment described in Example 3 below. [Modes for carrying out the invention]

[0012] This disclosure provides a method for separating biomolecules from cell cultures or biological solutions, as illustrated in Figure 1, thereby solving, or at least mitigating, problems associated with existing methods for separating biomolecules. (a) A step of providing magnetic particles containing ligands that can bind to biomolecules, (b) A step of bringing a cell culture or biological solution containing biomolecules into contact with magnetic particles to obtain magnetic particles containing bound biomolecules, (c) A step of holding magnetic particles in a magnetic separator using a magnetic field, (d) A step of optionally cleaning magnetic particles with a cleaning solution, (e) A step of stirring magnetic particles on at least one surface of the magnetic separator to form a fluid bed of magnetic particles within the magnetic separator, (f) A step of eluting bound biomolecules from magnetic particles by using a magnetic field to hold magnetic particles in a magnetic separator and providing a flow of eluent in a direction substantially perpendicular to at least one surface. Includes.

[0013] A notable advantage of the method disclosed herein is that biomolecules are eluted using an elution volume of remarkably low total elution volume, more specifically, an elution volume of up to 10 bed volumes, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 bed volume, preferably up to 4 bed volumes, more preferably up to 3 bed volumes. Herein, “bed volume” is understood as the volume of settled magnetic particles, usually expressed in milliliters (ml or mL) or liters (L). A smaller total elution volume required to elute biomolecules results in a higher concentration and lower dispersion of the eluted biomolecular sample. Higher biomolecular concentrations are desirable in any subsequent polishing step to avoid handling large sample volumes.

[0014] As described above, further improvements to the method of this disclosure may relate to further purification of biomolecules by membrane chromatography after magnetic separation. Therefore, this disclosure provides a method for separating biomolecules from cell cultures or biological solutions, as illustrated in Figure 6, but this method is (a) A step of providing magnetic particles containing ligands that can bind to biomolecules, (b) A step of bringing a cell culture or biological solution containing biomolecules into contact with magnetic particles to obtain magnetic particles containing bound biomolecules, (c) A step of holding magnetic particles in a magnetic separator using a magnetic field, (d) A step of optionally cleaning magnetic particles with a cleaning solution, (e1) A step of using a magnetic field to hold magnetic particles in a magnetic separator while providing a flow of eluent through the magnetic separator to elute bound biomolecules from the magnetic particles, (f1) A step of transporting biomolecules eluted from the magnetic separator to a membrane chromatography device, (g1) A step of separating biomolecules from impurities and / or contaminants by membrane chromatography. Includes.

[0015] Adding membrane chromatography after magnetic separation provides an extremely simple and rapid process that yields highly purified biomolecules.

[0016] The term “biomolecule” has its conventional meaning in the field of bioprocessing, where biomolecules are produced (often by recombination) by cells in a cell culture and purified from the cell culture by any separation and purification means. Alternatively, biomolecules are present in biological solutions that do not necessarily originate from a cell culture. Non-limiting examples of biomolecules are biomacromolecules, which are large biological polymers composed of linked monomers, such as enzymes, antibodies and antibody fragments, and peptides and proteins (which may be natural products or recombinants), including but not limited to carbohydrates and nucleic acid sequences, such as DNA and RNA. Other non-limiting examples of biomolecules are plasmids and viruses. Biomolecules or biomacromolecules may include, but not limited to, biomacromolecules intended to be used as biopharmaceuticals, i.e., pharmaceutical compounds. In this specification, biomolecules separated from the remaining cell culture or biological solution by the methods of this disclosure may also be referred to as “target biomolecules” or “targets.” The term "biomolecule" is intended to refer to a type of biomolecule, and even in its singular form, it is understood to encompass any of the many biomolecules.

[0017] In this specification, the term “cell culture” means a culture of cells or a group of cells being cultured, in which case the cells may be any type of cell, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. A cell culture may be ambiguous, i.e., it may contain multiple cells, or it may be in a cell-depleted state, i.e., the culture contains no or very few cells, but contains biomolecules released from the cells before the cells are removed. Furthermore, an ambiguous cell culture, such as those used in the methods of this disclosure, may include untreated cells, disrupted cells, cell homogenates, and / or cell lysates.

[0018] The term "biomedial solution" is intended to mean a solution of biological origin that contains biomolecules or mixtures of several biomolecules. Examples of biomedial solutions include any type of bodily fluid derived from humans or animals, such as plasma, blood, sputum, urine, and milk.

[0019] The term “magnetic particle” is defined herein as a particle that can be attracted by a magnetic field. At the same time, the magnetic particles used in the methods of this disclosure must not aggregate in the absence of a magnetic field. In other words, the magnetic particles must behave like superparamagnetic particles. The particles may have any symmetric shape, such as spherical or cubic, or any asymmetric shape. Spherical magnetic particles are often called magnetic beads. The terms “magnetic particle,” “magnetic bead,” “Mag particle,” “Mag bead,” “Mag particle,” and “Mag bead” are understood herein to be interchangeable and their scope is not limited to magnetic particles having a spherical shape.

[0020] Magnetic particles suitable for use in the methods of this disclosure are described in International Publication No. 2018122089, which is incorporated herein by reference as is. In particular, the magnetic particles may have a volume-weighted median diameter (d50, v) in the range of 8 to 300 μm, for example, 8, 9, 10, 15, 20, 25, 30, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 95, 100, 105, 110, 150, 200, 250, or 300 μm, preferably in the range of 20 to 200 μm, more preferably in the range of 37 to 100 μm. Furthermore, magnetic particles suitable for use in the methods of this disclosure may have an average density of 1.05 to 1.20 g per 1 ml of settled magnetic particles.

[0021] Magnetic particles suitable for use in the methods of the present disclosure may comprise a porous matrix, preferably a porous polymer matrix, and one or more magnetic granules embedded in the porous matrix. The magnetic particles may preferably comprise about 5 to 15% by mass of magnetic granules. Each magnetic granule embedded in the porous matrix of a magnetic particle may have a volume-weighted median diameter (d50, v) of about 1 to about 5 μm. Furthermore, the magnetic particles may preferably comprise a concentration of magnetic granules in the central region of the magnetic particle that is at least 200% of the concentration in the surface region of the magnetic particle, where the central region is defined as being at a distance greater than 0.2 times the particle radius from the surface of the magnetic particle, and the surface region is defined as being at a distance less than 0.2 times the particle radius from the surface of the magnetic particle.

[0022] The magnetic particles used in the methods of this disclosure include ligands that can bind to biomolecules. The ligands can be covalently coupled to the porous matrix of the magnetic particles. The type of ligand and its affinity constant k for biomolecules are described. off / k onIt is understood that the selection is based on the type of biomolecules to be separated from the cell culture or biological solution. Furthermore, it is understood that the ligand concentration per magnetic particle depends on, or is related to, for example, the concentration of biomolecules in the cell culture or biological solution, the dimensions of the magnetic particles, and / or the total volume of magnetic particles added to the magnetic separator.

[0023] In a currently preferred embodiment of this disclosure, the magnetic particles used in the method of this disclosure for separating biomolecules are Mag Sepharose® PrismA (GE Healthcare Bio-Sciences AB, art. no. 17550000).

[0024] Figure 2 schematically illustrates a non-limiting example of separation system 1 that may be used to carry out a method according to this disclosure. System 1 comprises a magnetic separator 5. The term “magnetic separator” has its conventional meaning in the field of separation processes and refers to a device for separating magnetic particles from a liquid. In the currently preferred embodiments of this disclosure, the magnetic separator used in the method for separating biomolecules is a high-gradient magnetic separator and is alternatively referred to as a high-gradient magnetic separation system (HGMS) as described in U.S. Patent No. 7,506,765,B2, which is incorporated herein by reference as is. The portion of separation system 1 shown in Figure 2 is similar to the portion of the system described in International Publication No. 2018,122,246, which is incorporated herein by reference as is. The magnetic separator 5 comprises an inlet 5a for receiving a feed from a cell culture 3 or biological solution 3 containing the biomolecules and magnetic particles containing ligands that can bind to these biomolecules. The magnetic separator 5 is configured to separate the magnetic particles containing the bound biomolecules from the remaining feed. The magnetic separator 5 includes a portion of magnetic material or magnetizable material inside the magnetic separator, which attracts magnetic particles when a magnetic field is applied.

[0025] The separation system 1 shown in Figure 2 comprises a capture cell 9 connected to the inlet 5a of the magnetic separator 5. The capture cell 9 shown in Figure 2 comprises a cell culture / biological solution inlet 9a for receiving a feed derived from the cell culture 3, and at least one magnetic particle inlet 9b for receiving magnetic particles. The capture cell 9 is configured to mix the feed from the cell culture or biological solution, so that the magnetic particles allow target biomolecules to bind to the magnetic particles before being transported to the magnetic separator 5. However, the capture cell 9 is understood to be an optional component of system 1. The cell culture 3 or biological solution 3 can function equally well as a capture cell if the subsequent step involves adding magnetic beads to the cell culture 3 or biological solution, respectively, and providing the magnetic separator 5 with a feed derived from a mixture of cell culture and magnetic particles, or a mixture of biological solution and magnetic particles, containing bound biomolecules. Another alternative is to add the magnetic beads directly to the magnetic separator 5 instead. In all embodiments, it is possible to add cell cultures or biological solutions separately, and to add magnetic beads directly into the magnetic separator.

[0026] Therefore, step (b) of the method of this disclosure is, (i) A step of adding magnetic particles to a magnetic separator, and thereafter providing a feed derived from a cell culture or biological solution to the magnetic separator, (ii) A process of providing a magnetic separator with a feed derived from a mixture of cell culture and magnetic particles, or a mixture of a biological solution and magnetic particles, which contains bound biomolecules. It may include.

[0027] After step (b), that is, the step of bringing a cell culture or biosolution containing biomolecules into contact with magnetic particles to bind the biomolecules to the magnetic particles, the magnetic particles described in steps (c), (d), and (e) are understood to contain the bound biomolecules.

[0028] According to step (d) described above, the method of the present disclosure may optionally include a step of washing the magnetic particles with a cleaning solution. For this purpose, the magnetic separator 5 is preferably connected to a cleaning device 13 configured to wash out components other than those magnetically coupled to the magnetic material portion from the magnetic separator 5. The cleaning device 13 comprises at least one cleaning buffer supply device 15 connected to the inlet 5a of the magnetic separator via a pump and possibly a capture cell 9, and a cleaning buffer collection device 17 connected to the outlet 5b of the magnetic separator 5. The cleaning device 13 is configured to flow the cleaning buffer through the magnetic separator 5 to wash out components other than those coupled to the magnetic portion from the feed.

[0029] According to the method of the present disclosure, magnetic beads can be washed in batch mode in a magnetic separator, wherein the magnetic separator is configured to emit a magnetic field when the magnetic particles are washed in batch mode. Alternatively and advantageously, the above method includes a step of applying at least one washing step in continuous mode, in which case step (d) of the above method is the following substep: (d1) A step of stirring magnetic particles on at least one surface to form a fluid bed of magnetic particles, (d2) A step of removing cell cultures or biological solutions by providing a flow of washing solution substantially perpendicular to at least one surface while holding magnetic particles in a magnetic field. Includes.

[0030] In this specification, the idiomatic phrase "substantially perpendicular to at least one face" is interpreted to mean an angle of 80 to 90° with respect to at least one face.

[0031] Furthermore, step (d) of the method of this disclosure is a sub-step as follows: (i) A process to remove the magnetic field, (ii) A step of resuspending magnetic particles, (iii) A step of bringing magnetic particles into contact with a portion of the cleaning solution, (iv) A step of holding magnetic particles using a magnetic field, (v) A step of removing the cleaning solution from the retained magnetic particles. It may include.

[0032] Alternatively or additionally, step (d) of the method of the present disclosure may be repeated at least once, for example, 1, 2, 3, etc., or more times, before proceeding to step (e) or alternatively to step (e1).

[0033] System 1, as shown in Figure 2, further comprises a collection cell 7 for collecting eluate and waste. In step (f) of the method of the present disclosure, it is understood that a flow of eluate is provided through the magnetic separator 5 by enabling the eluate to enter the magnetic separator 5 via the inlet 5a and enabling the eluate to be discharged from the magnetic separator 5 via the outlet 5b. The eluted biomolecules are discharged from the magnetic separator 5 together with the eluate and can be collected in the collection cell 7 connected to the outlet 5b of the magnetic separator 5.

[0034] Accordingly, the method of the present disclosure may optionally further include, after step (f), step (g), a step of collecting eluted biomolecules discharged from the magnetic separator along with the eluate, into a collection cell while using a magnetic field to hold the magnetic particles in the magnetic separator. The collection cell is connected to the outlet of the magnetic separator.

[0035] According to an alternative embodiment, instead of the collection cell 7, System 1, as shown in Figure 2, comprises a membrane chromatography device 101 for further purifying (also called polishing) the biomolecules. In this embodiment, in step (e1) of the method of the present disclosure, a flow of eluate is provided through the magnetic separator 5 by allowing the eluate to enter the magnetic separator 5 via the inlet 5a and by allowing the eluate to be discharged from the magnetic separator 5 via the outlet 5b. The eluted biomolecules are discharged from the magnetic separator 5 with the eluate and can be further purified by separation from impurities and / or contaminants in the membrane chromatography device 101 connected to the outlet 5b of the magnetic separator 5.

[0036] In the system 1 shown in Figure 2, the cell culture 3 or biological solution 3, magnetic separator 5, and collection cell 7 or alternatively membrane chromatography device 101 may be connected by pre-sterilized flexible tubing and sterile connectors. Furthermore, the collection cell 7 or alternatively membrane chromatography device 101 may be pre-sterilized and disposable. A closed, sterile separation system for single use is provided herein.

[0037] A magnetic separator used to carry out the method of the present disclosure further comprises an agitator (not shown in Figure 2; one non-limiting example is shown in Figure 3, as will be further described below). The agitator is configured to apply an agitation action to the magnetic particles, causing the magnetic particles to move around in a plane perpendicular to the direction of flow through the magnetic separator. The agitator may include at least one component, or a plurality of components configured to vary the strength of a magnetic field, for example, by applying an oscillating magnetic field, i.e., by regularly varying the strength near a central point or plane. Accordingly, step (e) of the method of the present disclosure, or alternatively step (e1), may include agitating the magnetic particles by varying the strength of a magnetic field on at least one face of the magnetic separator, for example, by applying an oscillating magnetic field on at least one face of the magnetic separator. Alternatively, the agitator may include at least one agitator, e.g., multiple agitators, in which case step (e) of the method of the present disclosure, or alternatively step (e1), includes agitating the magnetic particles by applying an agitator or by switching it on. In this specification, the term “agitator” means any type of device or substance / material having the ability to agitate, e.g., rotate or stir, the magnetic particles within the magnetic separator. Non-limiting examples of suitable agitators include devices attached to at least one fixed structure of the magnetic separator, e.g., a central rotatable carrier shaft forming a rotor, e.g., a rotatable separation structure, e.g., a rotatable disk or rotatable blade. The separation structure may consist, for example, a wire mesh, perforated metal foil, or perforated metal sheet.

[0038] Advantageously, the agitator may contain a magnetizable material, in which case the agitator can act as a portion of the magnetic material inside the magnetic separator, which attracts magnetic particles when a magnetic field is applied.

[0039] Figure 3 schematically shows a cross-sectional view of a non-limiting example of a magnetic separator 5 that may be used to carry out a method according to the present disclosure. The arrows in Figure 3 illustrate the flow of a liquid (e.g., eluate) that may enter the magnetic separator 5 from the inlet 5a and be discharged from the outlet 5b of the magnetic separator 5. The magnetic separator 5 comprises a housing 6 and further comprises a hollow, cylindrical electromagnet 11 configured to generate a magnetic field within the magnetic separator when the electromagnet is activated. The magnetic separator further comprises an agitator 13 comprising a rotor 15 to which a plurality of rotating disks 17 are mounted. The agitator 13 is configured to agitate magnetic particles 21 on at least one surface of the magnetic separator 5 according to step (e) of the method according to the present disclosure, or alternatively step (e1), to form a fluid bed of magnetic particles within the magnetic separator. In Figure 3, the magnetic separator 5 further comprises a plurality of stationary disks 19 mounted on the housing 6. The rotating disks 17 and stationary disks 19 are arranged alternately within the housing 6. The rotating disk 17 and / or the stationary disk 19 may include a magnetizable material. Furthermore, the rotating disk 17 and / or the stationary disk 19 may be perforated to allow magnetic particles and cell cultures / biological solutions to pass through the disk. The configuration of the rotating disk and / or the stationary disk may differ from the configuration shown in Figure 3. For example, the stationary disk 19 may be connected to the rotor 15 by, for example, a sealing material. Furthermore, the rotating disk may or may not be bonded to the wall of the housing 6. It is further understood that the agitator may include other components other than those shown in Figure 3. For example, instead of having a portion of magnetizable material connected to a fixed structure of the magnetic separator, or in addition to that, it may be considered to include a portion of magnetic material or magnetizable material, such as a large or large amount of steel wool or wire wool, inside the magnetic separator, which is not connected to any fixed structure of the magnetic separator and has the ability to attract magnetic particles when a magnetic field is applied and to retain the magnetic particles in the magnetic field within the magnetic separator.

[0040] Step (e) of the method of the present disclosure, or alternatively step (e1a), may include agitating magnetic particles in at least one plane of the magnetic separator, for example, in a plurality of substantially parallel or parallel planes, to form a plurality of fluidized beds of magnetic particles. In this specification, “substantially parallel planes” is interpreted as planes arranged at an angle of 0 to 10° relative to each other. In the case of a plurality of substantially parallel or parallel planes, step (f) or alternatively step (e1b) includes providing a flow of eluent in a direction substantially perpendicular or perpendicular to the plurality of planes. Not to be bound by theory, if the agitator includes a plurality of agitators in the form of rotatable components, arranged parallel to each other within the magnetic separator and configured to agitate magnetic particles in a plurality of parallel planes, it is considered possible to form a fluidized bed between each pair of agitators.

[0041] In the method of the present disclosure, the speed of the agitator in step (e), or alternatively in step (e1), may be in the range of 15 to 1500 rpm, for example 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500, preferably in the range of 50 to 300 rpm.

[0042] According to the alternative embodiment, steps (f) and (g) following step (e) are replaced by steps (f1) and (g1) following step (e1), (f1) A step of transporting biomolecules eluted from the magnetic separator to a membrane chromatography device, (g1) A step of separating biomolecules from impurities and / or contaminants by membrane chromatography. Includes.

[0043] The location of the membrane chromatography device 101 within System 1, as shown in Figure 2, is indicated above. Step (g1) may be alternatively referred to as biomolecular polishing.

[0044] The term "membrane chromatography" has its traditional meaning in the field of bioprocessing. Membrane chromatography involves the process of binding liquid components, such as individual molecules, aggregates, or particles, to a solid-phase surface in contact with the liquid. Molecules can access the active surface of the solid phase via convection. An advantage of membrane adsorbents over packed chromatography columns is their suitability for operation at fairly high flow rates. This is also known as convection chromatography. Convection chromatography matrices include any matrix where a water pressure difference between the inlet and outlet sides of the matrix provides the force for perfusion, resulting in substantially convective transport (which occurs very rapidly at high flow rates) of substances into or out of the matrix. Convective chromatography and membrane adsorbents are described, for example, in U.S. Patents 20140296464A1, 20160288089A1, International Publication Nos. 2018011600A1, 2018037244A1, 2013068741A1, 2015052465A1, and U.S. Patent No. 7867784B2, which are incorporated herein by reference as such.

[0045] In the method of this disclosure, the membrane chromatography device may include a chromatography material comprising one or more electrospun polymer nanofibers that, when used, form a stationary phase having a plurality of pores through which the mobile phase can pass. The stationary phase may be in the form of a membrane.

[0046] Alternatively, the membrane chromatography device may include at least one adsorbent membrane. The adsorbent membrane may include polymer nanofibers. Optionally, the membrane, for example, the adsorbent membrane, may include a nonwoven web of polymer nanofibers.

[0047] Polymer nanofibers can have an average diameter of 10 nm to 1000 nm. Polymer nanofibers with an average diameter of 200 nm to 800 nm are suitable for some applications. Polymer nanofibers with an average diameter of 200 nm to 400 nm may be suitable for specific applications.

[0048] Chromatographic materials or membranes, such as adsorbent membranes, may include polymers selected from the group consisting of polysulfone, polyamide, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, and polyethylene oxide, and mixtures thereof. Alternatively or additionally, the polymer may be cellulosic polymers selected from the group consisting of, for example, cellulose and partial derivatives of cellulose, particularly cellulose esters, such as cellulose acetate, crosslinked cellulose, graft-bound cellulose, or ligand-bound cellulose.

[0049] In the methods of the present disclosure, the membrane chromatography device may include a chromatography material functionalized by (i) a positively charged group, such as a quaternary amino group, a quaternary ammonium group, or an amine group, or (ii) a negatively charged group, such as a sulfonate group or a carboxylate group. Alternatively or additionally, the membrane chromatography device may include a chromatography material functionalized by a multimodal ligand selected from the group consisting of multimodal anion exchange ligands and multimodal cation exchange ligands. The multimodal anion exchange ligand may be an N-benzyl-N-methylethanolamine ligand coupled to a support, the support being linked to the nitrogen atom of the ligand via a linker. Alternatively or additionally, the membrane chromatography device may include a chromatography material functionalized by (i) an ion exchange group, (ii) an affinity peptide / protein-based ligand, (iii) a hydrophobic interaction ligand, (iv) an IMAC ligand, or (v) a DNA-based ligand, such as oligo-dT.

[0050] For convective membrane adsorbents, the size of the convection pores in the convection matrix can be reduced, for example, by using nanofibers as described above, in order to increase their surface area and volume. However, this increases the resistance to flow. Therefore, obtaining high flow rates through a chromatography device containing a high-volume convection matrix requires a chromatography device and design that can withstand high operating pressure.

[0051] In one embodiment of the methods of this disclosure relating to membrane chromatography, a membrane chromatography device such as that described in detail in a previously filed patent application, Indian Patent No. 201911019289, internal reference number 502800-IN-1, which is incorporated herein by reference as is, may be used.

[0052] As illustrated in Figure 7, the membrane chromatography device 101 includes a chromatography material unit 103 provided within a cassette 105. The membrane chromatography device 101 further includes a liquid delivery system 107 configured to deliver liquid to the inside and outside of at least one chromatography material unit 103. The chromatography material unit 103 is sandwiched between a delivery device 109a and a collection device 109b of the liquid delivery system 107 as described above. The delivery device 109a and the collection device 109b are identical, but not required, in the embodiment shown in Figure 7. In the embodiment shown in Figure 7, the cassette 105 includes the chromatography material unit 103 and the liquid delivery system 107 in the form of a delivery device 109a and a collection device 109b. In another embodiment, the liquid delivery system 107 can be provided separately from the cassette 105, for example, as a separation unit, or integrated into the housing 113 of the chromatography device 101.

[0053] The membrane chromatography device 101 further includes a housing 113, which houses at least one chromatography material unit 3 and a cassette 105. The housing comprises a top plate 125 and a bottom plate 127. An inlet 115 for receiving a liquid feed is provided on the top plate 125, and an outlet 119 for transferring the liquid outflow from the chromatography device is provided on the bottom plate 127. The top plate 125 and the bottom plate 127 are interconnected such that the inlet fluid channel 117 connects the inlet 115 to the chromatography material unit 103 via a liquid delivery system 107, and the outlet fluid channel 121 connects the outlet 119 to the chromatography material unit 103 via a liquid delivery system 107.

[0054] The delivery device 109a of the liquid delivery system 107 may include a plate (not shown) provided adjacent to the inlet surface 153a of the chromatography material unit 103, in which case the plate has several openings for delivering a liquid feed supplied from the inlet 115 of the chromatography device 101 to the chromatography material unit 103, wherein the total area of ​​the openings in the plate is smaller than the remaining area of ​​the plate, or less than 20% or less than 10% of the remaining area of ​​the plate, and the openings are connected to the delivery device inlet 157a via one or more fluid conduits (not shown) provided in the delivery device 109a.

[0055] Furthermore, the collection device 9b of the liquid delivery system 107 may include a plate (not shown) provided adjacent to the outlet surface 153b of the chromatography material unit 103, in which case the plate has several openings for collecting liquid from the chromatography material unit 103, the total area of ​​the openings in the plate being smaller than the remaining area of ​​the plate, or less than 20% or less than 10% of the remaining area of ​​the plate, and the openings being connected to the collection device outlet 157b via one or more fluid conduits (not shown) provided in the collection device 109b.

[0056] At least one chromatography material unit 103 may include at least one adsorbent membrane and / or any one or more chromatography materials described separately herein. At least one chromatography material unit may include at least one adsorbent membrane sandwiched between at least one top spacer layer (not shown) and at least one bottom spacer layer (not shown), or at least two adsorbent membranes stacked on top of each other with gaps provided using spacer layers (not shown), and sandwiched between at least one top spacer layer and at least one bottom spacer layer.

[0057] At least some parts of the chromatography device 101 can be sealed together, while at least the inlet 115 and outlet 119 remain open. In some embodiments, at least some parts of the chromatography device 101 can be sealed together with plastic or elastomer. Alternatively or additionally, at least some parts of the chromatography device 101 can be overmolded and sealed together. Furthermore, the cassette 105 and / or housing 113 can be overmolded. Overmolding is a method of creating a seal and providing stability to the device, thereby providing a robust membrane chromatography device 101. After overmolding, the chromatography device 101 can withstand an operating pressure of at least 10 bar or at least 15 bar.

[0058] In step (f) or alternatively step (e1b) of the method of the present disclosure, the linear velocity in a direction substantially perpendicular to at least one face, or alternatively substantially perpendicular to multiple faces, is in the range of 10 to 3000 cm / hour, preferably in the range of 50 to 600 cm / hour. Similar velocities, i.e., in the range of 10 to 3000 cm / hour, preferably in the range of 50 to 600 cm / hour, may be applied where applicable in other steps of the method of the present disclosure, such as, but not limited to, steps (d), (e) or alternatively step (e1), and / or step (g) or alternatively step (f1), and / or step (g1).

[0059] In step (g1) of the method of this disclosure, the time the biomolecules reside in the membrane chromatography device may be in the range of about 0.5 seconds to about 6 minutes, preferably about 1 second to about 30 seconds. This is equivalent to the time the biomolecules reside in the membrane or chromatography material being in the range of about 0.5 seconds to about 6 minutes, preferably about 1 second to about 30 seconds.

[0060] Therefore, the flow rate of the eluate passing through the membrane chromatography device may be in a range corresponding to the residence time in the membrane chromatography device in step (g1) (approximately 0.5 seconds to approximately 6 minutes, preferably in the range of approximately 1 second to approximately 30 seconds).

[0061] The flow of eluate through the membrane or chromatographic material can be a normal flow or a tangential flow.

[0062] Furthermore, the membrane chromatography in step (g1) may be flow-through membrane chromatography.

[0063] The method of the present disclosure may further include a step between step (e1) and step (f1) to adjust the pH of the eluate, dilute the eluate, or adjust the electrical conductivity of the eluate. In the method of the present disclosure, at least steps (c) to (f), or alternatively steps (c) to (e1), for example, steps (b) to (f) or alternatively steps (b) to (e1), etc., may be carried out within a magnetic separator.

[0064] Alternatively, each of steps (b) to (f) or (b) to (e1) may be carried out in a system comprising a bioreactor vessel fluidly connected to a contactor, and a contactor fluidly connected to a magnetic separator. The bioreactor vessel contains the cell culture / biological solution before initiating the separation of biomolecules from the cell culture / biological solution. Contact between the magnetic particles and the cell culture / biological solution may be carried out in a container to which the cell culture / biological solution and magnetic particles are transported (e.g., pumped, incorporated into a liquid flow, or fed by gravity), and may also be carried out in a contactor that can be agitated to some extent to provide rapid mass transport into the magnetic particles. The bioreactor vessel or contactor may be, for example, a flexible bag, such as a flexible plastic bag with one or more inlet and outlet ports.

[0065] The separation system used in the method of this disclosure may further include a pressurizing device, such as a peristaltic pump configured to produce a flow through a magnetic separator. Alternatively, the flow through the magnetic separator may be produced solely by gravity. Generally, the pressure applied to the system to make the method of this disclosure function may be considerably lower than the pressure applied to a high-performance liquid chromatography (HPLC) system. Advantages relevant to this specification include a simpler configuration and lower cost. Non-limiting examples of a preferred range of pressures applied to the separation system are 0 to 0.5 bar, e.g., 0.01 to 0.5 bar.

[0066] In the method of this disclosure, the magnetic separator can be connected to a chromatography system, such as the AKTA® pilot system (GE Healthcare), etc. (i.e., it replaces the chromatography column in the chromatography system). Although the pump pressure performance of the chromatography system is not required, it may be advantageous to use buffer control characteristics including a dual pump for gradient generation, a valve system, and a detector of the chromatography system.

[0067] The systems and / or magnetic separators and / or membrane chromatography devices within them used in the methods of this disclosure may be configured for automated, semi-automatic, or manual operation. They are usable in both pilot-scale and production-scale processes, with a magnetic particle volume of at least 0.1 L to 10 L. They are particularly applicable to capturing biopharmaceuticals from pre-clarification feeds, such as cell cultures, biological solutions, or cell lysates.

[0068] A device "comprising" one or more enumerated elements may also include other elements not specifically enumerated. The term "comprising" includes, as a subset, "consisting essentially of," meaning that a device has the listed components but not any other characteristics or components presented. Similarly, a method "comprising" one or more enumerated steps may also include other steps not specifically enumerated.

[0069] The singular forms "a" and "an" must be interpreted as also including the plural form.

[0070] The magnetic separator used in the embodiments described below is a high-gradient magnetic separation system (HGMS), more specifically the MES 100 RS (Andritz KMPT GmbH). However, it is understood that any type of magnetic separator configured as detailed above may be used to carry out the methods of this disclosure.

[0071] Similarly, as described above, any type of membrane chromatography device having a design capable of withstanding high operating pressures can be used to carry out the methods of this disclosure, and is therefore understood to be not limited to cellulose fiber chromatography (known as Fibro chromatography) cassettes / units, such as those used in some of the examples below. Fibro chromatography (GE Healthcare Life Sciences) is an ultrafast chromatographic purification method that utilizes high flow rates and high volume cellulose fibers to shorten process time and increase productivity (https: / / www.gelifesciences.com / en / us / solutions / bioprocessing / products-and-solutions / downstream-bioprocessing / fibro-chromatography).

[0072] Furthermore, the membrane chromatography materials used in membrane chromatography devices are not limited to so-called Fibro adhere materials, as used in some of the examples below. Fibro adhere (GE Healthcare Life Sciences) is a material containing cellulose nanofibers and can be derivatized with a potent ion-exchange multimodal ligand. Another non-limiting example of a suitable membrane chromatography material is Capto® adhere (GE Healthcare Life Sciences), which is based on a rigid agarose matrix that allows for the use of high fluid velocities. The agarose matrix is ​​derivatized with a multimodal anion-exchange ligand, N-benzyl-N-methylethanolamine. [Examples]

[0073] the purpose The objective of this study was to bind and elute IgG using Mag Sepharose® PrismA and a high-gradient magnetic separator (HGMS) system, MES100RS (Andritz KMPT GmbH). The objective was to investigate whether it is possible to elute IgG from the HGMS system using flow elution.

[0074] material Polyclonal IgG (Gammanorm®), Octapharma NaH2PO4H2O, pa, Merck Na2HPO42H2O, pa, Merck Sodium acetate trihydrate, PA, Merck Acetic acid, PA, Merck Tween (trademark) 20, Merck Ltd. Tris base, PA, Merck Mag Sepharose(TM) PrismA, GE Healthcare

[0075] device HGMS equipment, MES 100 RS, Andritz KMPT GmbH 25L Plastic Buffer Tray Mixed rotor, RW20, Janke & Kunkel 125mL sterile plastic bottle, Nalgene Spectrophotometer, GENESYS(TM) 10S UV-Vis, Thermo Scientific Glass filter, G3, approx. 700mL, Scott Duran. Centrifuge, 5810R, Eppendorff

[0076] buffer 20 mM sodium phosphate + 0.15 M NaCl pH 7.4 11.35g NaH2PO4H2O, 74.3g Na2HPO 4、 And 219.2 g of NaCl were diluted and mixed in a 25 L buffer container with 25 L of dH2O. 100mM NaOAc pH3.2 140 mL of acetic acid and 7.4 g of NaOAc 3H2O were diluted and mixed in a 25 L buffer container with 25 L of dH2O. 1M NaOH 40 g of NaOH was diluted and mixed with 1000 mL of milli-Q water. PBS containing 0.05% tween Two Medicago PBS tablets were mixed with 2000 mL of MilliQ water. 1 mL of Tween 20 was pipetteed and mixed with the solution. 100mM NaOAc pH2.9 5.7 mL of acetic acid and 0.02 g of NaOAc 3H2O were mixed with 1000 mL of milli-Q water.

[0077] Protocol Preparation of Mag Sepharose PrismA 200 mL of Mag Sepharose PrismA was washed with 5 cv PBS on a glass filter (G3 pore size) before use. IgG preparation (2 mg / ml in 6 L) 12 g (73 ml at 165 mg / ml) of human IgG, gammanorm, was diluted with 6 L of 20 mM sodium phosphate + 0.15 M NaCl pH 7.4. The total volume was 6080 ml, and the IgG concentration was analyzed using UV280 and found to be 1.9 mg / ml. Incubation of Mag Sepharose PrismA 37-100 containing IgG 400 mL of washed beads were transferred to a bucket with 6 L of a solution containing IgGg, and then mixed for 1 hour.

[0078] HGMS process Cooling water, pneumatic pressure, and distilled water were supplied to the MES 100 RS system. The system's peristaltic pump was initially calibrated using a 1 L volumetric flask of water at room temperature to clean the tubing and remove air from the system, and then using the prepared buffer.

[0079] [Table 1]

[0080] After incubation, the following program was used on the magnetic separator: 2 GE-IgG-purif: Main process 1: Loading of magnetic particles (MP) Sub-1: Load: Stop everything before starting, process duration 250 seconds, activate magnet, pump setting value +50%, open valves XV01 and XV14. The remaining feed was manually pumped through the tubes using PBS buffer to clean them. Main process 2: Recirculation Sub-1: Recirculation: Process duration 30 seconds, magnet activated, pump setting +30%, valves XV08 and XV13 opened. Main process 3: Buffer washing Sub-1: Buffer 1 feed: Process duration 60 seconds, magnet activated, pump setting +70%, valves XV02 and XV14 open. Sub-2: Buffer mixing: Stop everything before starting, process duration 20 seconds, mixer setting 60% Sub-3: Trap: Process time 10 seconds, magnet activated. Sub-4: Recapture: Process duration 60 seconds, magnet activated, pump setting value +30%, valves XV08 and XV13 opened. Sub-5: Transfer: Process duration 1 second, magnet activated, valves XV02, XV08, XV13, and XV14 opened. Main process 4: H2O washing Sub-1: Buffer 2 feed: Process duration 60 seconds, magnet activated, pump setting +70%, valves XV03 and XV14 open. Sub-2: Buffer mixing 2: Stop everything before starting, process duration 20 seconds, mixer setting 60% Sub-3: Trap: Process time 10 seconds, magnet activated. Sub-4: Recapture: Process duration 60 seconds, magnet activated, pump setting value +30%, valves XV08 and XV13 opened. Sub-5: Transfer: Process duration 1 second, magnet activated, valves XV02, XV08, XV13, and XV14 opened. Main process 5: Elution Sub-1: Trap: Process time 10 seconds, magnet activated Sub-2: Recapture: Process duration 60 seconds, magnet activated, pump setting value +30%, valves XV08 and XV13 opened. Sub-3: Transfer: Process duration 1 second, magnet activated, valves XV07, XV08, XV13, and XV16 opened. Sub-4: Elutate feeding: Process duration 4200 seconds, magnet activated, mixer setting 10%, pump setting +5%, valves XV07 and XV16 open. The eluate was manually collected in approximately 100 ml fractions into pre-weighed 125 ml plastic bottles. After elution, each bottle was weighed, and the actual eluted volume in each bottle was determined by subtracting the total volume of the empty bottle (51.5 mg).

[0081] analysis The eluate was neutralized to pH 5 using 2M Tris base. The titer of all fractions was determined by UV280 using a spectrophotometer (ThermoFisher GENESYS).

[0082] Results, analysis, and conclusions The IgG concentration was calculated by dividing the UV280 value in a 1 cm cuvette by the extinction coefficient of 1.36 to obtain the concentration in mg / ml. If the UV280 value was greater than 1, the sample was diluted. The concentration and cumulative yield were plotted against the cumulative volume of elution. The graph in Figure 4 shows the characteristic elution peak when the yield is 98% and the total magnetic bead volume is 2.7 × CV (total magnetic bead volume 0.4 L).

[0083] conclusion The mixing and flow rates were set to prevent the MagBeads from flowing out of the magnetic chamber during the elution period. By mixing while simultaneously switching on the magnet, the MagBeads are suspended within the magnetic chamber, enabling continuous elution instead of batch elution of IgG. This experiment demonstrates that IgG can be eluted in continuous mode while mixing with Mag Sepharose PrismA 37-100 μm in a magnetic chamber, and that a yield of 98% can be obtained with 2.7 volumes of Mag Sepharose beads. [Examples]

[0084] the purpose The objective of this study was to clear and purify CHO cell cultures containing IgG1 monoclonal antibody using Mag Sepharose PrismA (37-100 μm) and a high-gradient magnetic separator (HGMS) system, MES100RS (Andritz KMPT GmbH). The goal was to investigate whether it was possible to elute mAbs from the HGMS system using flow elution without any loss of Mag Sepharose.

[0085] material CHO cell culture, GE Healthcare NaH2PO4H2O, pa, Merck Na2HPO42H2O, pa, Merck Sodium acetate trihydrate, PA, Merck Acetic acid, PA, Merck Tween 20, Merck Tris base, PA, Merck Mag Sepharose PrismA, 37~100μm, GE Healthcare mAb standard, 8.82mg / mL, GE Healthcare

[0086] device HGMS equipment, MES 100 RS, Andritz KMPT GmbH 25L Plastic Buffer Tray Mixed rotor, RW20, Janke & Kunkel 125mL sterile plastic bottle, Nalgene HPLC, 1260 Infinity, Agilent Technologies Glass filter, G3 pore size, approximately 700 mL, Scott Duran. Centrifuge, 5810R, Eppendorff pH meter, 913 pH meter, Metrohm Corporation MabSelect SuRe HiTrap, 29-0491-04, GE HEALTHCARE Superdex 200 Increase 10 / 300 GL, GE HEALTHCARE 0.2 μm syringe filter, Sterivex HV, Millipore 10-100 μL pipette, Mettler Toledo 100-1000 μL pipette, Eppendorff. Cellbag(TM) 10L, BC11, Basic, GE HEALTHCARE

[0087] Conditions and measured values Solution preparations: 20 mM sodium phosphate + 0.15 M NaCl pH 7.4 11.35 g of NaH2PO4H2O, 74.3 g of Na2HPO4, and 219.2 g of NaCl were diluted and mixed with 25 L of dH2O in a 25 L buffer container. 100mM NaOAc pH3.2 140 mL of acetic acid and 7.4 g of NaOAc 3H2O were diluted and mixed with 25 L of dH2O in a 25 L buffer container. 1M NaOH 40g of NaOH was diluted and mixed with 1000mL of milliQ water. PBS containing 0.05% tween Two Medicago PBS tablets were mixed with 2000 mL of MilliQ water. 1 mL of Tween 20 was pipetteed and mixed with the solution. 100mM NaOAc pH2.9 5.7 mL of acetic acid and 0.02 g of NaOAc 3H2O were mixed with 1000 mL of milli-Q water.

[0088] Preparation of mAb feed Approximately 7 L of CHO cells containing mAb were used. The cell density was 23.81 MVC / mL, the viability was 66.4%, and the mAb titer was 2.7 mg / mL. The cells were pumped into a 10 L cell bag (basic).

[0089] Preparation for titer analysis, MabSelect SuRe HiTrap binding, and elution. A MabSelect SuRe HiTrap™ column was attached to the HPLC system. Standard curves using mAbs (mAb titer 8.82 mg / mL) were prepared in 250 μL plastic HPLC vials according to the table below.

[0090] [Table 2]

[0091] [Table 3]

[0092] Determination of cell culture titer Approximately 10 mL of cell suspension was centrifuged at 3000 rpm for 5 minutes, and the total volume of cell suspension and total solid volume were monitored to confirm that the volume of cell debris (solid) was 5%. The supernatant was filtered through a 0.2 μm filter into a 1 mL HPLC vial. The titer of the supernatant was determined according to the titration method described above.

[0093] [Table 4]

[0094] Preparation of Mag Sepharose PrismA 400 mL of Mag Sepharose PrismA was washed with 5 cv PBS on a glass filter (G3 pore size) before use.

[0095] Incubation of cell cultures with Mag Sepharose PrismA 400 mL of washed beads were transferred to a bottle with 6250 g of CHO cell suspension. The magnetic beads were mixed with the CHO cell suspension for 1 hour. 1 mL of the sample was centrifuged and analyzed using the mAbSelect SuRe Hitrap method (see above) to investigate any remaining mAbs in the feed (that did not bind to Mag Sepharose PrismA).

[0096] HGMS process Cooling water, air pressure, and distilled water were applied to the MES 100 RS system. The system's peristaltic pump was calibrated using a 1 L volumetric flask filled with water at room temperature. The inlet tubes and valves XV01, XV02, XV03, and XV07 used were primed with dH2O. Air bubbles in the magnetic separator were removed by priming the system until all air was removed, while simultaneously running the mixer at 50%. The hose between circulation valves XV08 and XV013 was filled using backflow in the system until no air was present. After priming with water, the system was primed with the actual running buffer. XV01 = Loading of Mag bead mixture (primed with 20 mM sodium phosphate + 0.15 M NaCl pH 7.4) XV02 = 20mM sodium phosphate + 0.15M NaCl pH 7.4 XV03 = dH2O XV07 = 100 mM sodium acetate pH 3.2 After incubation, the following program was used on the magnetic separator. Step 1. Load the mixture of magnetic particles and feed. The pump transfer time was set to 250 seconds, and 6 liters of feed mixture were pumped. The remaining feed was manually pumped in using PBS buffer to wash the mixture inlet tube. The system was set to recirculate, trapping all magnetic beads on the magnetizable disk within the isolation unit. Step 2. Wash with 20 mM sodium phosphate + 0.15 M NaCl, repeat 4 cycles. Sub-step 1. Feed the buffer (2L buffer) for 60 seconds using 70% pump speed. Sub-step 2. Mix the buffer and beads at 60% rotor speed for 20 seconds, then stop the operation of the magnet and valve. Sub-step 3. Activate the magnet to trap the beads. Sub-step 4. Re-capture. Recirculate using a 30% pump speed to trap all beads. Sub-process 5. Transfer. Open the valve for a short time (1 s) to avoid back pressure. Step 3. Wash with water, 1 cycle. Except for the fact that the water was pumped from valve XV03, the same sub-steps as those used for washing with 20 mM sodium phosphate + 0.15 M NaCl were used for the water washing. Step 4. Elution In this process, mAbs were eluted in a flow manner while the mixer and magnet were in operation. Sub-step 1. Trap. Activate the magnet. Sub-step 2. Recapture. Recirculate for 60 seconds using 30% pump speed. Sub-process 3. Transfer. Open the valve for a short time (1 s) to avoid back pressure. Sub-step 4. Elution feed: Elution was performed using valve XV07 with 100 mM NaOAc pH 3.2. The magnet was activated, the pump speed was set to 5% (140 mL / min), and the mixer was set to 10%. Elution was performed for 4286 seconds. The eluted material was collected manually in pre-weighed 150 mL plastic bottles in fractions of approximately 100 mL each. After elution, each bottle was weighed, and the actual elution volume in each bottle was determined by subtracting the total volume of the empty bottles. See Table 5 below.

[0097] [Table 5]

[0098] The eluate fraction was pale yellow to clear. The eluate was neutralized to pH 5 with 2M Tris base and then filtered through a 0.2 μm channel. The actual volume of 2M Tris base was included in the mass of the eluate. The titer of all fractions was determined by MagSelect SuRe HiTrap binding and elution assays. See above. Finally, fractions 7–18 were pooled, and 1 mL of this pool was diluted 20× in a 1.5 mL HPLC vial with 200 mM sodium phosphate pH 6.8. The results were then analyzed on an HPLC system using size exclusion chromatography (SEC) and compared to mAb standards (diluted 8× with the same buffer). The Mag Sepharose in the separator was released and pumped back into the container. The resin was washed with 2 cv of 1 M NaOH on a G3 glass filter for 30 minutes, equilibrated with 5 cv of PBS, and finally washed with 2 cv of 20% ethanol as a preservation solution. The resin was then returned to a plastic bottle with 20% EtOH for storage.

[0099] Purity according to the SEC method Injection volume: 10μL Flow rate: 0.8mL / min Column: Superdex 200 Increase 10 / 300 GL Stop time: 26 minutes Detected at 214 nm.

[0100] HCP analysis In the initial and pooled eluate samples, 50 μL of preservation solution was added to 450 μL of the sample before host cell protein (HCP) analysis. The analysis was performed using a third-generation CHO-HCP ELISA kit (Cygnus) on a Gyrolab workstation (Gyros Protein Technologies) equipped with software package 5.3.0.

[0101] Results, analysis, and conclusions Figure 5A shows the elution profile of mAbs from the magnetic separator. The pooled fractions (fractions 7-18) corresponding to 1.2 L of eluate demonstrate that an 87% mAb yield can be obtained using 3 bed volumes (1 bed volume = 400 mL) of elution buffer. The concentration of the pooled eluate was 10.7 mg / mL (4 × concentration). The binding yield in the incubation process was 94%, and the total elution yield was 91%. Pooled samples were analyzed by SEC and HCP assays and compared to pure mAb standards. The results are shown in Figure 5B (smaller scale) and Figure 5C (larger scale; magnified peak base). The solid line represents the pooled mAb eluate, and the dotted line represents the pure mAb standard. The chromatograms are standardized. The purity was determined to be 98.5% by integrating the aggregation peak, main peak, and post-main peak. [Examples]

[0102] the purpose The objective of this study was to investigate which mixing speeds (rotor speeds) and flow rates are usable for flow elution of IgG from Mag Sepharose PrismA while using fluidized magnetic beads without moving the beads. We also investigated how the IgG elution profile is affected by different mixing speeds, bead volumes, and bead sizes.

[0103] device HGMS separator, MES100RS, serial no. 400213239, Andritz GmbH. Incubation mixer, IL82274-3, Janke-Kunkel. UV reader, SpectraMax® plus, Molecular Devices Inc. UV reading plate, 96 wells, Lot 33918007, Corning Corporation Balance, PG 5002 DeltaRange, Mettler Plastic bottle, 130mL 25L Plastic Buffer Bottle Mag Sepharose PrismA 0~37μm Lot LS-033447, GE HEALTHCARE Mag Sepharose PrismA 37~100μm LS-32871, GE HEALTHCARE

[0104] material Human IgG, Gammanorm 165 mg / mL, Octapharma NaH2PO4H2O, pa, Merck Na2HPO42H2O pa, Merck NaCl, PA, Merck Acetic acid, PA, Merck Sodium acetate 3H2O, pa, Merck

[0105] Conditions and measured values Solution preparation: Buffer A, 20mM PO4+, 0.15M NaCl, pH 7.5

[0106] [Table 6]

[0107] B buffer, 0.1M sodium acetate, pH 3.2

[0108] [Table 7]

[0109] IgG 2 mg / mL 73 mL of IgG (Gammanorm) was diluted in 6 L of A buffer to obtain IgG with a concentration of approximately 2.0 mg / mL.

[0110] Rotor speed and flow velocity tests to investigate bead loss 100 mL, 300 mL, or 400 mL (moistened and settled resin) of Mag Sepharose PrismA 37-100 μm or 0-37 μm was pumped into the HGMS system while the magnet was running. The rotor was operated at different speeds, and the pump flow rate was also varied for buffer A, to investigate at what rotor speed and flow rate the magnetic beads began to move (visually) out of the HGMS chamber. The results are shown in Tables 6-10 (Tables 8-12) below. When loaded with 100 mL of Mag Sepharose 0-37 μm: the beads were trapped up to 2100 mL / min at a rotor speed of 150 rpm. At a rotor speed of 225 rpm, the beads began to move outside the HGMS. When loaded with 300 mL of Mag Sepharose 0-37 μm, the beads were trapped up to 560 mL / min at a rotor speed of 75 rpm. When loaded with 100 mL of Mag Sepharose 37-100 μm, the beads were trapped up to 2100 mL / min at a rotor speed of 150 rpm. When loaded with 300 mL of Mag Sepharose (37-100 μm): The beads were trapped up to 560 mL / min at a rotor speed of 150 rpm. The beads were still trapped at 140 mL / min at a rotor speed of 300 rpm. When loaded with 400 mL of Mag Sepharose 37-100 μm, the beads were trapped up to 1400 mL / min at a rotor speed of 75 rpm. At any load, operation at a higher flow rate was possible if the rotor speed was lower than the rotor speed at which the beads began to move out of the HGMS system. See table.

[0111] [Table 8]

[0112] [Table 9]

[0113] [Table 10]

[0114] [Table 11]

[0115] [Table 12]

[0116] IgG flow elution test Each 400 mL resin prototype was incubated with 6 L of IgG (2 mg / mL) sample for over 1 hour. 100 mL or 400 mL (1.6 L or 6.4 L of IgG resin mixture) of IgG-adsorbed Mag Sepharose PrismA was pumped into the system, the magnetic beads were trapped in a magnet, and the beads were washed three times with 1 L of A buffer. To investigate how bead volume and rotor speed affect elution performance, elution was performed at 140 mL / min with different rotor speeds. During elution, fractions of approximately 100–2100 mL were collected, and the absorption was determined for each fraction using a UV plate reader. The exact volume of each fraction was also determined by weighing. The UV of each fraction was plotted against the cumulative elution volume. See Figures 1–4. Generally, when the rotor rotation was 0%, high concentrations of IgG were still present in the eluate even after several column volumes (over 4000 mL) of elution buffer had been passed through, indicating insufficient IgG elution. Furthermore, the elution profile did not show a tendency for the concentration to decrease. For beads smaller than 37 μm, the smaller the bead volume, the less dependent the elution is on rotor speed. For 100 mL of beads, the elution peak shapes were similar at rotor speeds of 75 and 150 rpm (Figure 8A). For 400 mL of beads, the elution peak shapes differed more significantly between rotor speeds, and IgG elution was most effective at 150 rpm (Figure 8C). For beads measuring 37–100 μm, the smaller the bead volume, the less dependent the elution is on rotor speed. For 100 mL of beads, the elution peaks were similar at rotor speeds of 75 rpm and 150 rpm (Figure 8B). For 400 mL of beads, the elution peak shape differed dramatically between rotation at 150 rpm and rotation at 0 rpm. When rotated at 150 rpm, IgG eluted with a symmetrical elution peak, whereas at 0 rpm, IgG continued to elute even after 6500 mL of elution buffer had been passed through (Figure 8D). When comparing beads measuring 0–37 μm with those measuring 37–100 μm, the elution peak shapes were similar when using 100 mL of beads (see Figures 8A and 8B, respectively). With 400 mL of beads and a rotation speed of 150 rpm, the larger beads (37–100 μm) showed a significantly sharper elution peak than the beads smaller than 37 μm (400 mL). Using larger beads, it appeared that most IgG eluted before the elution buffer reached 2000 mL, whereas with beads smaller than 37 μm, the elution peak did not decrease to a stable baseline even after flowing 4000 mL (see Figures 8C and 8D, respectively). [Examples]

[0117] the purpose The objective of this study was to evaluate the effect of adding a membrane chromatography step to polish biomolecules after magnetic separation. More specifically, the study included purification using Mag Sepharose PrismA with a high-gradient magnetic separator system, followed by polishing of IgG1 monoclonal antibody (mAb) by membrane chromatography using a Fibro adhere unit prototype.

[0118] material Starting sample: IgG1 monoclonal antibody obtained from culturing CHO cells in XDR-10, viability 66.4%, concentration 2.7 g / L Superdex® 200 Increase 10 / 300 GL, GE Healthcare MagSepharose PrismA 37~100μm, 400ml, GE HEALTHCARE Fibro Adhere unit prototype, 0.4 ml: • Fibro Adhere 1: Using an adhesive reaction concentration of 167 g / liter, allyl glycidyl ether (AGE) was added, followed by crosslinking of the material with divinyl sulfone (DVS). The titer was 563 μmol / g. • Fibro Adhere 2: Using an adhesive reaction concentration of 167 g / liter, the material was crosslinked with DVS before adding AGE. The titer was 212 μmol / g. • Fibro Adhere 3: Using an adhesive reaction concentration of 500 g / liter, the material was crosslinked with DVS before adding AGE. The titer was 478 μmol / g.

[0119] device AKTA(TM) Pure 25, GE HEALTHCARE HPLC Infinity 1200, Agilent technologies

[0120] buffer 25 mM sodium phosphate + 0.15 M NaCl pH 6.3 4.88 g of NaH2PO4H2O, 2.6 g of Na2HPO4, and 17.5 g of NaCl were diluted and mixed with 2 L of dH2O. 25 mM sodium phosphate, pH 7 1.16 g of NaH2PO4H2O and 2.367 g of Na2HPO4 were diluted and mixed with 1 L of dH2O. 25 mM sodium phosphate + 1 M NaCl pH 7 0.464 g of NaH2PO4H2O, 3.851 g of Na2HPO4, and 58.44 g of NaCl were diluted and mixed with 1 L of dH2O. 25 mM sodium phosphate, pH 7.5 0.73 g of NaH2PO4H2O and 3.508 g of Na2HPO4 were diluted and mixed with 1 L of dH2O. 100 mM acetic acid, pH 3.0 6 ml of glacial acetic acid was diluted and mixed with 1 L of dH2O.

[0121] Conditions and measured values As described in Example 2, the eluate obtained from MagSepharose PrismA using a high-gradient magnetic separator system was used for further polishing by membrane chromatography using a Fibro adhere unit prototype. The Fibro Adhere unit was connected to the AKTA Pure 25 system and evaluated under three different 25 mM Na-phosphate buffer conditions (pH and electrical conductivity). See Table 11 (Table 13). 50 unit volumes (20 ml) were equilibrated at a flow rate of 16 ml / min, then 4 ml of mAb sample was applied at 16 ml / min, and the pass-through fraction was collected. The Fibro Adhere unit was washed with 20 unit volumes (8 ml), then cleaned with 25 unit volumes (10 ml) using 100 mM acetate pH 3.0 at a flow rate of 16 ml / min, and then re-equilibriumated with 50 unit volumes (20 ml). UV was continuously monitored at 280 nm.

[0122] [Table 13]

[0123] Results, analysis, and conclusions Under different buffer conditions, chromatograms obtained by polishing mAbs with a Fibro Adhere showed high UV values ​​during the sample application period in all three buffer conditions. The pass-through fractions of the samples during the application period were collected and further analyzed. See Table 12 (Table 14). The mAb concentration and the percentage of aggregates (%) were analyzed for each sample for 26 minutes at a flow rate of 0.8 ml / min using 0.2 M sodium phosphate buffer with SEC-HPLC, Superdex 200 Increase 10 / 300 GL. Host cell protein levels in the pass-through fractions were analyzed using the Cygnus 3rd generation CHO HCP ELISA kit.

[0124] [Table 14]

[0125] In conclusion, mAb polishing resulted in high yields (93–100%) and a substantial reduction in host cell proteins (HCPs). The reduction in HCPs was maximized at pH 7.5 and electrical conductivity 3.5 mS / cm, resulting in a yield of 93–99%, and the reduction in HCPs was also best achieved using the Fibro Adhere prototype and the highest ligand density.

[0126] This disclosure is not limited to the exemplary embodiments described above, and it is understood that several possible modifications to this disclosure may be made within the scope of the claims below. [Explanation of symbols]

[0127] 1. Separation system, system (Figure 2) 3. Cell cultures, biological solutions (Figure 2) 3. Chromatography material unit (Figure 7) (*Possible error) 5. Magnetic separator (Figure 2) 5a Inlet (Figure 2) 5b Outlet (Figure 2) 6. Enclosure (Figure 3) 7. Collection Cell (Figure 2) 9. Capture cell (Figure 2) 9a Cell culture / biological solution inlet (Figure 2) 9b Magnetic particle inlet (Figure 2) 9b Data collection device (Figure 7) (*Possible error) 11. Electromagnet (Figure 3) 13. Washing device (Figure 2) 13. Stirring device (Figure 3) 15. Washing buffer supply device (Figure 2) 15. Rotor (Figure 3) 17. Washing buffer collection device (Figure 2) 17. Rotating disk (Figure 3) 19 Static disk (Figure 3) 21 Magnetic particles (Figure 3) 101 Membrane chromatography device (Figure 2) (Figure 7) 103 Chromatography material unit (Figure 7) 105 Cassette (Figure 7) 107 Liquid delivery system (Figure 7) 109a Delivery device (Figure 7) 109b Collection device (Figure 7) 113 Enclosure (Figure 7) 115 Inlet (Figure 7) 117 Inlet fluid channel (Figure 7) 119 Outlet (Figure 7) 121 Outlet fluid channel (Figure 7) 125 Top plate (Figure 7) 127 Bottom plate (Figure 7) 153a Inlet surface (Figure 7) 153b Outlet surface (Figure 7) 157a Delivery device inlet (Figure 7) 157b Collection device outlet (Figure 7)

Claims

1. A method for separating biomolecules from cell cultures or biological solutions, (a) A step of providing magnetic particles containing a ligand that can bind to the biomolecule, (b) A step of bringing a cell culture or biological solution containing the biomolecules into contact with the magnetic particles to obtain magnetic particles containing the bound biomolecules, (c) A step of holding the magnetic particles in a magnetic separator using a magnetic field, (d) A step of cleaning the magnetic particles with a cleaning solution, (e1) A step of using the magnetic field to hold magnetic particles in the magnetic separator and providing a flow of eluent through the magnetic separator to elute the bound biomolecules from the magnetic particles, (f1) A step of transporting the biomolecules eluted from the magnetic separator to a membrane chromatography device, (g1) A step of separating the biomolecules from impurities and / or contaminants by membrane chromatography. Includes, Step (e1) is (e1a) A step of stirring the magnetic particles on at least one surface of the magnetic separator to form a fluid bed of magnetic particles within the magnetic separator. Includes, Step (e1a) includes a step of stirring the magnetic particles by changing the strength of the magnetic field, method.

2. Step (e1) is (e1b) A step of providing a flow of eluent perpendicular to at least one surface to elute the bound biomolecules from the magnetic particles. The method according to claim 1, including the method described in claim 1.

3. The method according to claim 2, wherein step (e1a) includes a step of stirring the magnetic particles by applying an oscillating magnetic field.

4. The method according to any one of claims 1 to 3, wherein in step (g1), the time during which the biomolecule resides in the membrane chromatography device is in the range of about 0.5 seconds to about 6 minutes.

5. The method according to any one of claims 1 to 4, wherein the magnetic separator is connected to a chromatography system that provides a flow of eluent and a flow of washing through the membrane chromatography device.

6. The method according to any one of claims 1 to 5, wherein the membrane chromatography device comprises a chromatography material comprising one or more electrospun polymer nanofibers that, when used, form a stationary phase having a plurality of pores through which the mobile phase can pass.

7. The method according to claim 6, wherein the stationary phase is in the form of a membrane.

8. The method according to any one of claims 1 to 7, wherein the membrane chromatography device comprises at least one adsorbent membrane.

9. The method according to claim 8, wherein the adsorbent membrane includes polymer nanofibers.

10. The method according to claim 8 or 9, wherein the membrane comprises a nonwoven fabric web of polymer nanofibers.

11. The method according to any one of claims 6 to 7 and 9 to 10, wherein the polymer is selected from the group consisting of polysulfone, polyamide, nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, and polyethylene oxide and mixtures thereof.

12. The method according to any one of claims 6 to 7 and 9 to 10, wherein the polymer is a cellulosic polymer selected from the group consisting of cellulose and partial derivatives of cellulose, particularly cellulose esters, crosslinked cellulose, graft-bound cellulose, or ligand-bound cellulose.

13. The method according to any one of claims 1 to 12, wherein the membrane chromatography device comprises a chromatography material functionalized with (i) a positively charged group or (ii) a negatively charged group.

14. The method according to any one of claims 1 to 13, wherein the membrane chromatography device comprises a chromatography material functionalized with a multimodal ligand selected from the group consisting of multimodal anion exchange ligands and multimodal cation exchange ligands.

15. The method according to claim 14, wherein the multimodal anion exchange ligand is an N-benzyl-N-methylethanolamine ligand coupled with a support, and the support is linked to the nitrogen atom of the ligand via a linker.

16. The method according to any one of claims 1 to 12, wherein the membrane chromatography device comprises a chromatography material functionalized with (i) an ion exchange group, (ii) an affinity peptide / protein-based ligand, (iii) a hydrophobic interaction ligand, (iv) an IMAC ligand, or (v) a DNA-based ligand.

17. The method according to any one of claims 1 to 16, wherein the membrane chromatography in step (g1) is flow-through membrane chromatography.

18. The method according to any one of claims 6 to 17, wherein the flow of the eluate through the membrane or chromatographic material is a normal flow or a tangential flow.

19. The method according to any one of claims 1 to 18, further comprising the step of adjusting the pH of the eluate, diluting the eluate, or adjusting the electrical conductivity of the eluate between step (e1) and step (f1).

20. The membrane chromatography device (101) A chromatography material unit (103) comprising a convection-based chromatography material, A liquid delivery system (107) configured to deliver liquid to the inside and outside of at least one chromatography material unit (103), Inlet (115) and, A liquid delivery system (107) connects the inlet to at least one inlet fluid channel (117) to at least one chromatography material unit (103), Outlet (119), At least one outlet fluid channel (121) connecting an outlet (119) to at least one chromatography material unit (103) via a liquid delivery system (107), wherein at least one inlet (115) and outlet (119) remain open, and at least some portions of the membrane chromatography device (101) are sealed together, and The method according to any one of claims 1 to 19, including the method described in any one of claims 1 to 19.

21. The method according to any one of claims 1 to 20, wherein step (b) or step (c) includes adding the magnetic particles to a magnetic separator including at least one agitator, and step (e1a) includes stirring the magnetic particles by turning on a switch on the agitator.

22. Process (d) is the following sub-process: (d1) A step of stirring the magnetic particles on at least one surface to form a fluid bed of magnetic particles, (d2) A step of removing the cell culture by providing a flow of washing solution perpendicular to at least one surface while holding the magnetic particles in the magnetic field. The method according to any one of claims 1 to 21, including the method described in any one of claims 1 to 21.

23. The method according to any one of claims 1 to 22, wherein step (e1a) includes a step of stirring the magnetic particles on a plurality of parallel surfaces of the magnetic separator to form a plurality of fluid beds of magnetic particles, and step (e1b) includes a step of providing a flow of the eluent in a direction perpendicular to the plurality of surfaces.

24. The method according to any one of claims 1 to 23, wherein the biomolecule is eluted using an eluent with a maximum bed volume of 10 beds.

25. The method according to any one of claims 1 to 24, wherein at least steps (c) to (e1) are performed within the magnetic separator.

26. Step (b) is, (i) the step of adding the magnetic particles to a magnetic separator, and thereafter providing a feed derived from the cell culture to the magnetic separator, (ii) A step of providing a magnetic separator with a feed derived from a mixture of the cell culture and the magnetic particles, which includes the bound biomolecules. The method according to any one of claims 1 to 25, including the method described in any one of claims 1 to 25.

27. The method according to any one of claims 1 to 26, wherein steps (b) to (e1) are carried out within an integrated bioreactor container / contactor and a magnetic separator.

28. Process (d) is the following sub-process: (i) the step of removing the magnetic field and (ii) A step of resuspending the magnetic particles, (iii) A step of bringing the magnetic particles into contact with a portion of the cleaning solution, (iv) A step of holding the magnetic particles using a magnetic field, (v) A step of removing the cleaning solution from the retained magnetic particles The method according to any one of claims 1 to 27, including the method described in any one of claims 1 to 27.

29. The method according to any one of claims 1 to 28, wherein step (d) is repeated at least once before proceeding to step (e1).

30. The method according to any one of claims 1 to 29, wherein the linear flow velocity of the eluent in step (e1) is in the range of 10 to 3000 cm / hour.

31. The method according to any one of claims 22 to 30, wherein the speed of the agitator in step (e1a) is in the range of 15 to 1500 rpm.

32. The method according to any one of claims 1 to 31, wherein the magnetic separator is connected to a chromatography system.

33. The method according to any one of claims 1 to 32, wherein the magnetic particles have a volume-weighted median diameter (d50, v) in the range of 8 to 300 μm.

34. The method according to any one of claims 1 to 33, wherein the magnetic particles have an average density of 1.05 to 1.20 g per 1 ml of settled particles.

35. The method according to any one of claims 1 to 34, wherein each of the magnetic particles comprises a porous polymer matrix and one or more magnetic granules embedded in the porous polymer matrix.

36. The method according to claim 35, wherein each of the magnetic particles contains 5 to 15% by mass of the magnetic granules.

37. The method according to claim 35 or 36, wherein the magnetic granules have a volume-weighted median diameter (d50, v) of 1 to 5 μm.

38. The method according to any one of claims 35 to 37, wherein each of the magnetic particles contains a concentration of magnetic granules in the central region of the particle that is at least 200% of the concentration in the surface region of the particle, the central region is defined as being at a distance greater than 0.2 times the particle radius from the particle surface, and the surface region is defined as being at a distance of less than 0.2 times the particle radius from the particle surface.

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