Method 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 in particular to methods for separating biomolecules from cell cultures or from biological solutions in magnetic separators. [Background technology]

[0002] The use of magnetic adsorption beads to separate biomolecules is known in the art. For example, US Patent No. 7,506,765 B2 describes a high gradient magnetic separator for selectively separating magnetic particles from a suspension, which is carried out through a matrix of plate-like separation structures of magnetic materials arranged in a magnetic field. Furthermore, US 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 materials on a micro separation column, which includes the release of biological materials from magnetic carriers and their elution from the micro separation column, while the magnetic carriers are still magnetically held inside the micro separation column by a matrix of ferromagnetic particles. WO2018122246 describes a method for separating biomolecules from a cell culture, comprising the steps of binding the biomolecules to magnetic beads, separating the magnetic beads with the bound biomolecules from the remaining cell culture by using a magnetic separator, conveying the magnetic beads with the bound biomolecules as a slurry with added buffer to separate eluted cells, and eluting the biomolecules from the magnetic beads in the eluted cells. WO2018122089 also 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 way to purify target biomolecules directly from raw 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 raw feed for a certain time so that they specifically bind to all target biomolecules in the feed. After binding, the magnetic beads are trapped by a magnet while the cells and impurities are decanted. A wash buffer is added to the magnetic beads, and the magnet is released and mixed with the wash buffer and magnetic beads. After mixing, the magnet is turned on to trap the beads, and the wash buffer is decanted. After several washes, an elution buffer is added to the magnetic beads as in the case of the wash buffer, and the target biomolecules are released from the magnetic beads using several batch elution steps. Since all the wash and elution steps are performed in batch mode, and several batch elution steps need to be performed to achieve a high elution yield, the target is more diluted using this technique compared to column chromatography. Also, when using batch elution, it is more difficult to optimize the elution conditions because the volume of washing buffer needs to be set to reach the proper elution conditions for releasing the target biomolecules from the 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 collect the elution pool using fractionation with one optimized elution buffer. However, this requires additional equipment and column loading, 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 Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need in the art for improved methods for separating biomolecules, including efficiently capturing and purifying biomolecules from raw feeds while achieving reduced process times and more cost-effective processes. [Means for solving the problem]

[0007] The above object of providing an improved method for separating biomolecules is achieved by the present disclosure, which relates to a method for separating biomolecules by simultaneously applying flow elution and agitation in a magnetic separator. As shown herein, this allows for direct flow elution of target biomolecules from the magnetic separator with surprisingly high yields and purity using small elution volumes. The accuracy of the method is equivalent to that of column chromatography, while the process time required is substantially shorter.

[0008] More specifically, the present disclosure provides a method for isolating a biomolecule from a cell culture or from a biological solution, comprising: (a) providing magnetic particles comprising a ligand capable of binding to a biomolecule; (b) contacting the cell culture or biological solution containing the biomolecules with the magnetic particles to obtain the magnetic particles containing the bound biomolecules; (c) retaining the magnetic particles within the magnetic separator using a magnetic field; (d) optionally washing the magnetic particles with a washing solution; (e) agitating the magnetic particles on at least one side of the magnetic separator to form a fluidized bed of magnetic particles within the magnetic separator; (f) using a magnetic field to retain the magnetic particles within the magnetic separator while providing a flow of elution fluid substantially perpendicular to at least one surface to elute the bound biomolecules from the magnetic particles; The present invention relates to a method comprising the steps of:

[0009] A further improvement realized by the inventors is that magnetic separation can be followed by membrane chromatography to further purify the biomolecule. Thus, the present disclosure provides a method for separating a biomolecule from a cell culture or from a biological solution, comprising: (a) providing magnetic particles comprising a ligand capable of binding to a biomolecule; (b) contacting the cell culture or biological solution containing the biomolecules with the magnetic particles to obtain the magnetic particles containing the bound biomolecules; (c) retaining the magnetic particles within the magnetic separator using a magnetic field; (d) optionally washing the magnetic particles with a washing solution; (e1) providing a flow of elution fluid through the magnetic separator while retaining the magnetic particles within the magnetic separator using a magnetic field to elute the bound biomolecules from the magnetic particles; (f1) transporting the biomolecules eluted from the magnetic separator to a membrane chromatography device; (g1) separating the biomolecules from impurities and / or contaminants by membrane chromatography; The present invention provides a method comprising:

[0010] Preferred aspects of the disclosure are set forth below in the detailed description and the dependent claims. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a flow chart of a method for separating biomolecules according to the present disclosure. [Diagram 2] FIG. 1 illustrates a schematic diagram of a system suitable for use in the methods of the present disclosure. [Diagram 3] FIG. 1 shows a schematic diagram of a non-limiting embodiment of a magnetic separator suitable for use in the methods of the present disclosure. [Figure 4] FIG. 1 shows a graph depicting the elution curves obtained from the polyclonal IgG experiment described in Example 1 below. [Figure 5A] FIG. 1 shows a graph depicting elution curves obtained from monoclonal antibody (mAb) experiments described in Example 2 below. [Figure 5B]FIG. 1 shows a graph depicting elution curves obtained from monoclonal antibody (mAb) experiments described in Example 2 below. [Figure 5C] FIG. 1 shows a graph depicting elution curves obtained from monoclonal antibody (mAb) experiments described in Example 2 below. [Figure 6] FIG. 1 shows a flow chart of an alternative method for separating biomolecules according to the present disclosure. [Figure 7] FIG. 1 shows a cross-section of a membrane chromatography device that may be used according to one embodiment of an alternative method for separating biomolecules according to the present disclosure. [Figure 8A] FIG. 1 shows a graph depicting elution curves obtained from monoclonal antibody (mAb) experiments described in Example 3 below. [Figure 8B] FIG. 1 shows a graph depicting elution curves obtained from monoclonal antibody (mAb) experiments described in Example 3 below. [Figure 8C] FIG. 1 shows a graph depicting elution curves obtained from monoclonal antibody (mAb) experiments described in Example 3 below. [Figure 8D] FIG. 1 shows a graph depicting elution curves obtained from monoclonal antibody (mAb) experiments described in Example 3 below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present disclosure solves or at least alleviates problems associated with existing methods for isolating biomolecules by providing a method for isolating biomolecules from a cell culture or from a biological solution, as illustrated in FIG. 1, the method comprising: (a) providing magnetic particles comprising a ligand capable of binding to a biomolecule; (b) contacting the cell culture or biological solution containing the biomolecules with the magnetic particles to obtain the magnetic particles containing the bound biomolecules; (c) retaining the magnetic particles within the magnetic separator using a magnetic field; (d) optionally washing the magnetic particles with a washing solution; (e) agitating the magnetic particles on at least one side of the magnetic separator to form a fluidized bed of magnetic particles within the magnetic separator; (f) using a magnetic field to retain the magnetic particles within the magnetic separator while providing a flow of elution fluid substantially perpendicular to at least one surface to elute the bound biomolecules from the magnetic particles; Includes.

[0013] A notable advantage of the disclosed method is that the biomolecules are eluted with a surprisingly low total elution volume, more specifically with an elution volume of up to 10 bed volumes of elution solution, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 bed volumes, preferably up to 4 bed volumes, more preferably up to 3 bed volumes. In this specification, "bed volume" is understood as the volume of the settled magnetic particles, usually expressed in milliliters (ml or mL) or liters (L). The smaller the total elution volume required to elute the biomolecules, the more concentrated and less dispersed the eluted biomolecule sample will be. A high concentration of biomolecules is desirable in any subsequent polishing steps to avoid handling large volumes of samples.

[0014] As mentioned above, a further improvement of the disclosed method may involve further purification of the biomolecules by membrane chromatography after magnetic separation. Thus, the present disclosure provides a method for isolating biomolecules from cell cultures or biological solutions, as illustrated in FIG. 6, which comprises: (a) providing magnetic particles comprising a ligand capable of binding to a biomolecule; (b) contacting the cell culture or biological solution containing the biomolecules with the magnetic particles to obtain the magnetic particles containing the bound biomolecules; (c) retaining the magnetic particles within the magnetic separator using a magnetic field; (d) optionally washing the magnetic particles with a washing solution; (e1) providing a flow of elution fluid through the magnetic separator while retaining the magnetic particles within the magnetic separator using a magnetic field to elute the bound biomolecules from the magnetic particles; (f1) transporting the biomolecules eluted from the magnetic separator to a membrane chromatography device; (g1) separating the biomolecules from impurities and / or contaminants by membrane chromatography; Includes.

[0015] The addition of membrane chromatography after magnetic separation provides a very simple and rapid process that yields highly pure biomolecules.

[0016] The term "biomolecule" has its conventional meaning in the field of bioprocessing, where a biomolecule is produced (often recombinantly) by cells in cell culture and purified from the cell culture by any separation and purification means. Alternatively, the biomolecule is present in a biological solution that does not necessarily originate from a cell culture. Non-limiting examples of biomolecules are biopolymers, which are large biological polymers composed of monomers linked together, such as enzymes, antibodies and antibody fragments, and carbohydrates, and nucleic acid sequences, such as DNA and RNA, including but not limited to peptides and proteins (which may be natural or recombinant). Other non-limiting examples of biomolecules are plasmids and viruses. A biomolecule or biopolymer may be a biomolecule, including but not limited to, a biological polymer intended to be used as, for example, a biopharmaceutical, i.e., a pharmaceutical compound. Herein, a biomolecule that is separated from the remaining cell culture or biological solution by the methods of the present disclosure may alternatively be referred to as a "target biomolecule" or "target". "Biomolecule" is intended to mean a type of biomolecule, and it is understood that the singular form of the term can encompass each of a number of biomolecules.

[0017] As used herein, the term "cell culture" refers to a culture of cells or a group of cultured cells, where the cells can be any type of cell, such as bacterial cells, viral cells, fungal cells, insect cells, or mammalian cells. The cell culture can be fuzzy, i.e., containing a plurality of cells, or cell-depleted, i.e., the culture contains no or few cells, but contains biomolecules released from the cells prior to cell removal. Additionally, fuzzy cell cultures as used in the methods of the present disclosure can include intact cells, disrupted cells, cell homogenates, and / or cell lysates.

[0018] The term "biological solution" is intended to mean a solution of biological origin that contains a biological molecule or a mixture of several types of biological molecules. Examples of biological solutions are any type of body fluid of human or animal origin, such as plasma, blood, sputum, urine, and milk.

[0019] The term "magnetic particles" is defined herein as particles that can be attracted by a magnetic field. At the same time, the magnetic particles used in the disclosed method 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 referred to as magnetic beads. It is understood that the terms "magnetic particles", "magnetic beads", "Mag particles", "Mag beads", "Mag particles", and "Mag beads" may be used interchangeably herein, and their scope is not limited to magnetic particles having a spherical shape.

[0020] Suitable magnetic particles for use in the methods of the present disclosure are described in WO2018122089, which is hereby incorporated by reference in its entirety. In particular, the magnetic particles may have a volume-weighted median diameter (d50, v) in the range of 8-300 μm, such as 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 20-200 μm, more preferably in the range of 37-100 μm. Furthermore, the magnetic particles suitable for use in the methods of the present disclosure may have an average density of 1.05-1.20 g per ml of sedimented magnetic particles.

[0021] The magnetic particles suitable for use in the disclosed method 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 5-15% by weight of magnetic granules. The magnetic granules embedded in the porous matrix of each 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, with the proviso that the central region is defined as having a distance from the magnetic particle surface that is greater than 0.2 times the particle radius, and the surface region is defined as having a distance from the magnetic particle surface that is less than 0.2 times the particle radius.

[0022] The magnetic particles used in the disclosed method include a ligand capable of binding to a biomolecule. The ligand may be covalently coupled to the porous matrix of the magnetic particle. The type of ligand and its affinity constant k for the biomolecule are off / k onIt is understood that the ligand concentration per magnetic particle is selected based on the type of biomolecule to be separated from the cell culture or biological solution. It is further understood that the concentration of ligand per magnetic particle depends on or correlates with, for example, the concentration of the biomolecule in the cell culture or biological solution, the size of the magnetic particle, and / or the total volume of magnetic particles added to the magnetic separator.

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

[0024] FIG. 2 shows diagrammatically a non-limiting example of a separation system 1 that can be used to carry out the method according to the present disclosure. The 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 that separates magnetic particles from a liquid. In a currently preferred embodiment of the present disclosure, the magnetic separator used in the present method for separating biomolecules is a high gradient magnetic separator, alternatively referred to as a high gradient magnetic separation system (HGMS), as described in US Pat. No. 7,506,765 B2, which is hereby incorporated by reference in its entirety. The part of the separation system 1 shown in FIG. 2 is similar to the part of the system described in WO2018122246, which is hereby incorporated by reference in its entirety. The magnetic separator 5 comprises an inlet 5a for receiving a feed from a cell culture 3 or a biological solution 3 containing the biomolecule and for receiving magnetic particles containing a ligand capable of binding to the biomolecule. The magnetic separator 5 is configured to separate the magnetic particles containing the bound biomolecule from the rest of the feed. Magnetic separator 5 includes portions of magnetic or magnetizable material within the magnetic separator that attract magnetic particles when a magnetic field is applied.

[0025] The separation system 1 shown in FIG. 2 comprises a capture cell 9 connected to an inlet 5a of a magnetic separator 5. The capture cell 9 shown in FIG. 2 comprises a cell culture / biological solution inlet 9a for receiving a feed from a 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 the target biomolecules to bind to the magnetic particles before being transferred to the magnetic separator 5. However, it is understood that the capture cell 9 is an optional component of the system 1. The cell culture 3 or biological solution 3 can equally well function as a capture cell if magnetic beads are added to the cell culture 3 or biological solution, respectively, followed by providing a feed from the mixture of cell culture and magnetic particles or from the mixture of biological solution and magnetic particles, containing the bound biomolecules, to the magnetic separator 5. Another alternative is to add the magnetic beads directly to the magnetic separator 5 instead. Addition of each cell culture or biological solution separately and addition of the magnetic beads directly into the magnetic separator is possible for all embodiments.

[0026] Thus, step (b) of the method of the present disclosure comprises: (i) adding magnetic particles to a magnetic separator and then providing a feed from a cell culture or biological solution to the magnetic separator; or (ii) providing a feed from a mixture of cell culture and magnetic particles or from a mixture of biological solution and magnetic particles, containing bound biological molecules, to a magnetic separator; may include.

[0027] After step (b), i.e., contacting a cell culture or biological solution containing biological molecules with magnetic particles to bind the biological molecules to the magnetic particles, the magnetic particles described in steps (c), (d) and (e) are understood to contain bound biological molecules.

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

[0029] According to the method of the present disclosure, the magnetic beads can be washed in a batch mode in a magnetic separator, the magnetic separator being configured to emit a magnetic field when the magnetic particles are washed in batch mode. Alternatively and advantageously, the method comprises applying at least one washing step in a continuous mode, in which case step (d) of the method comprises the following substeps: (d1) agitating the magnetic particles in at least one plane to form a fluidized bed of the magnetic particles; (d2) providing a flow of wash fluid substantially perpendicular to at least one surface while retaining the magnetic particles within the magnetic field to remove the cell culture or biological solution; Includes.

[0030] In this specification, the phrase "substantially perpendicular to at least one surface" is interpreted to mean an angle of 80 to 90 degrees to at least one surface.

[0031] Further, step (d) of the method of the present disclosure may further comprise the following substeps: (i) removing the magnetic field; (ii) resuspending the magnetic particles; (iii) contacting the magnetic particles with a portion of the wash solution; (iv) retaining the magnetic particles using a magnetic field; (v) removing the wash solution from the retained magnetic particles; may include.

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

[0033] The 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 through the magnetic separator 5 is provided by allowing the eluate to enter the magnetic separator 5 via inlet 5a and by allowing the eluate to exit the magnetic separator 5 via outlet 5b. Eluted biomolecules may exit the magnetic separator 5 together with the eluate and be collected in a collection cell 7 connected to the outlet 5b of the magnetic separator 5.

[0034] Therefore, the method of the present disclosure may optionally further comprise, after step (f), a step (g) comprising using a magnetic field to retain the magnetic particles within the magnetic separator while collecting eluted biomolecules that exit the magnetic separator with the eluate in a collection cell, the collection cell being connected to the outlet of the magnetic separator.

[0035] According to an alternative embodiment, instead of the collection cell 7, the system 1 as shown in Fig. 2 comprises a membrane chromatography device 101 for further purification (also called polishing) of the biomolecules. In this embodiment, in step (e1) of the disclosed method, 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 exit the magnetic separator 5 via the outlet 5b. The eluted biomolecules exit the magnetic separator 5 together 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 Fig. 2, it is understood that the cell culture 3 or biological solution 3, the magnetic separator 5, and the collection cell 7 or alternatively the membrane chromatography device 101 may be connected by pre-sterilized flexible tubing and sterile connectors. Furthermore, the collection cell 7 or alternatively the membrane chromatography device 101 may be pre-sterilized and disposable. A closed and sterile separation system for single use may be provided hereby.

[0037] The magnetic separator used to carry out the method of the present disclosure further comprises a stirring device (not shown in FIG. 2; one non-limiting example is shown in FIG. 3, as described further below). The stirring device is configured to apply a stirring action to the magnetic particles, moving them around in a plane perpendicular to the direction of flow through the magnetic separator. The stirring device may include at least one component, such as a plurality of components configured to vary the strength of the magnetic field, such as by applying an oscillating magnetic field, i.e., by regularly varying the strength around a central point or plane. Thus, step (e) or alternatively step (e1) of the method of the present disclosure may include a step of stirring the magnetic particles by varying the strength of the magnetic field at at least one surface of the magnetic separator, such as by applying an oscillating magnetic field at at least one surface of the magnetic separator. Alternatively, the stirring device may include at least one stirrer, such as a plurality of stirrers, in which case step (e) or alternatively step (e1) of the method of the present disclosure includes a step of stirring the magnetic particles by applying or switching on the stirrer. As used herein, the term "stirrer" refers to any type of device or substance / material capable of stirring, such as rotating or stirring, the magnetic particles in the magnetic separator. Non-limiting examples of suitable stirrers are devices attached to a central rotatable carrier shaft, such as a rotor, connected to at least one fixed structure of the magnetic separator, such as a rotatable separating structure, such as a rotatable disk or a rotatable blade. The separating structure may be composed of, for example, a wire mesh, a perforated metal foil, or a perforated metal sheet.

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

[0039] FIG. 3 shows diagrammatically a cross-sectional view of a non-limiting example of a magnetic separator 5 that can be used to carry out the method according to the present disclosure. The arrows in FIG. 3 illustrate the flow of liquid (e.g., eluate) that can enter the magnetic separator 5 from the inlet 5a and exit 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 number of rotating disks 17 are attached. The agitator 13 is configured to agitate the magnetic particles 21 on at least one side of the magnetic separator 5 to form a fluidized bed of magnetic particles within the magnetic separator according to step (e) or alternatively step (e1) of the method according to the present disclosure. In FIG. 3, the magnetic separator 5 further comprises a number of stationary disks 19 attached to the housing 6. The rotating disks 17 and the stationary disks 19 are arranged alternately within the housing 6. The rotating disk 17 and / or the stationary disk 19 may comprise a magnetizable material. Additionally, the rotating disk 17 and / or the stationary disk 19 may be perforated to allow the magnetic particles and the cell culture / biological solution to pass through the disk. It is understood that the configuration of the rotating disk and / or the stationary disk may differ from that shown in FIG. 3. For example, the stationary disk 19 may be connected to the rotor 15, for example, by a sealing material. Additionally, the rotating disk may or may not be attached to the wall of the housing 6. It is further understood that the stirring device may include other components than those shown in FIG. 3. For example, instead of or in addition to having a portion of magnetizable material connected to a fixed structure of the magnetic separator, it is contemplated to include a magnetic material or a portion of magnetizable material, such as a quantity or number of steel wool or wire wool, within the magnetic separator that is not connected to any of the fixed structures of the magnetic separator and that has the ability to attract magnetic particles when a magnetic field is applied and to hold the magnetic particles within the magnetic separator in the magnetic field.

[0040] Step (e) or alternatively step (e1a) of the method of the present disclosure may comprise agitating the magnetic particles in at least one plane of the magnetic separator, such as in a plurality of substantially parallel or parallel planes, to form a plurality of fluidized beds of magnetic particles. As used herein, "substantially parallel planes" is understood as planes that are disposed at an angle of 0-10° relative to one another. In the case of a plurality of substantially parallel or parallel planes, step (f) or alternatively step (e1b) comprises providing a flow of eluate in a direction substantially perpendicular or orthogonal to the plurality of planes. Without wishing to be bound by theory, it is believed that when the agitation device comprises a plurality of agitators in the form of rotatable components arranged in parallel within the magnetic separator and configured to agitate the magnetic particles in a plurality of parallel planes, it is 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 15-1500 rpm, such as 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500, etc., preferably in the range of 50-300 rpm.

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

[0043] The position of the membrane chromatography device 101 in the system 1 shown in Figure 2 is described above. Step (g1) may alternatively be referred to as polishing the biomolecule.

[0044] The term "membrane chromatography" has its conventional meaning in the field of bioprocessing. In membrane chromatography, there is a step of binding components of a liquid, such as individual molecules, associations, or particles, to a solid surface in contact with the liquid. The active surface of the solid phase is accessible to the molecules by convective transport. The advantage of membrane sorbents over packed chromatography columns is that they are suitable for operation at fairly high flow rates. This is also called convective chromatography. Convective chromatography matrices include any matrix in which the imposition of a hydraulic pressure difference between the inlet and outlet sides of the matrix provides the force to perfuse the matrix, providing substantially convective transport of substances into or out of the matrix, which occurs very rapidly at high flow rates. Convective chromatography and membrane adsorbents are described, for example, in U.S. Patent Nos. 20140296464A1, 20160288089A1, WO 2018011600A1, 2018037244A1, 2013068741A1, 2015052465A1, and U.S. Patent No. 7,867,784B2, which are hereby incorporated by reference in their entireties.

[0045] In the methods of the present disclosure, the membrane chromatography device may include a chromatography material comprising one or more electrospun polymeric nanofibers that, in use, form a stationary phase with a plurality of pores through which a mobile phase can pass. The stationary phase may be in the form of a membrane.

[0046] Alternatively, the membrane chromatography device can include at least one adsorptive membrane. The adsorptive membrane can include polymeric nanofibers. Optionally, the membrane, e.g., the adsorptive membrane, can include a nonwoven web of polymeric nanofibers.

[0047] The polymer nanofibers may have an average diameter of 10 nm to 1000 nm. In some applications, polymer nanofibers having an average diameter of 200 nm to 800 nm are suitable. Polymer nanofibers having an average diameter of 200 nm to 400 nm may be suitable for certain applications.

[0048] The chromatographic material or membrane, e.g., the adsorptive membrane, may comprise a polymer 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 a cellulosic polymer, e.g., selected from the group consisting of cellulose and partial derivatives of cellulose, in particular cellulose esters, e.g., cellulose acetate, cross-linked cellulose, graft-bound cellulose, or ligand-bound cellulose.

[0049] In the method of the present disclosure, the membrane chromatography device may comprise a chromatographic material functionalized with (i) positively charged groups, such as quaternary amino groups, quaternary ammonium groups, or amine groups, or (ii) negatively charged groups, such as sulfonate groups or carboxylate groups. Alternatively or additionally, the membrane chromatography device may comprise a chromatographic material functionalized with a multimodal ligand selected from the group consisting of a multimodal anion exchange ligand and a multimodal cation exchange ligand. 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 comprise a chromatographic 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, such as oligo dT.

[0050] To increase the surface area and capacity of a convective membrane adsorbent, the size of the convective pores in the convective matrix can be reduced, for example by using nanofibers as described above. As a result, however, resistance to flow increases. Thus, obtaining high flow rates through a chromatography device containing a high-capacity convective matrix requires a chromatography device and design that can withstand high operating pressures.

[0051] In one embodiment of the method of the present disclosure involving membrane chromatography, a membrane chromatography device may be used as detailed in earlier filed patent application Indian Patent No. 201911019289, Internal Reference No. 502800-IN-1, which is hereby incorporated by reference in its entirety.

[0052] As illustrated in FIG. 7, the membrane chromatography device 101 comprises a chromatography material unit 103 arranged in a cassette 105. The membrane chromatography device 101 further comprises a liquid delivery system 107 configured to deliver liquid to the interior and exterior 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 in the embodiment shown in FIG. 7, however, this does not have to be the case. In the embodiment shown in FIG. 7, the cassette 105 comprises 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, said liquid delivery system 107 can be provided separately from the cassette 105, for example as a separate unit, or can be integrated into the housing 113 of the chromatography device 101.

[0053] The membrane chromatography device 101 further comprises a housing 113 in which at least one chromatography material unit 3 and a cassette 105 are provided. The housing comprises a top plate 125 and a bottom plate 127. An inlet 115 for receiving a liquid feed is provided in the top plate 125 and an outlet 119 for transporting a liquid output stream from the chromatography device is provided in the bottom plate 127. The top plate 125 and the bottom plate 127 are interconnected such that an inlet fluid channel 117 connects the inlet 115 to the chromatography material unit 103 via a liquid delivery system 107 and an 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) adjacent to the inlet surface 153a of the chromatography material unit 103, in which case the plate has several openings for delivering the liquid feed supplied from the inlet 115 of the chromatography device 101 to 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 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) arranged 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 are connected to the collection device outlet 157b via one or more fluid conduits (not shown) arranged in the collection device 109b.

[0056] At least one chromatography material unit 103 may include at least one adsorptive membrane and / or any one or several of the chromatography materials described elsewhere herein. At least one chromatography material unit may include at least one adsorptive membrane sandwiched between at least one top spacer layer (not shown) and at least one bottom spacer layer (not shown), or may include at least two adsorptive membranes stacked together and spaced apart with 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 may be sealed together, leaving at least the inlet 115 and outlet 119 open. In some embodiments, at least some parts of the chromatography device 101 may be sealed together with a plastic or elastomer. Alternatively or additionally, at least some parts of the chromatography device 101 may be overmolded and sealed together. Furthermore, the cassette 105 and / or the housing 113 may be overmolded. Overmolding is a method that creates a seal and provides 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 flow velocity in a direction substantially perpendicular to at least one surface, or alternatively substantially perpendicular to the surfaces, is in the range of 10-3000 cm / h, preferably in the range of 50-600 cm / h. Similar flow rates, i.e. flow rates in the range of 10-3000 cm / h, preferably in the range of 50-600 cm / h, may be applied, where applicable, in other steps of the method of the present disclosure, such as, but not limited to, for example, step (d), step (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 the present disclosure, the residence time of the biomolecule 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, which equates to the residence time of the biomolecule 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) (in the range of about 0.5 seconds to about 6 minutes, preferably about 1 second to about 30 seconds).

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

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

[0063] The method of the present disclosure may further include a step of adjusting the pH of the eluate, diluting the eluate, or adjusting the electrical conductivity of the eluate between steps (e1) and (f1). In the method of the present disclosure, at least steps (c) to (f), or alternatively steps (c) to (e1), such as steps (b) to (f) or alternatively steps (b) to (e1), may be performed in a magnetic separator.

[0064] Alternatively, each of steps (b)-(f) or (b)-(e1) may be performed in a system comprising a bioreactor vessel in fluid communication with a contactor, and a contactor in fluid communication with a magnetic separator. The bioreactor vessel contains the cell culture / biological solution prior to commencing separation of biomolecules from the cell culture / biological solution. Contact between the magnetic particles and the cell culture / biological solution may be performed in a contactor, which may be a vessel in which the cell culture / biological solution and the magnetic particles are transported (e.g., pumped, entrained in a liquid stream, or gravity fed) and may be agitated to some degree 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 the present disclosure may further include a pressurizing device, such as a peristaltic pump configured to generate a flow through the magnetic separator. Alternatively, the flow through the magnetic separator may be generated solely by gravity. In general, the pressure applied to the system to function the method of the present disclosure may be significantly lower than that applied to a high performance liquid chromatography (HPLC) system. Advantages associated with the present disclosure include simpler construction and lower cost. Non-limiting examples of suitable ranges of pressure applied to the separation system are 0-0.5 bar, such as 0.01-0.5 bar.

[0066] In the methods of the present disclosure, the magnetic separator may be interfaced with (i.e., replaces) a chromatography system, such as the AKTA™ Pilot System (GE Healthcare). Although the pump pressure capabilities of a chromatography system are not required, it may be advantageous to use the dual pumps for gradient generation, the valve system, and the buffer control features including the detector of the chromatography system.

[0067] The systems and / or magnetic separators and / or membrane chromatography devices therein used in the methods of the present disclosure may be configured for automated, semi-automated, or manual operation. They can be used in both pilot and manufacturing scale processes, with magnetic particle volumes of at least 0.1 L to 10 L. They are particularly applicable to capture biopharmaceuticals from pre-clarified feeds, such as cell cultures, biological solutions, or cell lysates.

[0068] A device "comprising" one or more recited elements may also include other elements not specifically recited. The term "comprising" includes, as a subset, "consisting essentially of," meaning that the device has the listed components and no other stated features or components. Similarly, a method "comprising" one or more recited steps may include other steps not specifically recited.

[0069] The singular forms "a" and "an" should be construed as including the plural as well.

[0070] The magnetic separator used in the examples below is a High Gradient Magnetic Separation System (HGMS), more specifically a MES 100 RS (Andritz KMPT GmbH). However, it is understood that any type of magnetic separator configured as detailed above may be used to practice the methods of the present disclosure.

[0071] Similarly, as noted above, it is understood that any type of membrane chromatography device having a design capable of withstanding high operating pressures can be used to practice the disclosed methods and is therefore not limited to cellulose fiber chromatography (known as Fibro chromatography) cassettes / units as used in some of the examples below. Fibro chromatography (GE Healthcare Life Sciences) is an ultra-fast chromatographic purification method that utilizes high flow rates and high capacity cellulose fibers to reduce process times and increase productivity (https: / / www.gelifesciences.com / en / us / solutions / bioprocessing / products-and-solutions / downstream-bioprocessing / fibro-chromatography).

[0072] Furthermore, the membrane chromatography material used in the membrane chromatography device is not limited to the so-called Fibro adhere material, as used in some of the examples below. Fibro adhere (GE Healthcare Life Sciences) is a material that contains cellulose nanofibers and can be derivatized with strong ion-exchange multimodal ligands. 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 the use of high fluid speeds. The agarose matrix is ​​derivatized with the multimodal anion-exchange ligand, N-benzyl-N-methylethanolamine. EXAMPLES

[0073] the purpose The aim 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 aim was to investigate whether it was possible to elute IgG from the HGMS system using flow elution.

[0074] material Polyclonal IgG (Gammanorm™), Octapharma NaH 2 PO 4 H 2 O, pa, Merck Na 2 HPO 4 2H 2 O, pa, Merck Sodium acetate trihydrate, pa, Merck Acetic acid, pa, Merck Tween 20, Merck Tris base, pa, Merck Mag Sepharose(TM) PrismA, GE Healthcare

[0075] device HGMS equipment, MES 100 RS, Andritz KMPT GmbH 25L Plastic Buffer Tray Mixing 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 20mM Na phosphate + 0.15M NaCl pH 7.4 11.35g NaH 2 PO 4 H 2 O, 74.3g Na 2 HPO 4、 and 219.2 g of NaCl in a 25 L buffer container with 25 L of dH 2 Dilute and mix with O. 100mM NaOAc pH3.2 140 mL of acetic acid and 7.4 g of NaOAc 3H 2 Add 25 L of dHO to a 25 L buffer container. 2 Dilute and mix with O. 1M NaOH 40 g of NaOH was diluted with 1000 mL of milliQ water and mixed. PBS containing 0.05% Tween Two tablets of Medicago PBS were mixed with 2000 mL MilliQ water. 1 mL of Tween 20 was pipetted and mixed with the solution. 100mM NaOAc pH2.9 5.7 mL of acetic acid and 0.02 g of NaOAc 3H 2 O was mixed with 1000 mL of milli-Q water.

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

[0078] HGMS process Cooling water, air pressure, and distilled water were loaded into the MES 100 RS system. The peristaltic pump of the system was first calibrated with water filling a 1 L volumetric flask at room temperature, followed by the prepared buffer, to rinse the tubing and remove air from the system.

[0079] [Table 1]

[0080] After incubation on the magnetic separator the following program was used: 2 GE-IgG-purified: Main step 1: Loading of magnetic particles (MP) Sub1: Load: Stop all before starting, process duration 250 seconds, magnet running, pump setpoint +50%, valves XV01 and XV14 open. The remaining feed was manually pumped using PBS buffer to wash the tubing. Main process 2: Recirculation Sub 1: Recirculation: Process duration 30 seconds, magnet running, pump setpoint +30%, valves XV08 and XV13 open. Main step 3: Buffer washing Sub 1: Feed buffer 1: Process duration 60 seconds, magnet running, pump set value +70%, valves XV02 and XV14 open. Sub 2: Buffer mixing: Stop everything before starting, process time 20 seconds, mixer setting 60% Sub 3: Trap: Process time 10 seconds, magnet operation, Sub 4: Recapture: Process duration 60 seconds, magnet running, pump setpoint +30%, valves XV08 and XV13 open. Sub 5: Transfer: Process time 1 second, magnet operating, valves XV02, XV08, XV13, and XV14 open. Main process 4: H2O cleaning Sub 1: Buffer 2 feed: process time 60 seconds, magnet running, pump set value +70%, valves XV03 and XV14 open. Sub 2: Buffer mixing 2: Stop everything before starting, process time 20 seconds, mixer setting 60% Sub 3: Trap: Process time 10 seconds, magnet operation, Sub 4: Recapture: Process duration 60 seconds, magnet running, pump setpoint +30%, valves XV08 and XV13 open. Sub 5: Transfer: Process time 1 second, magnet operating, valves XV02, XV08, XV13, and XV14 open. Main Step 5: Elution Sub 1: Trap: Process time 10 seconds, magnet working Sub2: Recapture: Process duration 60 seconds, magnet running, pump setpoint +30%, valves XV08 and XV13 open. Sub 3: Transfer: Process time 1 second, magnet operating, valves XV07, XV08, XV13, and XV16 open. Sub 4: Feed eluate: Process duration 4200 seconds, magnet running, mixer set value 10%, pump set value +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 elution volume in each bottle was determined by subtracting the total volume (51.5 mg) from the empty bottle volume.

[0081] analysis The eluate was neutralized with 2 M Tris base to pH 5. For all fractions, titers were determined at UV280 using a spectrophotometer (ThermoFisher GENESYS).

[0082] Results, Analysis, and Conclusions Concentrations were calculated by dividing the UV280 value in a 1 cm cuvette by the extinction coefficient of 1.36 to obtain the concentration of IgG in mg / ml. Samples were diluted if the UV280 value was greater than 1. Concentrations and cumulative yields were plotted against the cumulative volume of elution. The graph in Figure 4 shows a characteristic elution peak at a yield of 98% and 2.7 x CV (total magnetic bead volume of 0.4 L).

[0083] conclusion Mixing and flow rates were set so that the MagBeads did not flow out of the magnetic chamber during the elution period. By mixing with the magnet switched on at the same time, the MagBeads were suspended within the magnetic chamber, allowing for continuous elution instead of batch elution of IgG. This experiment shows that it is possible to elute IgG in continuous mode while mixing with Mag Sepharose PrismA 37-100 μm in a magnetic chamber, giving a yield of 98% with 2.7 volumes of Mag Sepharose beads. EXAMPLES

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

[0085] material CHO cell culture, GE Healthcare NaH 2 PO 4 H 2 O, pa, Merck Na 2 HPO 4 2H 2 O, 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 Mixing rotor, RW20, Janke & Kunkel 125mL sterile plastic bottle, Nalgene HPLC, 1260 Infinity, Agilent Technologies Glass filter, G3 pore size, approx. 700mL, Scott Duran Centrifuge, 5810R, Eppendorff pH meter, 913 pH meter, Metrohm MabSelect SuRe HiTrap, 29-0491-04, GE HEALTHCARE Superdex 200 Increase 10 / 300 GL, GE HEALTHCARE 0.2 μm syringe filter, Sterivex HV, Millipore 10-100μL pipette, Mettler Toledo 100-1000μL pipette, Eppendorff Cellbag(TM) 10L, BC11, Basic, GE HEALTHCARE

[0087] Conditions and measured values Preparation of solutions: 20mM Na phosphate + 0.15M NaCl pH 7.4 11.35g NaH 2 PO 4 H 2 O, 74.3g Na 2 HPO 4 100 g of NaCl and 219.2 g of NaCl were diluted in 25 L dH2O in a 25 L buffer container. 2 Mixed with O. 100mM NaOAc pH3.2 140 mL of acetic acid and 7.4 g of NaOAc 3H 2 Dilute 25 L of dHO and add 25 L of dHO in a 25 L buffer container. 2 Mixed with O. 1M NaOH 40 g of NaOH was diluted and mixed with 1000 mL of milliQ water. PBS containing 0.05% Tween Two tablets of Medicago PBS were mixed with 2000 mL of MilliQ water. 1 mL of Tween 20 was pipetted and mixed with the solution. 100mM NaOAc pH2.9 5.7 mL of acetic acid and 0.02 g of NaOAc 3H 2O was 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, viability: 66.4%, and mAb titer was 2.7 mg / mL. The cells were pumped into a 10 L cell bag (basic).

[0089] Titer assay, MabSelect SuRe HiTrap binding and elution preparation A MabSelect SuRe HiTrap™ column was interfaced to the HPLC system. A standard curve was prepared with mAbs (mAb titer 8.82 mg / mL) in 250 μL plastic HPLC vials according to the table below.

[0090] [Table 2]

[0091] [Table 3]

[0092] Titer determination of cell cultures Approximately 10 mL of cell suspension was centrifuged at 3000 rpm for 5 minutes, and the cell debris (solid) volume was confirmed to be 5% by monitoring the total cell suspension volume and total solid volume. 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 on a glass filter (G3 pore size) with 5 cv of PBS before use.

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

[0096] HGMS process Cooling water, air pressure, and distilled water were loaded into the MES 100 RS system. The peristaltic pump of the system was calibrated using water to fill a 1 L volumetric flask at room temperature. The inlet tubing and valves used, XV01, XV02, XV03, XV07, 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 until no air was present in the system. After priming with water, the system was primed with working running buffer. XV01= Loading Mag Beads Mixture (primed with 20mM Na phosphate + 0.15M NaCl pH 7.4) XV02 = 20mM Na phosphate + 0.15M NaCl pH 7.4 XV03= dH 2 O XV07 = 100mM Na Acetate pH 3.2 After incubation on the magnetic separator the following program was used: Step 1. Loading the mixture of magnetic particles and feed The pump time was set to 250 seconds and 6 L of the feed mixture was pumped. The remaining feed was manually pumped using PBS buffer to wash the mixture inlet tube. The system was set to recirculation to trap all the magnetic beads on the magnetizable disk within the separation unit. Step 2. Wash with 20 mM Na phosphate + 0.15 M NaCl, repeat for 4 cycles Substep 1. Using 70% pump speed, feed buffer for 60 seconds (2 L buffer). Substep 2. Mix the buffer and beads for 20 seconds at 60% rotor speed and turn off the magnet and valves. Substep 3. Activate the magnet to trap the beads. Substep 4. Recapture. Recirculate using 30% pump speed to trap all beads. Substep 5. Transfer. Open the valve for a short time (1 s) to avoid back pressure. Step 3. Wash with water, 1 cycle The same sub-steps were used for the water wash as for the 20 mM Na phosphate + 0.15 M NaCl wash, except that the water was pumped through valve XV03. Step 4. Elution In this step, the mAb was flow eluted with the mixer and magnet running. Sub-step 1. Trap. Activate the magnet. Substep 2. Recapture. Recirculate for 60 seconds using 30% pump speed. Sub-step 3. Transfer. Open the valve for a short time (1 s) to avoid back pressure. Sub-step 4. Elution feed: 100 mM NaOAc pH 3.2, elute using valve XV07. Magnet running, pump speed 5% (140 mL / min), mixer set to 10%. Elution was carried out for 4286 seconds. The eluted material was manually collected in approximately 100 mL fractions into pre-weighed 150 mL plastic bottles. After elution, each bottle was weighed and the actual eluted volume in each bottle was determined by subtracting the total volume from the empty bottle volume, see Table 5 below.

[0097] [Table 5]

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

[0099] Purity by SEC method Injection volume: 10μL Flow rate: 0.8mL / min Column: Superdex 200 Increase 10 / 300 GL Stop Time: 26 minutes Detection at 214 nm.

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

[0101] Results, Analysis, and Conclusions Figure 5A shows the elution profile of the mAb from the magnetic separator. Pooled fractions (fractions 7-18) corresponding to 1.2 L of eluate show that a mAb yield of 87% can be obtained by using 3 bed volumes (1 bed volume = 400 mL) of elution buffer. The concentration of the pooled eluate was 10.7 mg / mL, which is 4x the concentration. The binding yield in the incubation step was 94% and the total elution yield was 91%. The pooled sample was analyzed by SEC and HCP assay and compared to a pure mAb standard. The results are shown in Figure 5B (smaller scale) and Figure 5C (larger scale; base of peak is enlarged). The solid line is the pooled mAb eluate and the dotted line is the pure mAb standard. The chromatograms are normalized. The aggregate peak, main peak, and peak after the main peak were integrated and the purity was determined to be 98.5%. EXAMPLES

[0102] the purpose The objective of this study was to investigate what mixing speeds (rotor speeds) and flow rates could be used to flow-elute IgG from Mag Sepharose PrismA using fluidized magnetic beads, but without moving the beads. It was also investigated how the IgG elution profile was affected when different mixing speeds, bead volumes, and bead sizes were used.

[0103] device HGMS separator, MES100RS serial no 400213239, Andritz Gmbh Incubation mixer, IL82274-3, Janke-Kunkel UV reader, SpectraMax™ plus, Molecular Devices UV reading plate, 96 well, 3635 Lot 33918007, Corning 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 165mg / mL, Octapharma NaH 2 PO 4 H 2 O, pa, Merck Na 2 HPO 4 2H 2 O pa, Merck NaCl, pa, Merck Acetic acid, pa, Merck Sodium acetate 3H 2 O, pa, Merck

[0105] Conditions and measured values Preparation of solutions: A buffer, 20 mM PO 4 + 0.15M NaCl pH7.5

[0106] [Table 6]

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

[0108] [Table 7]

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

[0110] Testing rotor speed and flow rate to investigate bead loss 100 mL, 300 mL, or 400 mL (wet settled resin) of Mag Sepharose PrismA 37-100 μm or 0-37 μm were pumped into the HGMS system with the magnet running. To investigate at what rotor speed and flow rate the magnetic beads start to move (visually) out of the HGMS chamber, the rotor was run at different speeds and the pump flow rate for A buffer was also varied. The results are shown in Tables 6 to 10 (Tables 8 to 12) below. Loaded with 100 mL of Mag Sepharose 0-37 μm: beads were trapped up to 2100 mL / min at a rotor speed of 150 rpm. At a rotor speed of 225 rpm, beads start to migrate out of the HGMS. Loaded with 300 mL of Mag Sepharose 0-37 μm: beads were trapped up to 560 mL / min at a rotor speed of 75 rpm. Loaded with 100 mL of Mag Sepharose 37-100 μm: beads were trapped up to 2100 mL / min at a rotor speed of 150 rpm. Loaded with 300 mL of Mag Sepharose 37-100 μm: beads were trapped up to 560 mL / min at a rotor speed of 150 rpm. Beads were still trapped at 140 mL / min with a rotor speed of 300 rpm. Loaded with 400 mL of Mag Sepharose 37-100 μm: beads were trapped up to 1400 mL / min at a rotor speed of 75 rpm. It was possible to run higher flow rates at any load, provided the rotor speed was lower than the rotor speed at which the beads began to migrate out of the HGMS system, see table.

[0111] [Table 8]

[0112] [Table 9]

[0113] [Table 10]

[0114] [Table 11]

[0115] [Table 12]

[0116] Flow elution test for IgG 400 mL of each resin prototype was incubated with 6 L of IgG (2 mg / mL) sample for >1 h. 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 and the magnetic beads were trapped by the 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 different rotor speeds at 140 mL / min. During elution, fractions of approximately 100-2100 mL were collected and the absorbance was determined for each fraction using a UV plate reader and the exact volume of each fraction was also determined using a weighing machine. The UV for each fraction was plotted against the cumulative elution volume. See Figures 1-4. In general, at 0% rotor speed, high concentrations of IgG were still present in the eluate even after several column volumes (>4000 mL) of elution buffer, indicating incomplete elution of IgG, and the elution profile did not show a trend toward a decrease in concentration. For beads smaller than 37 μm, the smaller the bead volume, the less dependent it is on rotor speed. For 100 mL beads, the elution peak shapes were similar at rotor speeds of 75 and 150 rpm (Figure 8A). For 400 mL beads, the elution peak shapes differed more significantly between rotor speeds, with IgG elution being most effective when 150 rpm was used (Figure 8C). For 37-100 μm beads, the smaller the bead volume, the less dependent on rotor speed. For 100 mL of beads, the elution peaks at rotor speeds of 75 rpm and 150 rpm were similar (Figure 8B). For 400 mL of beads, the elution peak shapes were dramatically different between 150 rpm and 0 rpm rotation. When spun at 150 rpm, IgG eluted with a symmetric elution peak, whereas at 0 rpm, IgG still eluted after 6500 mL of elution buffer was run (Figure 8D). Comparing 0-37 μm beads with 37-100 μm beads, the elution peak shapes are similar when 100 mL of beads are used (see Figures 8A and 8B, respectively). At 400 mL of beads and 150 rpm rotation, the larger beads (37-100 μm) showed a much sharper elution peak than the <37 μm beads (400 mL). With the larger beads, it appears that the majority of the IgG eluted before 2000 mL of elution buffer was reached, whereas for the <37 μm beads, the elution peak did not drop to a stable baseline even after 4000 mL of flow (see Figures 8C and 8D, respectively). EXAMPLES

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

[0118] material Starting sample: IgG1 monoclonal antibody obtained from XDR-10 culture on CHO cells, viability 66.4%, concentration 2.7g / L Superdex(TM) 200 Increase 10 / 300 GL, GE HEALTHCARE MagSepharose PrismA 37~100μm, 400ml, GE HEALTHCARE Fibro Adhere unit prototype, 0.4ml: Fibro Adhere 1: The material was crosslinked with divinyl sulfone (DVS) after the addition of allyl glycidyl ether (AGE) using an Adhere reaction concentration of 167 g / liter. The titer was 563 μmol / g. Fibro Adhere2: Material was cross-linked with DVS prior to the addition of AGE using an Adhere reaction concentration of 167 g / liter. Titer 212 μmol / g. Fibro Adhere3: Material was cross-linked with DVS before adding AGE using an Adhere reaction concentration of 500g / liter. Titer 478μmol / g.

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

[0120] buffer 25mM Na phosphate + 0.15M NaCl pH 6.3 4.88g NaH 2 PO 4 H 2 O, 2.6 g Na 2 HPO 4 , and 17.5 g NaCl in 2 L of dH 2 Dilute with 0 and mix. 25mM Sodium Phosphate pH7 1.16g NaH 2 PO 4 H 2 O and 2.367g Na 2 HPO 4 Add 1L of dHO 2 Dilute with 0 and mix. 25mM Na phosphate + 1M NaCl pH7 0.464g NaH 2 PO 4 H 2 O, 3.851g Na 2 HPO 4 , and 58.44 g of NaCl in 1 L of dH 2 Dilute with 0 and mix. 25mM Sodium Phosphate pH 7.5 0.73g NaH 2 PO 4 H 2 O and 3.508g Na 2 HPO 4 Add 1L of dHO 2 Dilute with 0 and mix. 100mM Acetic Acid pH3.0 Dissolve 6 ml of glacial acetic acid in 1 L of dHO. 2 Dilute with 0 and mix.

[0121] Conditions and measured values The eluate eluted from MagSepharose PrismA using a high gradient magnetic separator system as described in Example 2 was used for further polishing by membrane chromatography using a prototype Fibro adhere unit. The Fibro Adhere unit was connected to an AKTA Pure 25 system and three different 25 mM Na-phosphate buffer conditions (pH and conductivity) were evaluated. See Table 11. Equilibration was performed at a flow rate of 16 ml / min for 50 unit volumes (20 ml), after which a 4 ml mAb sample was applied at 16 ml / min and the flow-through was collected. The Fibro Adhere unit was washed with 20 unit volumes (8 ml) and cleaned with 100 mM acetic acid pH 3.0 at a flow rate of 16 ml / min for 25 unit volumes (10 ml), before re-equilibration for 50 unit volumes (20 ml). UV was constantly monitored at 280 nm.

[0122] [Table 13]

[0123] Results, Analysis, and Conclusions Chromatograms obtained after polishing mAb with Fibro Adhere under different buffer conditions showed high UV values ​​during the sample application period in all three buffer conditions. Flow through fractions of samples during the sample application period were collected and further analyzed; see Table 12. mAb concentration and aggregate percentage were analyzed by SEC-HPLC, Superdex 200 Increase 10 / 300 GL, using 0.2M Na phosphate buffer at a flow rate of 0.8 ml / min for 26 min per sample. Host cell protein levels in the flow through fractions were analyzed using Cygnus 3rd generation CHO HCP ELISA kit.

[0124] [Table 14]

[0125] In conclusion, polishing of mAbs resulted in high yields (93-100%) and substantial reduction of host cell proteins (HCPs). HCP reduction was greatest at pH 7.5 and conductivity 3.5 mS / cm, resulting in yields of 93-99%, but also highest when using the Fibro Adhere prototype and the highest ligand density.

[0126] It is understood that the present disclosure is not limited to the exemplary embodiments thereof described above, and that possible modifications thereto are possible within the scope of the following claims. [Explanation of symbols]

[0127] 1 Separation system, system (Fig. 2) 3 Cell cultures and biological solutions (Figure 2) 3 Chromatography material unit (Figure 7) (※ Possible error) 5 Magnetic separator (Figure 2) 5a Inlet (Fig. 2) 5b Outlet (Fig. 2) 6 Housing (Fig. 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 Collection device (Fig. 7) (※ Possible error) 11 Electromagnet (Fig. 3) 13 Cleaning equipment (Fig. 2) 13 Stirring device (Fig. 3) 15 Washing buffer supply device (Figure 2) 15 Rotor (Fig. 3) 17 Washing buffer collection device (Figure 2) 17 Rotating disk (Fig. 3) 19 Stationary disk (Fig. 3) 21 Magnetic particles (Figure 3) 101 Membrane Chromatography Device (Figure 2) (Figure 7) 103 Chromatography Materials Unit (Figure 7) 105 Cassette (Fig. 7) 107 Fluid Delivery System (Figure 7) 109a Delivery device (Figure 7) 109b Collection device (Figure 7) 113 Housing (Fig. 7) 115 Inlet (Fig. 7) 117 Inlet fluid channel (Figure 7) 119 Outlet (Fig. 7) 121 Outlet fluid channel (Figure 7) 125 Top plate (Fig. 7) 127 Bottom Plate (Fig. 7) 153a Inlet surface (Fig. 7) 153b Outlet surface (Fig. 7) 157a Delivery Device Inlet (Figure 7) 157b Collection device outlet (Figure 7)

Claims

1. 1. A method for isolating a biomolecule from a cell culture, comprising: (a) providing magnetic particles comprising a ligand capable of binding to said biomolecule; (b) contacting a cell culture containing said biomolecules with said magnetic particles to obtain magnetic particles containing bound biomolecules; (c) retaining the magnetic particles within a magnetic separator using a magnetic field; (d) optionally washing the magnetic particles with a washing solution; (e) agitating the magnetic particles on at least one side of the magnetic separator to form a fluidized bed of magnetic particles within the magnetic separator; (f) using the magnetic field to retain the magnetic particles within the magnetic separator while providing a flow of elution fluid substantially perpendicular to the at least one surface to elute the bound biomolecules from the magnetic particles; The method includes:

2. The method of claim 1 , wherein step (e) comprises agitating the magnetic particles by varying the strength of the magnetic field, such as by applying an oscillating magnetic field.

3. 1. A method for isolating a biological molecule from a cell culture or from a biological solution, comprising the steps of: (a) providing magnetic particles comprising a ligand capable of binding to said biomolecule; (b) contacting a cell culture or a biological solution containing said biomolecules with said magnetic particles to obtain magnetic particles containing bound biomolecules; (c) retaining the magnetic particles within a magnetic separator using a magnetic field; (d) optionally washing the magnetic particles with a washing solution; (e1) providing a flow of elution fluid through the magnetic separator while retaining the magnetic particles within the magnetic separator using the magnetic field to elute the bound biomolecules from the magnetic particles; (f1) transporting the biomolecules eluted from the magnetic separator to a membrane chromatography device; (g1) separating said biomolecules from impurities and / or contaminants by membrane chromatography; The method includes:

4. Step (e1) is (e1a) agitating the magnetic particles on at least one side of the magnetic separator to form a fluidized bed of magnetic particles within the magnetic separator; (e1b) providing an elution fluid flow in a direction substantially perpendicular to the at least one surface to elute the bound biomolecules from the magnetic particles; The method of claim 3 , comprising:

5. The method of claim 4 , wherein step (e1a) comprises agitating the magnetic particles by varying the strength of the magnetic field, such as by applying an oscillating magnetic field.

6. The method according to any one of claims 3 to 5, wherein in step (g1), the residence time of the biomolecule in the membrane chromatography device is in the range of about 0.5 seconds to about 6 minutes, preferably about 1 second to about 30 seconds.

7. 7. The method of any one of claims 3 to 6, wherein the magnetic separator is connected to a chromatography system that provides an elution flow and optionally a wash flow through the membrane chromatography device.

8. 8. The method of any one of claims 3 to 7, wherein the membrane chromatography device comprises a chromatographic material comprising one or more electrospun polymeric nanofibers that, in use, form a stationary phase with a plurality of pores through which a mobile phase can pass.

9. The method of claim 8 , wherein the stationary phase is in the form of a membrane.

10. The method of any one of claims 3 to 9, wherein the membrane chromatography device comprises at least one adsorptive membrane.

11. The method of claim 10 , wherein the adsorptive membrane comprises polymeric nanofibers.

12. The method of claim 10 or 11, wherein the membrane comprises a nonwoven web of polymeric nanofibers.

13. 13. The method of any one of claims 8-9 and 11-12, 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.

14. 13. The method according to any one of claims 8 to 9 and 11 to 12, wherein the polymer is a cellulosic polymer such as selected from the group consisting of cellulose and partial derivatives of cellulose, in particular cellulose esters, crosslinked cellulose, grafted cellulose, or ligand-bound cellulose.

15. 15. The method of any one of claims 3 to 14, wherein the membrane chromatography device comprises a chromatographic material functionalized with (i) positively charged groups, such as quaternary amino groups, quaternary ammonium groups, or amine groups, or (ii) negatively charged groups, such as sulfonate groups or carboxylate groups.

16. 16. The method of any one of claims 3 to 15, wherein the membrane chromatography device comprises a chromatographic material functionalized with a multimodal ligand selected from the group consisting of a multimodal anion exchange ligand and a multimodal cation exchange ligand.

17. 17. The method of claim 16, wherein the multimodal anion exchange ligand is a N-benzyl-N-methylethanolamine ligand coupled to a support, the support being linked to a nitrogen atom of the ligand via a linker.

18. 15. The method of any one of claims 3 to 14, wherein the membrane chromatography device comprises a chromatographic material functionalized with (i) ion exchange groups, (ii) affinity peptide / protein based ligands, (iii) hydrophobic interaction ligands, (iv) IMAC ligands, or (v) DNA based ligands, such as oligo dT.

19. 19. The method of any one of claims 3 to 18, wherein the membrane chromatography in step (g1) is flow-through membrane chromatography.

20. 20. The method of any one of claims 8 to 19, wherein the flow of the eluate through the membrane or chromatographic material is normal flow or tangential flow.

21. The method according to any one of claims 3 to 20, further comprising the step of adjusting the pH of the eluate, diluting the eluate, or adjusting the electrical conductivity of the eluate between steps (e1) and (f1).

22. The membrane chromatography device (101) At least one chromatography material unit (103) comprising a convection-based chromatography material; At least one liquid delivery system (107) configured to deliver liquid to the interior and exterior of the at least one chromatographic material unit (103); An inlet (115); at least one inlet fluid channel (117) connecting said inlet to at least one chromatographic material unit (103) by a liquid delivery system (107); Outlet (119) and at least one outlet fluid channel (121) connecting the outlet (119) to at least one unit of chromatography material (103) via a liquid delivery system (107), wherein at least some parts of the membrane chromatography device (101) are sealed together while at least the inlet (115) and the outlet (119) are left open; 22. The method of any one of claims 3 to 21, comprising:

23. 23. The method of any one of claims 1 to 22, wherein step (b) or (c) comprises adding the magnetic particles to a magnetic separator comprising at least one stirrer, such as a plurality of stirrers, and step (e) or (e1a) comprises stirring the magnetic particles by switching on a stirrer.

24. Step (d) comprises the following substeps: (d1) agitating the magnetic particles in at least one plane to form a fluidized bed of magnetic particles; (d2) providing a flow of wash fluid substantially perpendicular to the at least one surface to remove the cell culture while retaining the magnetic particles within the magnetic field; 24. The method of any one of claims 1 to 23, comprising:

25. 25. The method of any one of claims 1 to 24, wherein step (e) or step (e1a) comprises agitating the magnetic particles in a plurality of substantially parallel faces of the magnetic separator to form a plurality of fluidized beds of magnetic particles, and step (f) or step (e1b) comprises providing a flow of the eluate in a direction substantially perpendicular to the plurality of faces.

26. 26. The method of any one of claims 1 to 25, wherein the biomolecule is eluted with up to 10 bed volumes, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 bed volumes, preferably up to 4 bed volumes, more preferably up to 3 bed volumes of elution solution.

27. 27. The method according to any one of claims 1 to 26, wherein at least steps (c) to (f) or (c) to (e1), such as steps (b) to (f) or (b) to (e1), are carried out in a magnetic separator, preferably wherein the magnetic separator is a high gradient magnetic separation system.

28. Step (b) (i) adding said magnetic particles to a magnetic separator and then providing a feed derived from said cell culture to said magnetic separator; or (ii) providing a feed from the mixture of the cell culture and the magnetic particles, containing the bound biomolecules, to a magnetic separator.

28. The method of any one of claims 1 to 27, comprising:

29. 29. The method of any one of claims 1 to 28, wherein steps (b) to (f) or steps (b) to (e1) are carried out in an integrated bioreactor vessel / contactor and magnetic separator.

30. Step (d) comprises the following substeps: (i) removing the magnetic field; (ii) resuspending the magnetic particles; (iii) contacting the magnetic particles with a portion of a wash solution; (iv) retaining the magnetic particles using a magnetic field; (v) removing the wash solution from the retained magnetic particles; 30. The method of any one of claims 1 to 29, comprising:

31. 31. The method of any one of claims 1 to 30, wherein step (d) is repeated at least once before proceeding to step (e) or step (e1).

32. 32. The method according to any one of claims 1 to 31, wherein the linear flow velocity of the eluent in step (f) or (e1) is in the range of 10 to 3000 cm / h, preferably in the range of 50 to 600 cm / h in step (f).

33. 33. The process according to any one of claims 23 to 32, wherein the stirrer speed in step (e) or (e1a) is in the range of 15 to 1500 rpm, preferably in the range of 50 to 300 rpm.

34. 34. The method of any one of claims 1 to 33, wherein the magnetic separator is in communication with a chromatography system.

35. A method according to any one of the preceding claims, wherein the magnetic particles have a volume-weighted median diameter (d50, v) in the range of 8 to 300 μm, preferably in the range of 37 to 100 μm.

36. 36. The method of any one of claims 1 to 35, wherein the magnetic particles have an average density of 1.05 to 1.20 g / ml of sedimented particles.

37. 37. The method of any one of claims 1 to 36, wherein each of the magnetic particles comprises a porous polymer matrix and one or more magnetic granules embedded in the porous polymer matrix.

38. The method of claim 37, wherein each of the magnetic particles comprises 5 to 15% by weight of the magnetic granules.

39. The method according to claim 37 or 38, wherein the magnetic granules have a volume-weighted median diameter (d50, v) of 1 to 5 μm.

40. 40. The method of any one of claims 37 to 39, wherein each of the magnetic particles comprises a concentration of magnetic granules in a central region of the particle that is at least 200% of the concentration in a surface region of the particle, the central region being defined as having a distance from the particle surface that is greater than 0.2 times the particle radius, and the surface region being defined as having a distance from the particle surface that is less than 0.2 times the particle radius.

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