Magnetic separation

Abstract

Disclosed herein are systems, devices and methods for automated magnetic separation of magnetized targets in biological samples, wherein they include a magnetic field shield / barrier controllably operable to control a magnetic field by reaching and attracting the magnetized targets in the biological sample.

Description

Technical Field

[0001] The present invention relates to devices, methods and systems for automated magnetic separation of targets from biological samples.Such devices, methods and systems find use in a variety of clinical and laboratory settings. Background Art

[0002] Magnetic separation has been used as a method for separating magnetic impurities from fluids by applying a variety of different processes (US 3,985,646; US 4,054,513; and US 5,137,629). Magnetic separation techniques have also been applied to separate populations of biological materials using magnetic beads that have been coated with antibodies or polymers to bind to various biological targets, including viruses, bacteria, and cells (US 3,970,518; US 4,219,411; US ​​4,795,698; and US 5,385,707). The biological target can then be extracted from the fluid suspension using one of the previously developed magnetic separation devices described, for example, in US 4,710,472; US 5,691,208; US 6,193,892 and Zborowski et al., (Journal of Magnetism and Magnetic Materials, vol. 194, pp. 224-230, 1999). The magnetic field generated in the separation device exerts a force on the magnetic beads suspended therein, which can draw the magnetic beads out of the fluid suspension (as described in Shevkoplyas et al., (Lab on a Chip, vol.7, pp.1294-1302, 2007) and Warnke (IEEE Transactions on Magnetics, vol.39, issue 3, pp.1771-1777, 2003)) and any biological material bound to the magnetic beads. This allows the desired population to be separated by removing the desired population from the fluid suspension (known as positive selection), or by removing all other populations from the fluid suspension to leave only the non-magnetically bound target population (known as negative selection). Separating cells (such as T cells and stem cells) from heterogeneous cell populations is essential for developing cell therapies for treating various diseases.

[0003] A system uses static levitation (STEMCELL) in surrounding magnets EasySep TM Other automated systems that use magnetic beads to separate target populations are also known (from Milytenyi Biotec and from STEMCELL TM RoboSep by TechnologiesTM ).

[0004] There remains an unmet need for rapid and reliable magnetic separation of selected targets in biological samples, where the application of magnetic fields can be automated, customized, and controlled for isolating targets with desired high yield and high purity. Summary of the invention

[0005] The present invention provides a method for collecting target biological populations from biological samples in an automated cell culture system, the method comprising: a. combining the target biological population with magnetic particles; b. circulating the biological sample through one or more fluidic pathways of the automated cell culture system; c. exposing the target biological population combined with the magnetic particles to a magnetic field gradient; d. repeating steps bc one or more times; and e. collecting the target biological population combined with the magnetic particles.

[0006] The present invention also provides a method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising: a. binding the target biological population to magnetic particles; b. circulating the biological sample through one or more fluidic pathways of the automated cell culture system; c. exposing the target biological population bound to the magnetic particles to a magnetic field gradient to capture the target biological population bound to the magnetic particles; d. circulating unbound components of the biological sample through one or more fluidic pathways of the automated cell culture system; e. inserting a magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field to release the target biological population bound to the magnetic particles; f. circulating the target biological population bound to the magnetic particles through one or more fluidic pathways of the automated cell culture system; g. repeating steps bf once or more; and h. collecting the target biological population bound to the magnetic particles.

[0007] In another embodiment, a method for collecting a target biological population from a biological sample in an automated cell culture system is provided herein, the method comprising: a. binding a non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluidic pathways of the automated cell culture system; c. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient; d. repeating steps bc one or more times; and e. collecting the target biological population;

[0008] In still other embodiments, the present invention provides a method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising: a. binding a non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluidic pathways of the automated cell culture system; c. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient to capture the non-target biological population bound to the magnetic particles; d. circulating the target of the biological sample through one or more fluidic pathways of the automated cell culture system; e. inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field to release the non-target biological population bound to the magnetic particles; f. circulating the non-target biological population bound to the magnetic particles through one or more fluidic pathways of the automated cell culture system; g. repeating steps bf once or more; and h. collecting the target biological population. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following description of typical aspects described herein will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, present typical aspects are shown in the accompanying drawings. However, it should be understood that the invention is not limited to the precise arrangement and configuration of the aspects shown in the accompanying drawings. It should be noted that the same reference numerals refer to the same elements in different embodiments, as shown in the drawings and mentioned in the specification.

[0010] The description of this article will be more fully understood based on the following drawings:

[0011] FIG1 shows a magnetic field gradient used to move magnetic particles;

[0012] Figure 2 Magnetic beads of different sizes for cell separation applications are shown;

[0013] Figure 3 shows the effect of bead size on the net magnetic force acting on the bead in response to a magnetic field gradient;

[0014] FIG4 shows different conceptual approaches for generating an automatically controlled magnetic field;

[0015] Figure 5 Computational model showing the ability of high permeability and saturation materials to block magnetic flux density;

[0016] Figure 6 The incorporation of a magnetic separation tube on the cartridge is shown;

[0017] FIG. 7 shows a computer-aided design (CAD) model of a separator tube 701 aligned with a magnet array 704 consisting of rare earth magnets having alternating poles 702 and 703;

[0018] Figure 8CAD model assembly showing separation tube 701 in an "open" position aligned with magnet array 704, placed within magnetic field shield 801, which can be rotated using motor 802 and gear train 803;

[0019] Fig. 9 CAD assembly of a separation tube 701 aligned with a magnet array is shown in a "closed" position, with a magnetic field shield 801 located between the tube 701 and the magnet array 704;

[0020] FIG10 shows a cross-sectional view of the assembly in an “open” or “closed” position;

[0021] Fig.11 The complete assembly of a cassette 1101 consisting of a separation tube 701 aligned with a cell culture instrument 1102 containing a magnetic separation assembly (704 and 801) is shown;

[0022] FIG12 illustrates various non-limiting methods of generating local high magnetic field gradients (portions 1201-1208);

[0023] FIG13 shows the results obtained from Working Example 1, demonstrating the successful separation of cells from magnetic beads at different flow rates;

[0024] FIG14 shows the results obtained from Working Example 2, demonstrating the effect of different flow rates on the capture and release of positively selected cells;

[0025] FIG. 15 shows the results from Working Example 3 which added multiple passes through the separation tube 701 to the magnetic separation process in terms of capture and release of positively selected cells;

[0026] Figure 16 shows the results from Working Example 4, which increases the exposure of the magnetic beads to the magnetic field by increasing the waiting time in terms of capture and release of positively selected cells;

[0027] Fig.17 shows how various process modifications can modify the percentage of false positives obtained from Working Example 4;

[0028] FIG. 18 shows the results obtained from Working Example 5 for purifying a mixed cell population flowing through a separation tube 701 and passing through a magnet array 704;

[0029] FIG. 19 shows results obtained from Working Example 6, demonstrating how modifying the dimensions of magnets 702 and 703 can affect the properties of the magnetic field generated by magnet array 704 and cell capture; and

[0030] FIG. 20 shows the results obtained from Working Example 7 regarding the effect of adding rods / spacers 1201 to the box.

[0031] Fig.21 A schematic diagram of a recycling magnetic separation method according to an embodiment of the present invention is shown.

[0032] Figures 22A-22D Purification and recovery of waste cells according to an embodiment of the present invention are shown.

[0033] Figures 23A-23B The efficiency of release of beads from cells according to an embodiment of the present invention is shown.

[0034] 24A-24C illustrate the effect of intensity and multiple cycles of contact with a magnetic field.

[0035] Figures 24D-24E Shown is the capture and removal of cells in a separation tube as described herein.

[0036] 25A-25L show the results of positive and negative selection of target cells as described herein.

[0037] 26A-26D illustrate the recovery of cells in an automated cell culture system as described herein.

[0038] Figures 27A-27B Shown is the recovery of magnetic particles after washing in an automated cell culture system as described herein. DETAILED DESCRIPTION

[0039] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are merely provided for the purposes of providing the disclosed content prior to the filing date of the present application. Any content herein should not be construed as admitting that the present invention is not entitled to precede such disclosure by virtue of prior inventions. In addition, materials, methods and examples are illustrative only, not restrictive.

[0040] In the event of a conflict, the present specification (including definitions) will prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the subject matter of this article belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0041] As used herein, the articles "a" and "an" preceding an element or component are intended to be non-limiting to the number of instances (i.e., occurrences) of the element or component. Therefore, "a" or "an" should be understood to include one or at least one, and the singular form of an element or component also includes the plural unless the number is obviously intended to be singular.

[0042] As used herein, the term "invention" or "present invention" is a non-limiting term and is not intended to refer to any single aspect of the particular invention, but includes all possible aspects as described in the specification and claims.

[0043] As used herein, the terms "comprises and comprising", "includes and including", "having" and variations and conjugates thereof mean "including, but not limited to".

[0044] As used herein, the term "about" to modify the amount of the composition, component or reactant used refers to a change in the numerical amount, which can occur, for example, by typical measurements and liquid handling procedures for preparing concentrates or solutions. In addition, the change may occur due to unexpected errors in the measurement procedure, the manufacture of the composition or the implementation method, the difference in source or purity, etc. In one aspect, the term "about" refers to within 10% of the reported numerical value. In another aspect, the term "about" refers to within 5% of the reported numerical value. However, in another aspect, the term "about" refers to within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the reported numerical value.

[0045] If a range of values ​​is listed, this is only for convenience or brevity and includes all possible subranges and individual values ​​within and around the range boundaries. Unless otherwise specified, any numerical value includes actual approximate values, and integer values ​​do not exclude decimal values. Subrange values ​​and actual approximate values ​​should be considered as specifically disclosed values.

[0046] It should be understood that any element defined herein as included may be expressly excluded from the claimed invention by way of a restrictive clause or negative limitation.

[0047] As may be used herein, the terms "closely," "approximately," and "substantially" represent a corresponding relationship or measure or amount or quantity that has no adverse consequences or effects with respect to the referenced term or embodiment or operation or the scope of the invention.

[0048] As may be used herein, any terms referring to geometric relationships, such as "vertical," "horizontal," "parallel," "relative," "straight," "transverse," "parallel," "perpendicular," and other angular relationships, also represent approximate but functional and / or practical corresponding relationships.

[0049] As may be used herein, the terms "preferred," "preferably," "typical," "typically," or "optionally" do not limit the scope of the invention or embodiments thereof.

[0050] As may be used herein, the terms "substantial," "perceptible" (or their synonyms) mean, relative to the context, a measure or range or amount or degree that encompasses a substantial portion or majority of the referenced entity, or an extent that is at least moderate or substantially greater or larger or more effective or more significant relative to the referenced entity or relative to the referenced subject matter.

[0051] As used herein, the terms "negligible" and "slight" (or their synonyms) mean a corresponding relationship or measure or amount or quantity or degree that is sufficiently small to have no practical consequence with respect to the scope of the referenced term and the invention.

[0052] As used herein, the term "may" indicates an option or effect that is included or not included and / or used and / or implemented and / or occurs, however the option constitutes at least part of some embodiments of the present invention or a result thereof, without limiting the scope of the present invention.

[0053] As used herein, a "sample" may be any sample, and may be a "biological sample" which may be from a plant, human, animal or microbial source. A sample is typically a heterogeneous sample from which a target is selected for separation and collection. The target may be a cell, DNA, RNA, protein, peptide, microorganism, virus, etc. A biological sample comprises a target population.

[0054] Biological samples can include body fluid samples, body cell samples or biological tissue samples. Examples of biological samples or body fluid samples include urine, lymph, blood, plasma, serum, saliva, cervical fluid, cervical-vaginal fluid, vaginal fluid, breast fluid, breast milk, synovial fluid, semen, seminal fluid, feces, sputum, cerebrospinal fluid, tears, mucus, interstitial fluid, follicular fluid, amniotic fluid, aqueous humor, vitreous fluid, peritoneal fluid, ascites, sweat, lymph, lung sputum and lavage fluid or samples derived therefrom. Biological tissue samples are samples containing cell aggregates (usually specific species) and intercellular substances, which form one of the structural materials of human, animal, plant, bacterium, fungus or virus structure, including connective tissue, epithelial tissue, muscle and nervous tissue. Examples of biological tissue samples also include organs, tumors, lymph nodes, arteries and individual cells. For example, a sample can be a tissue sample suspected to be cancerous. The biological tissue sample can be first processed to separate cell aggregates.

[0055] In an embodiment, the biological sample is a blood cell, a white blood cell or a platelet. White blood cells (leukocytes) include neutrophils, lymphocytes (T cells, including T helper cells, cytotoxic T cells, T-killer cells, natural killer cells and B lymphocytes), monocytes, eosinophils, basophils, macrophages and dendritic cells.

[0056] As used herein, a "target cell" is a cell that is typically used to separate or concentrate from other cells (such as for examination or diagnosis), is of a specific type, or has a different characteristic relative to other cells (such as a selective mutual affinity for coupling to certain antibodies or other compounds or other particles). In a specific embodiment, the unique characteristic is a selective affinity for coupling or binding to magnetic beads to form a magnetic target cell.

[0057] As used herein, the term "patient sample" is defined as a biological sample taken from any animal for which diagnosis, screening, monitoring or treatment is contemplated. Animals include mammals. Patient refers to a subject, such as a mammal, primate, human or livestock subject suffering from a disease condition or a disease condition to be determined or treated. A patient sample can be a source of a source biological population.

[0058] As used herein, the term "antibody" is intended to include polyclonal and monoclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM) or antigen-binding portions thereof, including but not limited to F(ab) and Fv fragments such as scFv, single-chain antibodies, chimeric antibodies, humanized antibodies, recombinantly engineered antibodies and Fab expression libraries. Bispecific antibodies can also be immobilized on magnetic particles.

[0059] As used herein, a "labeling moiety" is one that is directly or indirectly detectable. The labeling moiety can be a detectable label and can be used in conjunction with a magnetic particle. Direct labeling moieties include radioisotopes; enzymes whose products are detectable (e.g., luciferase, β-galactosidase, etc.); fluorescent markers (e.g., fluorescein isothiocyanate (FITC), rhodamine, phycoerythrin, cyanine dyes, Cascade Blue, PerCP, Cy5, Cy7, allophycocyanin (APC), PECy5 or other tandem conjugates of different fluorescent dyes, Texas Red, etc.); fluorescent emitting metals, such as 152 Eu or other metals of the lanthanide series, which are linked to proteins through metal chelating groups such as EDTA; chemiluminescent compounds, such as luminol, isoluminol, acridinium salts, etc.; bioluminescent compounds, such as fluorescein, aequorin (green fluorescent protein), etc.; and metal compounds. Indirect labeling moieties include labeling molecules that bind to polypeptides, such as antibodies specific for polypeptides, wherein the labeled binding molecules are labeled as described above; and members of specific binding pairs, such as biotin, (members of the specific binding pair biotin-avidin), digoxigenin (members of the specific binding pair digoxigenin-antibody to digoxigenin), etc. Alternatively, the labeling moiety can be any suitable label, including but not limited to those described herein.

[0060] Magnetic particles labeled with binding partners such as antibodies, proteins or nucleic acid molecules are available from Miltenyi Biotec GmbH (Friedrich Ebert Str. 68, D-51429 Bergisch Gladbach, Germany). Methods for magnetically labeling biomolecules are known in the art; any known method can be used. For example, U.S. Pat. No. 6,020,210 describes methods for preparing magnetic particles and attaching biomolecules thereto. The first member of a specific binding pair can be associated with a magnetic particle, wherein the biomolecule to be modified includes a portion that binds to a member of the specific binding pair. Alternatively, the magnetic particle is coupled to, for example, an antibody or an immunoreactive fragment thereof, via a linker or spacer, such as, for example, a nucleic acid linker. The addition of a spacer or linker will allow the biomolecule to be presented in a more flexible manner, and sophisticated chemistry can connect the ligand in a specific orientation. There are a variety of chemistries for these couplings, as many companies have published protocols and will help those skilled in the art master the chemistry.

[0061] Examples of members of specific binding pairs that can be attached to magnetic particles include, but are not limited to, oligo dT (for binding to nucleic acid molecules including, for example, a poly-A tract at the 3' end); oligonucleotides having a specific nucleotide sequence (for binding to nucleic acid molecules including a complementary nucleotide sequence); avidins (e.g., streptavidin) (for binding to biotinylated biomolecules); antigen binding polypeptides, such as immunoglobulins (Igs) or epitope-binding fragments thereof (for binding to biomolecules including epitopes recognized by Igs); polynucleotide binding proteins (for binding to polynucleotides), such as transcription factors, translation factors, etc.; Ni or Co chelates (for immobilizing polyhistidine-tagged proteins); receptor-ligand systems or other specific protein-protein interaction pairs; aptamers (e.g., nucleic acid ligands for three-dimensional molecular targets); lectins (for binding to glycoproteins); lipids and phospholipids (for binding to lipid-binding proteins), such as phosphatidylserine and annexin V. Those skilled in the art will recognize other members of specific binding pairs that can be attached to magnetic particles.

[0062] Biomolecules can also be coupled to magnetic particles (covalently or non-covalently) by direct chemical conjugation or by physical association. Such methods are well known in the art. Biochemical conjugation is described in, for example, "Bioconjugate Techniques" Greg T. Hermanson, Academic Press. Non-covalent interactions, such as ionic bonds, hydrophobic interactions, hydrogen bonds and / or van der Waals forces can also be used to couple biomolecules to magnetic particles. For example, standard non-covalent interactions for binding biomolecules to chromatographic matrices can be used. A non-limiting example of such non-covalent interactions that can be used to bind biomolecules to magnetic particles is DNA bound to silica in the presence of chaotropic salts. Other such non-covalent bindings and conditions for achieving such non-covalent binding are known to those skilled in the art. See, for example, Molecular Cloning, Sambrook and Russell, Cold Spring Harbor Laboratory Press.

[0063] As used herein, "magnetic particles" are used as markers of biomolecular targets in biological samples (such as, but not limited to, antibodies, DNA, polypeptides, and cells) to help separate them from complex mixtures of samples. Magnetic particles can be classified according to size: microbeads of about <50 nm; nanobeads of about 100 to about 200 nm; and dynamic beads of about 1-5 μm. In addition, magnetic particles can be adapted for selective affinity (functionalization) to couple or bind to desired biomolecular targets such as to fluorescent markers, antibodies, nucleic acids, etc.

[0064] Different magnetic particles can be obtained from a variety of sources, including, for example, Dynal (Norway), Advanced Magnetics (Cambridge, Mass., USA), Immuncon (Philadelphia, USA), Immunotec (Marseilles, France), and Miltenyi Biotec GmbH (Germany). Preferred magnetic labeling methods include colloidal superparamagnetic particles with a size range of 5 to 200 nm, preferably 10 to 100 nm in size. These magnetic particles allow for quantitative magnetic labeling of cells, so that the amount of coupled magnetic labeling is proportional to the amount of bound product. For example, colloidal particles with various specificities are available from Miltenyi Biotec GmbH.

[0065] As used herein, "separation" includes separation or collection of accumulated target cells from a surrounding fluid mass, wherein the mass is a fluid mixture or suspension such as a cell emulsion or a combination thereof, and also means the concentration or enrichment of target cells relative to the surrounding mass or a provided cell sample (similar to precipitation or centrifugation to obtain a pellet).

[0066] As used herein, "depletion" with respect to separation refers to the removal of target cells from the bulk (similar to sedimentation or centrifugation to obtain a supernatant).

[0067] As used herein, "highly qualitative" (isolation, depletion) refers to high purity isolation of target cells that substantially excludes other cells or includes negligible amounts of other cells, such as about 10% to about 1% or less of isolated cells, and conversely depletion.

[0068] As used herein, "high quantitation" (separation, depletion) refers to high recovery separation of substantially all target cells or a very large number of target cells from a sample, such as about 80% to about 99% or more of the separated cells, and conversely depletion.

[0069] It should be noted that whenever reference is made herein to cells being attached or adhered or adhered to the wall of a tube, or terms similar thereto, it does not necessarily mean that the cells are directly attached to the wall, but that they are also indirectly connected or linked or attracted to the wall, such as through cell chains or groups of cells.

[0070] As used herein, "magnetic shielding" reduces and / or blocks a magnetic field in a space by blocking the magnetic field with a "magnetic field shield" (also referred to herein as a magnetic field shield / barrier, where the two terms are interchangeable).

[0071] As used herein, "HMPSM" refers to a high magnetic permeability and saturation material that produces a highly concentrated magnetic field within the material, effectively reducing and / or eliminating the effects of magnetic fields.

[0072] As used herein, a "magnetic field shield / barrier" is a structure that can be controlled with respect to the use of a magnetic field.

[0073] As used herein, an "electromagnet" is a type of magnet in which a magnetic field is created by an electric current. When the current is turned off, the magnetic field disappears. An electromagnet is typically made of a wire wound into a coil. The current passing through the wire creates a magnetic field that is concentrated in a hole in the center of the coil. The turns of wire are typically wound around a core made of a ferromagnetic or ferrimagnetic material such as iron; the core concentrates the magnetic flux and creates a more powerful magnet.

[0074] As used herein, a "permanent magnet" is a magnet that is permanently magnetic, as opposed to an electromagnet, which behaves like a magnet only when an electric current flows through it. Permanent magnets are made of substances such as magnetite (Fe3O4), the most magnetic naturally occurring mineral, or neodymium, a strongly magnetic synthetic substance.

[0075] As used herein, a "magnet array" is one or more magnets. The one or more magnets may be permanent magnets or electromagnets. The one or more permanent magnets may be a linear array, of different sizes, of different strengths, arranged in opposite magnetic pole orientations perpendicular to the axis of the linear array, or arranged to be rotated 90° from each other in a plane perpendicular to the axis of the linear array. Any number of magnets in the array may be held together physically or adhesively. The permanent magnets may be a material selected from iron, neodymium, samarium cobalt, or alnico.

[0076] A general non-limiting overview of the invention and implementation of the invention is given below. This overview summarizes exemplary practices of embodiments / aspects of the invention and provides a constructive basis for variations and / or alternative and / or different aspects / embodiments, some of which are subsequently described.

[0077] The present disclosure relates to devices, methods and systems for magnetically separating and collecting desired biomolecular targets in biological samples by positive or negative selection. As described herein, a magnetic field is generated substantially adjacent to a biological sample containing the desired magnetized biomolecular targets. The magnetic field can be switched "on" and "off" in an automatic manner to provide a magnetic field of desired intensity, continuous duration, intermittent duration, pulse duration and combinations thereof. This is achieved by introducing a magnetic field shield (also referred to herein as a magnetic field shield / barrier) to functionally control the application of the magnetic field encountered / applied to the biological sample. The magnetic field shield / barrier is located between the magnetic field source and the biological sample, and due to its high magnetic permeability and saturation material (HMPSM), a highly concentrated magnetic field is generated within it, which effectively reduces and / or eliminates the effects of the magnetic field on the biological sample containing the magnetized biomolecular targets.

[0078] In one aspect of the invention, cellular biomaterial is cultured in a bioreactor vessel and the desired cells are the biomolecule targets for magnetic separation and collection.

[0079] The devices, methods and systems described herein generally employ a method whereby a biological sample (a heterogeneous population of organisms) (which are typically, but not limited to, cells) has magnetic beads bound to specific biomolecular targets (specific cell types) in the sample, thereby producing "magnetized cells". A typical binding method may include: i) direct binding of magnetic beads conjugated to antibodies to the biological target; and ii) use of a multi-step process in which the biological target is bound to an antibody, which is conjugated to another antigen or binding pair. This antigen / binding pair is then bound to magnetic beads, which are conjugated to the corresponding antibody / binding pair. During magnetic separation, the magnetized cells (expressing the antigen; positively selected) that are target cells attached to the magnetic beads are attracted to a location near the magnet, while the cell population that is not attached to the beads (negatively selected) remains in the culture medium of the biological sample and is easily removed from the bound population. An alternative process to magnetic cell selection is to use antigen presenting magnetic microbeads to stimulate certain types of biological processes on the target (such as activation of T cells with anti-CD3 and anti-CD28 coupled to magnetic activation beads). After stimulation, the beads must be removed prior to downstream processing, which requires treating the cell suspension with an efficient magnet.

[0080] During separation, the magnetically tilted particles are subjected to a force vector F from an externally applied magnetic field B acting on the paramagnetic particles as shown in Equation 1 below (Pamme, 2006).

[0081]

[0082] where V is the volume of the particle, Δχ is the difference in magnetic susceptibility (ability to become magnetized) between the particle and the surrounding medium, μ0 is the magnetic permeability of vacuum, and is the dot product between the magnetic field and the gradient operator. From this equation it is clear that the success of a magnetic separation system depends on several parameters. The first is the particle size, where larger particles experience stronger magnetic forces. There are three typical size classifications for magnetic particles, i) < 50 nm (e.g. Miltenyi Biotec’s MicroBeads), ii) 100-200 nm (e.g. Nanobeads), or iii) 1-5 μm (e.g., Invitrogen ), they are easier to separate as their size increases. Next, increase the magnetic susceptibility of the magnetic bead relative to the surrounding medium. Since most beads are usually composed of an iron core and the surrounding medium is practically non-magnetizable, this value is usually already relatively large. Finally, increasing the magnetic field gradient can significantly increase the force applied to the magnetic beads. This is because the magnetic field gradient exerts an uneven force on the north and south poles of the magnetic particles due to the inhomogeneous spatial quality of the high gradient field ( Figure 1A). This non-uniform force on the particle causes the particle to move. In a perfectly uniform magnetic field, equal and opposite forces are generated on the poles of the magnetic particle, resulting in a net force of zero on the particle and no net motion ( Figure 1B ). In addition, for larger sized magnetic particles, the difference in magnetic force exerted by the gradient between the two poles is larger compared to smaller beads ( Figure 2 and Figure 3 ).

[0083] There are means of inducing "switchable" (can be turned on and off) magnetic fields that can create gradients to attract magnetic beads for automated separation, isolation, and collection. For example, an electromagnet is formed by wrapping a current-carrying wire around a rod of easily magnetized material (e.g., iron). Figure 4A ). They can be switched on or off by applying or removing, respectively, an electric current through a wire. Another approach is to use electropermanent magnets, which consist of a hard magnet with high magnetic coercivity (a high magnetic field that switches the magnetic poles) and a soft magnet with low coercivity ( Figure 4B ). Two magnets are connected to each other into a complete magnetic circuit by a paramagnetic material (such as iron). The soft magnet is wrapped in a current-carrying wire, and by pulsing a strong current in the wire, the magnetic poles of the soft magnet can be switched. When the poles are misaligned, a magnetic "current" flows through the paramagnetic material, and no external magnetic field is observed. However, when the poles are aligned, a magnetic current travels through the air and an external magnetic field is generated. The last method is to use permanent magnets to generate the magnetic field. By using a material with a high magnetic saturation, the magnetic field on one side of the magnet can be blocked ( Figure 4C ).

[0084] In one aspect of the invention, an array of strong permanent magnets ( Figure 4C ). This produces a strong magnetic gradient radially from the array, as well as a gradient linearly along the axis of the array. An improvement on this design is the Halbach array, in which the magnets in the array are rotated 90° relative to each other ( Figure 4D ). This results in a significant increase in the magnetic field on one side of the magnet, while the magnetic field on the other side decreases (Kang et al.).

[0085] The controllable magnetic field can be designed to perform sequential activities that control the isolation of target biological moieties containing the biomolecular target (through positive or negative selection), while enabling the removal and discarding of non-target biological moieties.

[0086] The ability to turn the magnetic field on and off (thus automating positive and negative cell selection) is a major operational requirement during the processing of biological samples using magnetic separation. The devices and methods described herein enable automated collection of target biological populations, thereby reducing overall process complexity and lowering operating costs.

[0087] The "on" and "off" switchable magnetic field described herein is to control the magnetic field encountered by biological samples labeled with magnetic particles (such as magnetic beads) by introducing a magnetic field shield / barrier. Through this controllable magnetic field, the sequential steps of target biological retention, subsequent secondary release and capture enable the production of a very compact and energy-efficient magnetic separation system.

[0088] The magnetic field shielding / barrier works due to the intrinsic properties of HMPSM. This high magnetic permeability and saturation result in a high concentration of magnetic fields in this material. Deploying HMPSM as a magnetic field shielding / barrier between a magnetic field source and a biological sample can virtually eliminate the effects of the magnetic field on the biological sample. Through this controllable activation of the magnetic barrier, magnetic separation of target populations from biological samples can be achieved with high reproducibility and relatively low cost.

[0089] Figure 1 shows the theory of how magnetic particles / beads (such as those used for cell separation and activation) respond to a magnetic field gradient. This is exemplified mathematically by equation 1. Each magnetic bead has a north pole and a south pole. When exposed to a magnetic field gradient, the magnetic forces acting on each pole of the particle (one attracting, one repelling) will be different, resulting in a net force ( Figure 1A ). In contrast, when there is no magnetic field gradient, the forces acting on each magnetic pole will be equal and opposite ( Figure 1B ). Therefore, no net force is exerted on the magnetic particles and no motion is induced. An array of magnets arranged so that the magnetic poles alternate between north (N) and south (S) can be used to generate these necessary magnetic field gradients. In addition, Figure 1C As shown, high magnetic field gradients can be generated by using an array of multiple strong small magnets, where the magnetic poles in the array alternate north-south orientations.

[0090] Figure 2 The sizes of magnetic beads commonly used for magnetic separation of cells are shown. There is currently a range of bead sizes, from Miltenyi's approximately 50 nm MicroBeads to Invitrogen, approximately 5 μm Increasing the size of the bead generally increases the sensitivity of the bead's response to a magnetic field because the difference in force acting on each pole of the bead increases.

[0091] Figure 3A method is outlined by which larger sized magnetic beads are more efficiently pushed out of a fluid in response to an induced magnetic field. In some embodiments, the beads are simultaneously exposed to the attractive and repulsive forces of the magnetic field on the corresponding magnetic poles of each bead. When moving away from the magnetic field source, the magnitude and gradient of the magnetic field decrease. Therefore, the net force on the beads depends on the distance from the magnetic field source and the diameter of the beads. Larger beads have a larger distance between the poles, resulting in a larger difference in the magnetic forces acting on the poles. Similarly, a stronger gradient (i.e., closer to the magnet) scales the difference in the forces acting on the corresponding magnetic poles of the beads. This increase in net force causes the beads to be attracted toward the magnet and out of the fluid more quickly.

[0092] FIG4 shows a method of using an automated control system to generate a magnetic field that can be turned on / off relative to application to a biological sample. Figure 4A In FIG. 1 , an electromagnet is shown in which a wire is wound around a ferromagnetic material such as iron. By applying an electric current to the wire, the magnetic field can be quickly and easily turned on and, conversely, off. However, the magnetic field thus generated can be low. In addition, the electromagnet generates a lot of heat, which is very problematic for a local cell culture environment. Figure 4B An electropermanent magnet consisting of a non-switchable rare earth magnet and an alnico permanent magnet with switchable magnetic poles is shown, both set in a ferromagnetic material forming a magnetic circuit. When the poles of the permanent magnets are aligned, the carbon steel adopts the same direction, creating a net magnetic flux in the air around the magnets (which can be used to pull the magnetic beads out of a fluid suspension). However, when the poles of the two permanent magnets are opposite, the magnetic flux is confined to the ferromagnetic material, preventing the extraction of the magnetic beads. The direction of the alnico magnet is switched by applying a current pulse of very high magnitude and short duration via a coil of wire surrounding it, resulting in a transient magnetic field of high magnitude. However, the magnetic gradient produced by this setup is also quite low compared to rare earth magnets, and this approach also produces electromagnetic interference, which may have unknown adverse effects on any surrounding electronic equipment. Figure 4C An array of permanent magnets with opposite magnetic pole orientations is shown with an HMPSM, such as Iron, Cobalt Iron, and Hiperco 50, on one side. By using such an HMPSM, the magnetic flux of the magnetic field will be amplified on the opposite side and reduced to negligible levels on the side with the HMPSM. By driving the HMPSM so that it is between the permanent magnet and the magnetic beads, the force acting on the beads can be effectively reduced to zero. Conversely, moving the HMPSM to the opposite side of the permanent magnet can induce a very strong magnetic force on the beads. Finally, in Figure 4D , a Halbach linear array is demonstrated in which a 90° rotation of the magnet allows an amplified magnetic field to be produced on one side of the array, while a significantly reduced or eliminated magnetic field is produced on the opposite side of the array.

[0093] Figure 5Computational modeling is shown demonstrating the function of an HMPSM (marked as x) on a permanent magnet. The magnetic flux is mainly transmitted through the air surrounding the magnetic field. However, the magnetic flux is not strong enough to be transmitted through the HMPSM. This results in no significant magnetic flux density on the side of the HMPSM opposite the magnet, while the magnetic flux density is mainly amplified at the poles of the permanent magnet.

[0094] Figure 6 The layout of an exemplary box commonly used in an automated cell culture system is shown, which has a separation tube for magnetic separation using magnetic beads extending along the length of the box surface. The tube is aligned with an array of permanent magnets located within the automated cell culture system. The separation tube is connected to various tubes and bags within the box, which are used for positive or negative binding of target cells. Using tubes that are as long as possible increases the volume that can be loaded into the separation tube, thereby reducing the processing time for magnetic separation to occur.

[0095] Figure 7 provides Figure 6 Attached or unattached magnetically bound beads to cells can be loaded into a separation tube 701 ( Fig. 7A ). In some aspects, the separator tube 701 is aligned with the permanent magnet array 704, such as Figure 7B As shown. In other aspects, the separation tube 701 is aligned with an electromagnet that replaces the permanent magnet array 704. The permanent magnet array 704 is composed of permanent magnets with alternating north poles (702) and south poles (703) to generate a magnetic flux gradient as high as possible. The magnetic beads in the separation tube 701 are attracted by the magnet array 704, thereby successfully separating the magnetic beads from the fluid suspension.

[0096] Figure 8An embodiment of a magnetic field shield / barrier when the magnetic field is turned on is shown. The separation tube 701 extends along the length of the permanent magnet array 704. Surrounding a portion of the permanent magnet array 704 is a magnetic field shield 801 (shown as a paramagnetic sheath 801) generated by the HMSPM. In some aspects, the magnetic field shield 801 is made of pure iron. In some aspects, the magnetic field shield 801 is made of a soft magnetic iron alloy (such as ferritic steel, silicon iron, nickel iron, or cobalt iron). In some aspects, the magnetic field shield 801 is made of a soft magnetic alloy of cobalt, vanadium, and iron (such as an alloy of about 49% cobalt, about 2% vanadium, and the remainder iron). In some aspects, the sheath 801 can be made of Hiperco50 or Hiperco 50A. In some aspects, the paramagnetic properties of the magnetic field shield 801 amplify the magnetic field gradient applied to the separation tube 701 by the magnet array 704. This actually increases the magnetic force acting on the beads, allowing them to be more efficiently removed from the fluid suspension, while non-magnetic matter will not be affected and will be able to pass through the separation tube unhindered. The permanent magnet array 704 and magnetic field shield 801 are also connected to a servo mechanism 802 and gear train 803, allowing the entire assembly to fully rotate, and be closed and opened as needed.

[0097] Fig. 9 Shown with Figure 8 The same components found in FIG. 701, but with the magnets in the "off" position. The separator tube 701 still extends along the permanent magnet array 704. However, in the off position, the magnet array 704 and the magnetic field shield 801 are rotated using a rotation servo mechanism 802 and a gear train 803 so that the magnetic field shield 801 is located between the separator tube 701 and the permanent magnet 704. As shown in FIG. 4 and Figure 5 As described above, the HMPSM material used for magnetic field shielding 801 blocks the passage of magnetic flux. Therefore, the magnetic flux gradient to which the separation tube 701 is exposed is significantly reduced, thereby losing the retention of the magnetic field on the magnetic beads. Applying a high flow rate of liquid or gas to the separation tube 701 can be used to flush the magnetic beads and magnetically bound cells.

[0098] FIG. 10 is a cross-section of the interface between the separator tube 701, the magnetic field shield 801 and the permanent magnet array 704. When in the "open" position, as shown in FIG. Fig. 10A As shown, there is a significant magnetic field acting on the separation tube 701, which can attract magnetic particles. When in the "closed" position, as shown in FIG. Fig. 10B As shown, the magnetic field acting on the separation tube 701 is negligible and previously bound cells and beads can be effectively removed.

[0099] Fig.11It is a side view of the interface of box 1101 and automated cell culture instrument 1102. Separation tube 701 is attached to box 1101 to allow magnetic or non-magnetic parts to move from different areas in box 1101 and move towards different areas in box 1101. On the contrary, magnetic separation assembly (composed of 704 and 801 together) is contained in automated cell culture instrument 1102. The control system associated with instrument 1102 can control servo mechanism 802 and gear train 803 to rotate magnet array 704 and magnetic field shield 801, so that the magnetic field is "turned on" or "turned off" when the magnetic field belongs to separation tube 701. When box 1101 and instrument 1102 interface together, separation tube 701 and separation assembly (704 and 801 together) are aligned to allow effective magnetic separation to be performed. In addition, using the peristaltic pump associated with instrument 1102, fluid (with or without magnetic beads and cells) can be transported or removed from separation tube 701.

[0100] 12 consists of a side view of a separation tube 701 positioned between the outer wall of a cassette 1101 and the outer wall of a cell culture instrument 1102 , with various means for increasing the number and magnitude of magnetic field gradients within the separation tube 701 . Fig. 12A A paramagnetic / non-magnetic spacer 1201 is shown extending along the length of the separation tube 701 on the outer surface of the cartridge 1101. The spacer 1201 serves to compress the tube 701 to minimize the distance between the magnetized elements flowing through the tube 701 and the magnet array 704. Likewise, magnetization of the spacer 1201 (if paramagnetic) from the magnet array 704 will cause another magnetic field gradient to form in the separation tube 701 on the side closest to the cartridge 1101. Additionally, cutting the spacer 1201 into smaller subsections along its length (not shown) can allow for the creation of a high gradient magnetic field at the point at the end of each spacer subsection 1201. Fig. 12B A paramagnetic wire mesh 1202 is shown within a separation tube 701 , which when exposed to the magnetic field generated by the permanent magnet array 704 , creates a high magnetic field gradient around the wires of the wire mesh 1202 . Fig. 12C Paramagnetic particles 1203 are shown within a separation tube 701 which, when exposed to a magnetic field from a permanent magnet array 704, produce a local magnetic field gradient. Fig.12D Paramagnetic rods 1204 are shown extending along the length of the separation tube 701 and being divided longitudinally into smaller sub-sections (not shown) and capable of producing high magnetic field gradients at the ends of each rod 1204 when exposed to the magnet array 704. Fig.12E A series of paramagnetic rods 1205 are shown extending along the length of the separation tube 701, wherein when the rods 1205 are exposed to the magnetic field from the array 704, a high magnetic field gradient is formed between the rods. Fig.12FA paramagnetic scaffold 1206 is shown extending along the length of the separation tube 701 , wherein when exposed to a magnetic field from the array 704 , a high magnetic field gradient is created within the bore. Figure 12G The paramagnetic coating 1207 of the separator tubes 701 is shown, consisting of small aberrations that produce a high magnetic field gradient between them when exposed to a magnetic field from the array 704. Fig.12H A paramagnetic filter 1208 is shown placed within the separation tube 701 which, when exposed to the magnetic field from the array 704, produces a high magnetic field gradient within the filter pores.

[0101] Optionally or additionally, in some aspects of the invention, various parameters are adjustable, such as magnetic field strength, spatial distribution (concentration) of biomolecular targets, and / or other parameters, such as temperature. For example, the flow rate and / or viscosity and / or elasticity of the biological sample fluid can be adjusted, such as to allow separation of target cells, but at least substantially prevent coagulation of non-target cells. In some embodiments, the fluid can be used to wash away separated target cells. The flow state and rate of the washing fluid are optionally adjusted to promote the removal of target cells from the tube wall (i.e., promote removal or release), which suddenly changes the flow, thereby inducing turbulence or shock, which helps to erode or destabilize the target cells on the tube wall.

[0102] In certain embodiments, the separated cells can be released from the separation tube by a combination of various methods and methods (e.g., demagnetization, bubbling, vibration, enzymes, sound waves and combinations thereof) before and / or simultaneously with the cells being washed out of the tube. Such peripheral processing can be performed to improve the properties of the separation and desired target population in terms of quality and / or quality. For example, the use of red blood cell (RBC) lysis methods may help to remove sticky RBCs, improve purity, and thereby make it easier to separate T cells from a general PBMC population.

[0103] Enzymes such as DNase can be used to assist in cell release.

[0104] Furthermore, when high mass or high purity depletion is desired (rather than collection of target cells), a sufficiently strong magnetic field can be applied that is stronger than the magnetic field used for collection, at the expense of non-target cells adhering to the wall and / or coagulating.

[0105] The devices, systems and methods can be embodied in kits and uses thereof for implementing one or more methods of the invention, the kits comprising one or more reagents, one or more magnetic particles, one or more binding partners, a magnet array, a magnetic field shield and / or instructions for use.

[0106] Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the present invention and practice the claimed method. Therefore, the following working examples specifically point out typical aspects of the present invention and should not be interpreted as limiting the remainder of the present disclosure in any way. Therefore, these examples are only for illustrative purposes and should not be used to limit the scope of the present invention in any way.

[0107] Examples:

[0108] Example 1 - Separation of beads from a fluid

[0109] CD3 / CD28 Activation using magnetic separation systems (704 and 801) (produced by Invitrogen) activated and expanded T cells (derived from PBMC from Lonza) were used to separate the T cells from the beads. 31 ml of beads-cells (1.6×10 6 Cells / ml) fluid suspension (T cell culture medium, 94% X-Vivo 15, 5% HS, 1% P / S, 10ng / ml IL-2) was prepared with the magnetic separation components (704 and 801) set to the "on" position. The fluid collected from the separation tube is referred to as the "cell fraction" because cells (non-magnetically bound) are less likely to be removed from the fluid suspension. The magnet assembly (704 and 801) was manually rotated (802 and 803 were not used) to the "off" position so that the paramagnetic material 801 was located between the separation tube 701 and the magnet array 704. Three flushing cycles (including alternating air and fluid flushing at 40ml / min, 4ml each time) were performed in the separation tube 701 to flush out and collect the and any cells attached to the walls of tube 701. This is called the "magnetic fraction" because it consists of all magnetically attracted cells and beads.

[0110] Using cell counts from both fractions, calculate The percentage of cells successfully separated from the 'cell fraction' (i.e. the percentage of total cells obtained in the 'cell fraction'). The percentage of cells successfully isolated in the two liquid fractions was about 95% (Figure 13A). The two liquid fractions were also counted using a hemacytometer to measure the number of cells in each corresponding fraction. At all flow rates, The percentage was at least 95% ( FIG. 13B ).

[0111] Example 2 - Continuous Flow of Bead-Bound Cells Through Separation Tubes

[0112] Using magnetic separation components (704 and 801), streptavidin nanobeads (from ) to the outside of cassette 1101 for positive selection. First, add blocking agent (5 μl / 10 7 cells, HumanTruStain FcX TM , ) Blocking cells (10 7 The blocking agent binds to Fc receptors on cells by incubating the cells and reagents together at room temperature for 10 minutes. Add the biotin-conjugated primary antibody mixture (10 μl / 10 7 cells, human CD14+ monocyte isolation, ), and the mixture was incubated at 2-8°C for 15 minutes. Similarly, streptavidin-coated nanobeads (10 μl / 10 7 cells) was added to the cell suspension at 2-8°C for an additional 15 minutes. The streptavidin on the nanobeads binds to the biotin on the antibodies of the target cells, magnetically binding the cells. To ensure the purity of the magnetically bound cell population, the bound cells were loaded into the EasySep TM (STEMCELL ) magnet for 5 minutes to pre-sort the bound cells, and then discard the supernatant filled with unbound cells.

[0113] In order to separate Jurkats using magnet array 704, Jurkats (1.5-2ml / min, 3ml) are passed through separation tube 701 at different flow rates (1-5ml / min), while magnetic separation components (704 and 801) are passed. The magnetically attracted cells are pulled out of the flowing suspension and adhered to the wall of tube 701 closest to magnet array 704. When the magnet is turned on, all cells not removed from the flow are captured as "negative fractions" (collected in a volume of 9-12ml). The magnet assembly (704 and 801) is manually rotated to the "off" position, and three flushing cycles (as described in Example 1) are performed to capture the "positive fraction". All of the above steps are performed using isolation buffer (98% DPBS, 2% FBS).

[0114] Using cell counts of the negative and positive fractions, the percentage of cells that failed to be captured (all cells in the "negative fraction" compared to the number of cells loaded), as well as the release efficiency of positively captured cells (captured cells that were subsequently successfully obtained in the "positive fraction") can be determined. Increasing the capture flow rate in the separation tube 701 resulted in an increase in the number of cells that the system failed to capture (Figures 14A and C), which was attributed to the reduced time that the bound cells were exposed to the magnetic field. However, increasing the capture flow rate to 5 ml / min greatly increased the release rate of bound cells, with a maximum of almost 100% (Figures 14B and D). This may be due to fewer cells leaving the suspension at higher flow rates and becoming trapped at various joints in the tubing loop. Changing the inner diameter of the separation tube (thick tube - 1 / 8" ID, thin tube - 3 / 32" ID) resulted in a slight increase in capture failures (Figures 14A and C), but a slight improvement in cell release (Figures 14B and D). These results may be due to the increased flow rate and wall shear stress in the thin tube.

[0115] Example 3 - Multiple passes of bead-bound cells through separation tubes

[0116] Jurkats were magnetically bound and preselected as described in Example 2. 6 Cells / ml, 3ml) are passed through the separation tube 701 at a flow rate of 5ml / min in isolation buffer, and magnetic separation components (704 and 801) are opened simultaneously. The cells not captured by the magnet array 704 are collected in the isolation buffer as the negative fraction (collection volume of 12ml) of the first pass. The cells are loaded into the separation tube 701 again with a capture flow rate of 5ml / min, and the cells not yet captured are collected as the negative fraction (collected as 12ml) of the second pass. After the second pass, the separation component (704 and 801) is transferred to the "off" position, and the cells captured in the separation tube 701 are exposed to 3 flushing cycles (as described in Example 1) to obtain the positive fraction. All of the above steps are carried out with isolation buffer.

[0117] As in Example 2, cells from each fraction were counted and quantified in terms of capture failure (for both passes, as a percentage of the total cells loaded into tube 701) and release efficiency. Adding additional passages of cells through separation tube 701 did successfully reduce the percentage of cells that failed to be captured (Figure 15A) and did not result in any negative reduction in release compared to a single passage (Figure 15B).

[0118] Example 4 - "Waiting Time" in Separation Tube

[0119] Jurkats were magnetically bound and preselected as described in Example 2. In order to effectively increase the duration of exposure of bound cells to the magnetic field, after loading into separation tube 701, Jurkats (1.5×10 6 , 3ml) remain stationary in different durations (1-5 minutes). Gently rinse test tube 701 at a speed of 5ml / min for 12ml, and collect the effluent as the negative fraction. After waiting for the duration, the separation components (704 and 801) are transferred to the "off" position, and three flushing cycles (described in Example 1) are applied to collect the positive fraction. The above steps are all performed using isolation buffer.

[0120] The collected negative and positive fractions were counted again and quantified based on capture failure and release rate. A significant trend was observed that increased waiting time resulted in reduced capture failure (Figure 16A), which may be due to the additional time that the magnetic particles can respond to the magnetic field. In addition, despite the increase in waiting time, the release rate for each waiting duration was close to 100% (Figure 16B), indicating that the increase in capture rate was not just due to the increase in cell loss at the connection in the pipeline loop.

[0121] As the waiting duration for the process is increased, negative cells are more likely to be captured by the process (hereinafter referred to as "false positives"). To quantify the percentage of false positives, Jurkats (3 × 10 6 Cells were loaded into a separation tube 701 (cells / ml, 3 ml). Similarly, the cells were waited for different durations (1 min or 3 min) and exposed to different "negative fraction" flushes, including high flow rate air or fluid flushes, with the magnetic components (704 and 801) still in the "on" position. Since the loaded cells are unbound, all cells are expected to appear in the "negative fraction" and any cells that do not appear are considered "false positives." Although using a more conventional capture sequence (capture flow rate of 5 ml / min, waiting time of 3 min) results in a poor false positive rate (30%), increasing the capture flow rate and shortening the waiting time both reduce it to about 20% ( Fig.17 In addition, the combination of significantly shortened waiting time and increased capture flow rate (condition 5) and the addition of 40 ml / min air flush (condition 7) can reduce the false positive rate to <3% ( Fig.17 ).

[0122] Example 5 - Isolation and purification of mixed cell populations

[0123] Thawed human peripheral blood mononuclear cells (PBMC) and Combined with CD3+ cells from heterogeneous cell populations by positive selection (from ThermoFisher Scientific). PBMC (10 7 cells / ml) and CD3 antibody (5μl / 10 7 cells, FlowComp TM Human CD3 antibody, Invitrogen) was incubated at 2-8°C for 10 minutes. Then, PBMCs were incubated with FlowComp TM Dynabeads (15 μl / 10 7 Cells (5-10×10 cells, Invitrogen) were allowed to bind for 15 minutes under shaking and tilting. Using different process parameters (flow rate, waiting time, number of passes, as described in FIG. 18 ), bead-bound cells (5-10×10 6 The magnet array 704 is loaded with 100% 100% 100% 100% 200% 400% 50% 200% 100% 50% 200% 100% 40% 2 ...

[0124] The viability (0.031 μl / 100 μl, Live / Dead) of the pre-isolation fraction and the post-isolation fraction (300k cells per well) was measured. TM Green, Invitrogen), CD3 (5 μl / 100 μl, PE mouse anti-human CD3, BD Biosciences), and in some experiments CD14 (0.625 μl / 100 μl, CD14 monoclonal antibody-Pacific Blue (Pacific Blue), Invitrogen) were fluorescently stained and analyzed using flow assisted cell sorting (FACS) to evaluate the phenotype of the obtained fractions. The initial loaded population was found to be heterogeneous, but mainly CD3+ (Figure 18-black line). For comparison, various process parameters were tested using the magnet array 704. It was observed that using Changing the waiting time from 0 to 2 min in conjunction with cells had limited effect on the purity of the output cells (Figure 18A, B and Figure 18F, G). However, further increasing the waiting time to 5 min significantly reduced the purity of the cells (Figure 18C, D). Between 3 ml / min and 10 ml / min, the flow rate also had limited effect on the purity of the cells (Figure 18G, H). The greatest substantial improvement in cell purity came from adding a second pass to the process, which increased the purity to 97.5% CD3+ cells (Figure 18E).

[0125] Example 6 - Magnets of different sizes for capturing magnetic beads

[0126] Permanent magnets (702 and 703) of different sizes (1 / 8" and 1" long) are used to assemble the magnet array 704. The magnitude of the magnetic field is measured at different specific distances from the magnet using a Gaussmeter (AlphaLab Inc.) (Figure 19A). From the magnitude measurements, an estimate of the magnetic field gradient is calculated (Figure 19B). It has been determined that in order to produce the strongest gradient, magnets of smaller lengths are more desirable. However, this stronger gradient degrades faster than longer magnets, thereby reducing the effective range of the magnet. This demonstrates the potential of using different magnet sizes in the magnet array 704 to achieve different separation goals, such as short-range separation of weakly bound targets and long-range separation of strongly bound targets.

[0127] To test the effect of the size of magnets 702 and 703 on cell separation, as described in Example 2, Nanobeads bound to Jurkat cells. Bead-bound cells (2.5 × 10 6 Cells / ml, 1.5ml) at a flow rate of 5ml / min and a waiting time of 5min flow through the magnet array 704 in the separation tube 701. Unbound cells are rinsed out of the tube 701 with 12ml of isolation buffer (98% PBS, 2% FBS, 2mM EDTA) at 5ml / min. The magnetic field generated by the array 704 is removed from the tube 701, and three flushing cycles (described in Example 1) are performed to remove the positive fraction from the tube. The results are compared with those obtained from typical magnet assemblies (704 and 801). It was observed that 1 / 8" long magnets 702 and 703 reduced capture failures compared to 1" long magnets 702 and 703 (Figure 19C). All of the above steps are performed using isolation buffer.

[0128] Example 7 - Add-ons to facilitate capture of weakly bound biological targets

[0129] One method to change or improve the ease of capture of weakly bound targets / beads is to reduce the distance between the magnet array 704 and the separation tube 701, thereby increasing the average magnetic field magnitude and gradient experienced within the tube 701, as demonstrated by Example 6. To this end, a 3 / 32" thick spacer 1201 is placed between the separation tube 701 and the cartridge 1101. As described in Example 2, the separation tube 701 is placed with the cartridge 1101. Nanobeads-bound Jurkat cells (2.5-5×10 6 Cells / ml, 1.5 ml) flowed into the separation tube 701 at a flow rate of 5 ml / min and allowed to wait for 3 or 5 minutes. The uncaptured cells were then allowed to flow out at 5 ml / min (11-13 ml) and three flushing cycles (as described in Example 1) were performed to collect the captured cell fraction. The results obtained by adding the spacer 1201 to the setup showed an improvement in the use of the spacer capture failure and the 3 minute wait time, reaching the level obtained using the 5 minute wait time (Figure 20A). The above steps were performed using isolation buffer.

[0130] Another method to reduce the distance between the biological target and the magnetic field is to include magnetizable objects (1202-1208) in the separation tube 701. The magnetic field generated by the magnet array 704 can be amplified by these objects (1202-1208), producing a magnetic field gradient that is inversely proportional to the size of the object (1202-1208) and has an effective range that is proportional to the size of the object (1202-1208). To demonstrate this, a paramagnetic mesh 1202 was added to the separation tube 701. Jurkat cells (1.25×10 7 cells / ml) and MicroBeads (20 μl / 10 7 After binding, the cells were flowed into a paramagnetic mesh containing 1202 (9×10 620B). The non-captured cells were rinsed from tube 701 with 12 ml of isolation buffer at 5 ml / min. The magnet array 704 was turned to the closed position, and three flushing cycles (described in Example 1) were applied to remove the positively captured cells from tube 701. In order to illustrate the non-specific capture by the net 1202, the results were normalized to the results obtained from the no-bead control (Jurkats not bound by MicroBeads), in which all captures were due to physical retardation from the net 1202. The use of paramagnetic nets resulted in a 10% relative increase in cells captured using beads compared to not using beads (Figure 20B). The above steps were performed using isolation buffer supplemented with 2 mM EDTA.

[0131] Method using recirculation and magnetic field

[0132] As described herein, in exemplary embodiments, methods for magnetic separation of targets within biological samples appropriately utilize sample recycling through multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) magnetic separation cycles. Through such recycling, the yield of the desired target is significantly increased.

[0133] The methods described herein are suitably performed in an automated cell culture system, and in embodiments may be performed in a cartridge within an automated cell culture system. Figure 6 An exemplary box is shown, and Fig.21 The position of the box in the flow chart of the automated cell culture system is shown. Fig.21 As shown, the automated cell culture system suitably comprises a cell proliferation chamber 2102 and several fluidic pathways 2104 and a magnetic field source 2106 , as well as input locations for reagents 2108 .

[0134] Fig.21 An exemplary embodiment is shown in which a recirculation path / line is shown in which a biological sample containing a target biological population is circulated multiple times through the recirculation path / line (bold line). Suitably, during each pass, the sample is exposed to a magnetic field gradient by a magnetic field source 2106 (e.g., a permanent magnet or an electromagnet).

[0135] The methods described herein for depleting a target population of organisms using recirculation of a biological sample may rely on positive or negative selection methods, or a combination of both.

[0136] Positive selection method

[0137] In an embodiment where a positive selection method is used to separate and capture a target biological population, a method for collecting a target biological population from a biological sample in an automated cell culture system is provided herein, the method comprising: binding the target biological population to magnetic particles; circulating the biological sample through one or more fluidic paths of the automated cell culture system; exposing the target biological population bound to the magnetic particles to a magnetic field gradient; repeating the cycling and exposure steps one or more times; and collecting the target biological population bound to the magnetic particles. In additional embodiments, the method may further comprise removing the target biological population from the bound magnetic particles.

[0138] Such positive selection methods rely on removing the target biological population directly from the biological sample, using a magnetic field to positively select the desired target population from the sample.

[0139] As described herein, suitably, the target biological population is combined with magnetic particles. This paper describes the method for combining magnetic particles with the target biological population, and suitably uses antibodies, proteins or nucleic acids. As described herein, the target biological population suitably comprises one or more cells, viruses, bacteria, proteins, DNA and / or RNA. In an exemplary embodiment, the target biological population is a colony of T cells, suitably T cells that have been produced to comprise required receptors. The biological sample from which the target population is removed may also include other undesirable cells, viruses, bacteria, proteins, DNA, RNA etc. (that is, non-target populations).

[0140] Other steps that may be included in the positive selection methods described herein include washing the biological sample (e.g., cell population), washing the magnetic particles, transferring the target biological population to a cell culture area (e.g., a proliferation chamber) and transferring the non-target biological population to a waste chamber, and finally removing from the automated cell culture system.

[0141] The biological sample is circulated through one or more fluidic pathways of an automated cell culture system, such as Fig.21 As shown. In embodiments, the biological sample may start as a cell culture sample in an input location 2108 of the system or in embodiments in a cell proliferation chamber 2102, after which it is transferred to an area where magnetic particles containing antibodies or other agents are provided so that the magnetic particles bind to the desired target population (e.g., cells). This binding to the magnetic particles may also occur in the proliferation chamber 2102 or any input location 2108 or chamber within the system.

[0142] Then, the biological sample is passed through the section of the automated cell culture system containing the magnetic field source 2106, so that the target biological population combined with the magnetic particles is exposed to the magnetic field gradient. As a result of this exposure, the target biological population (e.g., the colony of the desired cell) becomes combined with the magnetic field source (e.g., gathered against the side of the separation tube 701 or other similar devices), which is adjacent to the magnetic field source that generates the magnetic field. This separation pulls the target biological population (or at least a portion of the target biological population) out of the sample. Then the target biological population combined with the magnetic particles is appropriately collected. The exemplary method of collecting the target biological population is included in removing and washing the target biological population after being exposed to the magnetic field. In an embodiment, the target biological population is collected by circulating the gas phase fluid followed by the liquid phase fluid through the system once or multiple times. Suitably, the gas phase fluid includes one or more of air, nitrogen, oxygen and carbon dioxide. In a further embodiment, the liquid phase includes one or more of water, a buffered saline solution, a culture medium, animal serum, a chelating agent and an enzyme.

[0143] As described herein, it has been determined that recirculating a sample through multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) magnetic separation cycles increases the amount of target population removed from the sample. Thus, in suitable embodiments, the steps of a positive selection method in which a biological sample is circulated through one or more fluidic paths of an automated cell culture system and a target biological population bound to magnetic particles is exposed to a magnetic field gradient are suitably repeated two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. or more times). Fig.21 As shown, such recycling appropriately occurs throughout a recycling cycle, wherein the sample is passed adjacent to the magnetic field source 2106 to bind the target population, and then the sample is recycled and passed adjacent to the magnetic field source again to remove even more target populations that may not have been captured in the previous pass, before finally collecting the final target sample. This cycle can be repeated as many times as necessary until the target of the target population is reached, or it is determined statistically or by other means that additional cycles will not significantly increase the yield and / or purity of the target population. After collecting the target biological population, the method appropriately includes removing the target biological population from the bound magnetic particles so that the target population can be further processed or utilized in various procedures, etc., as described herein.

[0144] In further embodiments, the recycling methods described herein can include washing a target population bound to a magnetic source (e.g., a separation tube used in a magnetic-based method), followed by transferring the washed target population to a proliferation chamber for further processing and / or amplification. These elements of capture, washing, and transfer can then be performed with another biological sample containing a magnetically bound target population.

[0145] In embodiments, the magnetic field gradient to which the target population is exposed is provided by one or more permanent magnets. Exemplary materials that may be used in the permanent magnets are described herein and suitably include magnetite, neodymium, samarium cobalt, and / or alnico. As described herein, in embodiments, the permanent magnets are suitably configured in a linear array, such as Figure 7B The magnet array 704 in.

[0146] In further embodiments, the magnetic field gradient is provided by one or more electromagnets, as described herein.

[0147] In further embodiments, a positive selection method is provided herein, comprising collecting a target biological population from a biological sample in an automated cell culture system, the method comprising binding the target biological population to magnetic particles, circulating the biological sample through one or more fluidic paths of the automated cell culture system, exposing the target biological population bound to the magnetic particles to a magnetic field gradient to capture the target biological population bound to the magnetic particles, circulating unbound components of the biological sample through one or more fluidic paths of the automated cell culture system, inserting a magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field to release the target biological population bound to the magnetic particles, circulating the target biological population bound to the magnetic particles through one or more fluidic paths of the automated cell culture system, repeating the steps of circulating the biological sample to the circulating target biological population one or more times; and collecting the target biological population bound to the magnetic particles. In further embodiments, the positive selection method may further comprise removing the target biological population from the bound magnetic particles.

[0148] As described herein, such positive selection methods utilize a method in which a biological sample is passed, for example, through a separation tube 701 (such as Fig. 7A In the biological sample, the target biological population is bound to the magnetic particles. The method suitably comprises circulating the biological sample through one or more fluidic paths before or including the separation tube 701 and the magnetic source. The target biological population bound to the magnetic particles is suitably exposed to a magnetic field gradient to capture the target biological population bound to the magnetic particles (and is suitably recirculated through the magnetic field one or more times). For example, as Figure 8 As shown, the biological sample passes through a separation tube 701, and the target sample with bound magnetic particles is captured on the side of the tube by the magnetic field (see also Figures 24D-24E ).

[0149] Unbound components of the biological sample (ie, unwanted cells, proteins, DNA, or other structures) are then circulated through one or more fluidic pathways to remove them from the separation tube 701 .

[0150] The magnetic field shield / barrier is then appropriately inserted between the target biological population bound to the magnetic particles and the magnetic field to release the target biological population bound to the magnetic particles from the magnet. The target biological population bound to the magnetic particles is then circulated through one or more fluidic paths of the automated cell culture system and collected in a separate area of, for example, an automated cell engineering system. Various methods for collecting target biological populations are described herein.

[0151] Suitably, the steps of cycling the biological sample, exposing the sample (and the target biological population bound to the magnetic particles), inserting a magnetic shield / barrier between the target population and the magnetic field, and collecting the target biological population are repeated one or more times (suitably 2 or more times, 3 or more times, 4 or more times, 5 or more times, etc.). Each time through the cycle the yield of the target biological preparation is increased. As described herein, the target biological population is then suitably removed from the bound magnetic particles.

[0152] As described herein, suitably, the target biological population includes one or more of cells, viruses, bacteria, proteins, DNA and / or RNA, and in embodiments includes T cells. Methods and compounds for binding magnetic particles to target biological populations are described herein, and suitably include the use of antibodies, proteins or nucleic acids.

[0153] Exemplary magnetic fields are described herein, and are suitably produced by permanent magnets or electromagnets. Materials for making permanent magnets are described herein, and include, for example, magnetite, neodymium, samarium cobalt, and / or alnico. In embodiments, the permanent magnets are configured in a linear array. In embodiments where electromagnets are used, insertion of a magnetic field shield / barrier can be replaced by shutting down the electromagnet, for example by simply removing current from the electromagnet to stop the magnetic field.

[0154] As described throughout, in embodiments, the magnetic field shield / barrier suitably comprises a high magnetic permeability and saturation material. As described herein, in embodiments, the magnetic field shield / barrier is rotated to insert the magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field. Such embodiments are Figure 8 , 9 10A-10B and are described in detail herein.

[0155] Negative selection method

[0156] In an embodiment utilizing a negative selection method for a target biological population, provided herein is a method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising binding a non-target biological population to magnetic particles; circulating the biological sample through one or more fluidic paths of the automated cell culture system; exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient; repeating the cycle to the exposure step one or more times; and collecting the target biological population. The method may further comprise collecting the non-target biological population, suitable for elimination as waste.

[0157] As used herein, negative selection methods utilize the binding of magnetic particles to "non-target biological samples" (which refers to one or more cells, proteins, DNA, RNA, etc.) that are not contained in the "target biological population" and are therefore sought to be removed from the biological sample, leaving behind the target biological population. In such negative selection methods, magnetic separation is used to separate out the non-target biological population, thereby allowing the remaining target biological population to be collected from the sample.

[0158] As described herein, suitably, the non-target biological population is combined with magnetic particles. Methods for combining magnetic particles with non-target biological populations are described herein, and antibodies, proteins or nucleic acids are suitably used. As described herein, the non-target biological population suitably comprises one or more cells, viruses, bacteria, proteins, DNA and / or RNA. In an exemplary embodiment, the target biological population is a population of T cells, suitably T cells that have been produced to contain the desired receptors, and the non-target biological population includes any other cells, proteins, etc. in the sample to be removed, leaving the target population. The biological sample from which the target population is removed may also include other undesirable cells, viruses, bacteria, proteins, DNA, RNA, etc.

[0159] The biological sample is circulated through one or more fluidic paths of an automated cell culture system, such as Fig.21 In an embodiment, a biological sample may start as a cell culture sample at input location 2108, or cell proliferation chamber 2102, or elsewhere within the system, after which it is transferred to an area where magnetic particles containing antibodies or other agents are provided so that the magnetic particles bind to unwanted non-target populations (e.g., unwanted cells, proteins, DNA, etc.). This binding to magnetic particles may also occur within the proliferation chamber, at input location 2108, or elsewhere within the system.

[0160] The biological sample then passes through a section of an automated cell culture system comprising a magnetic field source 2106, so that the non-target biological population bound to the magnetic particles is exposed to the magnetic field gradient (the target biological population is also exposed to the magnetic field, but does not react to the magnetic field). Due to this exposure, the non-target biological population (e.g., a population of unwanted cells, proteins, etc.) becomes bound to the magnetic field source adjacent to the magnetic field (e.g., gathered on the side of a separation tube 701 or other similar device). This separation pulls the non-target biological population (or at least a portion of the non-target biological population) out of the sample. The target biological population that is not bound to the magnetic particles is then appropriately collected. An exemplary method for collecting a target biological population includes filtering, removing, and washing the target biological population (and thereby removing the non-target biological population) from the sample after exposure to the magnetic field.

[0161] In an embodiment, the target biological population is collected by circulating a gas phase fluid followed by a liquid phase fluid through the system one or more times. Suitably, the gas phase fluid comprises one or more of air, nitrogen, oxygen and carbon dioxide. In a further embodiment, the liquid phase comprises one or more of water, a buffered saline solution, a culture medium, an animal serum, a chelating agent and an enzyme.

[0162] As described herein, it has been determined that recirculating a sample through multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) magnetic separation cycles increases the amount and / or purity of the target population removed from the sample. Thus, in suitable embodiments, the steps of the negative selection method, in which the biological sample is circulated through one or more fluidic paths of the automated cell culture system and non-target biological populations bound to the magnetic particles are exposed to a magnetic field gradient, and the target biological population is collected, are suitably repeated two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. or more times). Fig.21 As shown, such recycling appropriately occurs throughout the recycling cycle, wherein the sample is passed adjacent to the magnetic field source 2106 to bind the non-target population, the target population is collected, and then the sample is recycled and passed adjacent to the magnetic field source again to remove even more non-target populations that may not have been captured in the previous pass. This cycle can be repeated as many times as necessary until the target population is reached, or it is determined statistically or by other means that additional cycles will not significantly increase the yield and / or purity of the target population. After collecting the target biological population, the method appropriately includes further processing, filtering or utilizing the target biological population in various procedures, etc., as described herein.

[0163] In embodiments, the magnetic field gradient to which the non-target population is exposed is provided by one or more permanent magnets. Exemplary materials that may be used in the permanent magnets are described herein and suitably include magnetite, neodymium, samarium cobalt, and / or alnico. As described herein, in embodiments, the permanent magnets are suitably configured in a linear array, such as Figure 7BThe magnet array 704 in.

[0164] In further embodiments, the magnetic field gradient is provided by one or more electromagnets, as described herein.

[0165] In a further embodiment, the present invention provides a negative selection method, which includes collecting a target biological population from a biological sample in an automated cell culture system, the method comprising binding a non-target biological population to magnetic particles; circulating the biological sample through one or more fluidic paths of the automated cell culture system; exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient to capture the non-target biological population bound to the magnetic particles; circulating the target biological population of the biological sample through one or more fluidic paths of the automated cell culture system; inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field to release the non-target biological population bound to the magnetic particles; circulating the non-target biological population bound to the magnetic particles through one or more fluidic paths of the automated cell culture system; repeating the step of circulating the biological sample to collect the target biological population one or more times; and collecting the target biological population.

[0166] As described herein, such negative selection methods utilize a design in which a target biological population is collected; the biological sample is passed, for example, through a separation tube 701 (such as Fig. 7A 701 ). In the biological sample, non-target biological populations are bound to magnetic particles. The method suitably comprises, before or including separation tube 701, circulating the biological sample through one or more fluidic paths. The non-target biological populations bound to the magnetic particles are suitably exposed to a magnetic field gradient to capture the non-target biological populations bound to the magnetic particles. For example, Figure 8 As shown, the biological sample passes through a separation tube 701, and the non-target sample with bound magnetic particles is captured on the side of the tube by the magnetic field (see also Figures 24D-24E ).

[0167] Unbound components of the biological sample (i.e., target biological populations, including cells, proteins, DNA or other structures of interest, including T cells) are then circulated through one or more fluidic paths and are appropriately collected by filtration or other mechanisms, for example, in a separate area of ​​an automated cell engineering system.

[0168] A magnetic field shield / barrier is then appropriately inserted between the non-target biological population bound to the magnetic particles and the magnetic field to release the non-target biological population bound to the magnetic particles from the magnet. The non-target biological population bound to the magnetic particles is then circulated through one or more fluidic paths of the automated cell culture system.

[0169] Suitably, the steps of cycling the biological sample, exposing the sample (and the non-target biological population bound to the magnetic particles), inserting the magnetic shield / barrier between the non-target population and the magnetic field, and collecting the target biological population are repeated one or more times (suitably 2 or more times, 3 or more times, 4 or more times, 5 or more times, etc.). Each time through such recycling the yield and / or purity of the target biological agent is increased to allow more and more of the non-target biological population to be removed, and more of the desired target biological agent to be isolated and collected.

[0170] Other steps that may be included in the negative selection methods described herein include washing the biological sample (e.g., cell population), washing the magnetic particles, transferring the target biological population to a cell culture area (e.g., a proliferation chamber) and transferring the non-target biological population to a waste chamber, and finally removing from the automated cell culture system.

[0171] As described herein, suitably, the non-target biological population includes one or more of cells, viruses, bacteria, proteins, DNA and / or RNA, and in embodiments, the target biological population includes one or more of cells, viruses, bacteria, proteins, DNA and / or RNA, and suitably comprises T cells. Methods and compounds for binding magnetic particles to non-target biological populations are described herein, and suitably comprise the use of antibodies, proteins or nucleic acids.

[0172] Exemplary magnetic fields are described herein, and are suitably produced by permanent magnets or electromagnets. Materials for making permanent magnets are described herein, and include, for example, magnetite, neodymium, samarium cobalt, and / or alnico. In embodiments, the permanent magnets are configured in a linear array. In embodiments where electromagnets are used, insertion of a magnetic field shield / barrier can be replaced by shutting down the electromagnet, for example by simply removing current from the electromagnet to stop the magnetic field.

[0173] As described throughout, in embodiments, the magnetic field shield / barrier suitably comprises a high magnetic permeability and saturation material. As described herein, in embodiments, the magnetic field shield / barrier is rotated to insert the magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and the magnetic field. Such embodiments are Figure 8 , 9 10A-10B and are described in detail herein.

[0174] In a further embodiment, provided herein is a method for washing and recovering magnetic particles in an automated cell culture system.

[0175] Suitably, the method comprises a. circulating magnetic particles through one or more fluidic paths of an automated cell culture system; b. exposing the magnetic particles to a magnetic field gradient to capture the magnetic particles; c. collecting the magnetic particles by applying a gas fluid phase followed by a liquid fluid phase; d. circulating the magnetic particles through one or more fluidic paths of an automated cell culture system; and e. repeating steps cd one or more times (e.g., 2 or more times, 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, 10 or more times, etc.).

[0176] As described herein, in embodiments, the magnetic particles are bound to the target biological population, which suitably occurs via antibodies, proteins or nucleic acids.

[0177] In further embodiments, the magnetic particles bind to non-target biological populations, including via antibodies, proteins or nucleic acids.

[0178] Exemplary non-target and target populations of organisms are described herein, and suitably the target population is any one or more of cells, viruses, bacteria, proteins, DNA and RNA, including T cells.

[0179] In an embodiment, the magnetic field gradient is provided by one or more permanent magnets, including permanent magnets comprising magnetite, neodymium, samarium cobalt, or alnico. As described herein, the permanent magnets can be configured as a linear array. In another embodiment, the magnetic field gradient is provided by an electromagnet.

[0180] Suitably, as described herein, a magnetic field shield / barrier is inserted between the magnetic particles and the magnetic field to allow the magnetic particles to be collected by blocking the magnetic field and to allow the magnetic particles that have been bound to the magnetic source to be released. Exemplary materials for magnetic field shields / barriers include high permeability and saturation materials. In embodiments where electromagnets are utilized, the current can be removed from one or more electromagnets to allow the magnetic particles to be collected, simply by turning off the source of the electromagnets.

[0181] Exemplary gas phase fluids that can be used in the method include one or more of air, nitrogen, oxygen and carbon dioxide. Exemplary liquid phase fluids that can be used include one or more of water, buffered saline solution, culture medium, animal serum, chelating agent and enzyme.

[0182] Bead recovery method

[0183] Data from negative and positive selection methods, as well as magnetic particle recovery, including multiple magnetic separation Leave

[0184] Figures 22A-22DThe binding of magnetic particles to target cells (purified cells) and waste cells (non-target population) is shown. As shown, the "improved method" using reagent amount optimization demonstrated high binding between magnetic particles and waste cells (non-target population for negative selection) with lower "false negatives".

[0185] Figures 23A-23B 23A shows the release of magnetic particles from cells. As shown, in FIG. 23A , the attachment mechanism used in the automated cell culture system (COCOON) shows a bead-cell release similar to the control, illustrating the ability to recover cells (or other target biological populations) according to the positive selection method. FIG. 23B shows the percentage of beads remaining in the target cells after release and removal of beads using the automated cell culture system.

[0186] To determine the benefits of multiple magnetic separations (i.e., recirculating the sample through an automated cell culture system to be exposed to a magnetic field 2, 3, 4, 5 times, etc.), experiments were performed while increasing the time of exposure to the magnet and decreasing the flow rate. As shown in FIG24A , increasing the magnet exposure time (from left to right) increased the percentage of bound cells captured from about 65% to at least 90%, which is equivalent to the control. Similarly, decreasing the flow rate (from right to left) in FIG24B illustrates an increase in bound cell capture from about 60% to about 85%, again similar to the control.

[0187] Table 1 below shows the increase in bound cell and total cell yield obtained using multiple passes utilizing the magnetic separation method described herein.

[0188] Table 1: Effect of yield - multiple passes through the magnetic field

[0189]

[0190]

[0191] Fig.24C The effect of the number of fluid flushing cycles to release captured biological sample from the separation line is shown, illustrating the significant recovery after the first two flushes.

[0192] Figures 24D-24E Figure 2 shows the use of a magnetic field to capture cells bound to magnetic particles in a separation tube (top) and the subsequent removal of the magnetic field and application of a fluid flushing cycle (according to Fig.24C ) followed by release of the cells (bottom), illustrating the effectiveness of the method described in this article.

[0193] exist Figures 25A-25FIn the present invention, positive and negative selection methods demonstrated high levels of purification of the target biological population (cells), with similar recoveries between the control and the automated cell culture system (COCOON) described herein for both positive and negative selection applications. Figure 25G-25I Cell purification using negative selection is shown, showing the improvement in purity by multiple passes using a recirculation line. The CD3+CD14+ population is the unwanted bead-bound monocyte-T cell aggregates. Figures 25J-25L Population purification using a negative selection process is shown. Multiple passes successfully removed unwanted bead-bound cells (highlighted by white circles).

[0194] Figures 26A-26C The advantages of magnetic separation in the automated cell culture system described herein are shown, with Figure 26A showing a significant reduction in process duration, Figure 26B showing low cell losses, and Figure 26C showing a reduction in volume loss compared to a bag and culture vessel based control. Fig.26D A population purification using a positive selection process is shown. Multiple passes through the recirculation line increased cell yield compared to a single pass.

[0195] Figures 27A-27B The ability to recover magnetic particles after washing in the automated cell culture system described herein is shown, wherein Figure 27A shows the effect of subsequent flushing of separation tube 701 on absolute recovery, and Figure 27B shows the effect of subsequent flushing of separation tube 701 on cumulative particle recovery relative to a traditional manual process.

[0196] The description of various embodiments and / or examples of the present invention has been presented for illustrative purposes, but is not intended to be exhaustive or limited to the disclosed embodiments and / or examples. Various modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terms used herein are selected to best explain the principles of the embodiments, practical applications, or technical improvements over technologies found on the market, or to enable those of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising: a. combining the target biological population with magnetic particles; b. circulating the biological sample through one or more fluidic paths of the automated cell culture system; c. passing the biological sample through a separation tube extending along the length of the magnet array; d. when the biological sample passes through the separation tube, exposing the target biological population bound to the magnetic particles to the magnetic field gradient generated by the magnet array to capture the target biological population bound to the magnetic particles; e. circulating the unbound components of the biological sample through the one or more fluidic paths of the automated cell culture system; f. rotating a magnetic field shield / barrier between the separation tube and the magnetic field gradient by a servo mechanism and a gear train so that the magnetic field shield / barrier is located between the separation tube and the magnet array to release the target biological population bound to the magnetic particles, wherein the magnet array and the magnetic field shield / barrier are connected to the servo mechanism and the gear train, thereby allowing the servo mechanism and the gear train to rotate the magnet array and the magnetic field shield / barrier; g. circulating the target biological population bound to the magnetic particles through the one or more fluidic paths of the automated cell culture system; h. Repeat steps bg one or more times; and i. Collecting the target biological population bound to the magnetic particles.

2. The method of claim 1, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA and RNA.

3. The method of claim 1 or claim 2, wherein the target biological population comprises T cells. The method of claim 1 , wherein the magnetic particles are bound to the target biological population via antibodies, proteins or nucleic acids. The method of claim 1 , wherein the magnet array comprises one or more permanent magnets.

6. The method of claim 5, wherein the one or more permanent magnets comprise magnetite, neodymium, samarium cobalt, or alnico. The method of claim 5 , wherein the one or more permanent magnets are configured in a linear array.

8. The method of claim 1, wherein the magnet array comprises one or more electromagnets.

9. The method of claim 1, wherein steps b- to b- are repeated at least twice.

10. The method of claim 1, wherein the magnetic field shield / barrier comprises a high permeability and saturation material.

11. The method of claim 1, wherein the target biological population is collected by circulating a gas phase fluid and then circulating a liquid phase fluid one or more times.

12. The method of claim 11, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen and carbon dioxide.

13. The method of claim 11, wherein the liquid phase fluid comprises one or more of water, a buffered saline solution, a culture medium, an animal serum, a chelating agent, and an enzyme.

14. The method of claim 1, further comprising removing the target biological population from the bound magnetic particles.

15. A method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising: a. Binding non-target biological populations to magnetic particles; b. circulating the biological sample through one or more fluidic paths of the automated cell culture system; c. passing the biological sample through a separation tube extending along the length of the magnet array; d. when the biological sample passes through the separation tube, exposing the non-target biological population bound to the magnetic particles to the magnetic field gradient generated by the magnet array to capture the non-target biological population bound to the magnetic particles; e. circulating the target biological population through one or more fluidic paths of the automated cell culture system; f. rotating a magnetic field shield / barrier between the separation tube and the magnetic field gradient by a servo mechanism and a gear train so that the magnetic field shield / barrier is located between the separation tube and the magnet array to release the non-target biological population bound to the magnetic particles, wherein the magnet array and the magnetic field shield / barrier are connected to the servo mechanism and the gear train to allow the servo mechanism and the gear train to rotate the magnet array and the magnetic field shield / barrier; g. circulating the non-target biological population bound to the magnetic particles through the one or more fluidic paths of the automated cell culture system; h. Repeat steps bg one or more times; and i. Collecting the target organism population.

16. The method of claim 15, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA and RNA.

17. The method of claim 15, wherein the target biological population comprises T cells.

18. The method of claim 15, wherein the non-target organism population is collected.

19. The method of claim 15, wherein the magnetic particles bind to the non-target biological population via antibodies, proteins or nucleic acids.

20. The method of claim 15, wherein the magnet array comprises one or more permanent magnets.

21. The method of claim 20, wherein the one or more permanent magnets comprise magnetite, neodymium, samarium cobalt, or alnico.

22. The method of claim 20, wherein the one or more permanent magnets are configured in a linear array.

23. The method of claim 15, wherein the magnet array comprises one or more electromagnets.

24. The method of claim 15, wherein steps bg are repeated at least twice.

25. The method of claim 15, wherein the magnetic field shield / barrier comprises a high permeability and saturation material.

26. The method of claim 15, wherein the target population of organisms is collected by circulating a gas phase fluid and then circulating a liquid phase fluid one or more times.

27. The method of claim 26, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen and carbon dioxide.

28. The method of claim 26, wherein the liquid phase fluid comprises one or more of water, a buffered saline solution, a culture medium, an animal serum, a chelating agent, and an enzyme.

29. A system for magnetically separating and collecting a target biological population from a biological sample, comprising: a. Magnetic field source; b. a separation tube extending along the length of the magnet array for flowing the biological sample, the separation tube being aligned with the magnetic field source; c. A magnetic field shield / barrier configured to be located between the magnetic field source and the separation tube; and d. an electromechanical drive assembly comprising a servomechanism and a gear train for rotating a magnetic field shield / barrier between the magnetic field source and the separation tube, wherein the magnetic field source and the magnetic field shield / barrier are connected to the servomechanism and the gear train to allow the servomechanism and the gear train to rotate the magnetic field source and the magnetic field shield / barrier; When the biological sample passes through the separation tube, when the target biological population is bound to multiple magnetic particles, the magnetic field shield / barrier is rotated by the servo mechanism and the gear train so that the magnetic field shield / barrier is located between the magnetic field source and the separation tube to release the target biological population.

30. The system of claim 29, wherein the system is part of an automated cell culture system.

31. The system of claim 29, wherein the magnetic field shield / barrier is a material having high magnetic permeability and saturation.

32. The system of claim 31, wherein the material having high magnetic permeability and saturation is selected from the group consisting of iron, iron alloys, ferritic steel, and Hiperco-50.

33. The system of claim 29, wherein the magnetic field source comprises one or more permanent magnets.

34. The system of claim 33, wherein the one or more permanent magnets comprise neodymium, samarium cobalt, or alnico.

35. The system of claim 29, wherein the magnetic field source comprises a plurality of permanent magnets configured in a linear array.

36. The system of claim 35, wherein the plurality of permanent magnets in a linear array are arranged with opposite magnetic pole orientations perpendicular to an axis of the linear array.

37. A method for collecting a target biological population from a biological sample in an automated cell culture system, the method comprising: a. combining the target biological population with magnetic particles; b. passing the biological sample through a separation tube extending along the length of the magnet array; c. when the biological sample passes through the separation tube, exposing the target biological population bound to the magnetic particles to the magnetic field gradient generated by the magnet array to capture the target biological population bound to the magnetic particles; d. removing unbound populations of the biological sample; e. rotating a magnetic field shield / barrier between the separation tube and the magnetic field gradient by a servo mechanism and a gear train so that the magnetic field shield / barrier is located between the separation tube and the magnet array to release the target biological population bound to the magnetic particles, wherein the magnet array and the magnetic field shield / barrier are connected to the servo mechanism and the gear train to allow the servo mechanism and the gear train to rotate the magnet array and the magnetic field shield / barrier; and f. Collecting the target biological population bound to the magnetic particles.

38. The method of claim 37, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA and RNA.

39. The method of claim 37, wherein the target biological population comprises T cells.

40. The method of claim 37, wherein the magnetic particles are bound to the target biological population via antibodies, proteins or nucleic acids.

41. The method of claim 37, wherein the magnet array comprises one or more permanent magnets.

42. The method of claim 41, wherein the one or more permanent magnets comprise magnetite, neodymium, samarium cobalt, or alnico.

43. The method of claim 41, wherein the one or more permanent magnets are configured in a linear array.

44. The method of claim 37, wherein the magnet array comprises one or more electromagnets.

45. The method of claim 37, wherein the magnetic field shield / barrier comprises a high permeability and saturation material.

46. ​​The method of claim 37, wherein the target population of organisms is collected by circulating a gas phase fluid and then circulating a liquid phase fluid one or more times.

47. The method of claim 46, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen and carbon dioxide.

48. The method of claim 46, wherein the liquid phase fluid comprises one or more of water, a buffered saline solution, a culture medium, an animal serum, a chelating agent, and an enzyme.

49. The method of claim 37, further comprising removing the target biological population from the bound magnetic particles.

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