Magnetic separation

Abstract

To provide a method for collecting a target biological population from a biological sample in an automated cell culture system.SOLUTION: This method includes a. binding a non-target biological population to magnetic particles; b. circulating a biological sample through one or more fluidics pathways of an automated cell culture system; c. exposing the non-target biological population bound to the magnetic particles to a magnetic field gradient; d. repeating steps b-c one or more times; and e. collecting the target biological population.SELECTED DRAWING: Figure 21

Description

[Technical Field]

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

[0002] Magnetic separation has been utilized as a method for separating magnetic impurities from fluids through the application of a variety of different processes (U.S. Patent Nos. 3,985,646, 4,054,513, and 5,137,629). Magnetic separation techniques have also been applied to the separation of populations of biological materials using magnetic beads coated with antibodies or polymers that bind to various biological targets, including viruses, bacteria, and cells (U.S. Patent Nos. 3,970,518, 4,219,411, 4,795,698, and 5,385,707). The biological targets can then be extracted from the fluid suspension using one of the previously developed magnetic separation devices described, for example, in U.S. Patent Nos. 4,710,472, 5,691,208, 6,193,892, and Zborowski et al. (Journal of Magnetism and Magnetic Materials, vol. 194, pp. 224-230, 1999). The magnetic field generated by the separation device exerts a force on magnetic beads suspended in a fluid suspension, allowing the beads to be drawn from the fluid suspension, as described by 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), as well as any biological material bound to the magnetic beads. This allows the isolation of a desired population, either by removal from the fluid suspension (also known as positive selection) or by removal of all other populations from the fluid suspension, leaving only the unbound population of interest (also known as negative selection). Isolation of cells, such as T cells and stem cells, from heterogeneous cell populations is essential for the development of cell therapies used to treat various diseases.

[0003] One system utilizes a quiescent suspension in a surrounding magnet (EasySep™ by STEMCELL Technologies®). Other systems are known that are automated and use magnetic beads to isolate target populations (AutoMACS™ by Milytenyi Biotec, and RoboSep™ by STEMCELL™ Technologies).

[0004] However, there remains an unmet need for rapid and reliable magnetic separation of selected targets within biological samples, where the application of the magnetic field can be automated, customized, and controlled to achieve the desired high yield and purity of target separation. Summary of the Invention

[0005] 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 the target biological population to magnetic particles; b. circulating the biological sample through one or more fluid paths of the automated cell culture system; c. exposing the target biological population bound to the magnetic particles to a magnetic field gradient; d. repeating steps b-c one or more times; and e. collecting the target biological population bound to the magnetic particles.

[0006] Also provided herein is 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 fluid paths 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 fluid paths of the automated cell culture system; e. inserting a magnetic field shield / barrier between the target biological population bound to the magnetic particles and a 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 fluid paths of the automated cell culture system; g. repeating steps b-f one or more times; and h. collecting the target biological population bound to the magnetic particles.

[0007] In additional embodiments herein, there is provided 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 fluid paths 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 b-c one or more times; and e. collecting the target biological population.

[0008] In further embodiments herein, there is also provided 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 fluid paths 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 fluid paths 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 a 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 fluid paths of the automated cell culture system; g. repeating steps b-f one or more times; and h. collecting the target biological population. [Brief explanation of the drawings]

[0009] The following description of exemplary embodiments described herein will be better understood when read in conjunction with the accompanying drawings. For purposes of illustrating the invention, embodiments that are presently exemplary are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. It should be noted that like reference numerals refer to like elements throughout the various embodiments shown in the drawings and referred to herein.

[0010] The present disclosure will be more fully understood in light of the following drawings. [Figure 1] FIG. 1 shows the magnetic field gradient used to move magnetic particles. [Figure 2] Figure 2 shows magnetic beads of various sizes used in cell separation applications. [Figure 3] FIG. 3 shows the effect of bead size on the net magnetic force acting on a bead in response to a magnetic field gradient. [Figure 4] FIG. 4 illustrates various conceptual methods for generating an automatically controlled magnetic field. [Figure 5] FIG. 5 shows a computational model of the ability of highly permeable and magnetically saturable materials to block magnetic flux density. [Figure 6] Figure 6 shows the incorporation of magnetic separation tubes on the cassette. [Figure 7] FIG. 7 shows a computer-aided design (CAD) model of a separation tube 701 aligned with a magnet array 704 consisting of rare earth magnets with alternating poles 702 and 703 . [Figure 8] FIG. 8 shows a CAD model assembly of a separation tube 701 aligned with a magnet array 704 in the “on” position, which is placed within a magnetic field shield 801 that can be rotated using a motor 802 and gear train 803. [Figure 9] FIG. 9 shows a CAD assembly of separation tube 701 aligned with the magnet array in the "off" position, with magnetic field shield 801 between tube 701 and magnet array 704. [Figure 10] FIG. 10 shows a cross-sectional view of the assembly in the "on" position or the "off" position. [Figure 11] FIG. 11 shows the complete assembly of a cassette 1101 consisting of a separation tube 701 aligned with a cell culture device 1102 containing a magnetic separation assembly (704 and 801). [Figure 12] FIG. 12 presents various non-limiting methods (portions 1201-1208) for generating localized high magnetic field gradients. [Figure 13] FIG. 13 shows the results obtained from Working Example 1, demonstrating the successful separation of cells from magnetic beads at various flow rates. [Figure 14] FIG. 14 shows the results obtained from Working Example 2, demonstrating the effect of different flow rates on the capture and release of positively selected cells. [Figure 15]FIG. 15 shows the results obtained from Working Example 3, which added multiple passes to the magnetic separation process through separation tube 701 for the capture and release of positively selected cells. [Figure 16] FIG. 16 shows the results from Working Example 4, where the exposure of the magnetic beads to the magnetic field was increased by adding a waiting time, for the capture and release of positively selected cells. [Figure 17] FIG. 17 shows how the false positive rate obtained from Working Example 4 can be modified by making various process modifications. [Figure 18] FIG. 18 shows the results obtained from Working Example 5 for the purification of a mixed cell population flowed through a separation tube 701 past a magnet array 704 . [Figure 19] FIG. 19 shows results from Working Example 6 illustrating how varying the size of magnets 702 and 703 can affect the magnetic field characteristics and cell capture generated by magnet array 704. [Figure 20] FIG. 20 shows the results from Working Example 7 regarding the effect of adding bars / spacers 1201 to the cassette. [Figure 21] FIG. 21 shows a schematic of a recirculation magnetic separation method according to an embodiment of the present disclosure. [Figure 22] 22A-22D show the recovery of purified and discarded cells according to embodiments herein. [Figure 23] 23A-23B show the efficiency of bead release from cells according to embodiments herein. [Figure 24AB] 24A-24C show the effect of contact strength and multiple cycles with the magnetic field. [Figure 24C] Same as above. [Figure 24DE] 24D-24E show the capture and removal of cells in a separation tube, as described herein. [Figure 25AF] 25A-25L show the results of positive and negative selection of target cells as described herein. [Figure 25GI]25A-25L show the results of positive and negative selection of target cells as described herein. [Figure 25JL] 25A-25L show the results of positive and negative selection of target cells as described herein. [Figure 26AC] 26A-26D show the harvesting of cells in an automated cell culture system as described herein. [Figure 26D] 26A-26D show the harvesting of cells in an automated cell culture system as described herein. [Figure 27] 27A-27B show the recovery of magnetic particles after washing in an automated cell culture system described herein. DETAILED DESCRIPTION OF THE INVENTION

[0011] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and patent applications discussed herein are presented solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In case of conflict, the present specification, including definitions, shall control. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the subject matter of this specification belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0012] As used herein, the articles "a" and "an" preceding an element or component are intended to be open-ended regarding the number of instances (i.e., occurrences) of that element or component. Thus, "a" or "an" should be interpreted as including one or at least one, and the singular form of that element or component also includes the plural unless the number is clearly intended to be singular.

[0013] As used herein, the term "invention" or "present invention" is an open-ended term and is not intended to refer to any one embodiment of a particular invention, but rather encompasses all possible embodiments described in the specification and claims.

[0014] As used herein, the terms "comprise," "including," "containing," "having," and their conjugations and cognates mean "including, but not limited to."

[0015] As used herein, the term "about," modifying the amount of an ingredient, component, or reactant employed, refers to variations in quantity that may occur through typical measuring procedures and liquid handling procedures used, for example, to make concentrates or solutions. Variations may also arise from inadvertent errors in measuring procedures, differences in the manufacture, source, or purity of ingredients employed to make a composition or perform a method, and the like. In one embodiment, the term "about" means within 10% of the reported numerical value. In another embodiment, the term "about" means within 5% of the reported numerical value. In yet another embodiment, the term "about" means within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reported numerical value.

[0016] When a range of values ​​is listed, it is merely for convenience or brevity and includes all possible subranges and individual values ​​within that range and around the boundaries of that range. All numerical values ​​include the actual approximation unless otherwise specified, and integer values ​​do not exclude fractional values. Subrange values ​​and actual approximation values ​​shall be considered to be specifically disclosed values.

[0017] It is understood that any element specified herein as being included may also be expressly excluded from the claimed invention for purposes of a disclaimer or negative limitation.

[0018] As used herein, the terms "approximately," "approximately," and "actually" mean a respective relation or measurement or amount or quantity or degree that has no adverse consequences or effects compared to a referenced term or embodiment or operation or scope of the invention.

[0019] As used herein, any term referring to an angular relationship, e.g., "vertical," "horizontal," "parallel," "opposite," "straight," "sideways," "parallel," "right angles," and other such terms, also refers to the respective angular relationship in approximately functional and / or practical terms.

[0020] As used herein, the terms "preferred," "preferably," "typical," "typically," or "optional" do not limit the scope of the invention or embodiments thereof.

[0021] As used herein, the terms "substantial," "substantial amount" (or synonyms thereof), as relevant in the context, mean a measure or range or amount or degree that encompasses most or the majority of the referenced entity, or at least moderately, or even higher, or larger, or more efficiently, or to a more significant extent, compared to the referenced entity or with respect to the referenced subject.

[0022] As used herein, the terms "negligible" and "insignificant" (or their equivalents) mean a sufficiently small association, measurement, amount, quantity, or degree, respectively, that has no practical consequence compared to the referenced term or to the scope of the invention.

[0023] As used herein, the term "may" means an option or effect that is not included and / or not used and / or not implemented and / or not occurring, but that constitutes at least part of some embodiments or results of the invention without limiting the scope of the invention.

[0024] As used herein, a "sample" may be any sample, including a "biological sample" that may be derived from a plant, human, animal, or microbial source. A sample is typically a heterogeneous sample from which targets are selected, separated, and collected. Targets may be cells, DNA, RNA, proteins, peptides, microorganisms, viruses, etc. A biological sample contains a target population.

[0025] Biological samples may include bodily fluid samples, somatic cell samples, or biological tissue samples. Examples of biological fluid samples or bodily fluid samples include urine, lymph, blood, plasma, serum, saliva, cervical mucus, cervical-vaginal fluid, vaginal fluid, milk, breast milk, synovial fluid, semen, feces, sputum, cerebrospinal fluid, tears, mucus, interstitial fluid, follicular fluid, amniotic fluid, aqueous humor, vitreous humor, peritoneal fluid and ascites, sweat, lymph, lung sputum, and lung lavage fluid, or samples derived therefrom. Biological tissue samples are samples containing aggregates of cells, usually specific types of cells and intercellular substances, which form one of the structural components of human, animal, plant, bacterial, fungal, or viral structures, including connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Examples of biological tissue samples also include organs, tumors, lymph nodes, arteries, and individual cells. For example, the sample may be a tissue sample suspected to be cancerous. The biological tissue sample may first be processed to dissociate cellular aggregates.

[0026] In some embodiments, the biological sample is blood cells, white blood cells, or platelets. White blood cells 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.

[0027] As used herein, a "target cell" is typically a cell intended to be separated or enriched from other cells (e.g., for testing or diagnosis), that is of a particular type or has a unique characteristic compared to other cells, such as a selective mutual affinity for binding to a particular antibody or other compound or other particle. In certain embodiments, the unique characteristic is a selective affinity that is coupled to or bound to a magnetic bead to form a magnetic target cell.

[0028] 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 anticipated. Animals include mammals. A patient refers to a subject, such as a mammal, primate, human, or livestock subject, suffering from a disease state or for which a disease state is to be determined or treated. A patient sample can be the source of a biological population from which it originates.

[0029] As used herein, the term "antibody" is intended to include monoclonal and polyclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), or antigen-binding portions thereof, including, but not limited to, F(ab), Fv fragments such as scFv, single-chain antibodies, chimeric antibodies, humanized antibodies, recombinant genetically engineered antibodies, and Fab expression libraries. Bispecific antibodies can also be immobilized on magnetic particles.

[0030] As used herein, "labeling moiety" can be directly or indirectly detectable. The labeling moiety can be a label that can be labeled and can be used in conjunction with magnetic particles. Direct labeling moieties include radioisotopes; enzymes whose products are detectable (e.g., luciferase, β-galactosidase, etc.); fluorescent labels (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, e.g., 152 Euor other members of the lanthanide series attached to proteins via metal chelating groups such as EDTA; chemiluminescent compounds such as luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds such as luciferin, aequorin (green fluorescent protein), and the like; and metal compounds. Indirect labeling moieties include labeled molecules that bind to the polypeptide, such as antibodies specific for the polypeptide, where the labeled binding molecule is labeled as described above and is labeled as one of a specific binding pair, such as biotin (one of the biotin-avidin specific binding pairs) or digoxigenin (one of the digoxigenin-antibody specific binding pairs). Alternatively, the labeling moiety may be any suitable label, including, but not limited to, those described herein.

[0031] For example, magnetic particles labeled with binding partners such as antibodies, proteins, or nucleic acid molecules are commercially available from Miltenyi Biotec GmbH (Friedrich Ebert Str. 68, D-51429, Bergisch Gladbach, Germany). Methods for magnetically labeling biomolecules are known in the art, and any known method can be used. For example, U.S. Patent No. 6,020,210 describes methods for preparing magnetic particles and attaching biomolecules to them. A first member of a specific binding pair can be associated with the magnetic particle, in which case the modified biomolecule contains a moiety that binds to the specific binding pair member. Alternatively, the magnetic particle can be linked to an antibody or its immunoreactive fragment via, for example, a linker or spacer (e.g., a nucleic acid linker). Adding a spacer or linker allows for more flexible presentation of the biomolecule, and careful chemical manipulation allows for the attachment of ligands in a specific direction. There are many chemical manipulation methods used for these linkages, and many companies have published protocols to assist those skilled in the chemical arts.

[0032] Examples of specific binding pair members that can be attached to magnetic particles include, but are not limited to, oligo-dT (e.g., for binding to nucleic acid molecules containing a 3'-terminal polyA moiety); oligonucleotides having specific nucleotide sequences (for binding to nucleic acid molecules containing complementary nucleotide sequences); avidin (e.g., streptavidin) (for binding to biotinylated biomolecules); antigen-binding polypeptides, such as immunoglobulins (Ig) or epitope-binding fragments thereof (for binding to biomolecules containing epitopes recognized by Ig); 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); and 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.

[0033] Biomolecules may be linked (covalently or non-covalently) to magnetic particles by direct chemical bonding or physical association. Such methods are known in the art. Biochemical binding is described, for example, in "Bioconjugate Techniques" by Greg T. Hermanson, Academic Press. Non-covalent interactions, such as ionic bonds, hydrophobic interactions, hydrogen bonds, and / or van der Waals forces, may also be used to link biomolecules to magnetic particles. For example, standard non-covalent interactions used to bind biomolecules to chromatography matrices may be used. One non-limiting example of such a non-covalent interaction that can be used to bind biomolecules to magnetic particles is DNA binding to silica in the presence of chaotropic salts. Those skilled in the art will recognize other such non-covalent bonds and conditions for achieving such binding. See, for example, Molecular Cloning, Sambrook and Russell, Cold Spring Harbor Laboratory Press.

[0034] As used herein, "magnetic particles" are used as labels for biomolecular targets in biological samples, such as, but not limited to, antibodies, DNA, polypeptides, and cells, to aid in the separation of the sample from a complex mixture. Magnetic particles may be classified according to size as follows: microbeads less than about 50 nm; nanobeads between about 100 and about 200 nm; and dynabeads between about 1 and 5 μm. Furthermore, magnetic particles may be adapted for selective affinity (functionalized) for linking or binding to desired biomolecular targets, such as fluorescent labels, antibodies, nucleic acids, etc.

[0035] A variety of magnetic particles are available from numerous sources, including, for example, Dynal (Norway), Advanced Magnetics (Cambridge, MA, USA), Immuncon (Philadelphia, USA), Immunotec (Marseille, France), and Miltenyi Biotec GmbH (Germany). A preferred magnetic labeling method involves colloidal superparamagnetic particles in the size range of 5-200 nm, preferably 10-100 nm. These magnetic particles allow for quantitative magnetic labeling of cells, whereby the amount of magnetic label attached is proportional to the amount of product bound. Colloidal particles with various specificities are available, for example, through Miltenyi Biotec GmbH.

[0036] As used herein, "separation" includes isolating or collecting target cells from a surrounding fluid bulk, where bulk can be, for example, a fluid mixture or suspension or emulsion of cells, or a combination thereof, and also includes concentrating or enriching the target cells relative to the surrounding bulk or a provided cell sample (similar to sedimentation or centrifugation to obtain a precipitate).

[0037] As used herein, "depletion" in relation to separation is the removal of target cells from the bulk (similar to sedimentation or centrifugation to obtain a supernatant).

[0038] As used herein, "highly qualitative" (separation, depletion) refers to the isolation of highly pure target cells, substantially free of other cells, or containing only trace amounts of other cells, e.g., less than about 10% to about 1% of the isolated cells, or vice versa in the case of depletion.

[0039] As used herein, "highly quantitative" (separation, depletion) refers to the high recovery or separation of a very large number of target cells from a sample, such as substantially all of the target cells, or about 80% to about 99% or more of the separated cells, or vice versa in the case of depletion.

[0040] It should be noted that whenever reference is made herein to the attachment or adhesion or adhesion of cells to the wall of a tube, or similar terms for such action, it does not necessarily mean that the cells are directly attached to the wall, but rather that they are indirectly connected, bound or attracted to the wall, for example by chains of cells or groups of cells.

[0041] As used herein, "magnetic shielding" refers to reducing and / or blocking a magnetic field in space by blocking the magnetic field with a "magnetic field shield" (also referred to herein as a magnetic field shield / barrier, and the terms are interchangeable).

[0042] As used herein, "HMPSM" means a material of high magnetic permeability and magnetic saturation that generates a highly concentrated magnetic field within itself, effectively reducing and / or eliminating the effects of the magnetic field.

[0043] As used herein, a "magnetic field shield / barrier" is a structure that can be controlled with respect to the use of a magnetic field.

[0044] As used herein, an "electromagnet" is a type of magnet in which a magnetic field is generated by an electric current. The magnetic field disappears when the current is turned off. Electromagnets usually consist of a wire wound into a coil. Current passing through the wire creates a magnetic field, which is concentrated in a hole in the center of the coil. The wire turns are often wound around a magnetic core made of a ferromagnetic or ferrimagnetic material, such as iron. The magnetic core concentrates the magnetic flux, creating a stronger magnet.

[0045] As used herein, a "permanent magnet" is a magnet that is permanent, as opposed to an electromagnet, which only behaves like a magnet when an electric current is passed through it. Permanent magnets are made from materials such as magnetite (Fe3O4), the most highly magnetic naturally occurring mineral, or neodymium, a highly magnetic synthetic material.

[0046] As used herein, a "magnet array" refers to one or more magnets. The one or more magnets may be permanent magnets or electromagnets. The one or more permanent magnets may be in a linear array, may be of different sizes, different strengths, and may be configured with opposite polar orientations perpendicular to the axis of the linear array, or may be configured rotated 90° relative to 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 magnets of a material selected from iron, neodymium, samarium-cobalt, or alnico.

[0047] A general, non-limiting overview of the invention and its implementation is provided below. The overview describes exemplary implementations of embodiments / aspects of the invention and provides an architectural basis for various and / or alternative and / or diverse aspects / embodiments, some of which are described below.

[0048] The present disclosure relates to devices, methods, and systems for magnetically separating and collecting desired biomolecular targets in a biological sample via positive or negative selection. As presented herein, a magnetic field is generated substantially adjacent to a biological sample containing the desired magnetized biomolecular targets. The magnetic field can be automatically switched "on" and "off," resulting in the generation of a magnetic field of desired intensity, continuous duration, intermittent duration, pulsed 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 facing / applied to the biological sample. The magnetic field shield / barrier is positioned between a magnetic field source and the biological sample and, as a function of its high magnetic permeability and saturation material (HMPSM), generates a highly concentrated magnetic field within itself, effectively reducing and / or eliminating the effect of the magnetic field on the biological sample containing the magnetized biomolecular targets.

[0049] In an embodiment of the invention, cellular biological material is cultured in a bioreactor tube, and the desired cells are the biomolecular targets for magnetic separation and collection.

[0050] The devices, methods, and systems described herein generally involve a biological sample (a heterogeneous biological population), typically, but not limited to, cells, having magnetic beads bound to specific biomolecular targets (specific cell types) in the sample, resulting in "magnetized cells." Typical binding methods can include: i) direct binding of magnetic beads bound to the antibody of the biological target; and ii) using a multi-step process in which the biological target is bound to an antibody or binding pair bound to another antigen. This antigen / binding pair is then bound to the magnetic beads associated with each antibody / binding pair. During magnetic separation, the magnetized cells (expressing the antigen; positively selected) that are the target cells bound to the magnetic beads are attracted to a location near the magnet, while the cell population without beads (negatively selected) remains in the 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 some type of biological process on the target cells (e.g., T cell activation using anti-CD3 and anti-CD28 coupled to magnetically activated beads). After stimulation, the magnetic beads must be removed, followed by downstream processing. This requires treating the cell suspension with an effective magnet.

[0051] During separation, the magnetically tilted particles are subjected to a force vector F from an applied magnetic field B, which acts on the paramagnetic particles as defined in Equation 1 below (Pamme, 2006).

number

[0052] where V is the volume of the particle and Δ Xis the difference in magnetic susceptibility (ability to be magnetized) between the particle and the surrounding medium, μ is the magnetic permeability of vacuum, and B·∇ 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 many parameters. First, particle size; larger particles experience stronger magnetic forces. There are three typical size categories for magnetic particles: i) less than 50 nm (e.g., Miltenyi Biotec's MACS® Microbeads), ii) 100-200 nm (e.g., BioLegend® Nanobeads), or iii) 1-5 μm (e.g., Invitrogen's Dynabeads®). The larger the size, the easier the separation. Second, increasing the magnetic susceptibility of the beads compared to the surrounding medium is typically already relatively large, since most beads often consist of an iron core and the surrounding medium is not actually magnetized. Finally, increasing the magnetic field gradient can dramatically increase the force exerted on the magnetic beads. This is because the spatial quality of a high-gradient magnetic field is sparse, causing the magnetic field gradient to exert unequal forces on the north and south poles of a magnetic particle (Figure 1A). This unequal force on the particle causes particle movement. In a perfectly uniform magnetic field, equal and opposite forces are generated on the two poles of a magnetic particle, resulting in zero net force on the particle and no net movement (Figure 1B). Furthermore, increasing the size of the magnetic particle increases the difference in the magnetic force exerted by the gradient between the two poles compared to small beads (Figures 2 and 3).

[0053] For automated separation, isolation, and collection, means exist for "switchably" inducing (turning on and off) magnetic fields that can generate gradients that attract magnetic beads. For example, electromagnets are formed by wrapping current-carrying wire around a magnetically susceptible material (e.g., iron) (Figure 4A). The electromagnet can be switched on or off by applying or removing current through the wire, respectively. Another approach is electro-permanent magnets, which consist of a hard magnet with high magnetic coercivity (high enough magnetic field to switch poles) and a soft magnet with low coercivity (Figure 4B). Both magnets are connected to each other with a paramagnetic material (e.g., iron) to complete a magnetic circuit. The soft magnet is wrapped with current-carrying wire, and pulsing a strong current through the wire allows the soft magnet's poles to be switched. When the poles are not aligned, the magnet's "current" flows through the paramagnetic material, and no external magnetic field is observed. However, when the poles are aligned, the magnetic current travels through the air, generating an external magnetic field. A final method is to use a permanent magnet to generate a magnetic field, and by using a material with high magnetic saturation, it is possible to block the field on one side of the magnet (Figure 4C).

[0054] In an embodiment of the present invention, an array of strong permanent magnets with alternating directionality is used (FIG. 4C). This generates a strong magnetic gradient that radiates from the array, as well as a gradient that is linear along the axis of the array. A modification of this design is the Halbach array, in which the magnets in the array are rotated 90° relative to each other (FIG. 4D). This induces a significant increase in the magnetic field on one side of the magnet, while weakening the magnetic field on the other side (Kang et al.).

[0055] This controllable magnetic field can be designed to perform sequential actions that control the isolation (either via positive or negative selection) of target biological fractions containing biomolecular targets, while simultaneously allowing non-target biological fractions to be removed and discarded.

[0056] The ability to switch the magnetic field on and off while processing biological samples using magnetic separation, allowing for automation of both positive and negative cell selection, is a key operational requirement. The devices and methods described herein enable automated collection of target biological populations, thereby reducing overall process complexity and operational costs.

[0057] The switchable "on" and "off" magnetic field described herein controls the magnetic field experienced by biological samples labeled with magnetic particles, e.g., magnetic beads, by introducing a magnetic shield / barrier. This controllable magnetic field-mediated biological retention of targets, followed by sequential steps of secondary release and capture, allows for the creation of highly compact and energy-efficient magnetic separation systems.

[0058] The magnetic shield / barrier functions due to the inherent properties of HMPSM. Its high permeability and magnetic saturation result in a highly concentrated magnetic field within the material. By placing the HMPSM as a magnetic shield / barrier between a magnetic field source and a biological sample, it is possible to virtually eliminate the effect of the magnetic field on the biological sample. Through the controllable activation of this magnetic barrier, magnetic separation of target populations from biological samples becomes feasible with high reproducibility and relatively low cost.

[0059] Figure 1 presents the theory of how magnetic particles / beads (e.g., those used in cell separation and activation) respond to a magnetic field gradient. This is mathematically demonstrated in Equation 1. Each magnetic bead has a north and a south pole. When exposed to a magnetic field gradient, a different magnetic force (one attractive, one repulsive) acts on each pole of the particle, resulting in a net force that can move the particle (Figure 1A). By comparison, in the absence of a magnetic field gradient, the forces acting on each pole are equal and opposite (Figure 1B). Therefore, no net force is exerted on the magnetic particle, leading to no movement. These necessary magnetic field gradients can be generated using an array of magnets arranged with alternating north and south poles. Furthermore, as shown in Figure 1C, high magnetic field gradients can be generated using an array of multiple strong, small magnets arranged with alternating north and south poles in the array.

[0060] Figure 2 shows the sizes of magnetic beads commonly used for magnetic cell separation. Current bead sizes range from Miltenyi's MACS® MicroBeads, which are approximately 50 nm, to Invitrogen's Dynabeads®, which are approximately 5 μm or smaller. Increasing bead size often increases the sensitivity of the beads to respond to magnetic fields, as does the difference in forces acting on each pole of the bead.

[0061] Figure 3 outlines how larger magnetic beads can more efficiently push fluid in response to an induced magnetic field. In some embodiments, the beads are simultaneously exposed to attractive and repulsive forces from the magnetic field at each of the beads' poles. The magnitude and gradient of the magnetic field decrease as the beads move away from the magnetic field source. The net force experienced by the beads therefore depends on their distance from the magnetic field source and their diameter. Larger beads have a greater distance between their poles, resulting in a greater difference in the magnetic forces acting on the poles. Similarly, as the gradient becomes stronger (i.e., closer to the magnet), the difference in the forces acting on each pole of the beads also increases. This increase in net force results in a faster attraction of the beads toward the magnet and out of the fluid.

[0062] Figure 4 presents a method for generating a magnetic field that can be rotated on and off for application to biological samples using an automated control system. In Figure 4A, an electromagnet is presented, in which a wire is wrapped around a ferromagnetic material, such as iron. By applying a current to the wire, the magnetic field can be quickly and easily turned on and off. However, the magnetic field generated is low. Furthermore, electromagnets generate significant levels of heat, which is very problematic for the local cell culture environment. Figure 4B presents an electro-permanent magnet consisting of both a non-switchable rare earth magnet and a pole-switchable alnico permanent magnet, both embedded in a ferromagnetic material that forms a magnetic circuit. When the poles of the permanent magnets are aligned, the carbon steel adopts the same direction, generating a net magnetic flux in the air surrounding the magnet (which can be used to extract magnetic beads from a fluid suspension). However, when the poles of the two permanent magnets are oppositely oriented, the magnetic flux is confined to the ferromagnetic material, preventing magnetic bead extraction. The orientation of an alnico magnet can be switched by applying a very large, short-duration current pulse through a coil of wire surrounding the alnico magnet, resulting in a transient, large magnetic field. However, the magnetic gradient resulting from such a configuration is also very low compared to rare-earth magnets, and this method also generates electromagnetic interference that may have unknown harmful effects on any surrounding electronics. Figure 4C shows an array of permanent magnets with opposite polarity, with an HMPSM (HMPSM), such as iron, cobalt-iron, or Hiperco50, on one side. Using this HMPSM, the magnetic flux of the magnetic field is amplified on the opposite side while being reduced to a negligible level on the HMPSM side. By operating the HMPSM so that it is between the permanent magnet and the magnetic beads, the force acting on the beads can be reduced to virtually zero. Conversely, by moving the HMPSM to the opposite side of the permanent magnet, a very strong magnetic force can be induced on the beads. Finally, Figure 4D presents a Halbach linear array, where a 90° rotation of the magnet generates an amplified magnetic field on one side of the array, while the magnetic field is significantly reduced or eliminated on the other side of the array.

[0063] Figure 5 presents computer modeling showing the function of the HMPSM (labeled x) on a permanent magnet. The magnetic flux is mainly transmitted through the air around the magnet. However, the magnetic flux is not strong enough to be transmitted through the HMPSM. Therefore, there is no significant magnetic flux density on the HMPSM opposite the magnet, but the magnetic flux density is mainly amplified at the two poles of the permanent magnet.

[0064] Figure 6 shows the layout of an example cassette often used in automated cell culture systems, with a separation tube for magnetic separation using magnetic beads extending along the entire length of the cassette face. This tube aligns with a permanent magnet array located within the automated cell culture system. The separation tube connects to various tubes or bags within the cassette, which are used for positive or negative selection of target cells. Using the longest possible tube increases the volume that can be loaded into the separation tube, thereby shortening the processing time for magnetic separation to occur.

[0065] FIG. 7 presents an embodiment of the separation process presented in FIG. 6. Magnetically coupled beads, bound or unbound to cells, can be flowed into a separation tube 701 (FIG. 7A). In some embodiments, the separation tube 701 is aligned with a permanent magnet array 704, as shown in FIG. 7B. In other embodiments, the separation tube 701 is aligned with an electromagnet that replaces the permanent magnet array 704. The permanent magnet array 704 consists of permanent magnets with alternating north poles (702) and south poles (703), creating the highest possible magnetic flux gradient. The magnetic beads in the separation tube 701 are attracted to the magnet array 704, thereby successfully separating the magnetic beads from the fluid suspension.

[0066] FIG. 8 illustrates an embodiment of a magnetic field shield / barrier with the magnetic field "on." A separation tube 701 extends along the entire length of a permanent magnet array 704. A magnetic field shield 801 (shown as paramagnetic sheath 801) made from HMSPM surrounds a portion of the permanent magnet array 704. In some embodiments, the magnetic field shield 801 is made from pure iron. In some embodiments, the magnetic field shield 801 is made from a soft magnetic iron alloy, such as ferritic steel, silicon-iron, nickel-iron, or cobalt-iron. In some embodiments, the magnetic field shield 801 is made from a soft magnetic alloy of cobalt, vanadium, and iron, such as an alloy of about 49% cobalt, about 2% vanadium, and the balance iron. In some embodiments, the sheath 801 may be made from Hiperco 50 or Hiperco 50A. In some embodiments, 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. In effect, this paramagnetism increases the magnetic force acting on the magnetic beads, so that they are more efficiently removed from the fluid suspension, while non-magnetic material is unaffected and can pass smoothly through the separation tube. The permanent magnet array 704 and magnetic field shield 801 are further connected to a servo 802 and gear train 803, so that the entire assembly can be fully rotated and turned off and on as needed.

[0067] Figure 9 shows the same assembly as in Figure 8, but with the magnets in the "off" position. Separation tube 701 still extends alongside permanent magnet array 704. However, in the off position, magnet array 704 and magnetic field shield 801 are rotated using rotation servo 802 and gear train 803, so that magnetic field shield 801 is between separation tube 701 and permanent magnet array 704. As shown in Figures 4 and 5, the HMPSM material used in magnetic field shield 801 prevents magnetic flux from passing through. This significantly reduces the magnetic flux gradient to which separation tube 701 is exposed, making it unable to hold the magnetic field on the magnetic beads. By applying a high-velocity liquid or gas flow to separation tube 701, the magnetic beads and magnetically bound cells can be flushed out.

[0068] 10 is a cross-sectional view of the interface between separation tube 701, magnetic field shield 801, and permanent magnet array 704. When in the "on" position, as shown in FIG. 10A, a large magnetic field acts on separation tube 701, attracting magnetic particles. When in the "off" position, as shown in FIG. 10B, a small magnetic field acts on separation tube 701, allowing previously bound cells and beads to be efficiently removed.

[0069] FIG. 11 is a side view of the interface between cassette 1101 and automated cell culture device 1102. Separation tube 701 is attached to cassette 1101, thereby moving magnetic and non-magnetic fractions to and from different regions within cassette 1101. Conversely, the magnetic separation assembly (consisting of 704 and 801) is contained within automated cell culture device 1102. A control system associated with device 1102 can control servo 802 and gear train 803 to rotate magnet array 704 and magnetic field shield 801, thereby turning the magnetic field "on" or "off" when associated with separation tube 701. When cassette 1101 and device 1102 are coupled, separation tube 701 and separation assembly (collectively 704 and 801) are aligned, allowing for efficient magnetic separation. Additionally, using a peristaltic pump associated with device 1102 , fluid (with or without magnetic beads and cells) can be transferred to or removed from separation tube 701 .

[0070] FIG. 12 is a side view of a separation tube 701 placed between the outer wall of cassette 1101 and the outer wall of cell culture device 1102, illustrating various means for increasing the number and magnitude of magnetic field gradients within the separation tube 701. FIG. 12A shows a paramagnetic / non-magnetic spacer 1201 extending along the entire length of the separation tube 701 on the exterior of cassette 1101. This spacer 1201 acts to compress the tube 701, minimizing the distance between the magnetized elements flowing through the tube 701 and the magnet array 704. Additionally, magnetization of the spacer 1201 (if paramagnetic) from the magnet array 704 creates another magnetic field gradient on the side of the separation tube 701 closest to cassette 1101. Furthermore, by cutting the spacer 1201 (not shown) into smaller pieces along its entire length, higher magnetic field gradients can be generated at the ends of each spacer piece 1201. FIG. 12 shows a paramagnetic wire mesh 1202 within a separation tube 701, demonstrating the generation of high magnetic field gradients around the strands of the mesh 1202 when exposed to a magnetic field generated by a permanent magnet array 704. FIG. 12C shows paramagnetic particles 1203 placed within the separation tube 701, generating localized magnetic field gradients when exposed to a magnetic field from the permanent magnet array 704. FIG. 12D shows paramagnetic rods 1204 extending along the entire length of the separation tube 701 and broken into small pieces (not shown) longitudinally, capable of generating high magnetic field gradients at the ends of each rod 1204 when exposed to the magnet array 704. FIG. 12E shows a series of paramagnetic rods 1205 extending along the entire length of the separation tube 701, generating high magnetic field gradients between the rods 1205 when exposed to a magnetic field from the array 704. Figure 12F shows a paramagnetic scanning bore 1206 extending along the length of separation tube 701, which creates a high magnetic field gradient within the bore when exposed to the magnetic field from array 704. Figure 12G shows a paramagnetic coating 1207 on separation tube 701 that consists of small aberrations, which creates a high magnetic field gradient between them when exposed to the magnetic field from array 704.FIG. 12H shows a paramagnetic filter 1208 placed within separation tube 701, which when exposed to the magnetic field from array 704, generates a high magnetic field gradient within the filter pores.

[0071] Optionally, or in addition, in some aspects of the present invention, various parameters can be adjusted, such as, for example, magnetic field strength, spatial distribution (concentration) of biomolecular targets, and / or other parameters, such as, for example, temperature. For example, the flow rate and / or viscosity and / or elasticity of the biological sample fluid can be adjusted to, for example, separate target cells while at least substantially preventing aggregation of non-target cells. In some embodiments, a fluid can be used to wash away the separated target cells. The flow pattern and speed of the washing fluid can optionally be adjusted to facilitate detachment (i.e., removal or release) of the target cells from the wall of the tube. Such abrupt changes in flow can induce turbulence or impacts, which can assist in the removal or destabilization of target cells on the wall of the tube.

[0072] In certain embodiments, release of separated cells from the separation tube by various methods (e.g., demagnetization, whisking, vibration, enzymes, sonication, and combinations thereof) and combinations of methods may be performed prior to and / or simultaneously with flushing of the cells from the tube. This peripheral processing may be performed to improve the separation and characterization of the desired target population with respect to quality and / or quantity. For example, red blood cell (RBC) lysis may be used to aid in the removal of sticky RBCs and improve purity, thereby facilitating the separation of T cells from the overall PBMC population.

[0073] Enzymes such as those described as DNase may be used to assist in cell release.

[0074] Furthermore, if high quality or high purity depletion (rather than collection of target cells) is the goal, a sufficiently strong magnetic field may be applied that is stronger than that used for collection, at the expense of non-target cells that are attached to the wall and / or clumped.

[0075] The devices, systems and methods may be embodied in kits that include 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, and in their use for practicing one or more of the present inventions.

[0076] Without further explanation, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and use the present invention and practice the claimed methods. The following working examples therefore specifically outline typical aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. The examples are therefore for illustrative purposes only, and are not to be used to limit the scope of the present invention in any way. [Example]

[0077] Example 1 - Separation of beads from a fluid T cells (derived from PBMCs from Lonza) activated and expanded using CD3 / CD28-activated Dynabeads® (Invitrogen) were processed using magnetic separation systems (704 and 801). The goal was to separate T cells from the beads. 31 ml of beads-cell (1.6 x 10 6A fluid suspension of 100 cells / ml (T cell medium, 94% X-Vivo 15, 5% HS, 1% P / S, 10 ng / ml IL-2) was flowed through the separation tube 701 at different flow rates (5, 10, and 20 ml / min). The magnetic separation assemblies (704 and 801) were set to the "on" position. Because it was unlikely that cells (not magnetically bound) were removed from the fluid suspension, the fluid collected from the separation tube was designated the "cell fraction." By manually rotating the magnet assemblies (704 and 801) to the "off" position (without using 802 and 803), the paramagnetic material 801 was positioned between the separation tube 701 and the magnet array 704. Three flushing cycles (each consisting of 4 ml alternating air and fluid rinses at 40 ml / min) were performed in the separation tube 701 to wash away and collect the Dynabeads® and all cells attached to the walls of the tube 701. This was named the "magnetic fraction" as it consisted of all magnetically attracted cells and beads.

[0078] Cell counts from both fractions were used to calculate the percentage of cells successfully separated from Dynabeads® (i.e., the percentage of total cells obtained in the "cell fraction"). At all applied flow rates, the percentage of cells successfully separated from Dynabeads® was approximately 95% (Figure 13A). Both fluid fractions were also counted using a hemocytometer to determine the number of Dynabeads® in each fraction. At all flow rates, the percentage of Dynabeads® removed from cells was at least 95% (Figure 13B).

[0079] Example 2 - Continuous flow of bead-bound cells through a separation tube Streptavidin nanobeads (BioLegend®) were bound to passaged Jurkats on the outside of cassette 1101 and positively selected using magnetic separation assemblies (704 and 801). Cells (10 7 cells / ml) were first treated with a blocking agent (5 μl / 10 7Non-specific binding was blocked by adding a biotin-conjugated primary antibody cocktail (10 μl / 10 μL) to the cells and incubating the cells and agents together at room temperature for 10 minutes. 7 Cells, human CD14+ monocyte isolation (Biolegend®), which bind to target cells, were added, and the mixture was incubated for 15 minutes at 2-8°C. Streptavidin-coated nanobeads (10 μl / 10 7 The nanobeads were then added to the cell suspension for an additional 15 minutes at 2-8°C. Streptavidin on the nanobeads binds to the biotin on the antibody of the target cells, thereby magnetically binding the cells. To ensure a pure population of magnetically bound cells, the bound cells were presorted for 5 minutes using an EasySep™ (STEMCELL Technologies®) magnet, after which the supernatant containing unbound cells was discarded.

[0080] To isolate Jurkats using the magnet array 704, Jurkats (1.5–2 ml / min, 3 ml) were passed through the separation tube 701 at different flow rates (1–5 ml / min) while the magnetic separation assemblies (704 and 801) were simultaneously turned on. Magnetically attracted cells emerged from the fluid suspension and adhered to the wall of the tube 701 closest to the magnet array 704. All cells not removed from the fluid by the turned-on magnet were captured as the "negative fraction" (collected in a volume of 9–12 ml). The magnet assemblies (704 and 801) were manually rotated to the "off" position, and three wash cycles (described in Example 1) were performed to capture the "positive fraction." All of the above steps were performed using isolation buffer (98% DPBS, 2% FBS).

[0081] Using cell counts of both the negative and positive fractions, it is possible to determine the percentage of cells that failed to be captured (all cells in the "negative fraction" relative to the number of cells that flowed through), as well as the release efficiency of positively captured cells (cells that were captured and then successfully entered the "positive fraction"). Increasing the capture flow rate in the separation tube 701 also increased the number of cells that failed to be captured by the system (Figures 14A and 14C). This is due to the shorter time that bound cells were exposed to the magnetic field. However, increasing the capture flow rate to 5 ml / min significantly improved the release rate of bound cells, reaching nearly 100% (Figures 14B and 14D). This is likely due to fewer cells leaving the suspension at the faster flow rate and becoming trapped at various connections in the tubing circuit. Modifying the inner diameter of the separation tube (thick tube - 1 / 8" ID (approximately 0.32 cm), thin tube - 3 / 32" ID (approximately 0.24 cm)) slightly increases capture failure (Figures 14A and 14C), but also slightly improves cell release (Figures 14B and 14D). These results are likely due to increased fluid velocity and shear stress in the walls of the thin tubes.

[0082] Example 3 - Bead-bound cells are passed multiple times through a separation tube Jurkats were magnetically bound and preselected as described in Example 2. As in Example 2, Jurkats (2 x 10 6 The magnetic separation assemblies (704 and 801) were turned on while 1000 cells / ml (3 ml) were passed through the separation tube 701 in isolation buffer at a flow rate of 5 ml / min. Cells not captured by the magnet array 704 were collected as the negative fraction of the first pass in isolation buffer (collected volume 12 ml). Again, cells were flowed through the separation tube 701 at a capture flow rate of 5 ml / min, and cells not yet captured were collected as the negative fraction of the second pass (again collected in 12 ml). After the second pass, the separation assemblies (704 and 801) were rotated to the "off" position, and the cells captured in the separation tube 701 were subjected to three wash cycles (described in Example 1) to obtain the positive fraction. All of the above steps were performed using isolation buffer.

[0083] As in Example 2, cells in each fraction were counted and quantified for capture failure (for both passes as a percentage of the total cells passed through tube 701) and release efficiency. Additional passage of cells past separation tube 701 successfully reduced the percentage of cells that failed to be captured (FIG. 15A) and did not result in a negative reduction in release compared to a single pass (FIG. 15B).

[0084] Example 4 - "Wait Time" in Separation Tubes Jurkats were magnetically bound and preselected as described in Example 2. To effectively increase the period of time that bound cells are exposed to the magnetic field, Jurkats (1.5x10 6 , 3 ml) was held stationary for various periods (1-5 min) while the magnetic assemblies (704 and 801) were rotated to the "on" position. Tube 701 was gently rinsed with 12 ml at 5 ml / min, and the effluent was collected as the negative fraction. After the waiting period, the separation assemblies (704 and 801) were rotated to the "off" position, and three rinse cycles (described in Example 1) were performed to collect the positive fraction. All of the above steps were performed using isolation buffer.

[0085] The collected negative and positive fractions were again counted and quantified for capture failure and release rate. A significant trend of decreased capture failure was observed with increasing waiting time (Figure 16A). This is likely due to the additional time allowed for the magnetic particles to respond to the magnetic field. Furthermore, despite the increased waiting time, the release rate at each waiting time was close to 100% (Figure 16B). This suggests that the improved capture rate is not simply due to increased cell loss at the tubing circuit connections.

[0086] Adding a waiting time to the process increases the likelihood that negative cells will be captured by the process (hereafter referred to as "false positives"). To quantify the rate of false positives, Jurkats that had not been bound to magnetic beads were flowed into the separation tube 701 at various capture flow rates (5 ml / min or 10 ml / min) (3 x 106 Similarly, cells were allowed to wait for various wait times (1 or 3 minutes) and various "negative fraction" washouts were performed. These washouts consisted of a rapid flow of air or fluid while the magnetic assemblies (704 and 801) were left in the "on" position. Because washed cells did not bind, all cells were expected to appear in the "negative fraction," and cells that did not appear were considered "false positives." Despite using a more standard capture sequence (5 ml / min capture flow rate, 3 min wait time), the false positive rate was poor (30%), but was reduced to approximately 20% by increasing the capture flow rate and decreasing the wait time (Figure 17). Further reductions in wait time and an accelerated capture flow rate (condition 5), combined with the addition of a 40 ml / min air washout (condition 7), reduced the false positive rate to less than 3% (Figure 17).

[0087] Example 5 - Separation of mixed cell populations and purification Lysed human peripheral blood mononuclear cells (PBMCs) were bound to Dynabeads®, and CD3+ cells were selected from a heterogeneous cell population by positive selection (ThermoFisher Scientific). 7 cells / ml) was first treated with CD3 antibody (5 μl / 10 7 The cells were incubated with FlowComp™ human CD3 antibody (Invitrogen) for 10 minutes at 2-8°C. The PBMCs were then incubated with FlowComp™ Dynabeads (15 μl / 10 7 The beads were bound to the cells (5-10x10 6A total of 1.5 ml of bead-bound CD3+ cells (1.5 ml / ml) was flowed through a separation tube 701 through a magnet array 704 using various process parameters (flow rate, wait time, and number of passes, as described in Figure 18). All cells not captured by the magnet (CD3-negative cells) were sent to waste and not characterized. The magnet array 704 was rotated to the "off" position, and tube 701 was subjected to three wash cycles to obtain the "positive fraction." This fraction consisted of bead-bound CD3+ cells. All of the above steps were performed in isolation buffer supplemented with 2 mM EDTA.

[0088] Pre- and post-separation fractions (300k cells per well) were fluorescently stained for viability (0.031 μl / 100 μl, Live / Dead™ 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, Invitrogen) and analyzed using flow-assisted cell sorting (FACS) to assess the phenotype of the resulting fractions. The initial flow-through population was found to be heterogeneous but predominantly CD3+ (Figure 18 - black line). For comparison, various process parameters were tested using the magnet array 704. It was observed that modifying the wait time from 0 to 2 minutes had only a limited effect on the purity of the output cells (Figures 18A, B and 18F, G). However, further increasing the wait time to 5 min resulted in a significant decrease in cell purity (Figure 18C, D). Flow rate also had a limited effect on cell purity between 3 ml / min and 10 ml / min (Figure 18G, H). The greatest improvement in cell purity was achieved by adding a second pass to the process, which improved purity to 97.5% CD3+ cells (Figure 18E).

[0089] Example 6 - Magnets of various sizes for magnetic bead capture Magnet array 704 was assembled using permanent magnets (702 and 703) of various sizes (1 / 8 inch and 1 inch long). The magnetic field magnitude was measured at various specific distances from the magnets using a Gaussmeter (AlphaLab Inc.) (Figure 19A). From the magnitude measurements, an estimate of the magnetic field gradient was calculated (Figure 19B). It was found that a shorter magnet length is desirable to produce the strongest gradient. However, the stronger gradient falls off more rapidly than with longer magnets, thereby reducing the effective range of the magnets. This demonstrates the feasibility of using different magnet sizes in magnet array 704 to achieve different separation objectives, such as short-range separation of weakly coupled targets or long-range separation of strongly coupled targets.

[0090] To examine the effect of the size of magnets 702 and 703 on cell separation, Jurkat cells were bound to BioLegend® nanobeads as described in Example 2. The bead-bound cells (2.5x10 6 Cells / ml (1.5 ml) were flowed past the magnet array 704 in the separation tube 701 at a flow rate of 5 ml / min with a wait time of 5 minutes. Unbound cells were washed out of the tube 701 with 12 ml of isolation buffer (98% PBS, 2% FBS, 2 mM EDTA) at 5 ml / min. The magnetic field generated by the array 704 was removed from the tube 701, and three wash cycles (described in Example 1) were performed to remove the positive fraction from the tube. The results were compared to those obtained from a typical magnet assembly (704 and 801). It was observed that the 1 / 8-inch long magnets 702 and 703 reduced capture failure compared to the 1-inch long magnets 702 and 703 (Figure 19C). All of the above steps were performed using isolation buffer.

[0091] Example 7 - Add-ons to enhance capture of weakly binding biological targets One way to alter or improve the ease of capture of weakly binding targets / small beads is to decrease the distance between the magnet array 704 and the separation tube 701, increasing the magnitude and gradient of the average magnetic field experienced within the tube 701, as described in Example 6. To accomplish this, a 3 / 32 inch thick spacer 1201 was placed between the separation tube 701 and the cassette 1101. Jurkat cells (2.5-5x10) bound to BioLegend® nanobeads as described in Example 2 were cultured. 6 A volume of 1.5 ml of cells / ml was flowed into the separation tube 701 at a flow rate of 5 ml / min, followed by a waiting period of 3 or 5 minutes. Untrapped cells were then washed away (11–13 ml) at 5 ml / min, and three wash cycles (described in Example 1) were performed to collect the trapped cell fraction. Adding the spacer 1201 to this setup resulted in an improvement in the capture failure rate from a waiting period of 3 minutes to the level achieved with a waiting period of 5 minutes ( FIG. 20A ). All of the above steps were performed using isolation buffer.

[0092] Another way to reduce the distance between the biological target and the magnetic field is to include magnetized 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). The magnetic field gradient is inversely proportional to the size of the objects (1202-1208), and the effective range is proportional to the size of the objects (1202-1208). To demonstrate this, a paramagnetic mesh 1202 was added to the separation tube 701. Jurkat cells (1.25x10 7 cells / ml) and MACS® MicroBeads (20 μl / 10 7 The cells were allowed to bind to CD3 microbeads (human, Miltenyi Biotec) by incubating the cells with the pre-bound beads for 15 minutes at 4-8°C. After binding, the cells were poured into a separation tube 701 containing a paramagnetic mesh 1202 at 5 ml / min (9 x 10 6The cells were then washed out of the tube 701 at 5 ml / min with 12 ml of isolation buffer. The magnet array 704 was rotated to the off position, and three wash cycles (described in Example 1) were performed to remove positively captured cells from the tube 701. To account for nonspecific capture by the mesh 1202, the results were normalized to those obtained from a no-bead control (Jurkat cells not bound to microbeads), in which all capture was due to physical constraint from the mesh 1202. The use of the paramagnetic mesh resulted in a relative increase in cell capture of 10% with beads compared to without beads (Figure 20B). The above steps were performed using isolation buffer supplemented with 2 mM EDTA.

[0093] Recirculation and magnetic field methods As described herein, in exemplary embodiments, methods for magnetically separating targets within a biological sample suitably utilize recirculation of the sample through multiple (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) magnetic separation cycles, which dramatically increases the yield of the desired target.

[0094] The methods described herein are suitably implemented in an automated cell culture system, and in some embodiments, can be performed in a cassette within the automated cell culture system. Figure 6 shows an example cassette, and Figure 21 shows the placement of this cassette in a flow diagram of the automated cell culture system. As shown in Figure 21, the automated cell culture system suitably includes a cell growth chamber 2102, several fluid paths 2104, as well as a magnetic field source 2106, and reagent input locations 2108.

[0095] 21 shows an example embodiment in which a recirculation path / line is illustrated, in which a biological sample containing a target biological population is circulated through the recirculation path / line (thick line) multiple times, and during each pass, the sample is suitably exposed to a magnetic field gradient provided by a magnetic field source 2106 (e.g., a permanent magnet or electromagnet).

[0096] The methods described herein utilize recirculation of a biological sample to remove target biological populations and may rely on positive or negative selection methods or a combination thereof.

[0097] Positive selection method In embodiments utilizing positive selection methods for isolating and capturing a target biological population, provided herein are methods for collecting a target biological population from a biological sample in an automated cell culture system, the method including binding the target biological population to magnetic particles, circulating the biological sample through one or more fluid paths of the automated cell culture system, exposing the target biological population bound to the magnetic particles to a magnetic field gradient, repeating the circulating and exposing steps one or more times, and collecting the target biological population bound to the magnetic particles. In additional embodiments, the method can further include removing the target biological population from the bound magnetic particles.

[0098] Such positive selection methods rely on the direct removal of a target biological population from a biological sample using a magnetic field to positively select the desired target population from the sample.

[0099] As described herein, the target biological population is suitably bound to magnetic particles. As described herein, a method for binding magnetic particles to a target biological population is suitably described, the method suitably using antibodies, proteins, or nucleic acids. As described herein, the target biological population suitably includes 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 a population of T cells produced containing a desired receptor. The biological sample from which the target population is derived may also include other unwanted cells, viruses, bacteria, proteins, DNA, RNA, etc. (i.e., non-target populations).

[0100] Additional 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 zone (e.g., growth chamber), and transferring the non-target biological population to a waste chamber and ultimately removal from the automated cell culture system.

[0101] A biological sample is circulated through one or more fluid paths of an automated cell culture system, such as that described in Figure 21. In some embodiments, the biological sample may begin as a cell culture sample at input location 2108 of the system, or in some embodiments, may begin as a cell culture sample in cell growth chamber 2102, and then be transferred to an area where magnetic particles containing antibodies or other agents are provided, and the magnetic particles bind to the desired target population (e.g., cells). This binding to magnetic particles may occur in growth chamber 2102 or any input location 2108 within the system.

[0102] The biological sample then passes through a section of the automated cell culture system that includes a source of magnetic field 2106, such that the target biological population bound to the magnetic particles is exposed to a magnetic field gradient. As a result of this exposure, the target biological population (e.g., a desired cell population) becomes bound to the source of the magnetic field (e.g., collects at the side of the separation tube 701 or other similar device), i.e., adjacent to the magnetic field source that generates the magnetic field. This separation draws the target biological population (or at least a portion of the target biological population) from the sample. The target biological population bound to the magnetic particles is then suitably collected. An exemplary method for collecting the target biological population includes removing and washing the target biological population after exposure to the magnetic field. In several embodiments, the target biological population is collected by circulating a gas phase fluid through the system, followed by one or more liquid phase fluids. Suitably, the gas phase fluid includes one or more of air, nitrogen, oxygen, and carbon dioxide. In further embodiments, the liquid phase includes one or more of water, buffered saline, culture medium, animal serum, a chelating agent, and an enzyme.

[0103] As described herein, it has been found 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 a suitable embodiment, the steps of the positive selection method in which a biological sample is circulated through one or more fluid paths of an automated cell culture system and the 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 or more times). As shown in Figure 21, this circulation suitably occurs through a recirculation cycle in which the sample passes adjacent to a source of a magnetic field 2106 that binds to the target population, after which the sample recirculates and again passes adjacent to the source of the magnetic field to remove further target populations that may not have been captured on the previous pass, before ultimately collecting the final target sample. This cycle may be repeated as many times as desired until the target population objective is achieved, or until it is determined through statistical or other means that additional cycles do not dramatically increase the yield and / or purity of the target population. After collection of the target biological population, the method suitably includes removing the target biological population from the bound magnetic particles, so that the target population can be further processed or utilized, for example, by various means described herein.

[0104] In additional embodiments, the recirculation methods described herein may include rinsing the target population bound to a magnetic source (e.g., a separation tube used in a magnetic-based method) and then transferring the washed target population to an expansion chamber for further processing and / or expansion. These capture, rinse, and transfer elements may then be performed on another biological sample containing the magnetically bound target population.

[0105] In some 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 utilized for the permanent magnets are described herein and suitably include magnetite, neodymium, samarium-cobalt, and / or alnico. As described herein, in some embodiments, the permanent magnets are suitably configured in a linear array, such as magnet array 704 of FIG. 7B.

[0106] In additional embodiments, the magnetic field gradient is provided by one or more electromagnets as described herein.

[0107] In a further embodiment, provided herein is a positive selection method 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 fluid 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 fluid 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 fluid paths of the automated cell culture system, repeating the steps of circulating the biological sample through circulating the target biological population one or more times, and collecting the target biological population bound to the magnetic particles. In additional embodiments, the positive selection method may further comprise removing the target biological population from the bound magnetic particles.

[0108] As described herein, the positive selection method utilizes a design in which a biological sample is passed through, for example, a separation tube 701 (e.g., as shown in FIG. 7A). Within the biological sample, target biological populations are bound to magnetic particles. The method suitably includes circulating the biological sample through one or more fluid paths preceding or including the separation tube 701 and a magnetic source. The target biological populations bound to the magnetic particles are suitably exposed to (and suitably recirculated through) a magnetic field gradient that captures the target biological populations bound to the magnetic particles. For example, as shown in FIG. 8, the biological sample is passed through the separation tube 701, and the bound magnetic particles and target sample are captured on the side of the tube by the magnetic field (see also FIGS. 24D-24E).

[0109] Unbound components in the biological sample (ie, unwanted cells, proteins, DNA, or other structures) are then circulated through one or more fluid paths and removed from separation tube 701 .

[0110] A magnetic field shield / barrier is then suitably inserted between the target biological population bound to the magnetic particles and the magnetic field, and the target biological population bound to the magnetic particles is released from the magnet. The target biological population bound to the magnetic particles is then circulated through one or more fluid paths of the automated cell culture system and collected, for example, in a separation area of ​​the automated cell manipulation system. Various methods for collecting the target biological population are described herein.

[0111] Suitably, the steps of circulating the biological sample, exposing the sample (and the target biological population bound to the magnetic particles), inserting a magnetic field shield / barrier between the target population and the magnetic field, and collecting the target biological population are repeated one or more times (suitably two or more, three or more, four or more, five or more, etc.). Each time this cycle is performed, the yield of the target biological product increases. As described herein, the target biological population is then removed from the appropriately bound magnetic particles.

[0112] As described herein, the target biological population suitably comprises one or more of cells, viruses, bacteria, proteins, DNA and / or RNA, and in some embodiments comprises T cells. Methods and compounds for binding magnetic particles to the target biological population are described herein, suitably comprising the use of antibodies, proteins or nucleic acids.

[0113] Exemplary magnetic fields are described herein, and are suitably generated by permanent magnets or electromagnets. Materials used in making permanent magnets are described herein, and include, for example, magnetite, neodymium, samarium-cobalt, and / or alnico. In some embodiments, the permanent magnets are configured in a linear array. In embodiments utilizing electromagnets, the insertion of a magnetic field shield / barrier can be replaced by turning off the electromagnet, e.g., by simply removing the current from the electromagnet, stopping the magnetic field.

[0114] As described throughout this specification, in some embodiments, the magnetic shield / barrier suitably comprises a highly magnetically permeable and magnetically saturable material. As described herein, in some embodiments, the magnetic shield / barrier rotates to insert the magnetic shield / barrier between the target biological population bound to the magnetic particles and the magnetic field. Such embodiments are illustrated in Figures 8, 9, and 10A-10B and described in detail herein.

[0115] Negative selection methods In embodiments utilizing negative selection methods for a target biological population, provided herein are methods for collecting a target biological population from a biological sample in an automated cell culture system, the method including binding a non-target biological population to magnetic particles, circulating the biological sample through one or more fluid 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 circulating and exposing steps one or more times, and collecting the target biological population. The method may further include collecting the non-target biological population for appropriate removal as waste.

[0116] As used herein, negative selection methods utilize the binding of magnetic particles to a "non-target biological sample," which refers to one or more cells, proteins, DNA, or RNA that are not included in the "target biological population" and therefore are sought to be removed from the biological sample to leave behind the target biological population. In such negative selection methods, magnetic separation is used to separate the non-target biological population and collect the remaining target biological population from the sample.

[0117] As described herein, the non-target biological population is suitably bound to magnetic particles. Methods for binding magnetic particles to the non-target biological population are described herein, suitably using antibodies, proteins, or nucleic acids. 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 generated to contain a desired receptor, while the non-target biological population comprises any other cells, proteins, etc. in the sample that is removed to leave behind the target population. The biological sample from which the target population is removed may also comprise other unwanted cells, viruses, bacteria, proteins, DNA, RNA, etc.

[0118] A biological sample is circulated through one or more fluid paths of an automated cell culture system, such as that described in Figure 21. In embodiments, the biological sample may begin as a cell culture sample at input location 2108, or in cell growth chamber 2102, or elsewhere in the system, and is then transferred to an area where magnetic particles containing antibodies or other agents are provided, which bind to unwanted, non-target populations (e.g., unwanted cells, proteins, DNA, etc.). This binding to magnetic particles may occur within the growth chamber, at input location 2108, or in another chamber in the system.

[0119] The biological sample then passes through a section of the automated cell culture system that includes a source of magnetic field 2106, so that non-target biological populations bound to the magnetic particles are exposed to the magnetic field gradient (the target biological population is also exposed, but does not react to the magnetic field). As a result of this exposure, the non-target biological populations (e.g., populations of undesired cells, proteins, etc.) become bound to the source of the magnetic field (e.g., collect on the side of the separation tube 701 or other similar device), i.e., adjacent to the magnetic field. This separation draws the non-target biological populations (or at least a portion of the non-target biological populations) from the sample. The target biological populations not bound to the magnetic particles are then appropriately collected. An exemplary method for collecting the target biological populations includes filtering, removing, and washing the target biological populations from the sample after exposure to the magnetic field (thus removing the non-target biological populations).

[0120] In some embodiments, the target biological population is collected by circulating a gas phase fluid followed by one or more liquid phase fluids through the system. Suitably, the gas phase fluid comprises one or more of air, nitrogen, oxygen, and carbon dioxide. In further embodiments, the liquid phase comprises one or more of water, buffered saline, culture medium, animal serum, chelating agents, and enzymes.

[0121] As described herein, it has been found 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 a suitable embodiment, the steps of the negative selection method in which a biological sample is circulated through one or more fluid paths of an automated cell culture system, non-target biological populations bound to 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 or more times). As shown in Figure 21, this circulation suitably occurs through a recirculation cycle in which the sample is passed adjacent to a source of a magnetic field 2106 that binds the non-target populations to collect the target population, after which the sample is recirculated and passed adjacent to the source of the magnetic field again to further remove non-target populations that could not be captured on the previous pass. This cycle can be repeated as many times as desired until the target population goal is achieved, or until it is determined through statistical or other means that additional cycles do not dramatically increase the yield and / or purity of the target population. After collection of the target biological population, the method may further include processing, filtering, or utilizing the target biological population, as appropriate, by various means as described herein.

[0122] In some 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 utilized for the permanent magnets are described herein and suitably include magnetite, neodymium, samarium-cobalt, and / or alnico. As described herein, in some embodiments, the permanent magnets are suitably configured in a linear array, such as magnet array 704 of FIG. 7B.

[0123] In additional embodiments, the magnetic field gradient is provided by one or more electromagnets as described herein.

[0124] In a further embodiment, provided herein is a negative selection method comprising collecting a target biological population from a biological sample in an automated cell culture system, the method comprising binding non-target biological populations to magnetic particles, circulating the biological sample through one or more fluid paths of the automated cell culture system, exposing the non-target biological populations bound to the magnetic particles to a magnetic field gradient to capture the non-target biological populations bound to the magnetic particles, circulating the target biological population of the biological sample through one or more fluid paths of the automated cell culture system, inserting a magnetic field shield / barrier between the non-target biological populations bound to the magnetic particles and the magnetic field to release the non-target biological populations bound to the magnetic particles, circulating the non-target biological populations bound to the magnetic particles through one or more fluid paths of the automated cell culture system, repeating the steps of collecting the biological sample through the target biological population one or more times, and collecting the target biological population.

[0125] As described herein, the negative selection method utilizes a collection of target biological populations; a design in which the biological sample is passed through, for example, a separation tube 701 (e.g., as shown in FIG. 7A). Within the biological sample, non-target biological populations are bound to magnetic particles. The method suitably includes circulating the biological sample through one or more fluid paths prior to or including the separation tube 701. The non-target biological populations bound to the magnetic particles are suitably exposed to a magnetic field gradient that captures the non-target biological populations bound to the magnetic particles. For example, as shown in FIG. 8, the biological sample is passed through the separation tube 701, and the bound magnetic particles and non-target sample are captured on the side of the tube by the magnetic field (see also FIGS. 24D-24E).

[0126] The unbound components of the biological sample (i.e., the target biological population containing desired cells, including T cells, proteins, DNA, or other structures) are then circulated through one or more fluid pathways and collected by filtration or other mechanism as appropriate, for example, in a separation area of ​​an automated cell manipulation system.

[0127] A magnetic field shield / barrier is then suitably inserted between the non-target biological population bound to the magnetic particles and the magnetic field, and the non-target biological population bound to the magnetic particles is released from the magnet, after which the non-target biological population bound to the magnetic particles is circulated through one or more fluid paths of the automated cell culture system.

[0128] Suitably, the steps of cycling the biological sample, exposing the sample (and non-target biological populations bound to magnetic particles), inserting a magnetic field shield / barrier between the non-target populations and the magnetic field, and collecting the target biological population are repeated one or more times (suitably two or more, three or more, four or more, five or more, etc.), with each cycle increasing the yield and / or purity of the target biological product, thereby allowing for the removal of increasingly more of the non-target biological populations and the separation and collection of more of the desired target biological product.

[0129] Additional 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 zone (e.g., growth chamber), and transferring the non-target biological population to a waste chamber and ultimately removal from the automated cell culture system.

[0130] As described herein, suitably the non-target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA and / or RNA, and in embodiments the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA and / or RNA, suitably comprising T cells. Methods and compounds for binding magnetic particles to non-target biological populations are described herein, suitably involving the use of antibodies, proteins or nucleic acids.

[0131] Exemplary magnetic fields are described herein, and are suitably generated by permanent magnets or electromagnets. Materials used in making permanent magnets are described herein, and include, for example, magnetite, neodymium, samarium-cobalt, and / or alnico. In some embodiments, the permanent magnets are configured in a linear array. In embodiments utilizing electromagnets, the insertion of a magnetic field shield / barrier can be replaced by turning off the electromagnet, e.g., by simply removing the current from the electromagnet, stopping the magnetic field.

[0132] As described throughout this specification, in some embodiments, the magnetic shield / barrier suitably comprises a highly magnetically permeable and magnetically saturable material. As described herein, in some embodiments, the magnetic shield / barrier rotates to insert the magnetic shield / barrier between the magnetic field and the non-target biological population bound to the magnetic particles. Such embodiments are illustrated in Figures 8, 9, and 10A-10B and described in detail herein.

[0133] In a further embodiment, provided herein is a method for washing and recovering magnetic particles in an automated cell culture system.

[0134] Suitably, the method comprises the steps of: a. circulating magnetic particles through one or more fluid 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 gaseous fluid phase and then a liquid fluid phase; d. circulating the magnetic particles through one or more fluid paths of the automated cell culture system, and repeating steps e.g., c., d ...

[0135] As described herein, in several embodiments, the magnetic particles are bound to the target biological population, suitably via an antibody, protein or nucleic acid.

[0136] In additional embodiments, the magnetic particles are bound to a non-target biological population, including binding via an antibody, protein, or nucleic acid.

[0137] Exemplary non-target and target biological populations are described herein, suitably the target population is any one or more of cells, viruses, bacteria, proteins, DNA and RNA, including T cells.

[0138] In some embodiments, the magnetic field gradient is provided by one or more permanent magnets, including permanent magnets comprising magnetite, neodymium, samarium-cobalt, or alnico. The permanent magnets may be configured in a linear array, as described herein. In additional embodiments, the magnetic field gradient is provided by an electromagnet.

[0139] Suitably, as described herein, a magnetic field shield / barrier is interposed between the magnetic particles and the magnetic field, and interrupting the magnetic field allows for collection of the magnetic particles and releases magnetic particles bound to the magnetic source. Exemplary materials for use in the magnetic field shield / barrier include materials with high magnetic permeability and magnetic saturation. In embodiments where electromagnets are utilized, the current may be removed from one or more electromagnets, simply interrupting the source of the electromagnet, allowing for collection of the magnetic particles.

[0140] Examples of gas phase fluids that may be utilized in the method include one or more of air, nitrogen, oxygen, and carbon dioxide. Examples of liquid phase fluids that may be used include one or more of water, buffered saline, culture medium, animal serum, chelating agents, and enzymes.

[0141] Bead recovery method Data on negative and positive selection methods, including multiple rounds of magnetic separation, and recovery of magnetic particles 22A-22D show the binding of magnetic particles to both target cells (purified cells) and waste cells (non-target population). As shown, the "improved process" utilizing optimization of reagent amounts shows high binding between magnetic particles and waste cells (negatively selected non-target population) and low "false negatives."

[0142] Figures 23A-23B show the release of magnetic particles from cells. As shown, in Figure 23A, the binding mechanism utilized in the automated cell culture system (COCOON) shows similar bead-cell release as the control, demonstrating the ability to recover cells (or other target biological populations) using positive selection methods. Figure 23B shows the percentage of beads remaining within the target cells after release and bead removal using the automated cell culture system.

[0143] To determine the benefit of performing multiple magnetic separations (i.e., recirculating the sample through the automated cell culture system and exposing it to the magnetic field two, three, four, or five times), experiments were performed assessing increasing magnet exposure time and decreasing flow rate. As shown in Figure 24A, increasing magnet exposure time (from left to right) increased the percentage of bound cells captured from approximately 65% ​​to at least 90%, similar to the control. Similarly, decreasing the flow rate (from right to left) in Figure 24B increased bound cells captured from approximately 60% to approximately 85%, similar to the control.

[0144] Table 1 below shows that multiple passes using the magnetic separation method described herein resulted in an increase in both the number of bound cells retained and the overall cell yield. [Table 1]

[0145] FIG. 24C shows the effect of the number of fluid flush cycles that expel the captured biological sample from the separation line, showing a high recovery after the first two flushes.

[0146] Figures 24D-24E show the capture of magnetic particle-bound cells in a separation tube using a magnetic field (top) and their release (bottom) after the magnetic field is turned off and multiple cycles of fluid flushing (as shown in Figure 24C), demonstrating the effectiveness of the methods described herein.

[0147] Figures 25A-25F show high levels of purification of the target biological population (cells) using positive and negative selection methods, demonstrating similar recovery between the automated cell culture system described herein (COCOON) and the control for both positive and negative selection methods. Figures 25G-25I show cell purification using negative selection, demonstrating the improvement in purity achieved by performing multiple passes using a recirculation line. The CD3+CD14+ population represents unwanted bead-bound monocyte-T cell aggregates. Figures 25J-25L show population purification using the negative selection process. Multiple passes successfully removed unwanted bead-bound cells (highlighted by white circles).

[0148] Figures 26A-26C show the benefits of magnetic separation in the automated cell culture system described herein. Figure 26A shows a significant reduction in process duration, Figure 26B shows reduced cell loss, and Figure 26C shows reduced volume loss compared to bag- and culture tube-based controls. Figure 26D shows population purification using a positive selection process. Multiple passes through the recirculation line improve cell yield compared to a single pass.

[0149] 27A-27B show the recovery of magnetic particles after washing in the automated cell culture system described herein. Compared to a typical manual process, FIG. 27A shows the effect of successive rinsing of the separation tube 701 on absolute recovery, and FIG. 27B shows the effect of successive rinsing of the separation tube 701 on cumulative particle recovery.

[0150] The description of various embodiments and / or examples of the present invention is presented for illustrative purposes and is not intended to be exhaustive or limited to the disclosed embodiments and / or examples. Many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the described embodiments. The terminology used herein has been selected to best explain the spirit of the embodiments, practical applications, or technical improvements over existing technology in the industry, or to enable others other than those skilled in the art to understand the embodiments disclosed herein.

[0151] A preferred embodiment of the present invention is as follows. [1] 1. A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding the target biological population to magnetic particles; b. circulating the biological sample through one or more fluid paths of the automated cell culture system; c. exposing the target biological population bound to the magnetic particles to a magnetic field gradient; d. repeating steps b-c one or more times; and e. collecting said target biological population bound to said 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 [1] or [2], wherein the target biological population comprises T cells. [4] The method according to any one of [1] to [3], wherein the magnetic particles are bound to the target biological population via an antibody, a protein, or a nucleic acid. [5] The method according to any one of [1] to [4], wherein the magnetic field gradient is provided by one or more permanent magnets. [6] The method of claim 5, wherein the permanent magnet comprises magnetite, neodymium, samarium-cobalt, or alnico. [7] The method according to any one of [5] to [6], wherein the permanent magnets are configured in a linear array. [8] The method according to any one of [1] to [4], wherein the magnetic field gradient is provided by one or more electromagnets. [9] The method according to any one of [1] to [8], wherein steps b and c are repeated at least twice.

[10] The method according to any one of [1] to [9], wherein the target biological population bound to the magnetic particles is collected by circulating a gas phase fluid and then a liquid phase fluid one or more times.

[11] The method according to claim 10, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen, and carbon dioxide.

[12] The method of claim 10, wherein the liquid phase comprises one or more of water, buffered saline, culture medium, animal serum, a chelating agent, and an enzyme.

[13] The method according to any one of [1] to

[12] , further comprising removing the target biological population from the bound magnetic particles.

[14] 1. A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding the target biological population to magnetic particles; b. circulating the biological sample through one or more fluid paths 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 fluid paths of the automated cell culture system; e. interposing a magnetic field shield / barrier between the target biological population bound to the magnetic particles and a 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 fluid paths of the automated cell culture system; g. repeating steps b through f one or more times; and h. collecting said target biological population bound to said magnetic particles.

[15] The method of claim 14, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA.

[16] The method of

[14] or

[15] , wherein the target biological population comprises T cells.

[17] The method according to any one of

[14] to

[16] , wherein the magnetic particles are bound to the target biological population via an antibody, a protein, or a nucleic acid.

[18] The method according to any one of

[14] to

[17] , wherein the magnetic field gradient is provided by one or more permanent magnets.

[19] 19. The method of claim 18, wherein the permanent magnet comprises magnetite, neodymium, samarium-cobalt, or alnico.

[20] The method according to any one of

[17] to

[19] , wherein the permanent magnets are configured in a linear array. 〔twenty one〕 The method according to any one of

[14] to

[16] , wherein the magnetic field gradient is provided by one or more electromagnets, and optionally the inserting step of step e is replaced by a step of removing current from the one or more electromagnets. 〔twenty two〕 The method according to any one of

[14] to

[21] , wherein steps b to f are repeated at least twice. 〔twenty three〕 The method according to any one of

[14] to

[22] , wherein the magnetic field shield / barrier comprises a material with high magnetic permeability and magnetic saturability. 〔twenty four〕 The method of any of

[14] to

[23] , wherein the magnetic field shield / barrier rotates to insert the magnetic field shield / barrier between the target biological population bound to the magnetic particles and the magnetic field. 〔twenty five〕 The method according to any one of

[14] to

[24] , wherein the target biological population is collected by circulating a gas phase fluid and then a liquid phase fluid one or more times.

[26] 26. The method of claim 25, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen, and carbon dioxide.

[27] The method of claim 25, wherein the liquid phase comprises one or more of water, buffered saline, culture medium, animal serum, a chelating agent, and an enzyme.

[28] The method according to any one of

[14] to

[27] , further comprising removing the target biological population from the bound magnetic particles.

[29] 1. A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. Binding a non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluid paths 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 b-c one or more times; and e. harvesting said target biological population.

[30] The method of claim 29, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA.

[31] The method of

[29] or

[30] , wherein the target biological population comprises T cells.

[32] The method according to any one of

[29] to

[31] , wherein the non-target biological population is collected.

[33] The method according to any one of

[29] to

[32] , wherein the magnetic particles are bound to the non-target biological population via an antibody, a protein, or a nucleic acid.

[34] The method according to any one of

[29] to

[33] , wherein the magnetic field gradient is provided by one or more permanent magnets.

[35] The method of claim 34, wherein the permanent magnet comprises magnetite, neodymium, samarium-cobalt, or alnico.

[36] The method according to any one of

[34] to

[35] , wherein the permanent magnets are configured in a linear array.

[37] The method according to any one of

[29] to

[33] , wherein the magnetic field gradient is provided by one or more electromagnets.

[38] The method according to any one of

[29] to

[37] , wherein steps b and c are repeated at least twice.

[39] The method according to any one of

[29] to

[38] , wherein the target biological population bound to the magnetic particles is collected by circulating a gas phase fluid followed by a liquid phase fluid one or more times.

[40] 39. The method of claim 39, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen, and carbon dioxide.

[41] The method of claim 39, wherein the liquid phase comprises one or more of water, buffered saline, culture medium, animal serum, a chelating agent, and an enzyme.

[42] 1. A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. Binding a non-target biological population to magnetic particles; b. circulating the biological sample through one or more fluid paths 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 said target of said biological sample through one or more fluid paths of said automated cell culture system; e. interposing a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and a 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 fluid paths of the automated cell culture system; and g. repeating steps b through f one or more times; and h. harvesting said target biological population.

[43] The method of claim 42, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA.

[44] The method of

[42] or

[43] , wherein the target biological population comprises T cells.

[45] The method according to any one of

[42] to

[44] , wherein the non-target biological population is collected.

[46] The method according to any one of

[42] to

[45] , wherein the magnetic particles are bound to the target biological population via an antibody, a protein, or a nucleic acid.

[47] The method according to any one of

[42] to

[46] , wherein the magnetic field gradient is provided by one or more permanent magnets.

[48] 48. The method of claim 47, wherein the permanent magnet comprises magnetite, neodymium, samarium-cobalt, or alnico.

[49] The method according to any one of

[47] to

[48] , wherein the permanent magnets are configured in a linear array.

[50] The method according to any one of

[42] to

[46] , wherein the magnetic field gradient is provided by one or more electromagnets, and optionally the step of inserting e is replaced by a step of removing current from the one or more electromagnets.

[51] The method according to any one of

[42] to

[50] , wherein steps b to f are repeated at least twice.

[52] The method according to any one of

[42] to

[51] , wherein the magnetic field shield / barrier comprises a material with high magnetic permeability and magnetic saturability.

[53] A method according to any one of

[42] to

[51] , wherein 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.

[54] The method according to any one of

[42] to

[53] , wherein the target biological population is collected by circulating a gas phase fluid followed by a liquid phase fluid one or more times.

[55] 54. The method of claim 54, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen, and carbon dioxide.

[56] 55. The method of claim 54, wherein the liquid phase comprises one or more of water, buffered saline, culture medium, animal serum, a chelating agent, and an enzyme.

[57] 1. A method for washing and recovering magnetic particles in an automated cell culture system, comprising: a. circulating the magnetic particles through one or more fluid paths of the automated cell culture system; b. exposing the magnetic particles to a magnetic field gradient to capture the magnetic particles; c. collecting said magnetic particles by applying a gaseous fluid phase followed by a liquid fluid phase; d. circulating the magnetic particles through one or more fluid paths of the automated cell culture system; and e. Repeating steps c-d one or more times.

[58] 58. The method of claim 57, wherein the magnetic particles are bound to a target biological population.

[59] 59. The method of claim 58, wherein the magnetic particles are bound to the target biological population via an antibody, protein, or nucleic acid.

[60] 58. The method of claim 57, wherein the magnetic particles are bound to a non-target biological population.

[61] 61. The method of claim 60, wherein the magnetic particles are bound to the non-target biological population via an antibody, protein, or nucleic acid.

[62] The method according to any one of

[58] to

[59] , wherein the target biological population is one or more of cells, viruses, bacteria, proteins, DNA, and RNA.

[63] The method of

[62] , wherein the target biological population is T cells.

[64] The method according to any one of

[57] to

[63] , wherein the magnetic field gradient is provided by one or more permanent magnets.

[65] 64. The method of claim 64, wherein the permanent magnet comprises magnetite, neodymium, samarium-cobalt, or alnico.

[66] The method according to any one of

[64] to

[65] , wherein the permanent magnets are configured in a linear array.

[67] The method according to any one of

[57] to

[67] , wherein a magnetic field shield / barrier is inserted between the magnetic particles and the magnetic field to enable collection of the magnetic particles.

[68] 68. The method of claim 67, wherein the magnetic field shield / barrier comprises a material with high magnetic permeability and magnetic saturability.

[69] The method of claim 57, wherein the magnetic field gradient is provided by one or more electromagnets.

[70] 69. The method of claim 69, wherein the current is removed from the one or more electromagnets, allowing the magnetic particles to be collected.

[71] The method according to any one of

[57] to

[70] , wherein the gas phase fluid includes one or more of air, nitrogen, oxygen, and carbon dioxide.

[72] The method according to any one of

[57] to

[71] , wherein the liquid phase fluid comprises one or more of water, buffered saline, culture medium, animal serum, a chelating agent, and an enzyme.

[73] The method according to any one of

[57] to

[72] , wherein steps c to d are repeated at least twice.

[74] 1. A system for magnetic separation and collection of a target biological population from a biological sample, comprising: a. Magnetic field source; b. a separation tube for flowing said biological sample, aligned with said magnetic field source; c. a magnetic field shield / barrier configured to be disposed between the magnetic field source and the separation tube; and d. A device for inserting the magnetic field shield / barrier between the magnetic field source and the separation tube.

[75] The system described in

[74] , wherein the system is part of an automated cell culture system.

[76] The system of

[74] or

[75] , wherein the magnetic field shield / barrier is a material with high magnetic permeability and magnetic saturation.

[77] The system of claim 76, wherein the material with high magnetic permeability and magnetic saturation is selected from the group consisting of iron, iron alloys, ferritic steel, and Hiperco-50.

[78] A system described in any one of

[74] to

[77] , wherein the magnetic field source includes one or more permanent magnets.

[79] The system of claim 78, wherein the permanent magnet comprises neodymium, samarium-cobalt, or alnico.

[80] A system described in any of

[74] to

[78] , wherein the magnetic field source includes a plurality of permanent magnets arranged in a linear array.

[81] The system of claim 80, wherein the plurality of permanent magnets in the linear array are arranged in an opposite polarity direction perpendicular to the axis of the linear array.

[82] A system described in any of

[74] to

[80] , wherein the device for insertion is a device for rotating the magnetic shield / barrier between the magnetic field source and the separation tube.

[83] The system of claim 82, wherein the device for rotation is a software-controlled electro-mechanical drive assembly.

[84] 1. A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. binding the target biological population to magnetic particles; b. 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; c. Removing the unbound population of said biological sample; d. interposing a magnetic field shield / barrier between the target biological population bound to the magnetic particles and a magnetic field to release the target biological population bound to the magnetic particles; and e. collecting said target biological population bound to said magnetic particles.

[85] The method of claim 84, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA.

[86] The method of

[84] or

[85] , wherein the target biological population comprises T cells.

[87] The method according to any one of

[84] to

[86] , wherein the magnetic particles are bound to the target biological population via an antibody, a protein, or a nucleic acid.

[88] The method according to any one of

[84] to

[87] , wherein the magnetic field gradient is provided by one or more permanent magnets.

[89] 88. The method of claim 88, wherein the permanent magnet comprises magnetite, neodymium, samarium-cobalt, or alnico.

[90] The method according to any one of

[84] to

[89] , wherein the permanent magnets are configured in a linear array.

[91] A method according to any of [84-90], wherein the magnetic field gradient is provided by one or more electromagnets, and optionally the inserting step of step d is replaced by a step of removing current from the one or more electromagnets.

[92] The method according to any one of

[84] to

[91] , wherein the magnetic field shield / barrier comprises a material with high magnetic permeability and magnetic saturability.

[93] A method according to any one of

[84] to

[92] , wherein 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.

[94] The method according to any one of

[84] to

[93] , wherein the target biological population is collected by circulating a gas phase fluid followed by a liquid phase fluid one or more times.

[95] The method of claim 94, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen, and carbon dioxide.

[96] 95. The method of claim 94, wherein the liquid phase comprises one or more of water, buffered saline, culture medium, animal serum, a chelating agent, and an enzyme.

[97] The method of any one of

[84] to

[96] , further comprising removing the target biological population from the bound magnetic particles.

[98] 1. A method for collecting a target biological population from a biological sample in an automated cell culture system, comprising: a. Binding a non-target biological population to magnetic particles; b. 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; c. collecting the target biological population; and d. inserting a magnetic field shield / barrier between the non-target biological population bound to the magnetic particles and a magnetic field to release the non-target biological population bound to the magnetic particles.

[99] The method of claim 98, wherein the target biological population comprises one or more of cells, viruses, bacteria, proteins, DNA, and RNA.

[100] The method of

[98] or

[99] , wherein the target biological population comprises T cells.

[101] The method according to any one of

[98] to

[100] , wherein the magnetic particles are bound to the non-target biological population via an antibody, a protein, or a nucleic acid.

[102] The method according to any one of

[98] to

[101] , wherein the magnetic field gradient is provided by one or more permanent magnets.

[103] The method of claim 102, wherein the permanent magnet comprises magnetite, neodymium, samarium-cobalt, or alnico.

[104] The method according to any one of

[102] to

[103] , wherein the permanent magnets are configured in a linear array.

[105] The method according to any one of

[98] to

[101] , wherein the magnetic field gradient is provided by one or more electromagnets, and optionally the step of inserting d is replaced by a step of removing current from the one or more electromagnets.

[106] The method according to any one of

[98] to

[105] , wherein the magnetic field shield / barrier comprises a material with high magnetic permeability and magnetic saturability.

[107] A method according to any one of

[98] to

[106] , wherein 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.

[108] The method according to any one of

[98] to

[107] , wherein the target biological population is collected by circulating a gas phase fluid followed by a liquid phase fluid one or more times.

[109] The method of claim 108, wherein the gas phase fluid comprises one or more of air, nitrogen, oxygen, and carbon dioxide.

[110] The method of claim 108, wherein the liquid phase comprises one or more of water, buffered saline, culture medium, animal serum, a chelating agent, and an enzyme.

Claims

[Claim 1] An invention substantially as described in the specification.

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