The present invention relates, in part, to magnetic support (e.g., bead) processing apparatus operable with select disposable consumable kits for selectively isolating and/or activating biological cells (e.g., mammalian cells, such as T cells, B cells, and NK cells), as well as purification of other biological materials. The present invention further relates to magnetic bead processing apparatus operable with select disposable consumable kits for selectively isolating and/or activating, for example, T-cells using magnetic beads or for separating the magnetic beads from the T-cell after activation of the T-cells.
A number of devices, methods and reagents have been developed that used magnetic attraction for the isolation and/or activation of cells. Often it is desirable to separate magnetic materials from cells at some point in workflows.
In part, provided herein are devices and reagents that allows for improvements in cell related workflows that involve the use of magnetic materials (e.g., magnetic supports, such as beads).
Some advantages of the bead processing assemblies and systems set out herein are that they are flexible in terms of steps and may be used in workflows that are scalable and automated. Further these magnetic processing systems may also be designed for closed-system and single-use workflows. One additional advantage that bead processing assemblies and systems set out herein have over many other types of magnetic processing system is possible to get the magnet closer to the magnetic particles the assemblies and systems set out herein. These advantages make these magnetic processing systems well suited for commercial manufacturing.
In one independent aspect of the present disclosure a bead processing assembly for use in attaching magnetic beads to biological cells and/or separating magnetic beads from biological cells is provided wherein the bead processing assembly comprises:
In one alternative embodiment, the base assembly further comprises a cover panel hingedly mounted to the housing assembly, the cover panel being movable between an open position wherein the front face of the support panel is openly exposed and a closed position wherein the cover panel covers the front face of the support panel.
In another alternative embodiment, the cover panel comprises:
In another alternative embodiment, the support panel comprises:
In another alternative embodiment, the front face of the support panel is disposed at an angle in a range between 30° and 70° when the housing assembly is resting on a horizontal surface.
In another alternative embodiment, the base assembly further comprises a plurality of pinch valves at least partially outwardly projecting from the front face of the support panel, wherein the plurality of pinch valves comprise at least 2, 3, 4, 6, or 8 pinch valves.
In another alternative embodiment, the first pump comprises a peristaltic pump.
In another alternative embodiment, the base assembly further comprises a first bubble sensor at least partially outwardly projecting from the front face of the support panel.
In another alternative embodiment, the base assembly further comprises a plurality of bubble sensors at least partially outwardly projecting from the front face of the support panel, the plurality of bubble sensors comprising at least 2, 3, or 4 bubble sensors.
In another alternative embodiment, the base assembly further comprises a pressure sensor at least partially outwardly projecting from the front face of the support panel.
In another alternative embodiment, the base assembly further comprises:
In another alternative embodiment, the base assembly further comprises a bead vial retainer including:
In another alternative embodiment, the body of the bead vial retainer comprises:
In another alternative embodiment, the mount assembly of the rocker assembly comprises a first riser and a spaced apart second riser mounted on the base assembly, the platform assembly being pivotably coupled to the first riser and the second riser so as to be at least partially disposed between the first riser and the second riser.
In another alternative embodiment, the means for repeatedly rocking the platform assembly relative to the mount assembly comprises:
In another alternative embodiment, the platform assembly comprises:
In another alternative embodiment, the means for selectively raising and lowering the magnet assembly comprises:
In another alternative embodiment, the scissor lift comprises:
An alternative embodiment further comprising:
In another alternative embodiment, when the magnet assembly is lowered to the deactivation position, the bottom surface of the support plate is at least 4 cm, 5 cm, or 6 cm away from the top surface of the magnet assembly.
In another alternative embodiment, the magnet assembly comprises a magnet.
In another alternative embodiment, the magnet assembly comprises:
In another alternative embodiment, the magnet comprises a plurality of separate and discrete magnets being disposed in a plurality of alternating orientations so as to produce a Halbach array.
In another alternative embodiment, the perimeter edge of the recess is inset from the perimeter edge of the casing at least 0.5 cm, 1 cm, 1.5 cm or 2 cm.
In another alternative embodiment, the casing and the magnet each have a rectangular configuration.
In another alternative embodiment, the platform assembly further comprises a cover assembly at least partially covering the platform and being movable relative to the platform.
In another alternative embodiment, the cover assembly comprises:
In another alternative embodiment, the cover plate is hingedly mounted to the cover housing.
Another alternative embodiment further includes a first latch for securing the cover plate to the cover housing when the cover plate is in the closed position.
Another alternative embodiment further includes means for resiliently restraining movement of the cover assembly away from the platform.
In another alternative embodiment, the means for resiliently restraining movement comprises:
Another alternative embodiment further includes a stop movable between a restraining position and a non-restraining position, in the restraining position, the stop is positioned to preclude some movement of the rod relative to the platform and in the non-restraining position the stop does not interfere with movement of the rod.
Another alternative embodiment further includes a solenoid valve mounted on the housing assembly, the solenoid valve moving the stop between the restraining position and a non-restraining position
Another alternative embodiment further includes:
Another alternative embodiment further includes a stop movable between a restraining position and a non-restraining position, in the restraining position, the stop is aligned with the flange so as to block some movement of the flange and the rod attached thereto, in the non-restraining position, the stop is not aligned with the flange and thus does not interfere with movement of the flange or rod attached thereto.
Another alternative embodiment further includes:
In another alternative embodiment, the cover housing further comprises a U-shaped channel formed at each opposing end of the recess, opposing ends of the clamp assembly being slidably disposed with the U-shaped channels.
In another alternative embodiment, the platform comprises:
A second independent aspect of the present disclosure includes a bead processing system for attaching magnetic beads to biological cells and/or separating magnetic beads from biological cells, the bead processing system comprising:
In one alternative embodiment, the base assembly further comprises a cover panel hingedly mounted to the housing assembly, the cover panel being movable between an open position wherein the front face of the tray is openly exposed and a closed position wherein the cover panel covers the front face of the tray.
In another alternative embodiment, the base assembly further comprises a plurality of pinch valves at least partially outwardly projecting from the front face of the support panel, wherein the plurality of pinch valves comprise at least 2, 3, 4, 6, or 8 pinch valves, wherein each of the plurality of pinch valves project through corresponding ones of the plurality of openings on the tray and engage with the flexible tubing.
In another alternative embodiment, the base assembly further comprises a first bubble sensor at least partially outwardly projecting from the front face of the support panel, the first bubble sensor projecting through a corresponding one of the plurality of openings on the tray and engage with the flexible tubing.
Another alternative embodiment further includes:
Another alternative embodiment further includes a bead vial retainer including:
In another alternative embodiment, the platform assembly comprises:
In another alternative embodiment, the platform assembly further comprises a cover assembly at least partially covering the platform and the processing bag thereon, the cover assembly being movable relative to the platform as the processing bag expands.
Another alternative embodiment further includes means for resiliently restraining movement of the cover assembly away from the platform.
In another alternative embodiment, the cover assembly comprises:
Another alternative embodiment further includes:
Another alternative embodiment further includes a first media bag fluid coupled to the tubing of the line set and housing a medium.
In another alternative embodiment, the bead processing assembly further comprises a stand upstanding from the base assembly and having a catch outwardly projecting therefrom, the media bag being supported on the catch.
In another alternative embodiment, the tubing of the line set is fluid coupled with a biological cell separator.
In another alternative embodiment, the tubing of the line set is fluid coupled with a biological cell expansion system.
In another alternative embodiment, the processing bag comprise a bead separation bag that comprises:
In a third independent aspect of the present disclosure, a consumable kit is provided for use with a magnetic bead processing assembly, the consumable kit including:
In one alternative embodiment, the air filter assembly comprises:
Another alternative embodiment further includes a mixing bag disposed on the front face of the tray and being fluid coupled with the tubing.
Another alternative embodiment further includes:
A third independent aspect of the present disclosure includes a method for operating a bead processing system for attaching magnetic beads to biological cells and/or separating magnetic beads from biological cells, the method comprising:
An alternative embodiment further includes fluid coupling a vial to the tubing of the line set, the vial housing a suspension comprising magnetic beads and a carrier liquid.
Another alternative embodiment further includes supporting a media bag housing liquid media on a bag stand of the bead processing assembly.
Another alternative embodiment further includes fluid coupling the tubing of the line set to a biological cell separator.
Another alternative embodiment further includes fluid coupling the tubing of the line set to a biological cell expansion system.
Another alternative embodiment further includes activating a computer processor of the bead processing assembly so that the computer processor facilitates rocking of the platform on which the processing bag is placed.
Another alternative embodiment further includes activating a computer processor of the bead processing assembly so that the computer processor facilitates raising and lowering a magnet relative to the platform on which the processing bag is placed.
Another alternative embodiment further includes activating a computer processor of the bead processing assembly so that the computer processor facilitates controlling the first pinch valve and the first pump to deliver a fluid to the processing bag on the platform.
Another alternative embodiment further includes activating a computer processor of the bead processing assembly so that the computer processor facilitates moving a vial fluid coupled with the tubing of the line set so as to mix a suspension disposed within the vial, the suspension comprising magnetic beads and a carrier liquid.
Another alternative embodiment further includes the biological cells being T cells.
In another alternative embodiment, the biological cells are attached to the magnetic beads through an antigen-antibody interaction.
In another alternative embodiment, the biological cells are detached from the magnetic beads by disruption of the antigen-antibody interaction.
In another alternative embodiment, the disruption of the antigen-antibody interaction is mediated by cleavage of the antibody.
In another alternative embodiment, the biological cells are detached from the magnetic beads by separation of the antibody from the magnetic beads.
In another alternative embodiment, the antibody is linked to the magnetic beads by a ligand.
In another alternative embodiment, the biological cells are detached from the magnetic beads by disruption of the ligand interaction with the antibody or the magnetic bead.
In another alternative embodiment, the biological cells are T cells.
In a fifth independent aspect of the present disclosure, a method is provided for separating biological cells of a first cell type from biological cells of a second cell type using a bead processing system, the method comprising:
In one alternative embodiment, the antibody has binding affinity for a protein selected from the group consisting of CD3, CD4, CD5, CD6, CD8, CD25, CD27, CD28, CD137, and CD278.
In another alternative embodiment, the antibody has binding affinity for a protein comprising CD3 or CD28.
In another alternative embodiment, the first cell type is T cells, B cells, or NK cells.
Another alternative embodiment includes detaching at least a majority of the biological cells of first cell type from the magnetic beads;
In another alternative embodiment, greater than 95% (e.g., from about 95% to about 99.999999%, from about 95% to about 99.9999%, from about 98% to about 99.999999%, from about 99% to about 99.999999%, from about 99.5% to about 99.999999%, from about 99.9% to about 99.999999%, etc.) of the magnetic beads are retained in place by the magnetic field while the at least a majority of the biological cells of the first cell type are washed away from the magnetic beads.
In another alternative embodiment, greater than 95% (e.g., from about 95% to about 99.999999%, from about 95% to about 99.9999%, from about 98% to about 99.999999%, from about 99% to about 99.999999%, from about 99.5% to about 99.999999%, from about 99.9% to about 99.999999%, etc.) of the magnetic beads are separated from the biological cells of the first cell type.
In another alternative embodiment, greater than 95% (e.g., from about 95% to about 99.999999%, from about 95% to about 99.9999%, from about 98% to about 99.999999%, from about 99% to about 99.999999%, from about 99.5% to about 99.999999%, from about 99.9% to about 99.999999%, etc.) of the magnetic beads detached from the biological cells of first cell type are separated from the biological cells of first cell type.
In another alternative embodiment, wherein the magnetic beads are from about 0.01 μm to about 3 μm, from about 0.01 μm to about 1 μm, from about 0.02 μm to about 2 μm, from about 0.04 μm to about 3 μm, from about 0.4 μm to about 1.5 μm, from about 0.5 μm to about 3 μm, from about 0.1 μm to about 3 μm, from about 0.2 μm to about 3 μm, from about 0.1 μm to about 20 μm, from about 0.1 μm to about 10 μm, from about 0.2 μm to about 5 μm, from about 0.3 μm to about 15 μm, from about 0.3 μm to about 10 μm, or from about 0.5 μm to about 3 μm in diameter.
Further provided herein, in part, are compositions and methods for separating a first biological material (e.g., a nucleic acid molecule, a protein, a cell, an extracellular vesicle, a virus like particle (VLP), etc.) from a second biological material (e.g., a cell lysate, a culture medium, an IVT reaction mixture, etc.) using bead processing systems. In some instances, such methods may comprise (a) contacting a sample comprising the first biological material and the second biological material with magnetic beads so that the magnetic beads attach to the first biological material through an affinity linkage, the sample and magnetic beads being disposed within a processing bag resting on a platform; (b) moving (e.g., raising) a magnet relative to the platform so that the magnet produces a magnetic field that securely fixes in place the magnetic beads relative to the platform, the magnetic beads having first biological material attached thereto; and (c) passing a first fluid through the sample and the magnetic beads while the magnetic field is applied thereto, thereby separating the first biological material from the second biological material. In many instances, the first fluid may be passed through with a force sufficiently strong to wash away the second biological material from the first biological material.
Methods such as those above may further comprise (d) passing a second fluid through the sample and past the magnetic beads while the magnetic field is applied thereto, wherein the second fluid induces release of the first biological material from the magnetic beads.
Methods such as those above may also further comprise (d) lowering the magnet relative to the platform so that the magnetic beads having the first biological material attached thereto are no longer securely fixed in place relative to the platform by the magnetic field of the magnet. In some instances, the magnetic beads may then be collected.
Further, when the first biological material is a nucleic acid molecule, the nucleic acid molecule may be deoxyribonucleic acid (DNA) (e.g., cDNA, genomic DNA, cell-free DNA, single-stranded DNA, double-stranded DNA, plasmid DNA, viral DNA, mitochondria DNA, etc.) or ribonucleic acid (RNA) molecule (e.g., messenger RNA (mRNA), ribosomal RNA, transfer RNA, guide RNA, tracr RNA, crRNA, etc.). Additionally, when the first biological material is a mRNA molecule, the mRNA molecule may encode one or more protein of a pathogenic agent (e.g., a virus). mRNA molecules encoding one or more protein of a pathogenic agent may be a component of a vaccine compositions. Thus, provided herein are methods for the production of mRNA molecules that may be used to produce vaccines.
When the first biological material is a cell or cells, these cells may be B cells, red blood cells, monocytes, stem cells, total T cells, helper T helper cells, regulatory T cells, cytotoxic T cells, natural killer cells, dendritic cells, thrombocytes, as well as other cell types. Further, cell may originate from a mammal (e.g., human, mouse, rat, pig, cow, gorilla, lama, camel, chimpanzee, etc.).
When the first biological material is a protein, the protein may be an antibody, enzyme, a receptor (e.g., a cell surface receptor), etc. Further, Protein A, Protein G, Protein L, or a functional variant of one of these proteins may be used to form affinity linkages with antibodies purified using methods set out herein.
When the first biological material is an extracellular vesicle, this vesicle may be an exosome, a microvesicles, apoptotic body, etc. Further, the vesicle (e.g., exosome) may be generated by a T cell such as a T cell engineered to express a chimeric antigen receptor (e.g., a CD19-CAR). Such vesicles (e.g., exosomes) may exhibit cytotoxic properties towards particular cells (e.g., tumor cells).
Vesicles (e.g., exosomes) generated by T cells may be separated from other biological materials using magnetic beads that form an affinity linkage with CD3, CD4 and/or CD8 receptors. In many instances, such methods may employ an anti-CD3 antibody, an anti-CD4 antibody, and/or an anti-CD8 antibody. Further, such antibodies may be, for example, monoclonal antibodies or variable heavy-heavy (VHH) antibodies. Additionally, such antibodies may be attached to a magnetic bead by a biotin or biotin derivative based linkage.
Further provided herein are compositions and methods for producing a purified ribonucleic acid (RNA) molecules. Such methods may include (a) fixing a first magnetic bead in place by a magnetic field, wherein an in vitro transcription (IVT) template is linked to the first magnetic bead; (b) contacting the first magnetic bead of step (a) with a reagent mixture suitable for IVT of the template under condition in which IVT occurs, thereby producing an RNA molecule, and (c) separating the RNA molecule from the first magnetic bead, thereby producing the purified RNA molecule.
In some instances, provided are methods further comprising (d) contacting the purified RNA molecule of step (c) with a second magnetic bead under conditions that allows for the purified RNA molecule to remain associated with the second magnetic bead during washing, (e) washing of the second magnetic bead while the second magnet bead is fixed in place by a magnetic field, and (f) releasing the purified RNA molecule from association with the second magnetic bead, thereby producing a highly purified RNA molecule.
In some instances, the IVT template may be produced by polymerase chain reaction (PCR). Further, one or more biotinylated primer may be used in the PCR, resulting in the formation of a biotinylated IVT template. Additionally, a biotinylated IVT template may be attached to the magnetic bead through an interaction between the biotin of the biotinylated IVT template and a group on the magnetic bead with affinity for biotin (e.g., avidin, streptavidin, etc.).
In some instances, the IVT template may comprise an open reading frame encoding a protein and a promoter operably connected to the open reading frame.
While the second magnetic bead may associate with RNA in a number of different ways, in some instance, free carboxylic acid groups may be present on the surface of these bead.
Further, purified RNA or highly purified RNA produced as set out herein may be messenger RNA (mRNA), such as mRNA encoding one or more protein of pathogen. Further provided herein are vaccine composition comprising such mRNA or protein encoded by such mRNA.
mRNA used in such vaccine compositions may be contained in, for example, lipid nanoparticles (see, e.g., WO 2021/159130 A2).
Various embodiments of the present invention will now be discussed with reference to the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope.
Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particularly exemplified apparatus, systems, methods, or process parameters that may, of course, vary. It is also to be understood that the terminology used herein is only for the purpose of describing particular embodiments of the present disclosure and is not intended to limit the scope of the disclosure.
All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Further, the following US patent documents are specifically incorporated by reference: U.S. Provisional Patent Application Nos. 63/090,399 (filed Oct. 12, 2020) and 63/137,389 (filed Jan. 14, 2021); U.S. Patent Publication No. 2017/0313772 A1; US Patent Publication No. 2019/0010435, published Jan. 10, 2019 and U.S. Pat. Nos. 9,567,346 and 10,196,631, and PCT Publication WO 2021/159130 A2.
The term “comprising” which is synonymous with “including,” “containing,” “having” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
It will be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a “port” includes one, two, or more ports.
As used in the specification and appended claims, directional terms, such as “top,” “bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “inner,” “outer,” “internal,” “external,” “interior,” “exterior,” “proximal,” “distal” and the like are used herein solely to indicate relative directions and are not otherwise intended to limit the scope of the disclosure or claims.
A “support,” as used herein, refers to any nonaqueous soluble material capable of having an antibody attached thereto and includes, without limitation, metals, glass, plastics, co-polymers, colloids, lipids, and the like. Essentially any nonaqueous soluble material capable of retaining an antibody bound or attached thereto.
As used herein, the term “magnetic support” refers to a structural material capable of being attracted to a magnetic field. In many instances, magnetic supports will comprise one or more magnetic metal (e.g., iron, cobalt, nickel, etc.). In many instances, a magnetic support will be a magnetic bead. Examples of commercially available magnetic beads are D
As used herein, the term “ligand” refers to a molecule that binds to one or more defined population of cells (e.g., members of T cell subpopulations). Ligand binding may be used for isolation of cells to which it binds or may induce a cellular response when bound alone or in conjunction with one or more additional ligands. For example, ligands that bind CD3 and CD28 receptors may be used to activate T cells.
Ligands may bind any cell surface moiety, such as a receptor, an antigenic determinant, or other binding site present on the target cell population. The agent may be a protein, peptide, antibody and antibody fragments thereof, fusion proteins, synthetic molecule, an organic molecule (e.g., a small molecule), or the like. Within the specification and in the context of T cell stimulation, antibodies are used as a prototypical example of such an agent.
As used herein, the term “separation” refers to dividing one component of a mixture from another component of the same mixture.
As used herein, “purified” means that the amount of a material that has been increased in relationship to another materials. By way of example if Protein X is processed in a manner in which the original concentration of Protein X was 1 μg/ml and at the end of the process the final concentration of Protein X is still 1 μg/ml but the concentration of Protein Y has gone from 2 μg/ml to 2 μg/ml, then Protein X has been purified. Further, “purification” refers to processes by which materials (e.g., proteins, nucleic acids, etc.) are purified.
As used herein, the term “antibody” includes: (a) any of the various classes or sub-classes of immunoglobulin (e.g., IgG, IgA, IgM, IgD or IgE derived from any animal e.g., any of the animals conventionally used, e.g., sheep, rabbits, goats, mice, camelids, or egg yolk), (b) monoclonal or polyclonal antibodies, (c) intact antibodies or fragments of antibodies, monoclonal or polyclonal, the fragments being those which contain the binding region of the antibody, e.g., fragments devoid of the Fc portion (e.g., Fab, Fab′, F(ab′) 2, scFv, VHH antibodies, VHH antibody fragments, as well as other single domain antibodies), the so called “half molecule” fragments obtained by reductive cleavage of the disulphide bonds connecting the heavy chain components in the intact antibody (Fv may be defined as a fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains), and (d) antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, fragments of antibodies, “humanized antibodies”, chimeric antibodies, or synthetically made or altered antibody-like structures.
Anti-CD3 antibodies that may be used in compositions and methods set out here include those expressed by the following clones: OKT3 (cBioscience, cat. no. 14-0037-82), BC3 (American Type Culture Collection, Deposit No. HB-10166), HIT3a (cBioscience, cat. no. 16-0039-81), F7.2.38 (Thermo Fisher Scientific, cat. no. MA5-12577), MEM-57 (Thermo Fisher Scientific, cat. no. MA1-19454), and UCHT1 (cBioscience, cat. no. 16-0038-81). Anti-CD28 antibodies that may be used in compositions and methods set out here include those expressed by the following clones: IC6 (Graves et al., Transplantation, 91:833-840 (2011), CD28.6 (Thermo Fisher Scientific, cat. no. 16-0288-81), CD28.2 (Thermo Fisher Scientific, cat. no. MA1-10166), 10F3 (Thermo Fisher Scientific, cat. no. CD2800), and BT3 (mIgG2a, antihuCD28, Diaclone, Besanqon, France).
As used herein, the term “VHH antibody” refers to antibodies that consists only of two heavy chains and, thus, lack light chains. Antibodies of this type can be produced by cartilaginous fish and camelids (e.g., alpacas, dromedaries, camels, llamas).
VHH antibodies many be engineered to such that both heavy domains are in the same protein molecule (a single chain antibody) and contain no constant regions. Engineered VHH antibodies may be relatively small in size (e.g., 12 to 15 kDa, about 120 amino acids) in comparison to monoclonal antibodies (see, e.g., Harmsen and De Haard, “Properties, production, and applications of camelid single-domain antibody fragments,” Applied Microbiol. Biotech., 77:13-22 (2007), U.S. U.S. Pat. No. 9,040,666). Such antibodies are also referred to herein as VHH antibodies. VHH antibodies may have one or two antigen binding sites and that may be monovalent or bivalent. Bivalents refer to having binding affinity to two different epitopes.
The term “Protein A,” As used herein, refers to the cell surface protein of Staphylococcus aureus composed of five highly similar domains (Domains E, D, A, B, and C), where each domain is composed of about 58 amino acids, and functional variants and functional derivatives thereof (including Domain Z). Protein A has the functional activity of binding to antibodies (e.g., IgG molecules) and may be used for antibody purification.
In many instances, Protein A is linked to a solid support (e.g., a bead) when used for antibody purification. This allows for antibodies bound to solid supports to be separated from unbound materials. Further, in many instances (e.g., commercial scale antibody production), it is desirable to be able to clean in place (CIP) Protein A bound to solid supports. In many instances, CIP is done using sodium hydroxide (e.g., 0.5M NaOH).
While full-length, wildtype Protein A exhibits some resistance to alkaline conditions, a number of modifications to various Protein A domains to enhance alkaline resistance (see, e.g., Linhult et. al., “Improving the Tolerance of a Protein A Analogue to Repeated Alkaline Exposures Using a Bypass Mutagenesis Approach,” Proteins, 55:407-416 (2004)). Further, alkaline sensitivity/resistance vary with the individual Protein A domains. Along these lines, Domain C has been found to be fairly alkaline resistant.
Proteins that contain repeats of one or more Protein A domains (e.g., five repeats of wild-type Domain B) that retain at least 50% of the binding capacity of wild-type Protein A molecules on a per domain basis are also considers to be Protein A molecules. Also considers to be Protein A molecules are proteins containing domains that share at least 90% sequenced identity to any stretch of 20 amino acids of any one of wildtype Protein A Domains E, D, A, B, and C and that retain at least 50% of the binding capacity of wild-type Protein A molecules on a per domain basis. By way of example, if wild-type Protein A molecules bind 50 units of antibody under a specified set of conditions and Protein A derivative with four repeated domains binds 20 units of antibody under the same conditions, then the Protein A derivative would be said to have 50% of the binding capacity of wild-type Protein A molecules.
As used herein, the term “virus-like particle”, or VLP, refers to viral protein complexes that resemble viruses but are replication deficient. VLPs can be used to deliver nucleic acid molecules to cells and include naturally occurring replication deficient viral protein complexes such as adeno-associated virus. VLPs may be enveloped or unenveloped. One example of enveloped lentiviral particles are those produced using the BLOCK-IT™ Lentiviral RNAi Zeo GATEWAY™ Vector Kit (Thermo Fisher Scientific, cat. no. V48820).
As used herein, a “Halbach array” is an arrangement of magnets (see
Where possible, like numbering of elements have been used in various figures. Furthermore, alternative configurations of a particular element may each include separate letters appended to the element number. Accordingly, an appended letter can be used to designate an alternative design, structure, function, implementation, and/or embodiment of an element or feature without an appended letter. For instance, an element “80” may be embodied in an alternative configuration and designated “80a.” Similarly, multiple instances of an element and or sub-elements of a parent element may each include separate letters appended to the element number. In each case, the element label may be used without an appended letter to generally refer to all instances of the element or any one of the alternative elements. Element labels including an appended letter can be used to refer to a specific instance of the element or to distinguish or draw attention to multiple uses of the element.
Various aspects of the present devices, systems, and methods may be illustrated with reference to one or more exemplary embodiments. As used herein, the term “embodiment” means “serving as an example, instance, or illustration,” and should not necessarily be construed as preferred or advantageous over other embodiments disclosed herein.
Various aspects of the present devices and systems may be illustrated by describing components that are coupled, attached, and/or joined together. As used herein, the terms “coupled”, “attached”, “connected” and/or “joined” are used to indicate either a direct connection between two components or, where appropriate, an indirect connection to one another through intervening or intermediate components. In contrast, when a component is referred to as being “directly coupled”, “directly attached”, “directly connected” and/or “directly joined” to another component, there are no intervening elements present.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, the preferred materials and methods are described herein.
Depicted in
The cell culture leaving cell separator 12 is then processed through a first bead processing system 14 for isolation and activation of the desired T-cells. That is, as will be discussed below in detail, first bead processing system 14 functions to mix the cell culture with magnet beads that are covalently coupled with specific antibodies (e.g., anti-CD3, anti-CD28, anti-CD137, etc.) so that the magnetic beads will automatically bind to and activate the desired T-cells. The T-cells coupled to the magnetic beads are then held in an isolation bag by a magnetic field while the remaining unwanted cells are washed away.
The isolated T-cells bound to the magnetic beads are then washed and released from the magnetic field and transferred to a cell expansion system 16. Cell expansion system 16 can comprise a conventional bioreactor system where the T-cells are grown in a growth medium under pre-establish conditions. Cell expansion system 16 can comprise a rocker or continuous stir bioreactor or any other bioreactor system, such as those using a gas permeable bag, where cells can be grown. Once the T-cells have grown to a desired density or reached another preestablished condition, the T-cells will either automatically separate from the magnetic beads or a detach reagent can be added to the suspension that causes separation of the magnetic beads from the T-cells. For example, activated T-cells will naturally downregulate CD3, and will therefore detach from the magnetic beads typically at around five days. In some embodiments, the suspension of T-cells, magnetic beads, and media can be manual or mechanical manipulated, such as within a bag, to help facilitate detachment of the T-cells from the magnetic beads. The suspension of T-cells, magnetic beads, and media is then transferred to a second bead processing system 18 where the desired T-cells are separated from the magnetic beads, as set out below in more detail. As discussed below in further detail, in one embodiment, bead processing system 18 can comprise reusing the apparatus of bead processing system 14 with a different consumable kit.
Finally, the T-cells separated from the magnetic beads can be transferred to a gene editing system 20 for genetically modifying the cells as desired. For example, DNA can be incorporated into the cells by, for examples, homologous recombination and/or non-homologous end joining. Gene editing may be mediated by site specific cleavage of intracellular nucleic acid (e.g., chromosomal nucleic acid). Further downstream processing and/or packaging of the T-cells can also be provided.
Instrument workflow system 8 can comprise a closed, sterile system where the biological sample can continuously flow between each component of the system under sterile conditions. Alternatively, the biologic sample can be separately transferred between each or select components of system 8. Each component of system 8 can be programmed to run automatically or can be operated manually. Depending on specific conditions, specific components of instrument workflow system 8 can be eliminated or additional components can be added.
A detailed description of the components and operation of bead processing systems 14 and 18 will now be described. In one embodiment, bead processing systems 14 and 18 can comprise the same reusable hardware but use different disposable, consumable components to achieve their different intended functions. This design has the advantage of reducing equipment cost and minimizing needed storage space. However, in other embodiments, separate types of bead processing systems can be used.
Bead processing system 14 comprises a reusable bead processing apparatus 22, as depicted in
Base assembly 32 comprises a housing 38 that extends between a front end 40 and an opposing back end 42 and that bounds a compartment 44 (
Housing 38 further includes opposing side panels 52A and 52B that upstand from and extend along opposing sides of floor panel 50. A front panel 54 is disposed at front end 40 of floor panel 50 and extends between side panels 52A and 52B. In one embodiment, front panel 54 is sloped at an angle relative to horizontal which is typically in a range between about 25° and 65° and more commonly in a range between 35° and 55°. Other angles can also be used. A screen 56 is disposed on front panel 54 and is in electrical communication with electrical circuitry 53. In one embodiment, screen 56 can comprise a touch screen to enable user input for operating bead processing apparatus 22. In alternative embodiments, screen 56 can simply comprise a display screen for displaying operation of bead processing apparatus 22. Optionally, an electrical interface port 58 and an activation switch 60 (
With continued reference to
Also formed on back panel 62 is a power inlet 66 configured for coupling with an electrical cable for providing electrical power to bead processing apparatus 22. Projecting forward toward front end 40 at an upper end of back panel 62 is a shelf panel 68. Shelf panel 68 is typically disposed horizontally and thus is commonly disposed parallel to floor panel 50 and/or perpendicular to back panel 62. Shelf panel 68 has an opening 70 passing therethrough in which rocker assembly 34 is disposed. Upstanding from a forward end of shelf panel 68 is a riser panel 72. Riser panel 72 terminates at a top edge 74 of bead processing apparatus 22 and extends between side panels 52. In one embodiment riser panel 72 can be disposed vertical so as to be parallel to back panel 62 and/or perpendicular to shelf panel 68.
Bag stands 36A and 36B upstand from shelf panel 68 on opposite sides of rocker assembly 34. Each bag stand 36 comprises a pole 98 having an upper end with a plurality of catches 99 formed thereat. Pole 98 can have a fixed length or be configured to telescopically lengthen or shorten. Catch 99 are configured to hold and support flexible bags housing a fluid and can be in the form of a hook or other structure that can retain a bag.
A stand 82 is disposed within compartment 44 and is mounted on floor panel 50. In one embodiment, stand 82 can be U-shaped and positioned to span over transformer 57 or other components disposed on floor panel 50. Rocker assembly 34 is disposed on stand 82 so as to pass through opening 70 on shelf panel 68. Further disposed within compartment 44 and mounted on floor panel 50 are a pair of triangular brackets 84A and 84B. Brackets 84 are disposed forward of stand 82 and extend to or toward front end 40. In one embodiment, each bracket 84 includes a vertical back rail 86, a horizontal base rail 88 that is secured to floor panel 50, and a front rail 90 that extends at an angle between a forward end of base rail 88 and an upper end of back rail 86. As discussed below in more detail, spanning between brackets 84, and more specifically between front rails 90, is a support panel 92. Support panel 92 is typically disposed at an angle relative to horizontal which is typically in a range between about 25° and 65° and more commonly in a range between 35° and 55°. Other angles can also be used.
With reference to
For case of discussion,
Depicted in
Returning to
Air filter assembly 218A comprises a stopcock 220 having an air filter 221 coupled thereto. In the depicted embodiment, stopcock 220 comprises a two-way stopcock that includes a stem 222 having ports 223A, 223B, and 223C outwardly projecting therefrom and fluid coupled therewith. A valve 224 is rotatably disposed within stem 222 and has an outwardly projecting handle 225 disposed therein. Rotation of handle 225 to a first position orientates valve 224 so that fluid can flow between ports 223A and 233B. Rotation of handle 225 to a second position orientates valve 224 so that fluid can only flow between ports 223A and 233C. Air filter 221 is coupled to port 223C and typically comprises an air sterilizing filter. In one embodiment, air filter 221 can have an average pore size of 0.2μ or smaller. As will be discussed below in more detail, handle 225 is configured to removably fit within keyed socket 97 of receiver 96 so that a secure engagement is formed therebetween. As a result, rotation of receiver 96 by drive motor 160 can facilitate rotation of handle 225 relative to stem 222 between the two operating positions. Rotational assembly 132B can have the same configuration and operation as rotational assembly 132A while air filter assembly 218B can have the same configuration and operation as air filter assembly 218A. As such, like elements between components are identified by like reference characters.
During assembly, drive motor 160 of rotational assemblies 132A and 132B can be secured to base panel 93/support panel 92 so that receivers 96 are received within corresponding openings 134A and 134B, respectively, of overlay panel 94. In this embodiment, as shown in
With continued reference to
Finally, a pressure sensor 140 can be mounted on support panel 92/base panel 93 and project through or be aligned with a corresponding opening 142 on overlay panel 94. Pressure sensor 140 is shown disposed between bubble sensors 136A and 136C but can be disposed at other locations. As will also be discussed below in more detail, pressure sensor 140 is used to detect the pressure of the fluid within the tubing of bead processing system 14. Monitoring the pressure can help detect operational failures and prevent over pressurizing the tubing and/or bags of consumable kit 170A. An example of a pressure sensor that can be used in the present disclosure is the PRO-3000-904 produced by INTROTEK INTERNATIONAL.
It is noted that overlay panel 94 primarily functions to cover fasteners and/or other element of disposed on base panel 93 for which access is not needed during normal operation. As such, in alternative embodiments, overlay panel 94 can be eliminated, such as is shown in
With continued reference to
Tray 172A also includes a plurality of spaced apart tube restraints 186 mounted on and outwardly projecting from top face 176. Although tube restraints 186 can have a variety of different configurations, in the depicted embodiment, each tube restraint 186 comprises a base 188 having a slot 190 extending therethrough. Slot 190 typically has a C-shaped or U-shaped transverse cross section that is configured to snuggly but removably received a tubing 200 of line set 174A. Tube restraints 190 function to contour and hold tubing 200 and can be formed at any desired location on tray 172A where line set 174A travels. However, tube restraints 186 are most commonly placed at or adject to where tubing 200 is being bent or manipulated. Tray 172A also includes a bag restraint 192 centrally upstanding from tray 172A. In the depicted embodiment, bag restraint 192 comprises a perimeter wall 194 upstanding from tray 172A and encircling a receiving area 196. Receiving area 196 is configured to receive a mixing bag 210 of line set 174A so that mixing bag 210 is supported and retained by perimeter wall 194.
As depicted in
During use, the assembled consumable kit 170A is placed on top of support panel 92 so that the mechanical components on support panel 92 are aligned with the related openings formed on tray 172A. As discussed below in more detail, tubing 200 is then manipulated to couple with each of pinch valves 128, pumps 124, bubble sensors 136 and pressure sensor 140 while air filter assemblies 218 are mounted on rotational assemblies 132.
Tubing 200 can have a variety of different configurations. In one embodiment, tubing 200 has an inside diameter in a range between 2.0 mm and 5.0 mm or between 2.5 and 5.0 mm or more commonly in a range between 3.0 mm and 5.0 mm. Other ranges can also be used. Tubing 200 is made of material that enables tubing 200 to be easily bent without plastic deformation and enables tubing 200 to be selectively pinched closed (such as by using pinch valves 128) to prevent fluid flow therethrough but will resiliently rebound to an open position to allow fluid to flow therethrough when the pinching force is released. Commonly, tubing 200 can be bent over an angle of at least 90°, 180°or 360° without plastic deformation. Examples of material that tubing can be made of include silicone, polyvinyl chloride (PVC) and thermoplastic elastomers (TPE) such as C-FLEX formulation 374 tubing. Different sections of tubing 200 can also be made of different materials depending on their intended function. In the present embodiment, tubing 200 is sized so that it can be received within slot 112 of pinch valves 128 (
Tubing 200 comprises a plurality of sections that are either integrally connected together or are connected together by fittings. For example, with reference to
Returning to
Tubing 200 also includes tubing sections 204D and 204E each having a first end that is fluid coupled with a mixing bag 210 positioned within bag restrainer 192. Mixing bag 210 can be permanently or removably fluid coupled to tubing sections 204D and 204E but they are all typically sterilized together in a closed state. Tubing section 204D couples with pinch valve 128E. Tubing section 204E couples with bubble sensor 136B, pinch valve 128F and air filter assembly 218B.
A tubing section 204F of tubing 200 terminates at an outlet end 211. Outlet end 211 is typically intended to couple with cell expansion system 16 (
A tubing section 204G of tubing 200 has a first end coupled with a collection bag 212. Collection bag 212 is used to collect the unwanted cells that are washed from the desired T-cells within isolation bag 206 and other negative fractions. Tubing section 204G is coupled with pinch valve 128J and connects with a second end of tubing section 204E. Tubing sections 204H, 204I, and 204J of tubing 200 each have a first end that is permanently or removably coupled to a separate media bag 216A, 216B, and 216C, respectively and an opposing send end that is fluid coupled, either directly or indirectly to tubing section 204G. Media bags 216 house a medium that is used for washing and other processing of the cells, as discussed later below. Tubing sections 205H, 205I, and 205J are coupled to pinch valves 128G, 128H and 128I, respectively. It is appreciated that the number of media bag 216 used can depend on the application and the size of bags 216. Thus, in other embodiments, one, two or four or more media bags 216 can be used with corresponding sections of tubing 200, rather than having three media bag 216.
A tubing section 204K of tubing 200 couples with a second end of tubing sections 204A, 204B, 204D, 204F and 204G. During assembly for use, tubing section 204K is coupled with pumps 124A and 124B and also with bubble sensors 136A and 136C and pressure sensor 140. Pumps 124A and 124B are used to facilitate controlled flow of fluid through line set 174A.
As previously discussed, the first end of tubing section 204C is connected to bead vial coupler 208. Turning to
As will be discussed below in more detail, beads 179 are used for the isolation and activation of desired T-cells. Beads 179 will typically be composed of a magnetic material and a polymer. While the actual structure of the bead can vary greatly, the beads may, as examples, have a magnetic core and a polymeric exterior or can be composed of a polymeric material with embedded magnetic particles. Exemplary magnetic beads are CTS™ D
Bead vial coupler 208 comprises a tubular stem 173 extending between first end 164 and opposing second end 165. First end 164 is configured to couple with neck 168 of vial 166 so that a secure fluid coupling is formed between compartment 175 of vial 166 and first end 164. Second end 165 is fluid coupled with tubing section 204C of tubing 200. In the depicted embodiment, bead vial coupler 208 further includes an expansion chamber 171 that outwardly projects from stem 173. Expansion chamber 171 is configured to automatically equalize the pressure within compartment 175 of vial 166 as bead mixture 177 is dispensed into line set 174A, thereby preventing the formation of a vacuum within chamber 171 that can interfere with fluid flow. Two examples of bead vial coupler 208 incorporating expansion chamber 171 are the PHASEAL™ drug vial access device produced by Becton Dickinson and a needle free vial spike valve available from OriGen Biomedical (cat. no. VSV). In alternative embodiments, bead vial coupler 208 need not include expansion chamber 171 and other configurations of bead vial couples can be used.
Turning to
Supported within compartment 44 of bead processing apparatus 22 is a drive motor 162 that is controlled by electrical circuitry 53 (
During assembly and use, bead vial coupler 208 having vial 166 attached thereto is advanced down through channel 156 of bead vial retainer 148 until shoulder 169 of vial 166 (FIG. 14) comes to rest on shoulder 157 of bead vial retainer 148. Body 150 of bead vial retainer 148 is configured to snugly and securely receive vial 166 within channel 156 under frictional engagement. During this assembly, expansion chamber 171 can project out through slot 159 of bead vial retainer 148.
Bead mixture 177 is more efficiently dispensed from vial 166 into line set 174A if beads 179 are suspended in carrier liquid 181. However, if inverted vial 166 mounted on bead vial retainer 148 remains stationary in a vertical orientation for a period, beads 179 can settle at neck 168 of vial 166 and thereby restrict or block flow out of vial 166. Accordingly, once bead vial coupler 208 with vial 166 are mounted on bead vial retainer 148 and it is desired to dispense bead mixture 177 into line set 174A, drive motor 162 can be activated to rotate drive shaft 163 so that bead vial retainer 148 is rotated to a forward position, as shown in
Further use and operation of consumable kit 170A will be discussed later after describing rocker assembly 34. Turning to
Platform assembly 232 is pivotally connected to upper end 242A. More specifically, platform assembly 232 includes, in part, a housing assembly 246 having a front wall 250A and an opposing back wall 250B with opposing lateral walls 248A and B that extend therebetween. Walls 240 and 250 upstand from a floor 252 (
Returning to
With continued reference to
In one embodiment of the present invention, means are provided for selectively rocking platform assembly 232/housing assembly 246. One example of the means for rocking is rocker drive 234. In alternative embodiments, rocker drive 234 could be replaced with a variety of different mechanical mechanisms that achieve the same function. For example, rocker drive 234 could be replaced with a cam follower system where motor 270 rotates a cam while a follower connected to the cam raises and lowers and end of platform assembly 232/housing assembly 246 as the cam rotates. In other embodiments, motor 270 could reciprocally dive a worm gear that selectively raises and lowers the end of platform assembly 232/housing assembly 246. In other embodiments, hydraulic or pneumatic systems could be used to raise or lower a piston that in turn selectively raises and lowers and end of platform assembly 232/housing assembly 246. Other conventional system can also be used.
With reference to
With continued reference to
Continuing with
In one embodiment, magnet 299 can comprise a single continuous magnet. In an alternative embodiment, magnet 299 can comprise a plurality of separate magnets that are adjacently disposed with recess 301. For example, in one embodiment, magnet 299 can comprise a plurality of magnets that are adjacently disposed so as to form a Halbach array. A Halbach array is a special arrangement of permanent magnets that augments the magnetic field on one side of the array while cancelling the field to near zero on the other side. This is achieved by having a spatially rotating pattern of magnetization. The rotating pattern of permanent magnets (on the front face; on the left, up, right, down) can be continued indefinitely and have the same effect. Depicted in
Returning to
Turning to
Second arm 326A also has a first end 342A and an opposing second end 344A. First end 342A is hingedly coupled to bottom surface 314 of shelf 310 at front end 316 by a hinge joint 345A. Second end 344A of second arm 326A is both slidably and hingedly coupled to floor 252 of housing assembly 246 at a back end by a guide 346A. That is, guide 332A is securely fixed to interior surface of 252 of floor 252 at the back end and includes an upwardly projecting flange 348A having an elongated, horizontally extending slot 350A formed thereon. A guide pin 352A orthogonally projects from second end 344A of second arm 326A, through slot 350A and toward second pair of scissor arms 322. In this configuration, second end 344A of second arm 326A can slide horizontally relative to floor 252 by guide pin 352A sliding within slot 350A and can freely rotate by either guide pin 352A rotating within slot 350A and/or second end 344A rotating relative to guide pin 352A.
Second pair of scissor arms 322 has the same configuration, structural elements and attachment to floor 252 and shelf 310 as first pair of scissor arms 320. As such, the above discussion is also applicable to second pair of scissor arms 322 and like elements between first pair of scissor arms 320 and second pair of scissor arms 322 are identified by like reference characters except that the reference characters for second pair of scissor arms 322 include the suffix “B”.
With continued reference to
By the controlled operation of motor 366, lift assembly 292 can be used to selectively control moving shelf 310/magnet assembly 294 between a raised position and a lowered position. For example, depicted in
For moving magnet assembly 294 lower relative to platform 290 to a deactivation position, motor 366 is activated so as to rotate threaded shaft 360 which advances collar 364 horizontally away from motor 366. In turn, collar 364 concurrently moves extensions 372 and guide pins 352 horizontally rearward along guides 346 which in turn moves second end 344A of first arm 324 horizontally away from first end 328A of second arm 326A. As ends 344A and 328A horizontally separate, arms 324 and 326 pivot relative to each other at axle 377 which causes the upper ends of arms 324 and 326 to both horizontally separate and lower, thereby lowering shelf 310 and magnet assembly 294 relative to support plate 380 as shown in
One embodiment of the present invention includes means at least partially disposed within the compartment of housing assembly 246 for selectively raising and lowering magnet assembly 294 relative to platform 290 between a raised activation position and a lowered deactivation position. Onc example of such means includes scissor lift 321 as described above. In alternative embodiments, scissor lift 21 can be replaced with a variety of alternative mechanism that can achieve the same function. By way of example and not by limitation, shelf 310 could be mounted on tracks that enable shelf 310 to vertically slide up and down relative to platform 290 while remaining horizontally disposed. Various mechanical system can then be used to move shelf 310 along the tracks. By way of example, motor 366 could be used to rotate a crank that raises and lowers a connecting arm connected to shelf 310. Alternatively, a cam follower system can be used where motor 366 rotate a cam while a follower connected to the cam and extending to shelf 310 raises and lowers shelf 310 as the cam rotates. In other embodiments, motor 366 could reciprocally dive a worm gear that selectively raises and lowers shelf 310. In other embodiments, hydraulic, pneumatic, cable or chain systems could be used to raise and lower shelf 310 along the tracks. Other conventional systems can also be used.
Turning to
Spring assembly 420A comprises a bracket 430A mounted to and outwardly projecting from housing assembly 246. Bracket 430A can also form a portion of housing assembly 246. Bracket 430A has a hole 432A that vertically extends therethrough. A rod 434A is slidably received within hole 432A and extends between a first end 436A and opposing second end 438A. First end 426A projects above bracket 430A and has a collar 440A securely coupled thereto. Collar 440 has an outer diameter larger than an outer diameter of hole 432A so that collar 440 prevents rod 434A from passing down through hole 432A. A flange 442A outwardly projects from second end 438A of rod 434A. A spring 444A extends between flange 442A and the bottom surface of bracket 430A such that as rod 434A is pushed upward through hole 432A, spring 444A is resiliently compressed so as to urge rod 434A downward. First end 436A of rod 434A terminates at an end face 446A having a threaded socket 448A formed thereon. As will be discussed below in greater detail, socket 448A is configured to receive a threaded fastener 450A. Spring assemblies 420B, 420C, and 420D are identical to spring assembly 420A and are each located adjacent to a corresponding corner of cover housing 422. As such, the above discussion of spring assembly 420A is likewise applicable to spring assemblies 420B-D. Furthermore, like elements of spring assembly 420A and spring assemblies 420B-D are identified by like reference characters except that each of the reference characters for spring assemblies 420B-D include the suffix B, C, and D, respectively.
Cover housing 422 includes an annular perimeter wall 454 having an interior surface 456 and an opposing exterior surface 458 that extend between an upper edge 460 and opposing lower edge 462. Interior surface 456 encircles an opening 464 that passes through perimeter wall 454 between upper edge 460 and lower edge 462. Opening 464 is configured to receive platform 290. Perimeter wall 454 comprises a front wall 466 and an opposing back wall 468 with lateral walls 470 and 472 extending therebetween. Outwardly projecting from exterior surface 458 of cover housing 422 are four mounts 474A-474D. Mounts 474A and 474B outwardly project from lateral wall 470 at a forward end and rearward end thereof, respectively. Likewise, mounts 474C and 474D outwardly project from exterior surface 458 of lateral wall 472 at a forward and rearward end thereof, respectively. Mounts 474 are positioned to align with spring assemblies 420 when cover housing 422 is received over platform 290. Mount 474A is configured to couple with first end 436A of rod 434A. For example, in one embodiment, mount 474A has a top surface 478A and an opposing bottom surface 480A. A recess 482A is formed on bottom surface 480A and is configured to receive first end 436A of rod 434A. An opening 484A is formed on top surface 478A that communicates with recess 482A. Opening 484A has a diameter smaller than the outer diameter of rod 434A as first end 436A. Fastener 450A is configured to pass down through opening 484A and thread into socket 448A so as to secure first end 436A of rod 434A to mount 474A. It is appreciated that a variety of alternative techniques can be used for securing together rod 434A and mount 474A. For example, the structures can be secured together by press-fit connection, adhesive, welding, or a variety of other types of mechanical fasteners. Mounts 474B-474D all have the same structural elements and configuration as mount 474A and are similarly configured to couple with rods 434B-D of spring assemblies 420B-D, respectively. Like elements between mount 474A and mounts 474B-D are identified by like reference characters except that reference characters for mounts 474B-D includes the suffixes B, C, and D, respectively.
Front wall 466 of cover housing 422 has a recess 486 formed thereon that extends down from upper edge 460. As discussed below, recess 486 is configured to receive a clamp assembly 402.
Sleeve 424 also includes an encircling perimeter wall 490 that is configured to encircle platform 290 and be received within opening 464 of cover housing 422. Sleeve 424 is coupled to cover housing 422 such as by fasteners and other conventional techniques so that sleeve 424 moves concurrently with cover housing 422. Sleeve 424 also has a recess 492 formed therein that aligns with recess 486 and is configured to received clamp assembly 402. Sleeve 424 can function, in part, to increase the structural integrity cover housing 422. For example, in one embodiment cover housing 422 can be made of a plastic while sleeve 424 is formed of a metal such as aluminum. This design reduces cost but achieves desired strength. In alternative embodiments, the structural integrity of cover housing 422 can be increased and sleeve 424 can be eliminated.
Lid 426 comprises a lid plate 499 having an inside face 500 and an opposing outside face 502 that extend between a front edge 504, a back edge 506, and opposing lateral edges 508 and 510. A pair of spaced apart hinges 512A and 512B extend between lateral edge 508 of lid plate 499 and lateral wall 470 of cover housing 422. As a result, lid 426/lid plate 499 is hingedly mounted to covering housing 422. Lid 426 further includes a handle 514 upwardly extends from lid plate 499 at or towards lateral edge 510. As better depicted in
Turning to
A stop assembly 526 can be associated with each spring assembly 420 or with only select spring assemblies. In the present depicted embodiment, a stop assembly 526B is associated with spring assembly 420D as depicted in
As will be discussed below in further detail, during operation, isolation bag 206 is positioned on top of platform 290. Lid 426 is then moved to the closed position and latches 528 are used to lock lid 426 in the closed position. In this position, fluid is delivered into and removed out of isolation bag 206. As isolation bag 206 expands and contracts by fluid flowing in and out of isolation bag 206, cover assembly 421 is moved between different positions relative to platform 290. For example, when isolation bag 206 is empty, springs 444 resiliently urge rods 434 downward away from brackets 430 so that collars 440 of each spring assembly 420 rests on top of brackets 430. In this configuration, a minimum gap is formed between lid plate 499 and platform 290. As fluid enters isolation bag 206, isolation bag 206 begins to expand which produces a force that pushes cover assembly 421 and rods 434 upward and away from platform 290. As rods 434 are raised, springs 444 are compressed so as to increasingly resist expansion of isolation bag 206 and upward movement of cover assembly 421. Using springs 444 to resiliently compress isolation bag 206 between cover assembly 421 and platform 290 as isolation bag 206 expands and contracts, helps to maintain isolation bag 206 with a more uniform thickness, as opposed to bulging in the center. As a result, beads 179 disposed within isolation bag 206 are maintained closer to platform 290 and thus closer to the magnetic field produced by magnet assembly 294, thereby improving retention of beads 179 by magnet assembly 294.
With stop 534A in the advanced restraining position, cover assembly 421 can only raise to a first elevated position relative to platform 290, as previously discussed with regard to
One embodiment of the present disclosure includes means for resiliently restraining movement of cover assembly 421 away from platform 290. One example of such means is spring assemblies 420 as discussed above and the alternatives thereof. However, it is appreciated that a variety of other structures can accomplish the same function. For example, in one alternative, springs 444 could be replaced with elastomeric sleeves that encircle rods 434 or other forms of elastomeric material. In another alternative, in contrast to compressing each spring 444 between flange 442 and bottom of bracket 430, springs 444 could extend between and connect to collar 440 and the top of bracket 430 so that springs 444 are resiliently stretched as rods 434 move vertically upward. In other embodiments, resilient members can extend between and connect to cover assembly 421 and housing assembly 246 either directly or indirectly at spaced apart locations. As such, the resilient members again resiliently stretch as cover assembly is raised relative to platform 290. The resilient members can comprise springs, clastic bands, or other forms of elastomeric material. Other configurations can also be used.
Returning to
Clamp assembly 402 comprises an elongated clamp base 403 having a top surface 548 and an opposing bottom surface 550 that extend between opposing ends 552A and 552B. Ends 552A and 552B are slidably received within channels 544A and 544B of cover housing 422, respectively, so that clamp base 403 can slide vertically within channels 544 but is retrained from lateral movement. Centrally recessed on top surface 548 of clamp base 403 is a lower capture groove 554. Clamp assembly 402 also includes a clamp closure 407 that can be removably coupled to clamp base 403. Clamp closure 407 has a top surface 556 and an opposing bottom surface 558. An upper capture groove 555 is centrally recess in bottom surface 558. Fasteners 410A and 410B, such as screws or bolts, pass through clamp closure 407 on opposing sides of upper capture groove 555 and can selectively secure to clamp base 403 by threaded or other engagement. Upper capture groove 555 aligns with lower capture groove 554 when clamp closure 407 secured to clamp base 403. Lower capture groove 554 and upper capture groove 555 each typically have a semi-cylindrical configuration that can be the same size and shape. As such, aligned capture grooves 554, 555 can form a cylindrical opening. In other embodiments, capture grooves 554, 555 can other shapes such as semi-polygonal configurations.
As previously mentioned, clamp assembly 402 is used in part for centering and securing isolation bag 206 on top of platform 290.
Sheets 412 and 413 are typically comprised of a flexible polymeric film. The film can be single ply but more commonly comprises multiple layers that are laminated or co-extruded. For example, each sheet can comprise between 3-9 layers. The film for sheets 412 and 413 commonly have a thickness in a range between 4 mil-15 mil with between 4 mil-10 mil being more common. Furthermore, the film is typically sufficiently flexible that it can be rolled into a tube without plastic deformation and/or can be folded over an angle of at least 90°, 180°, 270°, or 360° without plastic deformation. The film is typically approved for contact with living cells and can be sterilized by irradiation. One example of an extruded material that can be used in the present invention is the Thermo Scientific CX3-9 film available from Thermo Fisher Scientific. The Thermo Scientific CX3-9 film is a three-layer, 9 mil cast film produced in a cGMP facility. The outer layer is a polyester elastomer coextruded with an ultra-low-density polyethylene product contact layer. Another example is the Thermo Scientific CX5-14 cast film also available from Thermo Fisher Scientific. The Thermo Scientific CX5-14 cast film comprises a polyester elastomer outer layer, an ultra-low-density polyethylene contact layer, and an ethylene vinyl alcohol (EVOH) barrier layer disposed therebetween.
The above discussion with regard to the formation, materials and properties of isolation bag 206 is also applicable to the other bags disclosed herein including mixing bag 210, collection bag 212 and media bags 216. However, depending on the bag and the intended use, the bags can also have multiple ports and different port configurations. Furthermore, in some applications, the bags can be three dimensional bags as opposed to pillow type bags and can have different volumes.
Isolation bag 206 has an outer perimeter edge 419 that typically has a size and shape comparable to a perimeter edge 386 of support plate 380 of platform 290. Having this similar configuration makes is easy to properly position and align isolation bag 206 on support plate 380. For example, in one embodiment, when isolation bag 206 is centered on support plate 380, any gap between perimeter edges 386 and 419 is typically less than 10 mm, 8 mm, 6 mm 4 mm or 2 mm. However, other gaps can also be used.
During assembly, as depicted in
Once isolation bag 206 is properly positioned on platform 290 and secured to clamp assembly 402, lid 426 is moved to the closed positioned, as shown in
One or more bubble sensors, such as bubble sensors 562A and 562B, can also be secured to front wall 466 of cover housing 422. Bubble sensors 562 communicate with electrical circuitry 53 (
In some applications it can be desirable to use an isolation bag having a smaller volume. For example, where a small volume of cell culture is being processed, it can be desirable in some situations to concentrate the cells in an isolation bag having a volume smaller than isolation bag 206. By using smaller bags for smaller volumes of cell culture being processed, it is easier to control flow of the cell culture within the bag, thereby optimizing mixing of the cells and beads, as discussed below. The use of smaller bags can also assist in controlling flow of the cell culture out of the bag. Furthermore, there is a lower risk of dead space within the bag that can potentially be damaging to the cells.
Although an isolation bag having a smaller volume and corresponding smaller overall shape could be used, such a bag can be problematic in that it can be difficult to properly center and retain on platform 290. Furthermore, it can be expensive to design, produce and stock bags of different sizes. Accordingly, one of the unique features of one embodiment of the present disclosure is the design of isolation bags where all of the isolation bags have the same outer size and geometer but where some of the bag have been further processed to reduce the internal volume of compartment 415. For example, depicted in
Seal line 423 can be formed using a conventional film bonding technique, such as those discussed above with regard to sealing the perimeter edge of isolation bag 206. Furthermore, seal line 423 can be formed as part of the initial production process of forming isolation bag 206A or can be added later after initial isolation bag 206 has been formed. Isolation bag 206A also includes one or more vent holes 429 that pass through sheet 412 and/or 413 and into inactive compartment 452. Vent holes 429 permit air that may have been captured within inactive compartment 452 during production to escape so that isolation bag 206A will lay flat. Depending on the intended use, the volume of active compartment 451 can be selectively adjusted during production by moving seal line 423 toward front edge 427A or toward back edge 427B.
Other alternative isolation bags can be also be formed by placing one or more seal lines as different locations. For example, depicted in
Isolation bag 206B also includes at least one vent hole 429A that passes through sheet 412 and/or 413 and into inactive compartment 452A and at least one vent hole 429B that passes through sheet 412 and/or 413 and into inactive compartment 452B. Vent holes 429 permit air that may have been captured within inactive compartment 452A and 452B during production to escape so that isolation bag 206B will lay flat. Depending on the intended use, the volume of active compartment 451 can be selectively adjusted during production by moving seal line 423A and 423B toward each other or laterally outward toward side edges 428A and 428B.
In the depicted embodiment of isolation bag 206B, seal lines 423A and 423B are disposed in parallel alignment. Depicted in
In addition to the above, the various disclosed isolation bags also have a number of other unique features. For example, in some embodiments, each isolation bag is only formed with a single port 416 as opposed to having two or more ports. Having a single port through which all fluid flows in and out, helps to ensure that liquid, beads, and/or cells do not collect in an unused port where they can stagnate and/or potentially release when undesired. Furthermore, the seal lines, particularly those having the V-shaped configuration shown in
In light of the above discussion of the components of bead processing system 14, one example of the method of using bead processing system 14 will now be described. Initially, cover panel 100 of bead processing apparatus 22 is moved to the open position to expose support panel 92. A sterilized consumable kit 170A is then nested onto support panel 92. As part of this nesting process, tubing 200 of line set 174A is engaged with pinch valves 128, pumps 124 and bubble sensors 136. Likewise, air filter assemblies 218 are coupled with rotational assemblies 132. Vial 166 containing bead mixture 177 is fluid coupled with bead vial coupler 208 and is mounted to bead vial retainer 148. Lid 426 of rocker drive 234 is also moved to the open position and isolation bag 206 placed on platform 290/support plate 380 and secured to clamp assembly 402 for proper centering thereon. Lid 426 is then moved to the closed position and secured closed by latches 518. Cover panel 100 can also be moved to the closed position so as to cover consumable kit 170A. If not previously done, the desired number of media bags 216 containing media can be fluid coupled to tubing sections 204H-204J and suspended from catches 99 of bag stands 36 or otherwise suspended. Finally, inlet end 202 of tubing section 204A can be fluid coupled to a source for delivering a cell culture to line set 174A. The source can be cell separator 12 or to some other container or source. Outlet end 211 of tubing section 204F if fluid coupled with cell expansion system 16, a container, or some other downstream processing system for either colleting or processing the isolated and active T-cells that are produced by bead processing system 14. It is appreciated that each of the foregoing assembly steps can be performed in a variety of different orders.
Next, bead pressing apparatus 22 is activated such as through display screen 56 or some other user interface. Each of the following process steps can be performed either automatically through the control of pre-programmed electrical circuitry 53 or can be controlled manually through manual inputs to a user interface. Upon activation of bead processing apparatus 22, all of pinch valves 128 are typically moved to a closed position so as to preclude fluid flow through the tubing section coupled with the pinch valves. In the following method steps where it is discussed that select pinch valves are opened, it is understood that the remaining pinch valves remain closed to control fluid flow through line set 174A. The following step are primarily made with reference to
Step 1: Inject air into isolation bag 206 for partial inflating. This can be accomplished by opening pinch valve 128B and air filter assembly 218A and activating pump 124A. Pump 124A draws air into tubing section 204A through air filter 221A and then pumps the air up through tubing section 204B to isolation bag 206. Injecting air into isolation bag 206 can substantially improve liquid flow and movement within isolation bag 206 by decreasing contact between the liquid and the surface of isolation bag 206. As such, the injected air, as discussed below, can assist with mixing of the cells and magnetic beads.
Based on the operation of different pinch valves 128, it is appreciated that fluids can travel through a variety of different paths to achieve an intended function. As such, the described process steps set forth herein are only examples and other process steps can be used to achieve the same function.
Step 2: Prime tubing and isolation bag 206 with media. Pinch valves 128B and 128G can be opened while pump 124B is used to pass media from media bag 216C, through pump 124B, bubble sensors 136A and 136C, up through tubing section 204B and into isolation bag 206.
Step 3: Inject air into mixing bag 210. Air filter assembly 218B and pinch valve 128E can be opened and pump 124B used to pump air from air filter 221B, through pump 124B and pinch valve 128E to mixing bag 120. Again, injecting air within mixing bag 120 can help facilitate mixing of fluid therein.
Step 4: Suspend beads 179 within vial 166. This can be accomplished by activating motor 162 to facilitate rotation of vial retainer 148 and vial 166 as previously discussed. In other embodiments, such as where beads 179 are retained within a suspended bag or other container, it may not be necessary to manually or mechanically suspend beads 179 prior to injection.
Step 4: Inject suspended bead 179 from vial 166 into mixing bag 210. Pinch valves 128C, 128D, and 128F can be opened and pump 124B used to pump bead mixture 177 from vial 166, through pinch valves 128C, 128D, and 128F and into mixing bag 210.
Step 5: Inject media into mixing bag 210. Pinch valve 128G and 128E can be opened and pump 124B used to pump media from media bag 216C through pinch valves 128G and 128E and into mixing bag 210. It is appreciated that the media could have been drawn from any of media bags 216A-C or any combination thereof. For simplicity, media in the present steps will be drawn from media bag 216C with the understanding that it could likewise be drawn from any of media bags 216A-C.
In each of the steps disclosed herein wherein a fluid or suspension is passed through tubing and into a bag or container, quantities of fluid/suspension being delivered can be measured based on known factors such as lengths of tubing, size of tubing, duration of pump operation and detections made by the bubble sensors as to the transition between the flow of gas and liquid through the tubing. Based on these factors, it can be determined as to when to open one of air filter assemblies to dispense filtered air into the tubing. One or more of the pumps can then be used pump the air so as to push the measured quantity of fluid/suspension in the container while also assisting in removing liquid from the tubing. Using air to remove liquid from the tubing helps to facilitate the precise measurement of future quantities of liquid/suspension to be passed through the tubing and into a bag or container.
Step 6: Mix beads 179 within mixing bag 120. Pinches valves 128E and 128F can be opened and pump 124B used to continuously flow the mixture within mixing bag 210 out through tubing section 204E, through pump 124B and back into mixing bag 120 through tubing section 204D in a continuous loop. This flow homogeneous suspends beads 179 with the media.
Step 7: Transfer beads 179 from mixing loop into isolation bag 206. Pinch valves 128F and 128B can be opened and pump 124B used transfer the suspension containing beads 179 from mixing bag 210, through pinch valve 128F and pinch valve 128B into isolation bag 206. It is noted that the number of beads 179 dispensed into the isolation bag 206 can depend on the number of cells that are to be delivered from cell separator 12 or other container into isolation bag 206. Thus, not all of beads 179 within mixing bag 210 and/or vial 166 may be disposed into isolation bag 206. Rather, in one embodiment, the number of beads 179 delivered into isolation bag 206 may be substantially equal to or only slightly larger than the number of cells that are to be delivered into isolation bag 206. Furthermore, in one alternative embodiment, mixing bag 210 can be eliminated and beads 179 can be directly dispended into isolation bag 206 from vial 166 or from some other bag or container housing beads 179 and fluid coupled with the tubing. As isolation bag 206 begins to fill with fluid, isolation bag 206 expands between cover assembly 421 and platform 290/support plate 380 so as to raise cover assembly 421.
Step 8: Flush tubing containing beads 179 into isolation bag. Pinch valves 128B and 128G can be opened and pump 124B used to pass media from media bag 216C through tubing into isolation bag 206.
Step 9: Move magnet assembly 294 to raised activation position and activate rocketing. Lift assembly 292 is activated to raise magnet assembly 294 to the raised activation position relative to platform 290/support plate 380. Concurrently or consecutively with the movement of magnet assembly 294, rocker drive 234 is activated to facilitate repeated rocking of platform assembly 232 having isolation bag 206 mounted thereon. As beads 179 mix within isolation bag 206 by rocking, they are attracted to and held against platform 290/isolation bag 206 by the magnetic force produced to magnet assembly 294. If desired, stop 534A could be moved to the advanced restraining position prior to filling of isolation bag 206. Although this would increase the rate at which beads 179 are attracted to platform 290/support plate 380, it also would limit that amount of fluid that could be processed within isolation bag 206.
In one method of operation, the magnetic field can be applied after completion of the rocking. For example, prior to, concurrently with, or after pumping media into isolation bag 206, rocker drive 234 can be activated to facilitate repeated rocking of platform assembly 232 having isolation bag 206 mounted thereon. This rocking can help unbind any agglomeration of beads 179 and suspend any unwanted matter. Rocker drive 234 can then be deactivated and lift assembly 292 activated to raise magnet assembly 294 to the raised activation position relative to platform 290/support plate 380. Beads 179 settle under gravity and are attracted to and held against isolation bag 206/support plate 380 by the magnetic force produced to magnet assembly 294. In one embodiment, rocker drive 234 can rearwardly or negatively tilt platform assembly 232/isolation bag 206 so that port 416 of isolation bag 206 is elevated. A small amount of air or media can then be passed through port 416 and into isolation bag 206 to help ensure that no beads 179 are retained within port 416. The magnetic field can then be applied to isolation bag 206 while in this rearward tilt position.
Step 10: Transfer liquid from isolation bag 206 into collection bag 212. Rocker drive 234 can be controlled to forward or positive tilt platform 290 so that port 416 is disposed lower than the remainder of isolation bag 206. This orientation helps to ensure that fluid freely flows out of isolation bag 206 through port 416. Pinch valves 128B and 128J can be opened and pump 124 used to transfer fluid from within isolation bag 206, through pinch valves 128B and 128J and into collection bag 212. Beads 179 are retained within isolation bag 206 under the magnetic force of magnet assembly 294. The above steps are used as a pre-wash beads 179 to remove any free antibodies in the mix not covalently bound to beads 179. The above washing of beads 179 can be repeated as needed.
Step 11: Transfer media into isolation bag 206. Pinch valves 128B and 128G can be opened and pump 124B used to transfer a defined quantity of media from media bag 216C into isolation bag 206 so that beads 179 are diluted to a desired concentration.
Step 12: Mix beads 179 within isolation bag 206. Lift assembly 292 is activated to lower magnet assembly 294 to the deactivation position and rocker drive 234 is activated to repeatedly rock platform 290/support plate 380 and isolation bag 206 disposed thereon, thereby homogenously mixing beads 179 within the media.
Step 13: Transfer cell culture to isolation bag 206. Pinch valves 128A and 128B can be opened and pump 124A used to transfer the cell culture from cell separator 12 or some other container or source connected to inlet end 202 into isolation bag 206.
Step 14: Facilitate isolation and activation of desired T-cells. Rocker drive 234 can be activated or remains activated from Step 12 to facilitate rocking of platform 290/support plate 380 and isolation bag 206 thereon which mixes beads 179 with the cell culture containing the desired T-cells. Beads 179 having a desired antibody thereon will bind to and activate desired T-cells as beads 179 come in contact with the desired T-cells during the mixing process. Such mixing can occur for an extended period of time. By way of example, and not by limitation, the mixing can be between 15 minutes to 60 minutes and more commonly between 20 minutes to 40 minutes. Other time durations can also be used.
Step 15: Flush port 416. With rocker drive 234 deactivated, pinch valves 128B and 128G can be opened and pump 124B used to pump a small quantity of media from media bag 216 into isolation bag 206 so as to remove any cells and/or beads 179 that may have been caught within port 416. This flushing of port 416 can occur while rocker drive 234 rearwardly tilts platform 290/support plate 380 and isolation bag 206 so that port 416 is elevated.
Step 16: Capture T-cells bound with beads 179. Lift assembly 292 can then be activated to raise magnet assembly 294 relative to platform 290/support plate 380 to the activation position. The magnetic force produced by magnet assembly 294 causes beads 179 and T-cells bound to beads 179 to be held against platform 290/support plate 380 while retained within isolation bag 206.
Step 17: Transfer liquid from isolation bag 206 into collection bag 212. Rocker drive 234 can positively tilt platform 290 so that port 416 is downwardly positioned. Pinch valves 128B and 128J can be opened and pump 124B used to pump fluid from isolation bag 206 into collection bag 212 while beads 179 and the cells attached thereto remain securely retained within isolation bag 206 under the magnetic force produced by magnet assembly 294. This step is to remove the negative cell fraction, i.e., the cells that did not bind to a bead, from isolation bag 206.
Step 18: Deliver media to isolation bag 206. Pinch valves 128B and 128G can be opened and pump 124B used to pump media from media bag 216 into isolation bag 206.
Step 19: Wash cells bound with beads 179. Lift assembly 292 is activated to move magnet assembly 294 down to the deactivation position. Consecutively or concurrently, rocker drive 234 is activated to facilitate mixing of the beads 179 with bound T-cells in the freshly delivered media. This mixing can again occur for an extended period of time. However, the primary objective of this rocking/mixing is to free any cells or other biological material that may have been unintentionally captured within isolation bag 206 so that it can be removed.
Step 20: Remove liquid from isolation bag 206. Lift assembly 292 is activated to move magnet assembly 294 up to the activation position so as to again capture beads 179 and the T-cells bound thereto. Rocker drive 234 positively tilts platform 290 so that port 416 is downwardly positioned. Pinch valves 128B and 128G can be opened and pump 124B used to pump liquid from isolation bag 206 to collection bag 212. The washing set forth in Steps 18-20 can be repeated as many times as desired.
Step 21: Deliver media into isolation bag 206. Rocker drive 234 is controlled to disengage tilt and lift assembly 292 is activated to lower magnet assembly 294 to the disengaged position. Pinch valves 128B and 128G can be opened and pump 124B used to pump media from media bag 216 into isolation bag 206. The quantity of media delivered is dependent upon the desired concentration for the T-cells as they are dispensed out of the system.
Step 22: Mix cells within isolation bag 206. Rocker drive 234 is activated to mix beads 179 with T-cells attached thereto within the freshly delivered media so as to produce homogenous mixture.
Step 23: Transfer suspension within isolation bag 206 to cell expansion system 16 or other downstream processing equipment or collection container. Pinch valves 128A and 128K can be opened and pump 124B used to transfer suspension within isolation bag 206 to cell expansion system 16 or to other downstream processing equipment or collection container through tubing section 204F. In yet another alternative, the suspension could be returned to the container that originally held the cell culture prior to mixing with the beads and then subsequently moved to cell expansion system 16. The above steps 21-23 can be repeated until the cell concentration within the downstream equipment has reached a desired level.
Returning to
As depicted in
Line set 174B is also substantially similar to line set 174A and like elements between line sets 174A and 174B are identified by like reference characters. Line set 174B includes tubing section 204A that extends between inlet end 202 and tubing section 204K. Disposed at inlet end 202 is a connector 203C which, in this case, can be coupled with cell expansion system 16, e.g., a bioreactor or container thereof, or can be coupled with a separate container or separate source for receiving a culture comprised of cells, beads 179 and a liquid medium. Air filter assembly 218A is disposed on tubing section 204A. Tubing section 204B now has the first end coupled to a media bag 216D housing a liquid medium and an opposing second end coupled with tubing section 204K. Similarly, tubing section 204C has a first end coupled with a media bag 216E housing a liquid medium and an opposing second end that is now coupled directly with tubing section 204K rather than tubing section 204B.
Line set 174B also other new tubing sections. Specifically, line set 174B includes a tubing section 204L having a first end connect to a first port 568 of a bead separation bag 570 and an opposing second end connected to the end of tubing section 204K. A tubing section 204M has a first end connected to a second port 569 of bead separation bag 570 and an opposing second end connected to tubing section 204K upstream of the connection with tubing section 204L. Bead separation bag 570 will be discussed below in greater detail and is another example of a processing bag. A tubing section 204N has a first end connected to a bead waste bag 572 and an opposing second connected to tubing section 204M. Finally, tubing sections 204O and 204P are provided. Tubing section 204P has a first end coupled to a connector 203D and an opposing second end coupled with tubing section 204L while tubing section 204P has a first end coupled to a connector 203E and an opposing second end coupled with tubing section 204L. Connectors 203C-D can comprise any of the connectors previously discussed with regard to connector 203A. Connectors 203D and E can be coupled with gene editing system 20, a container, or other downstream processing equipment in which it is desired to receive the cells separated from beads 179. In alternative embodiments, only one of tubing sections 204O and 204P may be required. As with line set 174A, line set 174B is typically preassembled with tubing 200 and bags 216, 570, and 572, being sterilized, such as by irradiation, as a closed system. Tray 172B can be attached to line set 174B prior to or after sterilization. During use, sterile connection processes can be used to connect connectors 203C-203E to their corresponding containers or equipment. In alternative embodiments, one or more of bags 216, 570, 572 can be attached to tubing 200 using a sterile connection process after tubing 200 is sterilized. Bag 216, 570, and 572 can have the same properties and be produced using the same materials and processes previously described with regard to isolation bag 206.
Turning to
Partially sealed between sheets 412 and 413 on front edge 427A are ports 568 and port 569. Ports 568 and 569 are spaced apart and disposed on opposite sides of partition 578. Furthermore, ports 568 and 569 are tubular and communicate with compartment 415. In the depicted embodiment, each port 568 and 569 includes collar 417 and stem 418 outwardly projecting therefrom. Each stem 418 is typically barbed, although not required. During use, stem 418 of port 568 is coupled with tubing section 204L and stem 418 of port 569 is coupled with tubing section 204M of line set 174B (
Partition 578 has a first end 580 that is connected to perimeter seal 414 of front edge 427A and extends toward back edge 427B to a terminal second end 582. In one embodiment, partition 578 is linear and extends along a linear axis 584 that is centrally disposed between side edges 428A and 428B and bisects compartment 415. Partition 578 has opposing sides 586 and 588 that extend along a length L1 of partition between opposing ends 580 and 582. In one embodiment, at least a portion of opposing sides 586 and 588 are disposed in parallel alignment. In another embodiment, for at least a majority of the length L1 of partition, opposing sides 586 and 588 are disposed in parallel alignment. Second end 582 can comprise an enlarged area 590 that protrudes perpendicular to partition 578 that is at least 75%, 100%, 150%, 200% or 250% wider than a width between opposing sides 586 and 588 spaced from enlarged area 590. Enlarged area 590 is designed to deflect fluid traveling along the length of partition 578 away from the center of compartment 415. This deflection increases the distance that fluid needs to travel between ports 568 and 569. Using the schematic of
The length L1 of partition 578 can be dependent on a number of factors such as fluid flow rate, the size of ports 568 and 569 and the size of compartment 415. L1 is at least 5 mm, 10 mm, 20 mm, 40 mm, 50 mm, 60 mm, 65 mm, 70 mm, 75 mm, 80 mm, 100 mm, 150 mm, 175 mm, or 200 mm or is in a range between any two of the foregoing values. In another embodiment, compartment 415 has a length L2 extending between perimeter seal 414 at front edge 427A and perimeter seal 414 at back edge 427B. Length L1 of partition 578 can be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, or 60% of L2 or in a range between any two of the foregoing. In other embodiments, length L1 of partition 578 may extend from about 10% to about 90% (e.g., from about 25% to about 85%, from about 25% to about 80%, from about 25% to about 75%, from about 25% to about 70%, from about 25% to about 65%, from about 25% to about 60%, from about 25% to about 55%, from about 25% to about 50%, from about 25% to about 45%, from about 25% to about 40%, from about 25% to about 35%, from about 40% to about 90%, from about 40% to about 80%, from about 40% to about 70%, from about 50% to about 90%, from about 55% to about 85%, etc.) of the length L2 of compartment 415.
In one embodiment, partition 578 is formed by welding together overlapping sheets 412 and 413 using one or more of the above described processes for forming perimeter seal 414. In an alternative embodiment, an insert, such as a polymeric insert, can be positioned between overlapping sheets 412 and 413. Sheets 412 and 413 can then be secured to the opposing sides of the insert, such as by welding or adhesive, so as to form partition 578. Partition 578 can also be formed by releasably pressing sheets 412 and 413 together at the location of partition 578 so as to form a sealed engagement between sheets 412 and 413. For example, this could be accomplished through the use of a clamp. Regardless of the specific configuration, partition 578 is configured to restrict fluid communication between port 568 and port 569. Specifically, partition 578 is configured so that beads 179 cannot pass therethrough and, in more common embodiments, partition 578 is liquid tight so that beads 179, cells, liquid media and other biological components disposed within compartment 415 cannot pass through partition 578. Instead, a fluid or other component flowing into compartment 415 through inlet port 568 must pass (or flow) around partition 578, (e.g., second end 582) in order to exit compartment 415 through port 569. Thus, a direct, linear access between ports 568 and 569 is prevented by partition 578.
As a result of partition 578, compartment 415 has or forms a fluid pathway that extends from port 568, around second end 582 of partition 578, to port 569. As previously discussed, the extended length of the fluid pathway, caused by the addition of partition 578, increases the retention or residence time of fluid within compartment 415 as compared to a bag that does not include partition 578. As discussed below in more detail, this increased residence time induces and/or increases exposure of beads 179 disposed in the fluid to the magnetic field produced by magnet assembly 294 within compartment 415 and thus increases the capture of beads 179 within bead separation bag 570. It is appreciated that bead separation bag 570 and partition 578 thereof can have a variety of different configurations and be used in a variety of different ways. Examples of alternative configurations, materials, properties, designs, and uses of bead separation bag 570 are disclosed in US Patent Publication No. 2019/0010435, published Jan. 10, 2019.
The minimum distance of serpentine flow path 603 for a bead separation bag of the type set out in
Embodiments of the type shown in
Further, the bead separation bag may be designed such that the minimum distance of serpentine flow path 603 between first port 568 to second port 569 by way of serpentine flow path 603 is at least is at least twenty (e.g., from about twenty to about one thousand, from about thirty to about one thousand, from about forty to about one thousand, from about fifty to about one thousand, from about sixty to about one thousand, from about seventy to about one thousand, from about eighty to about one thousand, from about eighty to about five hundred, from about sixty to about three hundred, from about sixty to about two hundred, etc.) times the distance of D1.
Turning to
Turning to
Clamp assembly 605 also includes a clamp closure 612 that can be removably coupled to clamp base 602. Clamp closure 612 has a top surface 614 and an opposing bottom surface 616. A first upper capture groove 618A and a spaced apart second upper capture groove 618B are recessed into bottom surface 558. Fasteners 620A and 620C pass through clamp closure 612 on opposing sides of upper capture groove 618A and 618B and can selectively secure to clamp base 602 by threaded or other engagement. Likewise, a fastener 620B pass through clamp closure 612 between upper capture grooves 618A and 618B and can selectively secure to clamp base 602 by threaded or other engagement. Fasteners 620 can comprise bolts, screws, clamps, pins or other types of removably fasteners. During assembly, upper capture grooves 618A and 618B align with lower capture grooves 610A and 610B, respectively, as clamp closure 612 is secured to clamp base 602. Lower capture grooves 610 and upper capture grooves 618 each typically have a semi-cylindrical configuration that can be the same size and shape. As such, aligned capture grooves 610 and 618 can form a cylindrical opening. In other embodiments, capture grooves 610 and 618 can other shapes such as semi-polygonal configurations.
It is appreciated that the entirety of clamp assembly 605 is freely slidable within channels 544A and 544B. As such, clamp assembly 605 can be easily removed from cover housing 422 and replaced with different clamp assembly, such as clamp assembly 402 depending on the configuration of bag that is being positioned on platform 290.
During assembly, as depicted in
With consumable kit 170B coupled with bead processing apparatus 22, as discussed above, bead processing system 18 is activated, such as through display screen 56 or some other user interface. Each of the following process steps can be performed cither automatically through the control of pre-programmed electrical circuitry 53 or can be controlled manually through manual inputs to a user interface. Upon activation of bead processing system 18, all of pinch valves 128 are typically moved to a closed position so as to preclude fluid flow through the tubing section coupled with the pinch valves. In the following method steps where it is discussed that select pinch valves are opened, it is understood that the remaining pinch valves remain closed to control fluid flow through line set 174B.
Step 1: Lift magnet assembly 241. Lift assembly 292 is used to elevate magnet assembly 294 relative to platform 290/support plate 380 to the raised activation position. Stop 534A can be moved to the advanced retraining position so as to limit the ability of cover assembly 421 to rise relative to platform 290/support plate 380.
Step 2: Transfer suspension comprised of cells, beads 179 and media through bead separation bag 570. With reference to
In one modified version of Step 2, pinch valves 128G and 128H can remain closed until bead separation bag 570 is at least 30%, 40%, or 50% filled with the mixture of cells, beads 179 and media. Rocker drive 234 then tilts platform 290/support plate 380 so that ports 568 and 569 are upwardly tilted. Pinch valves 128G and 128H are then opened. As additional suspension is pumped into bead separation bag 570 through port 569, any air within bead separation bag 570 flows out through port 568. Once all of the air is removed, rocker drive 234 then tilts platform 290/support plate 380 horizontally. The remainder of the suspension is then be pumped through bead separation bag 570 with the debeaded fluid flowing into gene editing system 20 or some other container.
Step 3: Flow media to output. Pinch valves 128C, 128F, 128G and 128H can be opened. Pump 124A can be used to pump media from media bag 216E, through bead separation bag 570 and to gene editing system 20 or some other container or system where the cells have been collected. This process helps to flush out any remaining cells within bead separation bag 570 and/or the tubing.
Step 4: Transfer beads 179 to bead waste bag 572. Lift assembly 292 lowers magnet assembly 294 to the lowered deactivation position. Pinch valves 128E, 128G and 128B are opened and pump 124B is used to transfer media from media bag 216D, though pump 124B, along tubing section 204L and into bead separation bag 570 through port 568. The media then flows within compartment 415 of bead separation bag 570 around second end 582 of partition 578 and then out of compartment 415 through port 569. As the media is passing through compartment 415, beads 179 are no longer secured to platform 290 under the magnetic force. As such, beads 179 are carried away with the flowing media out through port 569. The media and beads 179 leaving bead separation bag 570 continue to flow through tubing section 204N and into bead waste bag 572. In one method of operation, rocker drive 234 can be activated so that platform 290/platform assembly 232 and bead separation bag 570 are continuously rocking as the media is pump through bead separation bag 570, thereby helping to detach and remove beads 179 from bead separation bag 570. Furthermore, once the media is finished pumping through bead separation bag 570, filtered air from air filter assembly 218A can be pumped through bead separation bag 570 to further help remove beads 179.
As an additional optional step, in one embodiment a collection container is coupled to connectors 203D and 203E where the debeaded cells are collected. Once above Step 4 is completed, pinch valves 128I, 128D and 128A can be opened and pump 124B can be used to pump the cells from the collection container back to the original container coupled to connector 203C. The above process Steps 1-4 can then be repeated to remove any residual beads 179 mixed with the cells.
Embodiments of the present disclosure have a number of unique advantages over conventional systems. By way of example, in contrast to conventional systems where magnetic beads and/or cells must be processed or transferred through multiple different apparatus to achieve cell activation and isolation, one embodiment of the present disclosure achieves isolation and activation of T-cells within a single apparatus. Further, some conventional systems require repeated manual manipulation of the beads and/or cells to achieve cell isolation and activation. In contrast, one embodiment of the present disclosure achieves all necessary steps for activation and isolation without any manual manipulation or input other than loading and starting the system.
One embodiment of the present disclosure also uses a single bead processing apparatus with multiple different disposable consumable kits to achieve different functions, e.g., activation and isolation of T-cells versus separation of T-cells from magnetic beads. Such a system minimizes cost and storage requirements by having multiple different uses for the same apparatus. Furthermore, the single use consumable kits which can be discarded eliminates the need for cleaning or sterilizing and avoids risk of cross contamination.
Furthermore, one embodiment of the disclosure provides a simple and elegant solution of combining the disposable consumable kit with the processing apparatus by designing the consumable kit to simply and easily nest on a front panel of the apparatus while needed components of the apparatus pass through the consumable kit. The assembly is simple and intuitive, thereby limiting errors in assembly and improving efficiency in use. Furthermore, the components passing through the consumable kit, e.g., pinch valves, pumps, and sensors, are easy accessed and inspected and can be easily coupled with the line set of the consumable kit.
The present disclosure provides a unique solution to a common problem of magnetic beads clogging when attempting to dispense from a vial, especially when the vial has set for an extended period. That is, automatically or selectively rotating the vial using the disclosed vial retaining system just prior to dispensing, resuspends the magnetic beads and helps to eliminate clogging.
The automated lift assembly of the present disclosure is also unique and advantages in that it enables easy activation and deactivation of the magnet for optimal processing of the magnet beads without the need for manual manipulation or transferring the beads to different apparatus.
The disclosed restraining apparatus used with the cover assembly is also a significant improvement over known art in that it passively restrains bulging of the processing bags which can hamper or produce irregular bead separation. That is, the springs help to keep the bag in a certain maximum height so as to ensure in some applications that the beads passing through the bag are subjected to a sufficient magnetic field produced by the magnet. Furthermore, use of springs in the system is beneficial in some embodiments in that the restraining force increases as the expansion of the bag increases. In addition, the resilient nature of the springs assists in applying a force to the bag that helps with dispensing fluid from the bag. Further, the springs may be used to keep the bag at a specified maximum height to ensure that all of the fluid in the bag is exposed to sufficient magnetic force to attract the magnetic beads therein.
The ability to selectively activate the stop is also particularly useful in some embodiments. Depending on the situation and the processing conditions, use of the stop can provide significant versatility to the system by enabling an operator to quickly and easily select between limiting bag expansion to a predefined thickness so as to optimize the application of the magnetic force to the beads or permitting free expansion of the bag so as to optimize the amount of fluid that can processed.
The clamp assembly of the present disclosure is also unique in that it is free floating to account for bag and port movement through expansion and contraction of the bag, provides a simply mechanism for centering the bag on the platform for optimal application of the magnetic force, and can easily be switched out for use with other clamp assemblies designed for use with different bags.
Numerous other advantages are also found within the disclosed systems and parts and uses thereof.
Depicted in
Base assembly 646 comprises a housing 652 that bounds a compartment. The same electrical components and other hardware, including electrical circuitry 53 (
Stage 656 outwardly projects from side panel 666B. Stage 656 includes a front face 668 on which user interface 56 and activation switch 60 are disposed. Screen 56 and activation switch 60 can have the same designs, alternatives, and functions as previously discussed with bead processing apparatus 22. In general, user interface 56 (with display and graphical user interface described with respect to
It is appreciated that bead processing apparatus 22 can similarly be described in terms of having a main housing with a stage projecting therefrom. Specifically, with reference to
As depicted in
With reference to
Turning to
As will be discussed below in further detail, spaced apart tubular sleeves 720 are disposed and secured within slot 714. As depicted in
During assembly, retention frame 694 is secured to housing assembly 246 so that opening 719 aligns with magnet assembly 294. For example, floor 716 or other portions of retention frame 694 can be secured to flange 391 of housing assembly 246.
Support plate 692 is secured within opening 719 of retention frame 694 and is used to directly support isolation bag 206 (
With reference to
Contact 688 is positioned on top of insulation seal 690 so as to be spaced apart from and, more specifically, elevated above support plate 692. Contact 688 also forms a continuous loop and can have a configuration similar to insulation seal 690. For example, contact 688 will typically have a width the same as or small than a width of insulation seal 690 so that when positioned, contact 688 does not project out beyond insulation seal 690. Contact 688 is also made from an electrically conductive material, such as the same types of materials of which support plate 692 can be made. A liquid tight seal can also be formed between insulation seal 690 and contact 688. The seal between insulation seal 690 and contact 688 can be a result of the material properties of insulation seal 690 and/or an adhesive or sealant placed therebetween.
One or more slots 730 can be formed extending through contact 688. Slots 730 can be configured so that when contact 688 is positioned on top of insulation seal 690, ribs 728 pass through or can be pressed through slots 730 so as to secure contact 688 on top of insulation seal 690, thereby preventing or limiting lateral movement of contact 688 relative to insulation seal 690.
As also depicted in
Restraint 686 functions in part to properly position and secure in position insulation scal 690 and contact 688. As shown in
As depicted in
Platform assembly 678 also includes a restraining assembly 760 (shown in
With reference to
Turning to
As also shown in
Middle cover 782 includes a rail 800 that partially encircles an opening 801. Rail 800 has a U-shaped transverse cross section that bounds a channel 807 formed on a bottom thereof (
With continued reference to
Turning to
Lid body 820 comprises a perimeter wall 826 formed in a continuous loop that encircles an opening 828 passing therethrough. As depicted in
Returning to
When lid 768 is in the closed position, as shown in
Rocker assembly 648 operates in substantially the same way as previously described rocker assembly 34. For example, initially lid 768 is moved to the open position and isolation bag 206, as depicted in
Once isolation bag 206 is properly positioned, lid 768 is manually moved to the closed position so that isolation bag 206 is positioned between lid 768 and support plate 692 and, more specifically, between lid plate 822 of lid 768 and support plate 692. Rocker assembly 648 can be configured so that lid 768 automatically locks when moved to the closed positioned or can require manual activation to lock. In the exemplary embodiment, the locking is achieved by latches 796 engaging with catch 824 and, more specifically, latch elements 798 engaging with catch elements 844. In alternative embodiments, a single latch element 798 and catch element 844 can be used or alternative locking structures can be used.
Once lid 768 is in the locked position, a liquid can be delivered into isolation bag 206 through tubing 200. As isolation bag 206 inflates with liquid, cover assembly 764 and track 777 are raised relative to support plate 692. Specifically, with lid 768 in the closed position, isolation bag 206 can be pressed between or be disposed directly adjacent to support plate 692 and lid plate 822. Accordingly, as isolation bag 206 inflates with liquid, isolation bag 206 outwardly pushes against lid 768/lid plate 822 causing all of cover assembly 764/track 777 to rise relative to support plate 692 by rods 770 moving upward. However, as previously discussed, as rods 770 move upward, springs 772 are compressed which produces a resilient downward force by lid 768/lid plate 822 onto isolation bag 206. This downward force helps to both secure isolation bag 206 to limit movement and flatten isolation bag 206, i.e., limit bulging in the middle, so that it has a more uniform thickness. As previously discussed with regard to rocker assembly 34, this flattening of isolation bag 206 can be helpful in the application of the magnetic field to the liquid within isolation bag 206.
In one exemplary embodiment of rocker assembly 648, restraining assembly 760 can also include stop assemblies 526A-D of rocker assembly 34 (
Rocker assembly 648 differs in part from rocker assembly 34 in that clamp assembly 402 (
In the same manner as previously discussed with regard to rocker assembly 34, rocker drive 234 of rocker assembly 648 can be selectively or automatically activated to tilt platform assembly 232/isolation bag 206 and/or to repeatedly rock platform assembly 232/isolation bag 206 relative to mount assembly 230, as needed for the intended use. In addition, in the same manner as previously discussed with regard to rocker assembly 34, lift assembly 292 of rocker assembly 648 can be selectively or automatically activated to raise and lower magnet assembly 294 relative to support plate 692, as needed for the intended use.
In one exemplary embodiment, cover assembly 764 of rocker assembly 648 can automatically detect leaking of liquid from isolation bag 206 or any other bag disposed on support plate 692 and capture a liquid leaking therefrom. For example, with reference to
It is appreciated that the leak detection components are optional and can be eliminated. In that case, insulation seal, contact, and/or restraint 686 can be eliminated. Furthermore, where the leak detection components are removed, support plate 692 need not be made from an electrically conductive material but could be made from other materials, such as plastics or composites. As liquid continues to leak from bag 848, it is captured within cavity 969 by being laterally restrained by restraint 686. As a result of lid plate 822 being translucent, the leaked liquid can be visually detected below lid plate 822. Channels 840 are formed through inner wall 830 of lid 768 so that as the liquid continues to rise with cavity 969, the liquid will eventually flow through channels 840 so as to be disposed on top of lid plate 822, i.e., within upper cavity 829. As a result, the leaked fluid can be more casily visually detected. Lid body 820 projecting above lid plate 822 restrains spilling of the leaked liquid outside of upper cavity 829. The sloping of retaining wall 838 provides improved visibility to upper cavity 829 for detecting the leaked liquid while also directing any liquid back toward lid plate 822 that may splash onto retaining wall 838.
Returning to
Catch 868 comprises a plate 870 having an upper end 882 and an opposing lower end 884. A pair of spaced apart C-shaped fingers 886A and 886B project from upper end 886 of plate 870. Finger 886A and 886B are configured to releasably snap fit onto stems 880A and 880B. This coupling enables catch 868 to be releasable secured to arm 876 to simplify the attachment and/or removal of bags from catch 868, as discussed below, and also enables catch 868 to pivot on arm 876 to facilitate attachment, removal, or manipulation of bag on catch 868 while catch 868 is retained on arm 876. Lower end 884 of catch 868 terminates at bottom edge 888 having a plurality of spaced apart notches 872 upwardly recessed therein. Plate 870 also includes a plurality of L-shaped fingers 874 with each finger 874 projecting into a corresponding notch 872. During use, bags used in the operation of bead processing apparatus 642, such as waste bags, collection bags, and/or bags containing media, biological product, beads and/or other product, can be adjacently supported on catch 868 by supporting a hanger of the bags on a corresponding finger 874. The hanger my extend from the bags or be formed by a hole extending through a perimeter of the bags. Each plate 870 can have at least 2, 4, 6, 8, 10 or more notches 872 with a corresponding finger 874 therein. Other numbers can also be used.
Returning to
Turning to
Tray 892A also includes previously discussed tube restraints 186 mounted on and outwardly projecting from the top face thereof. Tube restraints 186 support and secure the tubing of line set 894A. In contrast to tray 172A, tray 892A does not include bag restraint 192.
As depicted in
Line set 894A includes tubing sections 908A-9081 that extend between tray 892 and corresponding bags. For example, tubing section 908A connects to isolation bag 206, tubing sections 908B, 908C, 908E and 908F connect to media bags 216A-D, respectively, tubing section 908D connects to an output bag 928, tubing section 908G connects to an input bag 930, tubing section 908H connects to a bead bag 932, and tubing section 9081 connects to an output bag 934.
Bead processing apparatus 642 also includes a tubing restraint 910 that is used to releasably secure and organize tubing sections 908A-9081. Tubing restraint 910 is secured to an upper end of housing 652 above support panel 662. More specifically, with reference to
Bead processing apparatus 642 also distinguishes over bead processing apparatus 22 in that bead processing apparatus 642 eliminates bead vial retainer 148 and the associated use of vial 166 and bead vial coupler 208. In contrast to using vial 166 and related hardware, line set 894A incorporates the use of bead bag 932 coupled with tubing section 908H, as referenced above. Turning to
Prior to operation of bead processing system 14A, isolation bag 206 (or any other isolation bag disclosed herein) is enclosed within rocker assembly 648 and the remaining bags are typically secured to bag stands 650A and/or 650B, as previously discussed. The operation of assembled bead processing system 14A is then similar to the previously discussed operation of bead processing system 14. For example, primarily with reference to
Step 1: Inject air into isolation bag 206 for partial inflating. This can be accomplished by opening pinch valve 128J and activating pump 124 to pump air from air filter assembly 218B isolation bag 206. Injecting air into isolation bag 206 can substantially improve liquid flow and movement within isolation bag 206 by decreasing contact between the liquid and the surface of isolation bag 206. As such, the injected air, as discussed below, can assist with mixing of the cells and magnetic beads.
Based on the operation of different pinch valves 128, it is appreciated that fluids can travel through a variety of different paths to achieve an intended function. As such, the described process steps set forth herein are only examples and other process steps could be used to achieve the same function.
Step 2: Prime tubing and isolation bag 206 with media. Pinch valves 128A and 128F can be opened while pump 124 is used to pass media from media bag 216D, through pump 124B, and into isolation bag 206. It is appreciated media can be provided from any of media bags 216A, 216B, 216C or 216D. Thus, although media is primarily discussed herein as being drawn from media bag 216D, it is appreciated that it can commonly be drawn from one or more of the other media bags. Commonly, media will be drawn from one media bag until empty or close to empty and then be drawn from another media bag.
Step 3: Suspend beads within bead bag 932. As shown in
Step 4: Inject suspended beads 179 from bead bag 932 into isolation bag 206. Pinch valves 128A and 128H can be opened and pump 124 used to pump suspended beads 179 from bead bag 932 to isolation bag 206. In some embodiments, once a quantity of suspended beads 179 has been pumped out of bead bag 932, additional media can be pumped into bead bag 932 to resuspend any beads 179 that may have been retained within bead bag 932. This new suspension can then be pumped into isolation bag 206. The above process of adding media into bead bag 932 can be repeated multiple times to ensure that all of beads 179 are flushed out of bead bag 932 and the related tubing and into isolation bag 206.
Step 5: Pre-wash beads 179 within isolation bag 206. In one embodiment, beads 179 can be prewashed within isolation bag 206 to help remove any unwanted matter from within isolation bag 206. For example, this step can be used to remove any free antibodies in the mix not covalently bound to beads 179. In one exemplary embodiment, pump 124 can be used to pump media from one of media bag 216 into isolation bag 206. Prior to, concurrently with, or after pumping media into isolation bag 206, rocker drive 234 is activated to facilitate repeated rocking of platform assembly 232 having isolation bag 206 mounted thereon. This rocking can help unbind any agglomeration of beads 179 and suspend any unwanted matter. Rocker drive 234 can then be deactivated and lift assembly 292 activated to raise magnet assembly 294 to the raised activation position relative to support plate 692. Beads 179 settle under gravity and are attracted to and held against isolation bag 206/support plate 692 by the magnetic force produced to magnet assembly 294. In one embodiment, rocker drive 234 can rearwardly or negatively tilt platform assembly 232/isolation bag 206 so that port 416 of isolation bag 206 is elevated. A small amount of air or media can then be passed through port 416 to help ensure that no beads 179 are retained therein. The magnetic field can then be applied to isolation bag 206 while in this rearward tilt position. With magnet assembly 294 still in the raised activation position so that beads 179 are secured against isolation bag 206, rocker drive 234 can the tilt platform assembly 232/isolation bag 206 forward/positively so that port 416 is now lowered. This orientation helps to ensure that fluid freely flows out of isolation bag 206 through port 416. Pump 124 is then used to pump the liquid out of isolation bag 206 and into outlet bag 928, or other container, such as by opening pinch valves 128A and 128D. The above pre-washing process of beads 179 can be repeated any desired number of times, such as at last one, two, three or more time.
Step 6: Transfer media into isolation bag 206. Pump 124 is used to pump a defined quantity of media into isolation bag 206 so that beads 179 are diluted to a desired concentration. For example, pinch valves 128A and 128F can be opened and pump 124 used to pump media from media bag 216D into isolation bag 206.
Step 7: Suspend beads 179 within isolation bag 206. Lift assembly 292 is activated to lower magnet assembly 294 to the deactivation position and rocker drive 234 is activated to repeatedly rock platform 290 and isolation bag 206 disposed thereon, thereby homogenously suspending beads 179 within the media.
Step 8: Transfer cell culture to isolation bag 206. Pump 124 is used to pump the cell culture generated from cell separator 12 into isolation bag 206. In the depicted embodiment, the cell culture is disposed within input bag 930. Thus, pinch valves 128A and 128G can be opened and pump 124 used to pump the cell culture from input bag 930 to isolation bag 206. In other embodiments, pump 124 can be used to pump the cell culture directly from cell separator 12 or some other container.
Step 9: Facilitate isolation and activation of desired T-cells. Rocker drive 234 is activated or remains activated from Step 8 to facilitate rocking of platform 290 and isolation bag 206 thereon which mixes beads 179 with the cell culture containing the desired T-cells. Beads 179 having a desired antibody thereon will bind to and activate the desired T-cell as bead 179 comes in contact with the desired T-cell during the mixing process. Such mixing can occur for an extended period of time. By way of example, and not by limitation, the mixing can be between 15 minutes to 60 minutes and more commonly between 20 minutes to 40 minutes. Other time durations can also be used.
Step 10: Capture T-cells bound with beads 179. Lift assembly 292 is activated to raise magnet assembly 294 relative to support plate 692 to the activation position. The magnetic force produced by magnet assembly 294 causes beads 179 and T-cells bound to beads 179 to be held against isolation bag 206/support plate 692 while retained within isolation bag 206. As with the prewashing step, in one exemplary embodiment, rocker drive 234 can first rearwardly tilt platform assembly 232/isolation bag 206 so that port 416 of isolation bag 206 is elevated. A small amount of air or media can then be passed through port 416 to help ensure that no beads 179/cells are retained therein. The magnetic field can then be applied to isolation bag 206 while in this rearward tilt position.
Step 11: Transfer liquid from isolation bag 206 into collection bag 212. Rocker drive 234 can positively tilt platform 290 so that port 416 is downwardly positioned. Pump 124B can then be used to pump fluid from isolation bag 206 into output bag 928 while beads 179 and the cells attached thereto remain securely retained within isolation bag 206 under the magnetic force produced by magnet assembly 294. For example, pinch valves 128A and 128D can be opened and pump 124 used to pump the liquid from isolation bag 206 to output bag 928. This step is to remove the negative cell fraction, i.e., the cells that did not bind to a bead 179, from isolation bag 206.
Step 12: Wash cells bound with beads 179. Pump 124 can be used to pump media from one of media bags 216 into isolation bag 206. Lift assembly 292 is activated to move magnet assembly 294 down to the deactivation position. Consecutively or concurrently, rocker drive 234 is activated to facilitate mixing of beads 179 bound with T-cells in the freshly delivered media. This mixing can again occur for an extended period of time. However, the primary objective of this rocking/mixing is to free any cells or other biological material that may have been unintentionally captured within isolation bag 206 so that it can be removed. Lift assembly 292 is activated to move magnet assembly 294 up to the activation position so as to again capture beads 179 and the T-cells bound thereto. Again, in one embodiment, rocker drive 234 can rearwardly or negatively tilt platform assembly 232/isolation bag 206 so that port 416 of isolation bag 206 is elevated. A small amount of air or media can then be passed through port 416 to help ensure that no beads 179/cells are retained therein. The magnetic field can then be applied to isolation bag 206 while in this rearward tilt position. Rocker drive 234 is then positively tilts platform 290 so that port 416 is downwardly positioned. Pump 124 is then used to pump liquid from isolation bag 206 to collection bag 212. This washing step can be repeated as many times as needed, such as at least one, two, three or more times.
Step 13: Resuspend beads 179 with bound cells. Rocker drive 234 is controlled to disengage tilt and lift assembly 292 is activated to lower magnet assembly 294 to the disengaged position. Pump 124B is used to pump media from a media bag 216 into isolation bag 206. The quantity of media delivered is dependent upon the desired concentration for the T-cells as they are dispensed out of the system. Rocker drive 234 is activated to mix beads 179 with T-cells attached thereto within the freshly delivered media so as to produce homogenous mixture.
Step 14: Transfer suspension within isolation bag 206 to output bag 934 or directly to cell expansion system 16 or other downstream processing equipment or collection container. For example, pinch valves 128A and 128K can be opened and pump 124 used to transfer suspension within isolation bag 206 to output bag 934 or directly to some other downstream equipment or container. In yet another alternative, the suspension could be returned to the input bag 930. The above steps 13 and 14 can be repeated until all of the isolated cells have been removed and/or the cell concentration within the downstream bag/equipment has reached a desired level.
In the above described process, bead processing apparatus 22 uses magnetic beads 179 to isolate and activate desired T-cells. However, in an alternative embodiment, bead processing apparatus 22 can be used with magnetic beads 179 in an opposite process. That is, in contrast to having beads 179 bind to the desired cells, beads 179 can be designed to bind to cells that are not wanted within a mixture of cells. As a result, by using the above process, the unwanted cells can be bound to beads 179 and secured within isolation bag 206 by magnet assembly 294, while the desired cells are washed out of isolation bag 206 and transferred to a collection bag or other downstream apparatus for further processing.
As with bead processing apparatus 22, bead processing apparatus 642 can also be used in the formation of a bead processing system 18A which is an alternative bead processing system 18 (
As depicted in
Line set 894B includes tubing sections 954A-954F that extend between tray 892B and corresponding bags. For example, tubing sections 954A and 954B both connect to bead separation bag 570 (or any other bead separation bag disclosed herein), tubing sections 954C and 954E connect to media bags 216A and B, respectively, tubing section 954D connects to an output bag 928, tubing section 954F connects to an input bag 958.
Prior to operation of bead processing system 18A, bead separation bag 570 (or any other bead separation bag, such as bead separation bag 600) is enclosed within rocker assembly 648, the same as previously discussed with isolation bag 206, and the remaining bags are typically secured to bag stands 650A and/or 650B, as previously discussed. The operation of assembled bead processing system 18A is then similar to the previously discussed operation of bead processing system 18. For example, primarily with reference to
Step 1: Lift magnet assembly 241. Lift assembly 292 is used to elevate magnet assembly 294 relative to platform 290/support plate 692 to the raised activation position. In one embodiment, stop assemblies 526A-D can be activated so as to move to the advanced retraining position that limits the ability of rods 770 to rise and thereby limits the ability of cover assembly 421/lid plate 822 to rise relative to platform 290/support plate 692.
Step 2: Transfer suspension comprised of cells, beads 179 and media through bead separation bag 570. Pump 124 is used to transfer the culture comprised of cells, beads 179 and media that has been processed in cell expansion system 16 through separation bag 570. In the depicted embodiment, the processed cells, beads 179 and media are being dispensed from input bag 958. However, in other embodiments, they can be dispensed directly from cell expansion system 16 or some other container coupled to tubing section 954F. The suspension is pumped into bead separation bag 570 through port 569. The suspension then flows within compartment 415 of bead separation bag 570 (
In one modified version of Step 2, pinch valves 128B, 128I, and/or 128E can remain closed until bead separation bag 570 is at least 30%, 40%, or 50% filled with the mixture of cells, beads 179 and media. Rocker drive 234 then rearwardly tilts platform 290 so that ports 568 and 569 are upwardly tilted. Pinch valves 128B, 128I, and/or 128E are then opened. As additional suspension is pumped into bead separation bag 570 through port 569, any air within bead separation bag 570 flows out through port 568. Once all of the air is removed, rocker drive 234 then tilts platform 290 horizontally. The remainder of the suspension is then pumped through bead separation bag 570 with the debeaded fluid flowing into output bag 956 or some other container or machine.
Step 3: Flow media to output. Pump 124 is then used to pump media from media bag 216B, through bead separation bag 570 and to output bag 956. This process helps to flush out any remaining cells within bead separation bag 570 and/or the tubing.
Beads 179 can remain contained within separation bag 570 and be disposed of or otherwise further processed with line set 894B. Bead processing apparatus 642 has the same benefits and functional properties a previously discussed with regard to bead processing apparatus 22. However, bead processing apparatus 642 is simpler in design, safer to operate, and easier to operate than bead processing apparatus 22.
Provided herein are instruments and workflows for cell processing (see, e.g.,
Further, in some instances, the order of steps set out in
Further, some of all of the steps set out in
In many instances, workflows set out herein will be directed to the generation of CAR-T cell populations.
The first step in workflows of
Once the desired number of leukocytes have been harvested, the resulting cell population is generally washed (Step 2) to remove, for example, anticoagulant(s). In an early step, the cell population may be enriched for lymphocytes (Step 2) using, for example, a counterflow centrifugal elutriation system (e.g., a GIBCO™ CTS™ ROTEA™ Counterflow Centrifugation System, Thermo Fisher Scientific), which can separate cells by size and density.
Isolation of desired cells types (Step 3) (e.g., total T cell and T cell subsets, CD34+ stem cells, natural killer cells, as well as other cell types), may be performed using ligands having binding affinity for cell surface receptors. Examples of such cell surface receptors include CD3, CD4, CD5, CD6, CD8, CD25, CD27, CD28, CD137, and CD278 (ICOS). Further, isolation and activation may occur simultaneously. As an example, a mixed population of leukocytes may be exposed to anti-CD3 and anti-CD28 antibodies under conditions in which T cells are separated from other leukocytes and the combination of the anti-CD3 and anti-CD28 antibodies results in T cell activation.
By way of specific example, T cells may be isolated based upon the presence on their surfaces of CD3 markers. Some isolation methods use positive isolation of cells with the desired surface marker. An exemplary method for T cell isolation is as follows. A mixed leukocyte population is incubated (e.g., 20-30 minutes at 4° C.) with magnetic beads with anti-CD3 antibodies located on the bead surfaces (e.g., D
In many instances, once T cells have been isolated, these cells will be contacted with an anti-CD28 antibody capable of stimulating CD28 receptors, resulting in T cell activation. The anti-CD28 antibodies may be bound to magnetic beads. Further, as noted above, in some instances, the capture of T cells by anti-CD3 antibodies and stimulation of T cells by a combination of anti-CD3 and anti-CD28 antibodies may occur simultaneously. A commercially available product that may be used for both T cell isolation and activation is entitled CTS™ D
Samples of T cells exposed to anti-CD3 antibodies and/or anti-CD3 and anti-CD28 antibodies may be analyzed for activation levels. One type of assay for measuring activation is based upon screened T cells for CD25 (the alpha chain of the IL-2 receptor) expression levels. While the CD25 marker is found on a number of peripheral blood lymphocytes (e.g., regulatory and resting memory T cells), CD25 expression is generally considered to be a prominent T cell activation marker. Thus, methods provided herein include methods for measuring the percentage of activated T cells in a population. This percentage is calculated by comparing the number of nonactivated T cells with the number of activated T cells. Of course, the percentage of activated T cells will change with the duration of exposure to activation signals and as activated T cells expand.
Magnetic beads may be added to sample of biological cells by any number of means. In many instances, beads will be introduced into containers containing biological cells through interaction of the apparatus with a bead vial. However, beads may be introduced into containers by other means. For example, beads may be introduced into bags by injection through a syringe connected to a bag port. When magnetic beads are contacted with biological cells by means not using a vial, then the bead vial coupler 208, as well as other bead vial feature of the apparatus may not be present.
Step 4 in the exemplary workflow of
While expansion conditions may vary conditions, activated T cells may be cultured, for example, at 37° C. and 5% CO2 in cell culture medium (e.g., CTS™ OPTMIZER™ media without phenol red plus 2-5% CTS™ Immune Cell SR (Thermo Fisher Scientific, cat. nos. A3705001 and 15710-049). Further, cytokines and fresh medium may be added every 1-3 days to maintain a cell concentration of 0.5-2×106 cells/ml. T regulatory cells (Tregs) may be expanded in medium containing 100 ng/ml rapamycin (e.g., Thermo Fisher Scientific, cat. no. PHZ1235) and 300 IU IL-2/ml (e.g., Thermo Fisher Scientific, cat. no. PHC0027). CMV stimulated T cells may be expanded in 100 IU IL2/ml. Th17 cells may be expanded in medium containing polarizing cytokines (IL-6, IL-13, IL-23, and TGF-13, all, for example, from Thermo Fisher Scientific CA USA) in presence of anti-IL-4 and anti-IFN-γ neutralizing antibodies (both, for example, from Thermo Fisher Scientific, CA US) as described in Paulos et al. (Paulos et al., Science Transi. Med 55: 55ra78 (2010)). Further, 100 IU IL-2/ml may be added day 3 post-activation. IL2 may also be added one day 0, for example, during dilution of cells from the isolation bag to the output.
Expansion of cells (Steps 4 and 7) will generally occur under conditions suitable for cell division. Media that may be using for expansion include CTS™ OPTMIZER™ T-Cell Expansion SFM (Thermo Fisher Scientific, cat. no. A3705001) and LYMPHOONE™ T-Cell Expansion Xeno-Free Medium (Takara Bio, cat. no. WK552S).
In some instances, it may not be necessary or desirable to separate magnetic supports from cells. In such instances, Step 5 set out in
It will generally be desirable at some point in workflow to separate magnetic supports from cells. In some instances when the supports are bound to the cells, it will be necessary or desirable to disrupt the binding of the supports to the cells. For example, the cells and the magnetic supports may be associated with each other through conjugation of antibodies to magnetic supports.
Disruption of association of magnetic supports from cells may be accomplished by a number of means. Some exemplary cell release features are represented in
Another way that disruption of association of magnetic supports from cells may be accomplished is by competitive release. For example, “L” in
One processes that may be employed for the dissociation of cells and supports make use of anti-biotin antibodies. For example, a two antibody linking systems can be used where a first biotinylated antibody is used wherein the first antibody has binding affinity for a cell surface protein (e.g., a receptor). A second anti-biotin antibody may be conjugated to a support. Thus, the cells are associated with supports, in part, through the binding of the binding of the bead bound second antibody (anti-biotin antibody) to the first antibody (biotinylated, anti-cell surface protein antibody). Disruption of association between the supports and cells may be mediated by disruption of the binding of the second antibody to the biotin of the first antibody. This may be accomplished by the contacting the cell/bead complex with a releasing agent (e.g., biotin or biotin derivative). Compositions and methods related to the above are contained in U.S. Pat. No. 10,196,631.
It has been found that when beads bind to the surfaces of cells through antibodies, the bead are often released by the cell as the cells expand, as a result of downregulation of the cell surface marker bound to the antibody on the beads. Thus, in many instances, cells may be separated from beads without the performance of an active dissociation step. In many such instances, separation of cells from beads will occur after cells (e.g., T cells) have been expanded for from about 4 to about 21 days (e.g., from about 4 to about 21, from about 5 to about 21, from about 6 to about 21, from about 5 to about 14, from about 5 to about 12, from about 5 to about 10, from about 6 to about 14, from about 6 to about 12, from about 6 to about 10, etc. days). Also in many such instances, separation of cells from beads will occur after cells (e.g., T cells) after greater than 70% (e.g., from about 70% to about 99%, from about 70% to about 98%, from about 70% to about 95%, from about 70% to about 90%, from about 70% to about 85%, from about 75% to about 99%, from about 80% to about 99%, from about 85% to about 99%, from about 85% to about 95%, from about 85% to about 90%, from about 90% to about 99%, from about 90% to about 97%, etc.) of the cells are dissociated from beads. Of course, the user can separate the beads from the cells at any time point. However, in many instances, separation of the beads from the cells at an early time point will result of doing in a lower cell yield than if the beads are separated at a later time point. This is so because more beads will often be captured by the magnet at the earlier time point. Thus, in many instances, separation of cells from beads will occur will occur at a time point where cell yields are greater than 80% (e.g., from about 80% to about 99.5%, from about 85% to about 99.5%, from about 88% to about 99.5%, from about 90% to about 99.5%, from about 95% to about 99.5%, from about 98% to about 99.5%, from about 80% to about 98%, from about 85% to about 98%, from about 90% to about 98%, etc.). Cell yield is measured in such instances by the percentage of the total number of cells present being separated from the beads.
Cells not bound to or associated with magnetic supports may be separated from these supports using a magnetic field to capture the supports under conditions where the cells are not associated with a magnetic material. The examples below set out experiments and data using magnetic beads. Further, using instruments such as those set out herein, it has been shown to be possible to remove greater than 99% of the magnetic beads present in a bead/cell mixture. Thus, provided herein are methods for separation of magnetic beads from cells present in a bead/cell mixture, wherein greater than 95% (e.g., from about 95% to about 99.9999%, from about 97% to about 99.9999%, from about 98% to about 99.9999%, from about 99% to about 99.9999%, from about 98% to about 99.95%, from about 98% to about 99.90%, etc.) of the beads originally present are separated from the cells.
As set out in the examples below, debeading of a bead/cell mixture may be performed using bead processing system 18, as previously discussed herein, in conjunction with a bag of the type, for example, shown in
The flow rate through the bag is also a parameter that affects the debeading efficiency. It has been found the slower flow rates result in more efficient debeading and, hence, higher cell purity with respect to the number of beads present post-debeading.
Flow rates may vary from about 10 ml/min to 400 ml/min (e.g., from about 10 ml/min to 400 ml/min, from about 20 ml/min to 400 ml/min, from about 30 ml/min to 400 ml/min, from about 40 ml/min to 400 ml/min, from about 40 ml/min to 300 ml/min, from about 40 ml/min to 200 ml/min, from about 40 ml/min to 100 ml/min, from about 50 ml/min to 300 ml/min, from about 50 ml/min to 200 ml/min, from about 45 ml/min to 150 ml/min, etc.). Often the flow rate will be selected to allow for removal of at least 99% (from about 99% to about 99.9999%, from about 99% to about 99.99%, from about 99.5% to about 99.9999%, from about 99.8% to about 99.9999%, from about 99% to about 100%, etc.) of the beads. Thus, provided herein are methods for the separation of at least 99% of bead present in a bead/cell mixture. In many instances, such separation methods will result in the bead to cell ratio decreasing by a factor of at least 7 (e.g., a factor of from about 2 to about 7, from about 3 to about 7, from about 4 to about 7, from about 2 to about 7, from about 5 to about 7, from about 2 to about 6, from about 3 to about 6, from about 4 to about 6, from about 5 to about 6, from about 2 to about 5, from about 3 to about 5, from about 3 to about 5, etc.). By way of example, going from a bead to cell ratio of 3:1 to a bead to cell of 0.3:1 would be a one factor decrease. Further, going from a bead to cell ratio of 3:1 to a bead to cell of 0.03:1 would be a two factor decrease.
Example 3 shows data generated using bead processing systems and methods set out herein. These data show that over 99.99% of the beads were removed in the debeading process. It was further estimated that only 1 bead per 450,000 of the original beads present co-localized with the debeaded cells. Results from three replicates using conditions similar to those of Example 3 but with a 200 ml/min flow rate were 0, 1 and 2 total beads and, thus, 0 (0 beads), 13 (1 bead), a 27 (2 beads) co-localized beads per 3×106 lysed cells. Thus, in some instances, cell may be separated from as much as 100% of the originally present.
Step 6 set out in
T cells, for example, may be engineered to expression a chimeric antigen receptors (CARs). CARs are receptors that are designed to bind to cell surface proteins on target cells (e.g., human leukocyte antigen antigens. Further, T cells may be engineered to express CARs on their surface, allowing them to recognize specific antigens (e.g., tumor antigens). These CAR T cells can then be expanded by methods of the present invention and infused into the patient. Typically, this will occur after the T cells are washed (Step 8 in
In some instances, cells (e.g., a T cell) may be engineered to express a CAR wherein the CAR T cell exhibits an antitumor property. CARs can be designed to comprise an extracellular domain having an antigen binding domain fused to an intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). Such a CAR, when expressed in a T cell is able to redirect antigen recognition based on the antigen binding specificity.
The antigen binding moiety of a CAR may comprise a target-specific binding element otherwise referred to as an antigen binding moiety. The choice of moiety used will often depend on the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, the antigen moiety domain in the CAR of may be associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.
The expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
Some methods used to engineer cells employ replication deficient lentiviral vectors to deliver nucleic acids to the cells. For example, nucleic acid molecules encoding CARs are often introduced into cells using such vectors. In particular, lentiviral vectors with the VSV-G pseudotype allow for efficient transduction under automated manufacturing methods. Further, cell engineering may be performed using a number of viral systems, such as Moloney Murine Leukemia Virus (MMLV), gibbon ape leukemia virus (GALV), feline endogenous retrovirus (FERV), baboon endogenous retrovirus (BaEV), and various gamma or alpha retroviral vectors.
Cell engineering may also be performed by introduction of nucleic acid molecules into cells by transfection of electroporation. One instrument that may be used in such methods is the NEON™ Transfection System (Thermo Fisher Scientific, cat no. MPK10025), which has been found to allow for up to 90% transfection with difficult to transfect cells and can be used to transfect 6×106 cells per reaction.
Nucleic acid molecules into cells may remain episomal or may integrate into cellular nucleic acid molecules (e.g., chromosomal DNA, mitochondrial DNA, etc.). In many instances, integration of nucleic acid molecules will be mediated by nonhomologous end joining or homologous recombination. Further, in many instances, it will be desirable for nucleic acid molecules to integrate into cellular nucleic acid at a specific locus, such as a “safe harbor”. In such instances, a “nucleic acid cutting entity” capable of generating site single-stranded or double-stranded breaks in nucleic acids may be used. A number of nucleic acid cutting entities are known in the art. For example, in some embodiments the nucleic acid cutting entity includes one or more zinc finger proteins, transcription activator-like effectors (TALEs), CRISPR complex (e.g., Cas9 or CPF1), homing endonucleases or meganucleases, argonaute-nucleic acid complexes, or macronucleases.
Support (e.g., magnetic supports, such as beads) based purification of nucleic acid molecules may be based on liquid and stationary phases, allowing for selectively separation of nucleic acid molecules from each other as well as from other types of molecules.
Provided herein are compositions and methods for the purification of nucleic acid molecules. In many instances, such methods will involve the association of nucleic acid molecules with supports (e.g., magnetic supports, such as beads). These supports may then be held in place by a magnetic field allowing for separation of materials associated with the supports from materials not associated with the supports.
Nucleic acid molecules (as well as other type of molecules and sometimes cells comprising such molecules) may be associated with supports by, for example, covalent bonds, non-covalent bonds (e.g., ionic interactions), precipitation, or a combination of such processes. Exemplary methods for nucleic acid purification involve the use of supports composed of or containing (1) silica, (2) glass, (3) diatomaceous earth, (4) anion exchange materials, (5) cellulose, and (6) affinity association materials (e.g., oligo dT, biotin-streptavidin, etc.).
Nucleic acid molecule association mechanisms with various materials and groups vary. Silica for example, is believed to associate with nucleic acid molecules by attraction between negatively charged groups of nucleic acid molecules with negatively charged groups of silica. While the mechanism of action is not fully known, it is believed that salt bridges form between the negatively charged groups and/or the silica surface and the DNA become dehydrated.
Similar to silica, anion exchange materials associate with negatively charged nucleic acid molecules through negatively charged groups (e.g., carboxylic acid groups). Further, associated with a support (e.g., magnetic supports, such as beads), nucleic acid molecules (as well as other types of molecules, such as proteins) may be precipitated to enhance association with the support. Precipitation may be mediated by contacting the support with, for example, a high salt, alcohol solution (e.g., 70% ethanol), followed by washing with an alcohol solution to remove the salt. One advantage of nucleic acid molecule precipitation is that it allows for the removal of solutes (e.g., salts) from the support bound nucleic acid molecule prior to nucleic acid release by solubilization (e.g., solubilization by an aqueous solution without ethanol). In some instances, this will result in a low salt, soluble nucleic acid molecule solution for which there is it not necessary to remove salts (e.g., by dialysis). Examples of such processes are set out below in Examples 4 and 5.
Once nucleic acid molecules are associated with a support (e.g., magnetic supports, such as beads) and separated from other materials, it will generally be desirable to separate the associated nucleic acid molecules from the supports. This will normally involve release of the nucleic acid molecules from the supports, followed by physical separation of the support from the nucleic acid molecules. Release may be mediated in any number of way, including alteration of pH, ionic strength, and/or osmolality. Release may also be mediated by the use metal ion chelation (e.g., mediated by EDTA, EGTA, etc.) and competitive ligand binding.
When purification of total nucleic acid in sample is desired, then nucleic acid molecules will often be associated with supports by a mechanism that is not nucleic acid type or sequence specific. When purification of a specific type of nucleic acid in sample is desired, then nucleic acid molecules will often be associated with supports by a mechanism that results in association with one or more specific feature of the desired nucleic acid molecules. By way of example, mRNA may be separated from other materials, including other nucleic acid molecules (e.g., genomic DNA molecules, plasmid DNA molecules, ribosomal RNA molecules, tRNA molecules, etc.) by the presence of 3′ polyA regions. Thus, mRNA may be purified by contact with a support (e.g., magnetic supports, such as beads) capable of binding to polyA nucleic acid regions, followed by washing of support and the bound mRNA, then followed by release of the mRNA from the support, and then separation of the support from the mRNA. A number of supports that may be used in such processes are commercially available (e.g., Thermo Fisher Scientific, D
Methods for the production and purification of mRNA are set out in Examples 4 and 5. Initially in these examples, biotinylated DNA molecules is produced by a polymerase chain reaction (PCR) with a biotinylated primer. The resulting biotinylated DNA molecule encodes a mRNA molecule that is operably linked to a T7 promoter and is bound to magnetic bead with surface bound streptavidin. Once bound to the magnetic bead, the DNA molecule/bead complexes are separated from PCR reaction mixture components. Bead bound DNA is then transcribe by an in vitro transcription (IVT) reaction mixture. The resulting mRNA in not associated with the magnetic beads and, thus, may be separated from the beads by the beads being held in place by a magnetic field. The mRNA molecules are then separated from IVT reaction mixture components by association with magnetic beads comprising carboxylic acid groups. In this instance, mRNA molecules are held in place while IVT reaction mixture components are removed by washing. Thus, the first separation is a negative selection and the second separation is a positive selection.
Further, in many instances, IVT transcription templates may be reused a number of times. For example, it has been found that T7 promoter driven IVT transcription templates bound to supports may be reused multiple (e.g., from about 2 to about 15, from about 3 to about 15, from about 4 to about 15, from about 5 to about 15, from about 2 to about 10, from about 2 to about 8, etc.) times by re-adding fresh IVT reaction mixture components.
Bead processing assemblies and systems set out herein may be used for all or part of workflows involving processes such as those set out above and in Examples 4 and 5, as well as other processes set out herein. Again, using the processes set out in Examples 4 and 5 for illustration, a single solution container (e.g., bag) could be used for initial purification of mRNA. This is so because the magnetic supports (streptavidin beads) with the transcription template for purification is first held in place and then is held in place again when the transcription template is separated from the transcription product (i.e., the mRNA molecules). When additional purification of the mRNA is desired, then a second container (e.g., bag) will often be used. This is so because the mRNA molecule released from the magnetic supports and removed from the container may be further purified by associated with other magnetic supports (carboxylic acid beads) and, in many instance, these other magnetic supports may be held in place after washing and release of the mRNA molecules.
In many instances, reagent addition and removal may be performed again using bead processing assemblies and systems set out herein. Further, a heating pad or element may also be added to the bead processing assemblies and systems set out herein for altering reaction temperatures for various purposes, including enhancing the release of from solid supports and/or solubilization of molecules (e.g., precipitated nucleic acid molecules) after purification. Heating may also be useful for other purposes. For example, when bead processing assemblies and systems set out herein are used for in vitro transcription, it will generally be advantageous to heat the reaction mixture to 37° C. or other suitable temperature. Further, higher temperature (e.g., 65° C.) may be used to release nucleic acid molecule from solid supports (e.g., mRNA from magnetic beads with carboxylic acid groups).
Along these lines, bead processing assemblies and systems set out herein may contain heating and/or cooling elements for increasing or decreasing the temperature of containers (e.g., bags). These heating and/or cooling elements may be used to, for example, accelerate reactions, deaccelerate reactions, solubilize materials, or precipitate materials.
Further, in-line or reservoir heating and/or cooling maybe used in conjunction with or as part of bead processing assemblies and systems set out herein. Heating and cooling elements allow for heating or cooling of materials either prior to introduction into a container or after materials have come out of a container. Using the schematic shown in
In some instances, the biological material that is sought to be purified may be a protein. Any number of methods may be used for such purification, including for example, covalent bonds, non-covalent bonds (e.g., ionic interactions), precipitation, or a combination of such processes. In some instances, binding partner affinity may be used, with the binding partner (ligand) varying with the protein that is sought to be purified.
When a protein is purified using bead processing assemblies and systems set out herein, one binding partner will often be associated with a solid support (e.g., a magnetic bead) and the other binding partner will be associated with the protein to be purified.
Proteins that may purified by methods set out here may vary greatly. Proteins purified by methods set out here may also be bound to affinity reagents that bind to the protein either to a naturally occurring region of the protein or to an exogenously added tag. In addition to examples such as Protein A binding to antibodies, such methods include those where an antibody with specificity for the protein being purified is linked to a solid support. For example, when the protein is an antibody (e.g., an IgG, an IgA, an IgD, an IgM, an IgE, etc.) or a mixture of antibodies (e.g., IgG antibodies present in serum, etc.), the ligand may be Protein A, Protein G, Protein L, or one or more functional variant of one or more of these proteins.
Thus, proteins purification methods may also be based on association with an exogenously added affinity tag. By this it is meant that the affinity tag is not normally present in the naturally occurring protein. Exemplary tags and binding partners that may be present in compositions and used in methods set out herein include maltose-binding protein (MBP)/amylose, and the glutathione-S-transferase (GST)/glutathione tags, polyhistidine (His)/metal ions (e.g., copper and cobalt), streptavidin/biotin (e.g., N-ethyl-biotin), and antigen-antibody reactions (epitope) tags (e.g., c-Myc tag/anti-c-Myc antibody, FLAG/anti-FLAG antibody, and hemagglutinin (HA) tag/anti-HA antibody.
Methods in protein purification workflows, as well as other workflows, may vary widely but, in some instances, solid supports such as magnetic particles (e.g., magnetic beads) may be held in place by a magnetic field and then contacted with a protein binding partner. In other instances, supports such as magnetic particles (e.g., magnetic beads) may be contacted with a protein binding partner and then held in place by a magnetic field. In both such instances, the supports may be held in place and washed.
It may be desirable to release a protein from a support, for example, after a process by which the protein has been separated from other materials (e.g., cell debris). The process by which the protein is released from the support will be determined by the protein and/or the nature of the association between the protein and the support.
Protein release from a support may be mediated by the use of protease cleavage either within the protein or between the protein and an exogenously added tag. Exemplary proteases that may be used include, a rhinovirus 3C protease, a TVMV protease, a plum pox virus protease, turnip mosaic virus protease, tobacco etch virus (TEV) protease, thrombin, Factor Xa, and enteropeptidase.
One type of protein purification method that allows for both purification and release of the protein from supports uses biotin and biotin derivative (e.g., biotin, desthiobiotin, N-ethyl-biotin, etc.) in conjunction and biotin binding proteins.
Proteins may be biotinylated by a number of methods including chemical and enzymatical methods. Chemical protein biotinylation often results in nonspecific biotinylation of amine, carboxylic acid, and sulfhydryl groups. Enzymatic protein biotinylation may be designed to results in biotinylation of a specific groups within a protein. One example of an enzyme that may be used for protein biotinylation is the E. coli biotin holoenzyme synthetase, biotin ligase (BirA). This enzyme catalyzes transfer of biotin to an amino group of a specific lysine of the acetyl-CoA carboxylase biotin carboxyl carrier protein (BCCP) subunit.
In some instances, an antibody or other protein with ligand binding activity is associated with a support through a low affinity biotin derivative (e.g., desthiobiotin, N-ethyl-biotin, etc.)/biotin binding protein (e.g., avidin, streptavidin, neutravidin, etc.) association. Release of the low affinity biotin derivative from the biotin binding protein is mediated by competition with high affinity biotin (e.g., d-biotin).
Cell capture and release methods that may be used for protein purification, as well as for cells, viruses, virus like particles and other biological molecules, are those that use the CAPTURESELECT™ N-Ethyl Biotin (NEB) Anti-CD4 Conjugate (Thermo Fisher Scientific, cat. no. 7113762100) and CAPTURESELECT™ N-Ethyl Biotin (NEB) Anti-CD8 Conjugate (Thermo Fisher Scientific, cat. no. 7113772100). In this instance, a biotinylated anti-CD4 antibody or anti-CD8 antibody is bound to a magnetic bead through a biotin/streptavidin association. In summary, the methods set out in this product areas follow. First, anti-CD4 antibodies or anti-CD8 antibodies biotinylated with NEB are contacted with streptavidin coated magnetic beads. The bead are then contacted with CD4+ or CD8+ T cells under conditions that allows for binding of the T cells through an NEB/streptavidin association. The beads are then washed after a short incubation. In many instances, the beads will be held in place by a magnetic field during the washings. After washing, the beads are contacted with a release reagent containing d-biotin. The supernatant with the CD4+ or CD8+ T cells is then removed. In many instances, the beads will be held in place by a magnetic field during the washings. The result being a purified population of CD4+T or CD8+ T cells with few or no beads present.
Method such as the above many be performed in a closed system and automated and performed in a closed system. With respect to automation, all reactions steps may be performed in one or more containers (e.g., bags) with tubes connected for the addition and removal of reagents. By way of example, bead processing assemblies and systems such as those set out herein may be used to hold the beads in place after addition and incubation of the beads with a release reagent.
Also provided herein are cell/virus surface display methods. Using yeast (e.g., Saccharomyces cerevisiae) surface display as an example, nucleic acid encoding a protein of interest, or portion thereof, may expressed as a fusion with an S. cerevisiae cell surface protein under conditions in which the protein of interest, or portion thereof, is present on the exterior of the yeast cell. One yeast gene that can be used for this purpose encodes the A-agglutinin-binding subunit (Aga2p) cell surface protein. The result being, after expression, that the protein of interest, or portion thereof, is located on the surface of the yeast cell as a component of a cell surface fusion protein. These cells are then contacted with a support to which a binding partner is bound, after which unbound yeast cells are removed by washing. Nucleic acid encoding potential proteins of interested may then be isolated cloned and/or sequenced. In many, the protein of interest will be an antibody that isolated from a library of antibodies with specificity to a support bound antigen.
Similar phage display methods may also be performed using bead processing assemblies and systems provided herein.
Bead processing assemblies and systems provided herein may also be used to purify viruses and virus like particles. Viruses and virus like particles purified using methods set out here may be enveloped on non-enveloped virus and virus like particles.
Viruses and virus like particles may be isolated by association with supports (e.g., magnetic beads). The manner by which viruses and virus like particles associate with supports may vary with the structures of the individual viruses and virus like particles. By way of example, adeno-associated virus (AAV) is non-enveloped and antibodies have been developed with binding affinity to AAV capsid proteins. Further, AAV capsid variations (serotypes) are known and result in different AAV serotype having different cell and tissue specificities.
One commercially available AAV capsid binding antibody is a VHH antibody referred to as AAVX antibody (see CAPTURESELECT™ Biotin Anti-AAVX Conjugate, Thermo Fisher Scientific, cat. no. 7103522500). The AAVX antibody is a single antibody that has binding affinity for multiple capsid serotypes. The AAVX antibody, as well as other antibodies with specificity for AAV capsid proteins, may be used to purify AAV virus like particles. Of course, other AAV antibodies may also be used, many of which have activity more directed to specific AAV capsids (e.g., CAPTURESELECT™ Biotin Anti-AAV8 Conjugate, cat. no. 7103382500; CAPTURESELECT™ Biotin Anti-AAV9 Conjugate, cat. no. 7103332500; etc.). In many such methods, compositions containing AAV virus like particles may be with an anti-AAV capsid antibody that is bound to a support (e.g., magnetic supports, such as beads) for a sufficient period of time to allow for binding of AAV particles to the support. In many instances, the support will be washed to separate the solid support from unbound materials, after which AAV particles will be released from the solid support. One reagent that may be used for AAV particle release is 50 mM citric acid, pH 3.0. Resulting solutions containing AAV particles may then be neutralized using, for example, using 100 mM Tris, pH 9.0.
Supports for purification of some AAV serotypes (e.g., AAV2 and AAV6) may also comprise heparin (see Auricchio et al., “Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column”, Hum. Gene Ther., 12:71-76 (2001)). Thus, supports (e.g., magnetic supports, such as beads) comprising heparin may be used in methods set out herein.
Enveloped viruses and virus like particles may also be purified using compositions, methods and devices provided herein. Enveloped viruses and virus like particles, as well as cells and exosomes, may be purified, for example, through the use of affinity agents. As part of such methods, enveloped viruses and virus like particles many be purified based upon association of a ligand with a protein present in the envelop of the virus and virus like particles that one seeks to purify. An example of such a method is set out in Mekkaoui et al., “Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell”, Mol. Ther. Methods Clin. Dev., 11:155-165 (2018). In this method, packaging cells were genetically engineered to express a biotin-mimicking peptide fused to CD8a amino acid sequences, referred to as cTag8. Lentiviral particles generated by budding acquire an envelope comprising the cTag8. Enveloped lentiviral particles may then by association with supports comprising a biotin binding partner (e.g., streptavidin). One advantage of such methods is that biotin-mimicking peptide may be used that have low affinity from the biotin binding partner used (e.g., streptavidin, avidin, nitrated avidin, nitrated streptavidin) than biotin or biotin derivatives (e.g., desthiobiotin, N-ethyl-biotin, anti-biotin antibodies, etc.). Nitrated avidin and streptavidin are modified protein (e.g., with a nitro group added to a tyrosine) that allow for ligand release under milder conditions that their respective unmodified proteins (see Morag et al., “Immobilized nitro-avidin and nitro-streptavidin as reusable affinity matrices for application in avidin-biotin technology”, Anal. Biochem., 243:257-263 (1996)). Thus, biotin and/or biotin derivatives may be used to release the lentiviral particles by competitive binding. Of course, such tags may also be used to purify molecules to which they are associated with (e.g., proteins).
Provided herein are instruments, compositions, methods and workflows for the purification of viruses, virus like particles and vesicles (e.g., exosomes). In many instances, such methods will involve contacting compositions (e.g., cell cultures, cell culture supernatants, conditioned cell culture media, cell lysates, etc.) with supports (e.g., magnetic supports, such as beads) to which viruses, virus like particles and vesicles (e.g., exosomes) will associate under conditions that allow for the viruses and/or virus like particles to associate with the supports, followed by washing of the supports, and then inducing dissociation of the viruses and/or virus like particles from the supports.
The viruses, virus like particles and/or vesicles (e.g., exosomes) may associate with the supports may be mediated in any number of ways, including ionic interactions and ligand/binding partner interactions such as antibody/antigen interaction.
As set out above, release of viruses, virus like particles and/or vesicles (e.g., exosomes) associated with supports may be mediated in a number of ways. These include inducing dissociation by altering the pH, the ionic strength, the ionic charge, the temperature (e.g., increasing the temperature), and solution polarity.
In many instances, the supports will be held in place by a magnetic field during the washing and after dissociation of the viruses, virus like particles and/or vesicles (e.g., exosomes) from the supports.
Extracellular vesicles (e.g., exosomes, microvesicles, apoptotic bodies, etc.) and liposomes may be purified using methods similar to those set out herein for enveloped virus and virus like particles.
Extracellular vesicles are replication deficient lipid bilayer particles that are released from almost all types of cell. Extracellular vesicles range in size from about 20 to as large as 10 microns or larger, although the majority of extracellular vesicles are smaller than 200 nm.
Exosomes are type of extracellular vesicle, typically 30 to 150 nm. Exosomes are believed to be released upon fusion of multivesicular bodies with the cell membrane by a large number of mammalian cells. A wide variety of molecules have been found to be associated with extracellular vesicles and extracellular vesicles, such as exosomes, are believed to transport nucleic acids, proteins, and lipids for purposes such as intercellular communication and activation of target cell signaling pathways.
In cancers, extracellular vesicles, such as exosomes, are believed to participate in processes such as the regulation of immune responses and the promotion of angiogenesis. Extracellular vesicles, such as exosomes, derived from patient samples (e.g., blood) can be used for non-invasive early detection and diagnosis of cancers. Further extracellular vesicles, such as exosomes, may be used as natural drug delivery vehicles for, for example, cancer therapy.
Extracellular vesicles, such as exosomes, may be purified by association (e.g., non-affinity or affinity association) with supports (e.g., magnetic supports, such as beads). One example of a type of non-affinity exosome purification method is through the use of ion exchange groups. Anion exchange groups, similar to those used for nucleic acid molecule purification may be used for extracellular vesicles (e.g., exosomes) purification.
It has had been found that the D
A number of affinity based methods have been developed for the purification of exosomes. One type of method uses antibodies targeting tetraspanin proteins (e.g., CD9, CD63, and CD81), which are often enriched on exosome surfaces (see Liangsupree et al., “Modern isolation and separation techniques for extracellular vesicles”, J. of Chromatography A 1636:461773 (2021)). Two advantage of immunoaffinity purification of exosomes are (1) purification selectivity and (2) the ability to purify exosomes derived from different cell types. For example, epithelial cell adhesion molecule (EpCAM CD326) has been found to be highly expressed in certain cancer cells, including lung, stomach, colon, prostate, and ovarian cancer cells.
In some instances, affinity mediated purification of extracellular vesicles (e.g., exosomes) may be mediated by affinity reagents (e.g., antibodies) having binding affinity for one or more of the following proteins: CD1 (e.g., CD1a, CD1b, CD1c, CD1d, and CD1e), CD2, CD3 (e.g., CD3d, CD3e, and CD3g), CD4, CD5, CD6, CD7, CD8 (e.g., CD8a and CD8b), CD14, CD16, CD19, CD21 (Complement Receptor 2), CD23, CD24, CD27, CD28, CD29 (integrin beta 1), CD30, CD42 (e.g., CD42a, CD42b, CD42c, and CD42d), CD44, CD45, CD51, CD63, CD79 (e.g., CD79a and CD79b), CD80, CD81, CD86, CD94 (KLRD1), CD95, CD97, CD114 (G-CSF receptor), CD115 (CSF1 receptor), CD116, CD117, CD118, CD119, CD120 (e.g., CD120a and CD120b), CD121 (e.g., CD121a and CD121b), CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD130, CD131, CD132, CD134, CD135, CD137, CD138, CD140 (e.g., CD140a and CD140b), CD150, CD152, CD153, CD154, CD157, CD158 (e.g., CD158a, CD158b1, CD158b2, CD158b, CD158c, CD158d, CD158c1, CD158c2, CD158f1, CD158f2, CD158g, CD158h, CD158i, CD158j, and CD158k), CD160, CD161, CD167 (e.g., CD167a and CD167b), CD172 (e.g., CD172a, CD172b, and CD172g), CD179 (e.g., CD179a, CD179b, CD179c, and CD179d), CD181, CD182, CD183, CD191, CD194, CD200, CD202b, CD212, CD215, CD217, CD218 (e.g., CD218a and 218b), CD220, CD221, CD222, CD223, CD226, CD227, CD235a (Gly A), CD244, CD247 (CD3-Zeta), CD252, CD253, CD254, CD256 (APRIL), CD257 (BAFF), CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD263, CD264, CD265, CD266, CD267, CD272, CD273, CD274, CD275, CD276, CD278 (ICOS), CD279, CD304, CD305, CD314, CD326, CD331, CD332, CD333, CD335, CD336, CD337, CD357, CD358, CD360, and CD366.
An example of an affinity purification reagent that may be used to purify exosomes is the Exosome-Human CD81 Isolation Reagent (from cell culture) product available from Thermo Fisher Scientific (cat. no. 10616D).
Provided herein are compositions and methods for the generation and purification of extracellular vesicles (e.g., exosomes) from cells grown in culture. In some such methods, cells are grown in culture for sufficient period of time for extracellular vesicles (e.g., exosomes) formation, then the extracellular vesicles (e.g., exosomes) are separated from the cells (e.g., by centrifugation). Once separated from cells, the extracellular vesicles (e.g., exosomes) may then be separated from surrounding materials (e.g., proteins, culture media, etc.) by association with supports (e.g., magnetic beads) and processing using bead processing assemblies and systems set out herein. Of course, bead processing assemblies and systems may be designed to automate workflows such as those set out above or at least a number of steps of such workflows.
In some instances, the cells used for the generation and purification of extracellular vesicles (e.g., exosomes) will contain or express one or more molecule that is included in or a component of the extracellular vesicles (e.g., exosomes). Such cells may (1) naturally produce the molecule, (2) be exposed to the molecule under conditions in which the molecule is taken up by the cells, or (3) be engineered to produce the molecule (e.g., a protein, such as a chimeric antigen receptor (CAR).
In some instances, provided herein are methods for the generation and purification of exosomes for cells grown in culture, where the exosomes comprise a therapeutic agent. One example of such a method is where the cells are exposed to a therapeutic agent that is taken up by the cells, followed by purification of exosomes generated by these cells.
Extracellular vesicles (e.g., exosomes) may also be loaded with molecules (e.g., anti-cancer drugs such as paclitaxel, siRNA, proteins, etc.) by electroporation (see, e.g., Zhou et al., “Bone marrow mesenchymal stem cells-derived exosomes for penetrating and targeted chemotherapy of pancreatic cancer”, Acta Pharm. Sin. B., 10:1563-1575 (2020) . Extracellular vesicles (e.g., exosomes) may also be loaded with molecules by transfection (e.g., lipid mediated transfection). Further, exosomes may be loaded before or after support (e.g., magnetic supports, such as beads) mediated purification.
Provided herein are methods for the purification of extracellular vesicles (e.g., exosomes) obtained from cell engineering to contain or comprise a specific biological molecule. In some instances, the biological may be a genome editing complex or one or more components (e.g., a Cas9 protein, a guide RNA molecule/Cas9 protein complex, etc.) comprising and/or nucleic acid encoding such a complex (e.g., DNA or RNA encoding a Cas9 protein and/or a guide RNA molecule). In some instances, the biological may be an RNA molecule (e.g., an siRNA molecule, a microRNA molecule, a mRNA molecule, etc.).
One therapeutic application of extracellular vesicles (e.g., exosomes) is in cancer treatment. It has been shown that exosomes released by chimeric antigen receptor T cells (CAR-T cells) release exosomes that carry CARs on their surfaces. These exosomes have been found to contain cytotoxic molecules and cytotoxic activity against tumors (Fu et al., “CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity”, Nat. Commun., 10:4355 (2019) and Yang et al., “The exosomes derived from CAR-T cell efficiently target mesothelin and reduce triple-negative breast cancer growth”, Cell. Immunol., 360:104262 (2021)). Further, such exosomes are believed to induce fewer side effects in individuals (e.g., cancer patients) to which they are administered compared to CAR-T cells.
Provided herein, in part, are methods for the purification of extracellular vesicles (e.g., exosomes) generated by T cells. Such methods may comprise maintaining T cells in culture media for time period sufficient for the generation of extracellular vesicles (e.g., exosomes), followed by purification on these extracellular vesicles (e.g., exosomes). In many instances, the extracellular vesicles (e.g., exosomes) generated by the T cells will be engineered to express CAR prior to generation of the extracellular vesicles (e.g., exosomes). In some instances, such extracellular vesicles (e.g., exosomes) will be administered to an individual (e.g., a cancer patient).
In some instances, instruments, compositions, methods and/or workflows may involve the depletion of a sample of extracellular vesicles (e.g., exosomes). One example such depletion is where a sample contains exosomes derived from red blood cells. In such instances, a support with binding affinity for CD235a (Gly A) may be used to remove red blood cells and/or extracellular vesicles (e.g., exosomes) formed by red blood cells from the sample. A commercially available product that may be used in such workflows is CD235a (Glycophorin A) MicroBeads, Human (Miltenyi, cat. no. 130-050-501).
Method such as the above many be performed in a closed system and automated and performed in a closed system. With respect to automation, all reactions steps may be performed in one or more containers (e.g., bags) with tubes connected for the addition and removal of reagents. By way of example, bead processing assemblies and systems such as those set out herein may be used to hold the beads in place after conditions within the bag have been changed to induce release of viruses and/or virus like particles from the support.
The subject matter set out herein can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting. Further, portions of the following experiments were performing using the instruments and materials exemplified in the Figures. For example, the below T cell isolation and activation performed under the conditions of rocking was achieved using bead processing apparatus 22 with consumable kit 170A (
250×106 CD3+ cells were incubated with CTS™ D
To analyze CD3+ depletion from PBMC for isolation efficiency, samples were collected from the PBMCs prior to isolation and from the negative fraction, following the above approaches. Samples were stained with a fluorescently labeled anti-CD3 antibody and the number of CD3+ cells in the input and negative fraction was analyzed by flow cytometry. The isolation efficiencies (depletion of CD3+ cells from the negative fraction) were calculated by subtracting the fraction of CD3+ cells in the negative fraction from 1 and multiplied by 100%.
The average isolation efficiency from two replicates of an experimental run was 93% (Standard Deviation (SD) 0.47)
After CD3+ cell isolation, the T cells were transferred to a bioreactor and expanded for 6 days in CTS™ OPTMIZER™ T Cell Expansion SFM (Thermo Fisher Scientific, cat. no. A1048501), supplemented with 100 IU/mL IL-2 (Thermo Fisher Scientific, cat. no. PHC0021). To assess viability, a sample (2 ml) was aseptically collected from the bioreactor, resuspended, debeaded, and analyzed for percent viability using SYTOX™ Blue Dead Cell Stain (Thermo Fisher Scientific, cat. no. S34857) and flow cytometry.
T cell purity on day 1 and day 6 of expansion was assessed by flow cytometry.
T cells were stained using an anti-CD3+ antibody, monocytes were stained using an anti-CD14 antibody, B-cells were stained using an anti-CD19 antibody, and natural killer (NK) NK cells were stained using an anti-CD56 antibody. All antibodies were directly conjugated to a fluorochrome, allowing direct detection by flow cytometry.
Data obtained from two replicates of an experimental run are set out below in Table 2. These data were generated using cells that had been incubated under rocking and shaking conditions.
After 6 days of expansion, cells were debeaded by a continuous flow approach with a 46 continuous flow rate using a bag similar to that shown in
Following bead removal, sample containing 3×106 CD3+ cells are removed from the output bags, lysed, then concentrated to a small volume (20 μL). Beads in the entire lysates are counted to estimate the number of beads per 3×106 CD3+ cells. Beads are counting using a light microscope and KOVA™ Glasstic slide counting chamber (Fisher Scientific, cat no. 22-270141), according to the manufacturer's protocol. Resulting data from two replicates of an experimental run are set out in Table 3.
The number of beads present prior to the debeading process is estimated to be 9×106, based upon the beads being mixed with cell as a 3:1 ratio. Thus, at a flow rate of 46 ml/min, it is estimated that over 99.99% of the beads were removed in the debeading process. It is estimated that, on average, of an estimated 9,000,000 beads originally present, only 1 bead per 450,000 co-localized with the debeaded cells.
D
A biotinylated PCR product containing the T7-promoter upstream of the UTR and ORF, optionally with a defined polyA-tail in the end, was prepared. The forward primer was biotinylated and had a distance to the T7 promoter of at least 50-100 base pairs. The PCR product was then diluted to 20 ng/μL in 1×Streptavidin binding and washing buffer (the 2×D
Preparation of Streptavidin beads is performed as follows:
MEGAS
Immobilized IVT Template: 1 mg D
IVT Reaction Mixture: MEGAS
mRNA capture by precipitation onto D
Part 1. Template immobilization on D
For initial set up, load the buffers and reagents to in a glass bioreactor. Bags may also be used Thoroughly resuspend D
Outlines of the workflows set out in this example are shown in
Load the buffers and reagents to bags and prepare the MEGAscript MIX and keep on ice prior to the transfer to bag. MEGA
Load the buffers and reagents to bags, thoroughly resuspend D
Various alterations and/or modifications of the inventive features illustrated herein, and additional applications of the principles illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, can be made to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the claims, and are to be considered within the scope of this disclosure. Thus, while various aspects and embodiments have been disclosed herein, other aspects and embodiments are contemplated. While a number of methods and components similar or equivalent to those described herein can be used to practice embodiments of the present disclosure, only certain components and methods are described herein.
It will also be appreciated that systems, processes, and/or products according to certain embodiments of the present disclosure may include, incorporate, or otherwise comprise properties features (e.g., components, members, elements, parts, and/or portions) described in other embodiments disclosed and/or described herein. Accordingly, the various features of certain embodiments can be compatible with, combined with, included in, and/or incorporated into other embodiments of the present disclosure. Thus, disclosure of certain features relative to a specific embodiment of the present disclosure should not be construed as limiting application or inclusion of said features to the specific embodiment. Rather, it will be appreciated that other embodiments can also include said features without necessarily departing from the scope of the present disclosure.
Moreover, unless a feature is described as requiring another feature in combination therewith, any feature herein may be combined with any other feature of a same or different embodiment disclosed herein. Furthermore, various well-known aspects of illustrative systems, processes, products, and the like are not described herein in particular detail in order to avoid obscuring aspects of the example embodiments. Such aspects are, however, also contemplated herein.
The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. While certain embodiments and details have been included herein and in the attached disclosure for purposes of illustrating embodiments of the present disclosure, it will be apparent to those skilled in the art that various changes in the methods, products, devices, and apparatus disclosed herein may be made without departing from the scope of the disclosure or of the invention, which is defined in the appended claims. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Exemplary Subject Matter of the Invention is represented by the following clauses:
Clause 1: A bead processing assembly for use in attaching magnetic beads to biological cells and/or separating magnetic beads from biological cells, the bead processing assembly comprising: a base assembly comprising: a housing; a support panel disposed on the housing and having a front face; a first pinch valve at least partially outwardly projecting from the front face of the support panel; and a first pump at least partially outwardly projecting from the front face of the support panel; and a rocker assembly supported on the base assembly, the rocker assembly comprising: a mount assembly supported on the base assembly; a platform assembly pivotably secured to the mount assembly; and a rocker driver configured for rocking the platform assembly relative to the mount assembly.
Clause 2: The bead processing assembly as recited in clause 1, wherein the base assembly further comprises a cover panel hingedly mounted to the housing, the cover panel being movable between an open position wherein the front face of the support panel is openly exposed and a closed position wherein the cover panel covers the front face of the support panel.
Clause 3: The bead processing assembly as recited in clause 2, wherein the cover panel comprises: a perimeter frame hingedly mounted to the housing and encircling an opening; and a transparent window disposed within the opening.
Clause 4: The bead processing assembly as recited in any of clauses 1 to 3, wherein the support panel comprises: a base panel having a front face, the first pinch valve and the first pump being mounted on the base panel so as to at least partially outwardly project from the front face thereof; and an overlay panel being disposed on the front face of the base panel, the overlay panel having openings extending therethrough and through which at least portions of the first pinch valve and first pump project.
Clause 5: The bead processing assembly as recited in any of clauses 1 to 4, wherein the front face of the support panel is disposed at an angle in a range between 30° and 70° when the housing is resting on a horizontal surface.
Clause 6: The bead processing assembly as recited in any of clauses 1 to 5, wherein the base assembly further comprises a plurality of pinch valves at least partially outwardly projecting from the front face of the support panel, wherein the plurality of pinch valves comprise at least 2, 3, 4, 6, or 8 pinch valves.
Clause 7: The bead processing assembly as recited in any of clauses 1 to 6, wherein the first pump comprises a peristaltic pump.
Clause 8: The bead processing assembly as recited in any of clauses 1 to 7, wherein the base assembly further comprises a first bubble sensor at least partially outwardly projecting from the front face of the support panel.
Clause 9: The bead processing assembly as recited in any of clauses 1 to 8, wherein the base assembly further comprises a plurality of bubble sensors at least partially outwardly projecting from the front face of the support panel, the plurality of bubble sensors comprising at least 2, 3, or 4 bubble sensors.
Clause 10: The bead processing assembly as recited in any of clauses 1 to 9, wherein the base assembly further comprises a pressure sensor at least partially outwardly projecting from the front face of the support panel.
Clause 11: The bead processing assembly as recited in any of clauses 1 to 10, wherein the base assembly further comprises: an opening extending through the front face of the support panel; and a first rotational assembly mounted to the support panel, the first rotational assembly comprising: a receiver having a keyed socket formed thereon, the keyed socket being aligned with the opening; and a drive motor coupled to the receiver, the drive motor being configured to selectively rotate the receiver in opposite directions.
Clause 12: The bead processing assembly as recited in any of clauses 1 to 11, wherein the base assembly further comprises a bead vial retainer including: a body configured to receive a vial; an elongated arm extending from the body; and a motor at least partially disposed within the housing of the base assembly and connected to a free end of the arm, the motor being configured to vertically, reciprocally rotate the body attached thereto over an angle of at least 60°.
Clause 13: The bead processing assembly as recited in clause 12, wherein the body of the bead vial retainer comprises: an interior surface that bounds a C-shaped channel; and a shoulder that radially inwardly project from the interior surface.
Clause 14: The bead processing assembly as recited in any of clauses 1 to 13, wherein the mount assembly of the rocker assembly comprises a first riser and a spaced apart second riser mounted on the base assembly, the platform assembly being pivotably coupled to the first riser and the second riser so as to be at least partially disposed between the first riser and the second riser.
Clause 15: The bead processing assembly as recited in any of clauses 1 to 14, wherein the rocker driver comprises: a crank; a motor that selectively rotates the crank; and a connecting arm that extends from the crank to the platform assembly.
Clause 16: The bead processing assembly as recited in any of clauses 1 to 15, wherein the platform assembly comprises: a housing assembly bounding a compartment; a platform mounted to the housing assembly; a magnet assembly disposed between the housing assembly and the platform, the magnet assembly having a top surface and an opposing bottom surface; and a lift configured for selectively raising and lowering the magnet assembly relative to the platform between an activation position and a deactivation position.
Clause 17: The bead processing assembly as recited in clause 16, wherein the lift comprises: a scissor lift at least partially disposed within the compartment of the housing assembly; and a motor that operates the scissor lift.
Clause 18: The bead processing assembly as recited in clause 17, wherein the scissor lift comprises: a shelf, the magnet assembly being disposed on the shelf; a first pair of scissor arms extending between the housing assembly and the shelf; a second pair of scissor arms extending between the housing assembly and the shelf, the second pair of scissor arms being spaced apart from the first pair of scissor arms; a threaded shaft coupled to the motor; and a collar engaging the threaded shaft such that rotation of the threaded shaft by the motor facilitates linear movement of the collar along the threaded shaft, the collar also engaging with the first pair of scissor arms and the second pair of scissor arms.
Clause 19: The bead processing assembly as recited in any of clauses 16 to 18, further comprising: the platform comprising a support plate having a top surface and an opposing bottom surface; wherein when the magnet assembly is raised to the activation position, the bottom surface of the support plate is within 1 cm, 0.5 cm, or 0.2 cm of the top surface of the magnet assembly.
Clause 20: The bead processing assembly as recited in clause 19, wherein when the magnet assembly is lowered to the deactivation position, the bottom surface of the support plate is at least 4 cm, 5 cm, or 6 cm away from the top surface of the magnet assembly.
Clause 21: The bead processing assembly as recited in any of clauses 16 to 20, wherein the platform comprises: a support plate secured to the housing assembly and being formed of an electrically conductive material, the support plate have a top face; an insulative seal forming a continuous loop and being disposed on the top face of the support plate so as to encircle at least a portion of the support plate, the insulative seal being formed of an electrically insulative material; and a contact forming a continuous loop and being disposed on top of the insulative seal so as to be spaced apart from the support plate; and the support plate and the contact being in electrical communication with electrical circuitry that can produce an electrical potential between the contact and the support plate.
Clause 22: The bead processing assembly as recited in any of clauses 16 to 21, wherein the platform comprises: a support plate having a top face and being secured to the housing assembly; a restraint positioned above the top face of the support plate, the restraint comprising:
a boundary wall that partially encircles the top face of the support plate and extends between opposing ends; and a tray extending between the opposing ends of the boundary wall, the tray having an upwardly sloping floor the projects out away from the boundary wall and a pair of spaced apart posts upwardly projecting from the floor.
Clause 23: The bead processing assembly as recited in any of clauses 16 to 22, wherein the platform comprises: a retention frame secured to the support housing, the retention frame comprising an annular inner wall encircling an opening, and annular outer wall encircling the inner wall, and a slot being formed between the inner wall and the outer wall; a guide secured within the slot of the retention frame, the guide bounding a channel; and an electric latch slidably disposed within the channel of the guide.
Clause 24: The bead processing assembly as recited in any of clauses 16 to 23, wherein the magnet assembly comprises a magnet.
Clause 25: The bead processing assembly as recited in clause 24, wherein the magnet assembly comprises: a non-magnetic casing having a top surface and an opposing bottom surface that extend to a perimeter edge, a recess being formed on the top surface and having a perimeter edge; and the magnet being disposed within the recess of the casing.
Clause 26: The bead processing assembly as recited in clause 25, wherein the magnet comprises a plurality of separate and discrete magnets being disposed in a plurality of alternating orientations so as to produce a Halbach array.
Clause 27: The bead processing assembly as recited in clause 25, wherein the perimeter edge of the recess is inset from the perimeter edge of the casing at least 0.5 cm, 1 cm, 1.5 cm or 2 cm.
Clause 28: The bead processing assembly as recited in clause 25, wherein the casing and the magnet each have a rectangular configuration.
Clause 29: The bead processing assembly as recited in any of clauses 16 to 28, wherein the platform assembly further comprises a cover assembly at least partially covering the platform and being movable relative to the platform.
Clause 30: The bead processing assembly as recited in clause 29, wherein the cover assembly comprises: a cover housing that at least partially encircles an opening, at least a portion of the platform being aligned within the opening of the cover housing; and a lid being movably mounted to the cover housing between an open position and a closed position, the lid at least substantially covering the opening of the cover housing when in the closed position.
Clause 31: The bead processing assembly as recited in clause 30, wherein the lid is hingedly mounted to the cover housing.
Clause 32: The bead processing assembly as recited in clause 30, wherein the lid comprises: a perimeter wall the encircles an opening and that has an upper end and an opposing lower end; a lid plate having a top surface and an opposing bottom and being secured to the lower end of the perimeter wall so as to extend over the opening, at least a portion of the perimeter wall upstanding above the top surface of the lid plate so that an upper cavity is formed that is partially bounded by the lid plate and is encircled by the perimeter wall.
Clause 33: The bead processing assembly as recited in clause 32, wherein the perimeter wall has a channel extending therethrough adjacent to the top surface of the lid plate so as to communicate with the upper cavity.
Clause 34: The bead processing assembly as recited in any of clauses 30 to 33, further comprising a first latch for securing the lid to the cover housing when the lid is in the closed position.
Clause 35: The bead processing assembly as recited in any of clauses 29 to 34, further comprising means for resiliently restraining movement of the cover assembly away from the platform.
Clause 36: The bead processing assembly as recited in clause 35, wherein the means for resiliently restraining movement comprises:
a rod having a first end and an opposing second end, the first end being secured to and projecting from cover assembly; and a spring engaged with the rod such that as a force is used to move the cover assembly away from the platform the spring resiliently urges the cover assembly back toward the platform.
Clause 37: The bead processing assembly as recited in clause 36, further comprising a stop movable between a restraining position and a non-restraining position, in the restraining position, the stop is positioned to preclude some movement of the rod relative to the platform and in the non-restraining position the stop does not interfere with movement of the rod.
Clause 38: The bead processing assembly as recited in clause 37, further comprising a solenoid valve mounted on the housing assembly, the solenoid valve moving the stop between the restraining position and a non-restraining position.
Clause 39: The bead processing assembly as recited in any of clauses 36 to 38, further comprising: a hole formed on the housing assembly, the rod being slidably disposed within the hole; a flange outwardly projecting from the second end of the rod; and the spring extending between the flange and the housing assembly so that as the rod is concurrently lifted with the cover assembly, the springe is resiliently compressed.
Clause 40: The bead processing assembly as recited in clause 39, further comprising a stop movable between a restraining position and a non-restraining position, in the restraining position, the stop is aligned with the flange so as to block some movement of the flange and the rod attached thereto, in the non-restraining position, the stop is not aligned with the flange and thus does not interfere with movement of the flange or rod attached thereto.
Clause 41: The bead processing assembly as recited in any of clauses 30 to 40, further comprising: a recess formed on the cover housing and communicating with the opening of the cover housing; and a clamp assembly slidably disposed within the recess, the clamp assembly comprising: a base clamp having a lower clamp grove formed thereon; a clamp closure having an upper clamp groove formed thereon; and a fastener selectively securing the clamp closure to the base clamp so that that upper clamp groove is aligned with the lower clamp groove.
Clause 42: The bead processing assembly as recited in clause 41, wherein the cover housing further comprises a U-shaped channel formed at each opposing end of the recess, opposing ends of the clamp assembly being slidably disposed with the U-shaped channels.
Clause 43: The bead processing assembly as recited in any of clauses 16 to 42, wherein the platform comprises: a support plate having a top surface and an opposing bottom surface; and a sidewall projecting away from the support plate and being secured to the housing assembly, the support plate and sidewall at least partially bonding a cavity in which the magnet assembly is received when the magnet assembly is in the raised activated position.
Clause 44: A bead processing system for attaching magnetic beads to biological cells and/or separating magnetic beads from biological cells, the bead processing system comprising: a bead processing assembly comprising: a base assembly comprising: a housing; a support panel disposed on the housing and having a front face; a first pinch valve at least partially outwardly projecting from the front face of the support panel; a first pump at least partially outwardly projecting from the front face of the support panel; and a rocker assembly supported on the base assembly, the rocker assembly comprising: a mount assembly supported on the base assembly; a platform assembly pivotably secured to the mount assembly and comprising a platform; and a rocker driver configured for rocking the platform assembly relative to the mount assembly; a consumable kit comprising: a tray having a front face and an opposing back face with a plurality of openings extending therebetween, the tray being removably nested on the front face of the support panel so that the first pinch valve and the first pump project through corresponding ones of the plurality of openings; and a line set secured to the front face of the tray, the line set comprising: flexible tubing secured to the front face of the tray and engaging with the first pinch valve and the first pump; and a plurality of flexible bags fluid coupled with the tubing, the plurality of flexible bags comprising a processing bag supported on the platform of the platform assembly.
Clause 45: The bead processing system as recited in clause 44, wherein the base assembly further comprises a cover panel hingedly mounted to the housing assembly, the cover panel being movable between an open position wherein the front face of the tray is openly exposed and a closed position wherein the cover panel covers the front face of the tray.
Clause 46: The bead processing system as recited in clause 44 or 45, wherein the base assembly further comprises a plurality of pinch valves at least partially outwardly projecting from the front face of the support panel, wherein the plurality of pinch valves comprise at least 2, 3, 4, 6, or 8 pinch valves, wherein each of the plurality of pinch valves project through corresponding ones of the plurality of openings on the tray and engage with the flexible tubing.
Clause 47: The bead processing system as recited in any of clauses 44 to 46, wherein the base assembly further comprises a first bubble sensor at least partially outwardly projecting from the front face of the support panel, the first bubble sensor projecting through a corresponding one of the plurality of openings on the tray and engage with the flexible tubing.
Clause 48: The bead processing system as recited in any of clauses 44 to 47, further comprises: an opening extending through the front face of the support panel; and a first rotational assembly mounted to the support panel, the first rotational assembly comprising: a receiver having a keyed socket formed thereon, the keyed socket being aligned with the opening; and a drive motor coupled to the receiver, the drive motor being configured to selectively rotate the receiver in opposite directions; a stopcock fluid coupled with the tubing of the line set, the stopcock having a rotatable handle received within the keyed socket of the receiver; and an air filter fluid coupled with the stopcock.
Clause 49: The bead processing system as recited in any of clauses 44 to 48, further comprises a bead vial retainer including: a body having a channel configured to receive a vial; an elongated arm extending from the body; a motor at least partially disposed within the housing assembly of the base assembly and connected to a free end of the arm, the motor being configured to vertically, reciprocally rotate the body attached thereto over an angle of at least 60°; and a vial received within the channel of the body, the vial housing magnetic beads and a media, the tubing of the line set being fluid coupled with the vial.
Clause 50: The bead processing system as recited in any of clauses 44 to 49, wherein the platform assembly comprises: a housing assembly bounding a compartment; a platform mounted to the housing assembly; a magnet assembly disposed between the housing assembly and the platform, the magnet assembly having a top surface and an opposing bottom surface; and a lift disposed within the compartment of the housing assembly for selectively raising and lowering the magnet assembly relative to the platform between an activation position and a deactivation position.
Clause 51: The bead processing system as recited in clause 50, wherein the platform assembly further comprises a cover assembly at least partially covering the platform and the processing bag thereon, the cover assembly being movable relative to the platform as the processing bag expands.
Clause 52: The bead processing system as recited in clause 51, further comprising means for resiliently restraining movement of the cover assembly away from the platform.
Clause 53: The bead processing system as recited in clause 51, wherein the cover assembly comprises: a cover housing that at least partially encircles an opening, the processing bag being at least partially disposed within the opening of the cover housing; and a lid being movably mounted to the cover housing between an open and closed position, the lid at least substantially covering the opening of the cover housing when in the closed position.
Clause 54: The bead processing system as recited in clause 53, further comprising: a recess formed on the cover housing and communicating with the opening of the cover housing; a clamp assembly slidably disposed within the recess, the clamp assembly comprising: a base clamp having a lower clamp groove formed thereon; a clamp closure having an upper clamp groove formed thereon; and a fastener selectively securing the clamp closure to the base clamp so that that upper clamp groove is aligned with the lower clamp groove; wherein the processing bag disposed on the platform has a port that is at least partially disposed within the aligned upper clamp groove and lower clamp groove and is clamped between the base clamp and the clamp closure.
Clause 55: The bead processing system as recited in any of clauses 44 to 54, further comprising a first media bag fluid coupled to the tubing of the line set and housing a medium.
Clause 56: The bead processing system as recited in clause 55, wherein the bead processing assembly further comprises a stand upstanding from the base assembly and having a catch outwardly projecting therefrom, the media bag being supported on the catch.
Clause 57: The bead processing system as recited in any of clauses 44 to 56, wherein the tubing of the line set is fluid coupled with a biological cell separator.
Clause 56: The bead processing system as recited in any of clauses 44 to 57, wherein the tubing of the line set is fluid coupled with a biological cell expansion system.
Clause 58: The bead processing system as recited in any of clauses 44 to 58, wherein the processing bag comprise a bead separation bag that comprises: a collapsible bag body comprised of polymeric film and bounding a compartment; a pair of spaced apart ports coupled to the bag body and communicating with the compartment; a partition disposed within the compartment of the bag body at a location between the pair of spaced apart ports, the partition being secured to the bag body so that fluid entering one of the ports must flow around the partition before it can leave through the other port.
Clause 60: A consumable kit for use with a magnetic bead processing assembly, the consumable kit comprising: a tray having a front face and an opposing back face with a plurality of openings extending therebetween; and a line set secured to the front face of the tray, the line set comprising: flexible tubing secured to the front face of the tray so as to align with at least some of the plurality of openings; a plurality of flexible bags fluid coupled with the tubing; and an air filter coupled with the tubing.
Clause 61: The consumable kit as recited in clause 60, further comprising an air filter assembly coupled with the tubing, the air filter assembly comprising: a stop-cock fluid coupled with the tubing, the stop-cock comprising: a sleeve outwardly projecting from the front face of the tray; a valve rotatably disposed within sleeve; and a handle secured to the valve for selectively rotating the valve, the handle outwardly projecting from the back face of the tray; and the air filter fluid coupled with the sleeve.
Clause 62: The consumable kit as recited in clause 61, further comprising a mixing bag disposed on the front face of the tray and being fluid coupled with the tubing.
Clause 63: The consumable kit as recited in clause 61 or 62, further comprising: a bead vial coupler fluid coupled to the tubing of the line set; and a vial secured to the bead vial coupler, the vial housing a suspension comprising magnetic beads and a carrier liquid.
Clause 64: A rocker assembly comprising: a mount assembly; a platform assembly pivotably secured to the mount assembly; a rocker driver configured for rocking the platform assembly relative to the mount assembly; wherein the platform assembly comprises: a housing assembly bounding a compartment; a platform mounted to the housing assembly; a magnet assembly disposed between the housing assembly and the platform; a lift configured for selectively raising and lowering the magnet assembly relative to the platform between an activation position and a deactivation position; and a cover assembly at least partially covering the platform and being movable relative to the platform.
Clause 65: The rocker assembly as recited in clause 64, wherein the cover assembly comprises: a cover housing that at least partially encircles an opening, at least a portion of the platform being aligned within the opening of the cover housing; and a lid being movably mounted to the cover housing between an open position and a closed position, the lid at least substantially covering the opening of the cover housing when in the closed position.
Clause 66: A bead processing assembly for use in attaching magnetic beads to biological cells and/or separating magnetic beads from biological cells, the bead processing assembly comprising: a base assembly comprising: a housing; a support panel disposed on the housing and having a front face; a first pinch valve at least partially outwardly projecting from the front face of the support panel; and a first pump at least partially outwardly projecting from the front face of the support panel.
Clause 67: A method for operating a bead processing system for attaching magnetic beads to biological cells and/or separating magnetic beads from biological cells, the method comprising: removably nesting a tray on a front face of a support panel of a bead processing assembly so that a first pinch valve and a first pump projecting from the support panel pass through corresponding openings formed on the tray; engaging a tubing of a line set disposed on the tray to the first pinch valve and the first pump; and placing a processing bag fluid coupled with the line set on a platform of the bead processing assembly.
Clause 68: The method as recited in clause 66, further comprising fluid coupling a vial or a flexible bag to the tubing of the line set, the vial or the flexible bag housing a suspension comprising magnetic beads and a carrier liquid.
Clause 69: The method as recited in clause 66 or 67, further comprising supporting a media bag housing liquid media on a bag stand of the bead processing assembly.
Clause 70: The method as recited in any of clauses 67 to 69, further comprising fluid coupling the tubing of the line set to a biological cell separator.
Clause 71: The method as recited in any of clauses 67 to 70, further comprising fluid coupling the tubing of the line set to a biological cell expansion system.
Clause 72: The method as recited in any of clauses 67 to 71, further comprising activating a computer processor of the bead processing assembly so that the computer processor facilitates rocking of the platform on which the processing bag is placed.
Clause 73: The method as recited in any of clauses 67 to 72, further comprising activating a computer processor of the bead processing assembly so that the computer processor facilitates raising and lowering a magnet relative to the platform on which the processing bag is placed.
Clause 74: The method as recited in any of clauses 67 to 73, further comprising activating a computer processor of the bead processing assembly so that the computer processor facilitates controlling the first pinch valve and the first pump to deliver a fluid to the processing bag on the platform.
Clause 75: The method as recited in any of clauses 67 to 74, further comprising activating a computer processor of the bead processing assembly so that the computer processor facilitates moving a vial fluid coupled with the tubing of the line set so as to mix a suspension disposed within the vial, the suspension comprising magnetic beads and a carrier liquid.
Clause 76: The method as recited in any of clauses 67 to 75, wherein the biological cells are T cells.
Clause 77: The method as recited in any of clauses 67 to 76, wherein the biological cells are attached to the magnetic beads through an antigen-antibody interaction.
Clause 78: The method as recited in clause 77, wherein the biological cells are detached from the magnetic beads by disruption of the antigen-antibody interaction.
Clause 79: The method as recited in clause 78, wherein the disruption of the antigen-antibody interaction is mediated by cleavage of the antibody.
Clause 80: The method as recited in any of clauses 77 to 79, wherein the biological cells are detached from the magnetic beads by separation of the antibody from the magnetic beads.
Clause 81: The method as recited in any of clauses 77 to 80, wherein the antibody is linked to the magnetic beads by a ligand.
Clause 82: The method as recited in clause 81, wherein the biological cells are detached from the magnetic beads by disruption of the ligand interaction with the antibody or the magnetic bead.
Clause 83: The method as recited in any of clauses 67 to 82, wherein the biological cells are T cells.
Clause 84: A method for separating biological cells of a first cell type from biological cells of a second cell type using a bead processing system, the method comprising: (a) contacting a sample comprising the biological cells of the first cell type and the second cell type with magnetic beads so that the magnetic beads attach to the biological cells of the first cell type through an antibody linkage, the sample and magnetic beads being disposed within a processing bag resting on a platform; (b) raising a magnet relative to the platform so that the magnet produces a magnetic field that securely fixes in place the magnetic beads relative to the platform, the magnetic beads having the biological cells of the first cell type attached thereto; (c) passing a fluid through the sample and the magnetic beads while the magnetic field is applied thereto, the fluid passing with a force sufficiently strong to wash away the biological cells of the second cell type from the biological cells of the first cell type; and (d) lowering the magnet relative to the platform so that the magnetic beads having the biological cells of the first cell type attached thereto are no longer securely fixed in place relative to the platform by the magnetic field of the magnet.
Clause 85: The method as recited in clause 84, wherein the antibody has binding affinity for a protein selected from the group consisting of CD3, CD4, CD5, CD6, CD8, CD25, CD27, CD28, CD137, and CD278.
Clause 86: The method as recited in clause 85, wherein the antibody has binding affinity for a protein comprising CD3 or CD28.
Clause 87: The method as recited in clause 86, wherein the first cell type is T cells.
Clause 88: The method as recited in any of clauses 84 to 87, further comprising: detaching at least a majority of the biological cells of first cell type from the magnetic beads; applying a magnetic field to the magnetic beads so as to securely fix the magnetic beads in place; and passing a fluid through the magnetic beads and the separated biological cells of first cell type while the magnetic field is being applied, the fluid passing with a force sufficiently strong to wash away at least a majority of the biological cells of the first cell type from the magnetic beads while at least a majority of the magnetic beads remain fixed in place by the magnetic field.
Clause 89: The method as recited in clause 88, wherein greater than 95% of the magnetic beads are retained in place by the magnetic field while the at least a majority of the biological cells of the first cell type are washed away from the magnetic beads.
Clause 90: The method as recited in clause 89, wherein greater than 95% of the magnetic beads are separated from the biological cells of the first cell type.
Clause 91: The method as recited in any of clauses 88 to 90, wherein greater than 95% of the magnetic beads detached from the biological cells of first cell type are separated from the biological cells of first cell type.
Clause 92: The method as recited in any of clauses 84 to 91, wherein the magnetic beads are from about 0.5 μm to about 3 μm in diameter.
Clause 93: The method as recited in any of clauses 84 to 92, further comprising tilting the platform on which the processing bag is resting in a first direction relative to horizontal, the magnetic field being applied to the magnetic beads within the processing bag while the platform is tilted in the first direction.
Clause 94: The method as recited in clause 93, wherein the magnet is raised after the platform is tilted in the first direction.
Clause 95: The method as recited in clause 93 or 94, further comprising injecting a gas or a liquid through a port of the processing bag while the platform is tilted in the first direction.
Clause 96: The method as recited in any of clauses 93 to 95, further comprising: tilting the platform in a second direction opposite to the first direction so that the platform on which the processing bag is resting is angled relative to horizontal; and withdrawing liquid from the processing bag while the platform is tilted in the second direction.
Clause 97: A method for separating a first biological material from a second biological material using a bead processing system, the method comprising: (a) contacting a sample comprising the first biological material and the second biological material with magnetic beads so that the magnetic beads attach to the first biological material through an affinity linkage, the sample and magnetic beads being disposed within a processing bag resting on a platform; (b) raising a magnet relative to the platform so that the magnet produces a magnetic field that securely fixes in place the magnetic beads relative to the platform, the magnetic beads having first biological material attached thereto; and (c) passing a first fluid through the sample and the magnetic beads while the magnetic field is applied thereto, thereby separating the first biological material from the second biological material.
Clause 98: The method of clause 97, further comprising: (d) passing a second fluid through the sample and the magnetic beads while the magnetic field is applied thereto, wherein the second fluid induces release of the first biological material from the magnetic beads.
Clause 99: The method of clause 97 or 98, further comprising: (d) lowering the magnet relative to the platform so that the magnetic beads having the first biological material attached thereto are no longer securely fixed in place relative to the platform by the magnetic field of the magnet.
Clause 100: The method of any of clauses 97 to 99, wherein the second biological material is a cell lysate or a culture medium.
Clause 101: The method of any of clauses 97 to 100, wherein the first biological material is selected from the group consisting of: (a) a nucleic acid molecule; (b) a protein; (c) a cell; (d) an extracellular vesicle; and (c) a virus-like particle (VLP).
Clause 102: The method of clause 101, wherein the nucleic acid molecule is a ribonucleic acid (RNA) molecule.
Clause 103: The method of clause 101 or 102, wherein the cell is a mammalian cell.
Clause 104: The method of clause 101 or 103, wherein the cell is a T cell.
Clause 105: The method of clause 101, wherein the protein is an antibody.
Clause 106: The method of clause 105, wherein the affinity linkage with the antibody is formed by Protein A, Protein G, Protein L, or a functional variant of one of these proteins.
Clause 107: The method of clause 101, wherein the extracellular vesicle is an exosome.
Clause 108: The method of clause 107, wherein the exosome was generated by a T cell.
Clause 109: The method of clause 108, wherein the T cell was engineered to express a chimeric antigen receptor.
Clause 110: The method of clause 107, wherein the affinity linkage with the exosome is formed by an anti-CD3 antibody, an anti-CD4 antibody, and/or an anti-CD8 antibody.
Clause 111: The method of clause 110, wherein the anti-CD3 antibody, the anti-CD4 antibody, and/or the anti-CD8 antibody is attached to the magnetic beads by a biotin or biotin derivative linkage.
Clause 112: The method of clause 110, wherein one or more of the anti-CD3 antibody, the anti-CD4 antibody, and/or the anti-CD8 antibody is a variable heavy-heavy (VHH) antibody.
Clause 113: A method for producing a purified ribonucleic acid (RNA) molecule, the method comprising: (a) fixing a first magnetic bead in place by a magnetic field, wherein an in vitro transcription (IVT) template is linked to the first magnetic bead; (b) contacting the first magnetic bead of step (a) with a reagent mixture suitable for IVT of the template under condition in which IVT occurs, thereby producing an RNA molecule, and (c) separating the RNA molecule from the first magnetic bead, thereby producing the purified RNA molecule.
Clause 114: The method of clause 113, further comprising: (d) contacting the purified RNA molecule of step (c) with a second magnetic bead under conditions that allows for the purified RNA molecule to remain associated with the second magnetic bead during washing, (e) washing of the second magnetic bead while the second magnet bead is fixed in place by a magnetic field, and (f) releasing the purified RNA molecule from association with the second magnetic bead, thereby producing a highly purified RNA molecule.
Clause 115: The method of clause 113 or 114, wherein the IVT template is produced by polymerase chain reaction (PCR).
Clause 116: The method of clause 115, wherein one or more biotinylated primer is used in the PCR and results in the formation of a biotinylated IVT template.
Clause 117: The method of clause 116, wherein the biotinylated IVT template is attached to the magnetic bead through an interaction between the biotin of the biotinylated IVT template and a group on the magnetic bead with affinity for biotin.
Clause 118: The method of any of clauses 113 to 117, wherein the IVT template comprises an open reading frame encoding a protein and a promoter operably connected to the open reading frame.
Clause 119: The method of any of clauses 114 to 118, wherein free carboxylic acid groups are present on the surface of the second magnetic bead.
Clause 120: The method of clauses 113 or 114, wherein the purified RNA or the highly purified RNA is a messenger RNA (mRNA).
Clause 121: The method of clause 120, wherein the mRNA encodes a protein of pathogen.
Clause 122: A vaccine composition comprising the mRNA of any of clauses 113 to 121 or the protein encoded by this mRNA.
Clause 123: A non-transitory computer readable storage medium storing one or more programs, the one or more programs comprising instructions which, when executed by an electronic device with a display, cause the electronic device to:
display, on the display, a graphical user interface comprising a primary selection area comprising at least one primary virtual bioprocess parameter input and a secondary selection area comprising at least one secondary virtual input that overrides or duplicates the primary virtual bioprocess parameter input.
Clause 124: The storage medium of clause 123, wherein the secondary virtual input comprises a cancel-step, pause-step, stop-step, skip-step or duplicate-step input.
Clause 125: The storage medium of clause 123, wherein the primary virtual bioprocess parameter input comprises a bioprocess parameter that controls the operation of the bead processing assembly of any one of clauses 1-43 and 66.
Clause 126: The storage medium of clause 123, wherein the primary virtual bioprocess parameter input comprises a bioprocess parameter that controls the operation of the bead processing system of any one of clauses 44-59.
Clause 127: The storage medium of clause 123, wherein the primary virtual bioprocess parameter input comprises a bioprocess parameter that controls the operation of the rocker assembly of any one of clauses 64-65.
Filing Document | Filing Date | Country | Kind |
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PCT/US2021/054490 | 10/12/2021 | WO |
Number | Date | Country | |
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63137389 | Jan 2021 | US | |
63090399 | Oct 2020 | US |