Sample manufacturing composition, device, system and method

Abstract

This specification relates to compositions, apparatuses, devices, methods, kits, and systems for sample preparation (e.g., separation, reduction, or removal of small molecules from biomolecules in a sample). Exemplary small molecules that may be separated, reduced, or removed have a molecular weight range of < 2000 Da and may include, but are not limited to, dyes, biotin, affinity tags, crosslinking agents, reducing agents, labels, nanoparticles, radioactive ligands, mass tags, unreacted molecules, and combinations thereof, intermediates, and derivatives. Exemplary biomolecules present in the sample include, but are not limited to, proteins, glycoproteins, antibodies, peptides, nucleic acids, polysaccharides, carbohydrates, and lipids. The methods, compositions, kits, devices, devices, and systems of this disclosure may advantageously provide excellent separation of small molecule contaminants and additionally reduce the time and cost associated with the separation of small molecules from larger biomolecules in a sample. As described herein, separated biomolecules may be processed for better downstream processing.

Description

Technology Field

[0001] This specification relates to compositions, devices, apparatuses, methods, kits, and systems for sample preparation (e.g., separation of small molecules from larger molecules). In some embodiments, the compositions, apparatuses, devices, systems, methods, and kits described herein may be used for the separation, extraction, purification, reduction, or removal of small molecules from larger molecules, which are, but not limited to, biomolecules in a sample. Background Technology

[0002] Sample preparation techniques for isolating biomolecules aim to enable downstream analysis and processing of biomolecules by extracting them from other sample components and sample processing components. For example, during the preparation of samples of biomolecules such as proteins or nucleic acids, it is often necessary to label the biomolecules using dyes, affinity tags, radiolabels, mass tags, etc. In other instances, it is necessary to chemically modify the biomolecules through reduction, oxidation, cross-linking, methylation, etc. During these processes, some of the labeling agents or chemicals remain in the sample as unreacted labels / chemicals, or in the form of partially reacted intermediates or derivatives. These unreacted small molecules can cause various problems during downstream analysis or the use of the biomolecules. For example, free unreacted fluorescent dyes that are not conjugated to proteins or nucleic acids cause background issues during the fluorescence imaging of proteins or nucleic acids.

[0003] Another example is the preparation of tagged antibodies (e.g., biotinylated antibodies) typically used to detect corresponding antigens. A streptavidin-based support is used with the biotinylated antibody for the detection of the antigen. If free unreacted biotin is present in the sample of the biotinylated antibody, it will interact with the streptavidin support and impair the binding ability of the biotinylated antibody.

[0004] Some methods used to address the removal of small molecules, such as labels and chemicals, from larger molecules, such as biomolecules, include dialysis combined with ion-exchange chromatography, size exclusion chromatography, or desalination resins combined with ion-exchange resins. However, each of these methods has several disadvantages.

[0005] For example, dialysis is commonly used to remove unreacted dyes, biotin, and reducing agents from proteins. However, dialysis requires two to three buffer changes and takes 12 to 14 hours to remove small molecules.

[0006] Size exclusion chromatography is another method used to remove small molecules. This procedure requires complex and expensive chromatography instruments (e.g., AKTA system, GE). In these complex chromatography systems, samples containing small molecules pass through specific columns that are customized and set up for specific purposes and sample types. Typically, customized 1 ml, 5 ml, 10 ml, or larger columns are set up depending on the sample size. Larger biomolecules, such as antibody conjugates, are excluded by the column first, followed by the exclusion of smaller molecules passing through the pores. Therefore, small molecules require more time to elute from the column. The execution time of size exclusion chromatography can vary from 30 minutes to several hours, depending on column size, setup time, and other factors. Consequently, the size exclusion chromatography method is expensive and time-consuming.

[0007] Another procedure uses demineralizing resins, where various resins are used for buffer exchange and demineralization. However, demineralizing resins have a very limited ability to remove small molecules such as dyes, labels, and conjugates (which will also be described later in this specification).

[0008] Ion exchange resins combined with demineralization resins or dialysis have been used to separate small molecules interacting with ion exchangers on the resin. However, in addition to the disadvantages of the aforementioned dialysis and demineralization resin-based methods, protein recovery from ion exchange resins is poor because proteins must be eluted from the ion exchangers. Ion exchange methods for protein elution require multiple steps that are time-consuming and tedious when combined with the additional need for dialysis or demineralization resins.

[0009] Therefore, in methods such as but not limited to fluorescence imaging, bioconjugation, and immunoprecipitation, for the separation of larger biomolecules or larger molecules from small molecules such as free dyes, labels, reducing agents, crosslinking agents, etc., there is a need for better methods, compositions, systems, and apparatuses that enable cleaner downstream processing of biomolecules.

[0010] summation

[0011] In some embodiments, this specification relates to compositions, apparatuses, devices, methods, kits, and systems for sample preparation, for example, but not limited to the separation of small molecules from larger molecules (e.g., biomolecules, but not limited thereto) within a sample. In some embodiments, the compositions, apparatuses, devices, systems, methods, and kits described herein may be used for separation, extraction, purification, removal, reduction of the amount of small molecules, or removal of small molecules from larger molecules in a sample. In some embodiments, the compositions, apparatuses, devices, systems, methods, and kits described herein significantly reduce the amount of small molecules from biomolecules or larger molecules in a sample. In some embodiments, the compositions, apparatuses, devices, systems, methods, and kits described herein rapidly reduce the amount of small molecules from biomolecules or larger molecules in a sample. The terms biomolecule and larger molecule are used interchangeably in this specification. The term larger is used only in relation to the small molecules to be removed and does not specify or limit the biomolecule to any particular size or size range.

[0012] Some examples of one or more small molecules that can be separated, extracted, reduced, or removed by the compositions, apparatus, devices, systems, methods, and kits of the present disclosure include, but are not limited to, dyes, derivatives of dyes, biotin, biotin derivatives, crosslinking agents, reducing agents, oxidizing agents, methylating agents, protein preservatives, labels, nanoparticles, radioactive ligands, mass tags, unreacted molecules and combinations thereof, intermediates, and derivatives.

[0013] Dyes suitable for use are known to those skilled in the art, including pyrene, coumarin, cyanine, benzofuran, quinoline, quinazolinone, indole, benzazole, borapolyazindacen and xanthenes including fluorescein, rhodamine and rhodole, and literature (RICHARD P. HAUGLAND, MOLECμLAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (11 th Includes, but not limited to, other dyes described in edition, January, 2010), the entirety of which is incorporated herein by reference.

[0014] In some embodiments, small molecules that can be separated by the composition, apparatus, device, system, method, and kit of the present disclosure have a molecular weight range of < 2000 Da.

[0015] Exemplary larger molecules that can be separated from small molecules by the compositions, apparatuses, devices, systems, methods, and kits of the present disclosure include, but are not limited to, proteins, glycoproteins, antibodies, peptides, nucleic acids (DNA, genomic DNA, pDNA, RNA), polysaccharides, carbohydrates, lipids, toxins, nanoparticles, and derivatives of each of the molecules described above. Derivatives of molecules include, without limitation, tagged proteins or tagged nucleic acids; molecules labeled with various labels such as, but not limited to, dyes, fluorescent dyes, radiolabels, affinity labels, mass tags, metals, etc.; conjugated molecules including conjugated antibodies; molecules conjugated to nanoparticles such as gold nanoparticles; molecules conjugated to toxins such as biotin-labeled toxins by, in non-limiting examples, cholera-toxin-labeled compounds, etc.; and chemical derivatives of biomolecules such as, but not limited to, proteins having reduced proteins, oxidized proteins, methylated nucleic acids, or sulfhydryl-modified proteins. In some embodiments, larger molecules and biomolecules may be included in the sample.

[0016] One embodiment of the present disclosure relates to a composition for separating or extracting one or more small molecules from a sample. In some embodiments, the composition comprises (at least one) size exclusion support and one or more moiety capable of binding to one or more small molecules to separate the small molecules from the rest of the sample.

[0017] In some embodiments, contacting a sample with the composition of the present disclosure significantly reduces the amount of one or more small molecules from the sample.

[0018] Larger molecules present in the sample have a size excluded by the composition of the present disclosure. One or more small molecules are bonded to the composition through at least one moiety.

[0019] In some embodiments, contacting a sample with a composition of the present disclosure comprises, without limitation, one or more of the following: applying the sample onto the composition; passing the sample through the composition; causing the sample to flow through the composition by gravity or by using rotation or centrifugal force; and creating a positive pressure difference or a negative pressure difference to cause the sample to move through the composition.

[0020] In some embodiments, in the composition of the present disclosure, at least one moiety is coupled to a size exclusion support. In some embodiments, in the composition of the present disclosure, at least one moiety is fixed on the size exclusion support. In some embodiments, in the composition of the present disclosure, at least one moiety is attached to the size exclusion support.

[0021] In some embodiments, in the composition of the present disclosure, at least one moiety is bonded to one or more small molecules by charge interaction, hydrophilic interaction, hydrophobic interaction, affinity interaction, hydrogen bonding, Van der Waals force, or covalent bonding.

[0022] In some embodiments, the composition of the present disclosure may include at least two moiety, or at least three moiety, or at least four moiety, or at least five moiety, etc.

[0023] In some embodiments, the size exclusion support used in the composition of the present disclosure excludes molecules of 2 kDa or greater than 2 kDa from a sample. In some embodiments, the size exclusion support used in the composition of the present disclosure excludes molecules of 3 kDa or greater from a sample.

[0024] In some embodiments, the composition of the present disclosure may comprise one or more size exclusion supports. For example, the composition may comprise at least a second size exclusion support and at least one second moiety. In some embodiments, the composition of the present disclosure may further comprise a third size exclusion support, a fourth size exclusion support, a fifth size exclusion support, and the like.

[0025] Each exclusion support may be combined with the same or different moiety. For example, in some embodiments, a first-size exclusion support may be combined with one or more moietys, e.g., a first moiety, a second moiety, a third moiety, a fourth moiety, a fifth moiety, etc. In other exemplary embodiments, a first-size exclusion support may be combined with a first moiety, a second-size exclusion support may be combined with a second moiety, a third-size exclusion support may be combined with a third moiety, a fourth-size exclusion support may be combined with a fourth moiety, and a fifth-size exclusion support may be combined with a fifth moiety, etc. Other combinations are also considered.

[0026] The composition of the present disclosure may, in some embodiments, comprise different proportions of various combinations of size exclusion supports and moiety. For example, the composition may comprise a proportion of a first size exclusion support and at least one first moiety and one or more additional moietys, or one or more additional size exclusion supports and moietys. In other examples, the composition may comprise a proportion of a first size exclusion support and a first moiety and at least one second size exclusion support and at least one second moiety.

[0027] The compositions of the present disclosure may also comprise a blend of exclusion supports and moiety, for example, a blend of a first size exclusion support and a first moiety and a second size exclusion support and a second moiety, etc., or even a blend of a first size exclusion support and a first moiety, a second moiety (and a third moiety, etc.). Compositions comprising various combinations of one or more size exclusion supports and one or more moietys are considered.

[0028] In some embodiments, one or more moietyes of the composition of the present disclosure may include polysaccharides, dextrans, polyethylene glycol polymers, amine-containing polymers, polyamino acids, lipopolysaccharides, antibiotics, chelating groups, magnetic particles, paramagnetic particles, functional groups, ion-exchangers, and combinations thereof.

[0029] In some embodiments, the amine-containing polymer of the composition of the present disclosure is poly(ethylene glycol)diamine, polyethylenediamine, linear polyethyleneimine, or branched polyethyleneimine. In some embodiments, the linear polyethyleneimine is diethylenediamine.

[0030] In some embodiments of the composition of the present disclosure, at least one moiety is dextran. Various dextrans may be used. In some embodiments, the dextran used in the composition of the present disclosure has a molecular weight in the range of about 6 kDa to 2800 kDa. In some embodiments, the dextran used in the composition of the present disclosure has a molecular weight in the range of about 1500 kDa to 2800 kDa.

[0031] In some embodiments, the moiety used in the composition of the present disclosure is an ion-exchanger. The ion-exchanger is an anion-exchanger or a cation-exchanger. Non-limiting examples of ion-exchangers include a negatively charged hydroxyl group and a positively charged pentylamine group, diamine, or imine group.

[0032] In some embodiments, the moiety used in the composition of the present disclosure is a polyamino acid, e.g., polylysine, polyhistidine, and / or polyglutamate.

[0033] In some non-limiting exemplary embodiments, the composition of the present disclosure may comprise: a first size exclusion support is combined with a first moiety, the first moiety comprises, for example, an amine-containing polymer (e.g., polyethylene glycol diamine), and a second size exclusion support is combined with a second moiety, the second moiety comprises, for example, dextran.

[0034] In some other non-limiting exemplary embodiments, the composition of the present disclosure may comprise: a first size exclusion support is combined with a first moiety comprising, for example, poly(ethylene glycol)diamine, polyethylenediamine, linear polyethyleneimine or branched polyethyleneimine, and a second size exclusion support is combined with a second moiety comprising, for example, dextran.

[0035] In some other non-limiting exemplary embodiments, the composition of the present disclosure may comprise: at least a first size exclusion support bonded to a first moiety comprising poly(ethylene glycol)diamine, and the first or second size exclusion support bonded to a second moiety, the second moiety comprising dextran.

[0036] In some other non-limiting exemplary embodiments, the composition of the present disclosure may comprise: a first size exclusion support is bonded to a first moiety comprising N,N diethylethylenediamine, and a second size exclusion support is bonded to a second moiety, and the second moiety comprises, for example, dextran.

[0037] In some non-limiting exemplary embodiments, the composition of the present disclosure may comprise: a first size exclusion support bonded to a first moiety, the first moiety comprising, for example, an amine-containing polymer, and a second size exclusion support bonded to a second moiety, the second moiety comprising, for example, a polyethylene glycol polymer.

[0038] Compositions as described herein are incorporated in the apparatus, apparatus, system, and kit of the present disclosure and are used in one or more methods of the present disclosure that are further described in detail in the following paragraphs.

[0039] In some embodiments, the present disclosure provides an apparatus and / or device for separating, removing, or extracting one or more small molecules from a sample comprising: a) a container comprising: at least one size exclusion support and at least one moiety capable of binding to one or more small molecules; and b) a receptacle located below the container.

[0040] In some embodiments, the present disclosure provides a system for separating, removing, or extracting one or more small molecules from a sample comprising: a) a container comprising: a size exclusion support and one or more moiety capable of binding to one or more small molecules; and b) a reservoir located below the container. The system of the present disclosure is configured to have a force such as vacuum, gravity, or negative or positive pressure applied to the sample within the container, but is not limited thereto. In some embodiments, the system of the present disclosure includes means for applying a force such as vacuum, gravity, or negative or positive pressure, but not limited thereto, to a combination of the reservoir and the container.

[0041] In some embodiments of the apparatus, apparatus, or system of the present disclosure, the reservoir is attached to a column. In some embodiments, the reservoir is detachable from the column. The contents of the reservoir can be removed by a user. The reservoir of the apparatus collects a sample with significantly reduced small molecules. The apparatus, apparatus, or system of the present disclosure is operable to separate, reduce, or remove one or more small molecules from a sample in a single step. The apparatus, apparatus, or system of the present disclosure is operable to have a force such as vacuum, gravity, or negative or positive pressure applied to a sample within a container.

[0042] In some embodiments of the apparatus, apparatus or system of the present disclosure, it is configured to be operable to receive gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, and combinations thereof.

[0043] In some embodiments of the apparatus, apparatus or system of the present disclosure, the container is a columnar container, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate. Exemplary containers include, but are not limited to, a spin column, a multi-well plate, a multi-well filter plate, a micro-well plate, and a micro-well filter plate.

[0044] In embodiments, the systems, apparatuses, and devices of the present disclosure include a container comprising one or more compositions of the present disclosure as set forth in the paragraphs above and below.

[0045] In some embodiments, the present disclosure describes a kit for separating a biomolecule from one or more small molecules, comprising: an apparatus comprising: a) a container comprising a size exclusion resin and at least one moiety capable of binding to at least one small molecule and capturing said small molecule; and b) a reservoir located below the container, wherein the apparatus is configured to be operable by gravity flow, centrifugal force, positive pressure, negative pressure, vacuum and combinations thereof.

[0046] The kit of the present disclosure may comprise one or more compositions, one or more devices, apparatuses, and / or systems described herein. In some embodiments, the kit of the present disclosure comprises at least two moieties capable of binding to one or more small molecules.

[0047] In some embodiments of the kit of the present disclosure, the device is a spin column, a multi-well filter plate, or a multi-well plate. The kit may further comprise one or more buffers packaged in one or more individual containers or contained in a first container.

[0048] In some embodiments, the present disclosure describes a method for separating a biomolecule from one or more small molecules, comprising: a) applying a sample to a container comprising a size exclusion support and at least one moiety capable of binding to at least one small molecule; and b) subjecting the container to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof, wherein the biomolecule in the sample is excluded through the size exclusion support and collected as a flow-through, and the at least one small molecule is separated from the sample by binding to at least one moiety.

[0049] In some embodiments of the method of the present disclosure, separation of at least one small molecule from the remainder of the sample is performed in one step. In some embodiments of the method of the present disclosure, the effluent is collected in a storage located below the container.

[0050] The methods, compositions, kits, devices, apparatuses, and systems of the present disclosure advantageously provide excellent separation of small molecules and additionally reduce the time and cost associated with separating small molecules from larger biomolecules in a sample. As described herein, the separated larger biomolecules may be suitable for better downstream processing. While specific advantages have been disclosed above, it will be understood that various embodiments may include all or some of the previously disclosed advantages, or none of them. Other technical advantages may be readily apparent to those skilled in the art in light of the teachings of the present disclosure.

[0051] These and other features of this lesson will become more apparent from the detailed descriptions in the following paragraphs. Brief explanation of the drawing

[0052] One or more embodiments of the present disclosure may be better understood by referring to one or more of the drawings below. Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of this teaching in any way. FIG. 1a illustrates a schematic diagram of a size exclusion support that combines with a moiety to form an exemplary composition according to one embodiment of the present disclosure; FIG. 1b illustrates a schematic diagram of a composition of the present disclosure that combines with one or more small molecules according to one embodiment of the present disclosure; FIG. 2a illustrates a schematic diagram of a non-limiting exemplary composition of the present disclosure according to one embodiment of the present disclosure; FIG. 2b illustrates a schematic diagram of a non-limiting exemplary composition of the present disclosure according to one embodiment of the present disclosure; FIG. 2c illustrates a schematic diagram of a non-limiting exemplary composition of the present disclosure according to one embodiment of the present disclosure; FIG. 2d illustrates a schematic diagram of a non-limiting exemplary composition of the present disclosure according to one embodiment of the present disclosure; FIG. 2e illustrates a schematic diagram of a non-limiting exemplary composition of the present disclosure according to one embodiment of the present disclosure; FIG. 2f illustrates a schematic diagram of a non-limiting exemplary composition of the present disclosure according to one embodiment of the present disclosure; FIG. 3 illustrates an exemplary three-dimensional (3-D) view of a mechanism / device according to one embodiment of the present disclosure; FIGS. 4a and 4b illustrate three-dimensional (3-D) views of an exemplary mechanism / device according to one embodiment of the present disclosure; FIG. 5 illustrates the removal of exemplary small molecule, glass dye using a composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 6 illustrates the removal of exemplary small molecule, glass dye using a composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 7 illustrates the removal of an exemplary small molecule, reducing agent using a composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 8 illustrates the removal of exemplary small molecules, glass dyes using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 9 illustrates the removal of exemplary small molecules, glass dyes using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 10 illustrates the removal of exemplary small molecules, glass dyes using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 11 illustrates the removal of exemplary small molecules, glass dyes using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 12 illustrates the removal of an exemplary small molecule and two exemplary glass dyes using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 13 illustrates the removal of exemplary small molecules, reducing agents, and corresponding protein recovery using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 14 illustrates the removal of exemplary small molecules, free biotin, and corresponding protein recovery using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 15 illustrates the quantitative data of FIG. 13 for the removal of an exemplary small molecule, glass dye Alexa Fluor™ 555 and protein recovery using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 16 illustrates the quantitative data of FIG. 12 for the removal of exemplary small molecules, free dye fluorescein and protein recovery using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 17 illustrates the removal of an exemplary small molecule, BS3, bis(sulfosuccinimidyl)suberic acid and the recovery of a protein using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 18 illustrates the removal of an exemplary small molecule, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and the recovery of a protein using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure; FIG. 19 illustrates the removal of an exemplary small molecule, DTT (dithiothreitol), and protein recovery using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure. FIGS. 20a and 20b illustrate the removal of an exemplary small molecule, glass dye Alexa Fluor™ 647, and the recovery of a protein using a composition, apparatus, kit, and / or method according to one embodiment of the present disclosure, compared with the removal of the small molecule and the recovery of the protein by other existing products sold for similar uses; FIG. 21 illustrates the removal of an exemplary small molecule, biotin, and protein recovery using a composition, apparatus, kit, and / or method according to one embodiment of the present disclosure, compared with the removal of a small molecule and protein recovery by other existing products sold for similar uses; FIG. 22 illustrates the removal of an exemplary small molecule, reducing agent TCEP, using a composition, apparatus, kit, and method according to one embodiment of the present disclosure, compared with the removal of a reducing agent by other existing products sold for similar uses; FIG. 23 illustrates the removal of an exemplary small molecule, the free dye Alexa Fluor™ 647, and the recovery of a protein using a composition, spin column device, kit, and method according to one embodiment of the present disclosure, compared to the removal of the free dye Alexa Fluor™ 647 using a conventional dialysis method; FIG. 24 illustrates the removal of exemplary small molecules, biotin, and protein recovery using a composition, apparatus, kit, and method according to one embodiment of the present disclosure, compared with the removal of biotin using a conventional dialysis method; FIG. 25 illustrates the removal of an exemplary small molecule, glass dye Alexa Fluor™ 647, and protein recovery using a composition, spin plate device, kit, and method according to one embodiment of the present disclosure; FIG. 26 illustrates the removal of exemplary small molecules, free dye fluorescein dye, and protein recovery using a composition, spin column apparatus, kit, and method according to one embodiment of the present disclosure; FIGS. 27a and 27b illustrate immunofluorescence images of exemplary cells stained with a polyclonal antibody-Alexa Fluor™ 647 conjugate, and removal of excess free dye Alexa Fluor™ 647 by washing using a spin column comprising the composition of the present disclosure (Fig. 27b), (without using the spin column of the present disclosure not ) Shown in comparison with the immunofluorescence image of the same cells without washing (Fig. 27a); FIGS. 28a, 28b, and 28c illustrate immunofluorescence images of exemplary cells stained with a polyclonal antibody labeled with Alexa Fluor™ 647, and the removal of excess free dye Alexa Fluor™ 647 by washing using a spin column comprising the composition of the present disclosure (Fig. 28c) compared with an immunofluorescence image of the same cells without washing (without using the spin column of the present disclosure). not )(Fig. 28a); and, compared with an immunofluorescence image of the same cells washed using a spin column with the conventional product GE PD10 (Fig. 28b); FIGS. 29a and 29b illustrate immunofluorescence images of exemplary cells stained with a monoclonal antibody labeled with Alexa Fluor™ 488, and compare the removal of excess free dye Alexa Fluor™ 488 by a spin column having the composition of the present disclosure (Fig. 29b) with an immunofluorescence image of the same cells without washing (without using the spin column of the present disclosure hmm ) represents (Fig. 29a); FIGS. 30a and 30b illustrate a comparison of a protein recovery and dye removal composition according to one embodiment, the apparatus and method of the present disclosure (Loomba), and an ion exchange resin (Dowex); and FIGS. 31a and 31b illustrate a comparison of a protein recovery and dye removal composition according to one embodiment, the apparatus and method of the present disclosure (Loomba), and an ion exchange resin (Dowex). Specific details for implementing the invention

[0053] It should be understood that both the foregoing general description and the following detailed description are illustrative and descriptive, and are not intended to limit the scope of this teaching. In this application, the use of the singular includes the plural form unless specifically stated otherwise. For example, the singular forms used herein (“a,” “an,” and “the”) also include the plural form unless the context otherwise indicates. Similarly, any singular term used herein also means the plural or the opposite, unless the context otherwise indicates.

[0054] Furthermore, it is not intended to restrict the use of "comprise," "contain," and "include," or modifications of the root, e.g., "comprises," "contained," and "including." The use of "or" means "and / or" unless otherwise specified. "And / or" means that the preceding and succeeding terms may be taken together or separately. For example, "X and / or Y" may mean "X" or "Y" or "X and Y."

[0055] Whenever a range of values ​​is provided in this invention, the range means, unless specifically stated otherwise, a starting value and an ending value and any value or range of values ​​between them. For example, “0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; a range between 0.2-0.3, 0.3-0.4, 0.2-0.4; an increase between 0.25, 0.35, 0.225, 0.335, 0.49; an increase range between 0.26 and 0.39; etc.

[0056] As used herein, the term “or combinations thereof” refers to all permutations and combinations of the items listed prior to the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing from this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are explicitly included. Those skilled in the art will understand that, typically, there is no limit to the number of items or terms in any combination unless otherwise specified in the context.

[0057] The terms "separation," "extraction," "extracted," "removal," "reduction," "reduction in volume," or "purification" all refer to the action or process of removing or separating a substance (e.g., small molecules such as labels or chemicals, or larger molecules, or biomolecules such as DNA, RNA, or proteins) from a mixture (of various components, such as cellular components, or substances within a sample containing small molecules and / or biomolecules and / or larger molecules). The extracted substance or the sample from which the substance was extracted may have a significantly reduced amount of the present component, be significantly reduced, significantly removed, significantly pure, or pure (free of any contaminants), or be concentrated or significantly concentrated compared to before extraction.

[0058] The term "small molecule" generally refers to any molecule smaller than a large molecule (equivalent to, but not limited to, a biomolecule) used to process, derivatize, conjugate, crosslink, label, tag, or chemically or biologically modify larger molecules for further analysis. Larger molecules and / or biomolecules, including proteins, glycoproteins, antibodies, nucleic acids (DNA, genomic DNA, pDNA, RNA), polysaccharides, carbohydrates, lipids, and toxins, as well as some other larger molecules such as nanoparticles, are often derivatized by various treatments prior to further analysis or use. Derivatization involves labeling molecules with markers such as dyes, affinity tags, radiolabels, mass tags, metals, etc. Derivatization also involves chemically modifying molecules by reduction, oxidation, methylation, biological or biochemical modification, etc. Derivatization of biomolecules includes tagged proteins or nucleic acids; biomolecules labeled with various markers such as dyes, fluorescent dyes, radiolabels, affinity tags, mass tags, metals, etc.; conjugated biomolecules, including conjugated antibodies; and biomolecules conjugated to nanoparticles. Metals such as gold conjugated to nanoparticles; dyes or labels such as biotin conjugated to toxins; chemical derivatives of biomolecules such as but not limited to proteins having reduced proteins, oxidized proteins, methylated nucleic acids, or proteins modified by sulfhydryl, are included, but not limited to.

[0059] Derivatization methods often leave “small molecule” byproducts in samples, such as unreacted glass labels, partially reacted labels, derivatives of unreacted glass labels including glass dyes, derivatives of dyes, glass radioligands, intermediates of radioligands, glass mass tags, glass metals, biotin, biotin derivatives, crosslinkers and their derivatives, excess reducing agents or their derivatives, unreacted nanoparticles, other unreacted, partially reacted or intermediate molecules and combinations thereof, intermediates and derivatives. Because these unreacted small molecules can cause various problems during downstream analysis or use of larger molecules, it is necessary to separate, extract, remove, or reduce the amount of “small molecules” from larger molecules and their derivatives during sample preparation. In some embodiments, the small molecules that can be separated, extracted, or removed by the compositions, apparatus, instruments, systems, kits, and methods of the present disclosure are typically < 2 kDa.

[0060] The term “support” refers to an inert porous solid. The term “size exclusion support” describes an inert porous solid having a porosity that determines the size of molecules that can be included or excluded from entering or entering the pores. In some embodiments, the pore size of the size exclusion support is equal to 2 kDa. In some embodiments, the pores of the size exclusion support of the present disclosure have a molecular size cut-off of 2 kDa, so that the excluded molecules are 2 kDa or larger than 2 kDa. In some embodiments, the pore size of the size exclusion support is > 2 kDa. In some embodiments, the pores of the size exclusion support of the present disclosure have a molecular size cut-off of about > 2 kDa, so that the excluded molecules are molecules larger than 2 kDa. In one embodiment, the pore size of the size exclusion column has a molecular size cut-off size for excluding pore molecules of about 2 kDa to about 150 kDa, about 5 kDa to about 150 kDa, including but not limited to a range between, for example, 2 kDa, 3 kDa, 5 kDa, 7 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, and 150 kDa. In some embodiments, there is no upper limit on the size of proteins that may be excluded, and mega-dalton size molecules are also conceived to be separated from small molecule impurities or contaminants by the compositions, devices, systems, kits, and methods of the present disclosure.

[0061] Paragraph headings used herein are for organizational purposes only and are not to be interpreted as limiting the subject matter described in any way. All literature and similar materials cited in this application, regardless of the format of such literature and similar materials, including but not limited to patents, patent applications, articles, books, papers, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. If one or more of the incorporated literature and similar materials define or use a term in a manner inconsistent with the definition of such term in this application, this application limits it. The teachings are described with various embodiments, but the teachings are not intended to be limited to such embodiments. On the contrary, the teachings include various alternatives, modifications, and equivalents as will be understood by those skilled in the art in light of the teachings.

[0062] Composition:

[0063] One embodiment of the present disclosure describes a composition for separating, extracting, removing, and / or reducing the amount of one or more small molecules from a larger biomolecule. The composition comprises a size exclusion support and at least one moiety capable of binding to one or more small molecules, thereby separating the small molecules from other components (e.g., a larger biomolecule) present in a sample.

[0064] Size exclusion supports typically comprise spherical beads made of a gel or a gel-like material having pores. Some exemplary size exclusion supports are made of dextran polymers, agarose, polyacrylamide, cellulose materials, and derivatives thereof. The pore size range of the size exclusion support determines the size of molecules that can be included or excluded from entering the size exclusion support.

[0065] When a sample solution is applied to a size exclusion support, it moves down the support, and smaller sample components enter the pores. Sample components larger than the pore size cannot enter the pores. Therefore, larger sample components are excluded and eluted from the size exclusion column more rapidly than smaller components trapped within the pores.

[0066] The present composition comprises a combination of at least one size exclusion support and at least one moiety capable of binding to one or more small molecules. Accordingly, the composition of the present disclosure provides separation of small molecules from larger biomolecules of a sample by a combination of size exclusion and size inclusion, as well as separation by binding of small molecules onto at least one moiety. The inventors have discovered that the present composition provides a rapid, economical, and efficient separation of small molecules from larger biomolecules.

[0067] In some embodiments, the composition of the present disclosure comprises at least one moiety coupled to at least one size exclusion support. In some embodiments, the composition of the present disclosure comprises at least one moiety fixed on at least one size exclusion support.

[0068] FIG. 1a illustrates a schematic diagram of an exemplary size exclusion support (10) that combines with a moiety (20) to form an exemplary composition (30) according to one embodiment of the present disclosure. FIG. 1b illustrates a schematic diagram of a composition (30) of the present disclosure that combines with one or more small molecules (40) to form a complex (50) according to one embodiment of the present disclosure.

[0069] FIG. 2a illustrates a schematic diagram of another non-limiting exemplary composition (30') of the present disclosure, comprising an exemplary size exclusion support (10') coupled with a moiety (20') according to one embodiment of the present disclosure. FIG. 2b illustrates another non-limiting exemplary composition (30") of the present disclosure, comprising an exemplary size exclusion support (10') coupled with a moiety (20") according to one embodiment of the present disclosure. These figures are non-limiting exemplary compositions used to illustrate some possible compositions. In light of the description and drawings herein, those skilled in the art will recognize that various different combinations of compositions are considered by the present disclosure.

[0070] In some embodiments, immobilization is achieved by the formation of covalent bonds, e.g., amide bonds, alkylation, amination, amidation, covalent amine-forming bonds, or covalent amide-forming bonds between a size exclusion support and a moiety, but is not limited thereto. In some embodiments, the size exclusion support comprises dextran polymers, agarose, polyacrylamide, cellulose materials, and derivatives thereof. In some exemplary embodiments, the size exclusion support comprises hydroxyethylcellulose. In an exemplary immobilization, according to the present disclosure, hydroxyethylcellulose may be periodate oxidized to produce an aldehyde group. These generated aldehyde groups can react with terminal amines on exemplary moietyes (e.g., but not limited to pentylamine (which can be used as a spacer molecule) or poly(ethylene glycol)bisamine) to form Schiff base intermediates. Unstable Schiff base interactions are chemically stabilized by reduction using chemicals such as sodium cyanoborohydride to form secondary amine bonds.

[0071] In some embodiments, the pore size of the size exclusion support is equal to 2 kDa or > 2 kDa. In these exemplary compositions, the molecules excluded from the pores are molecules of 2 kDa or molecules of greater than 2 kDa. Biomolecules of 2 kDa or greater than 2 kDa are eluted from these compositions. In some exemplary embodiments, the pore size of the size exclusion support is about > 2 kDa to about 50 kDa, > 2 kDa to about 75 kDa, > 2 kDa to about 100 kDa, > 2 kDa to about 150 kDa, or > 2 kDa to about 200 kDa. In some embodiments, the pore size of the size exclusion support is about 7,000 Da to about 50,000 Da. In some embodiments, there is no upper limit on the size of proteins that may be excluded, and mega-dalton size molecules and larger ones are also conceived to be separated from small molecule impurities or contaminants by the compositions, devices, systems, kits, and methods of the present disclosure.

[0072] In some embodiments, the composition of the present disclosure (when in contact with a sample) significantly reduces the amount of one or more small molecules in the sample. In some embodiments, contacting the sample with the composition of the present disclosure comprises, without limitation, one or more of the following: applying the sample onto the composition, passing the sample through the composition, causing the sample to flow through the composition by gravity or by using rotation or centrifugal force, and causing the sample to move through the composition by creating a pressure difference.

[0073] In some embodiments, at least one moiety is bonded to one or more small molecules by charge interaction, hydrophilic interaction, hydrophobic interaction, affinity interaction, hydrogen bonding, van der Waals forces, or covalent bonding.

[0074] In some embodiments, the composition of the present disclosure may comprise at least two moiety, or at least three moiety, or at least four moiety, or at least five moiety, etc. FIG. 2c illustrates a schematic diagram of a non-limiting exemplary composition (30"') of the present disclosure comprising an exemplary size exclusion support (10') coupled with at least two moietys (20' and 20") according to one embodiment of the present disclosure. These drawings are non-limiting exemplary compositions used to illustrate some possible combinations of different numbers of moiety. In light of the descriptions and drawings herein, one of the art realizes that some different combinations of compositions are considered by the present disclosure and that the drawings do not limit the scope of the teachings of the present disclosure.

[0075] In some embodiments, the size exclusion resin excludes molecules from samples equal to 2 kDa. In some embodiments, the size exclusion resin excludes molecules from samples > 2 kDa. In some embodiments, the size exclusion resin excludes molecules from samples > 3 kDa. In some embodiments, the size exclusion resin excludes molecules from samples > 5 kDa.

[0076] In some embodiments, the composition of the present disclosure may further comprise at least a second size exclusion support. In some embodiments, the composition of the present disclosure may further comprise a third size exclusion support, a fourth size exclusion support, a fifth size exclusion support, etc. In some embodiments, the composition of the present disclosure may further comprise at least one second moiety, at least a third moiety, a fourth moiety, a fifth moiety, etc. One size exclusion support may comprise one or more different types of moiety. Alternatively, different size exclusion supports may comprise the same moiety or different types of moiety.

[0077] The compositions of the present disclosure, in some embodiments, comprise different proportions of various combinations of one or more size exclusion supports and one or more moietyes as described herein. For example, the compositions of the present disclosure comprise different proportions of a first size exclusion support and at least one first moiety and a second size exclusion support and at least one second moiety. Other exemplary compositions of the present disclosure comprise different proportions of a first size exclusion support and at least one first moiety and a first size exclusion support and at least one second moiety. Yet another exemplary composition of the present disclosure comprises different proportions of a first size exclusion support and at least one first moiety and a first size exclusion support and at least one first moiety and a second moiety (and a third moiety, etc.). A composition is considered capable of facilitating the separation or removal of one or more biomolecules present in a sample or one or more small molecules by the application of a sample to a single composition.

[0078] FIG. 2d illustrates a schematic diagram of a non-limiting exemplary composition comprising a blend of compositions 30" and 30" in a ratio n:n (where each n can independently be a number from 1 to 9, e.g., 1:1, 2:1, 3:4, 1:5). FIG. 2e illustrates a schematic diagram of another non-limiting exemplary composition comprising a blend of compositions 30"' and 30" in a ratio n:n (where each n can independently be a number from 1 to 9). FIG. 2f illustrates a schematic diagram of another non-limiting exemplary composition comprising a blend of compositions 30" and 30"' in a ratio n:n (where each n can independently be a number from 1 to 9). These figures are non-limiting exemplary compositions used to illustrate some possible compositions. In light of the description and drawings herein, those skilled in the art will recognize that several different blends of compositions and combinations of compositions are considered by the present disclosure.

[0079] In some embodiments, one or more moietyes of the composition of the present disclosure may include polysaccharides, polyethylene glycol polymers, amine-containing polymers, polyamino acids, antibiotics, chelating groups, magnetic particles, paramagnetic particles, functional groups, ion-exchangers, and combinations thereof.

[0080] Exemplary amine-containing polymers that may be used in the compositions of the present disclosure include, but are not limited to, poly(ethylene glycol)diamine, polyethylenediamine, linear polyethyleneimine, or branched polyethyleneimine. An exemplary linear polyethyleneimine is diethylenediamine.

[0081] In some exemplary compositions of the present disclosure, one or more polysaccharide moietys may be one or more dextrans. Various dextrans may be used. In some embodiments, the dextran used in the compositions of the present disclosure has a molecular weight in the range of about 6 kDa to 2800 kDa. In some embodiments, the dextran used in the compositions of the present disclosure has a molecular weight in the range of about 1500 kDa to 2800 kDa.

[0082] In some embodiments, the moiety used in the composition of the present disclosure is an ion-exchanger, such as an anion exchanger or a cation exchanger. An anion exchanger moiety may bind to a small molecule having a negative charge and leave a positively charged sample component. A cation exchanger may bind to a small molecule having a positive charge and leave a negatively charged sample component. Non-limiting examples of ion exchangers include negatively charged hydroxyl groups, positively charged pentylamine groups, diamines, and imines.

[0083] In some exemplary compositions of the present disclosure, one or more polyamino acid moietyes may be polylysine, polyhistidine, polyglutamine, polyasparagine, etc. In some embodiments, the polyamino acid moiety may remove or separate endotoxins from other larger biomolecules.

[0084] In some embodiments, the size exclusion support or moiety may include a reactive functional group. A functional group on the size exclusion support may be used to interact with and bind to one or more moietyes to form a composition. A functional group on the moiety may be used to interact with and bind to one or more small molecules so that they may be separated or extracted from a larger biomolecule. The functional group may include, but is not limited to, a group for binding or interacting with the moiety or small molecule, such as a hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, etc.

[0085] In another embodiment, the functional group represents a reactive functional group R attached to a moiety by a size exclusion support or a covalent bond L. x This, or reactive functional group R x (-LR xIt includes at least one reactor represented by ). The reactor functions as a site for binding, attachment, and / or interaction with a moiety or small molecule, wherein the reactor chemically reacts with a suitable reactive or functional group on the solid support, moiety, or small molecule. In an exemplary embodiment, the reactive group or functional group may be an acrylamide, an activated ester of a carboxylic acid, an acyl halide group, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a thioboronate group, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine, a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a sulfonyl halide, a thiol group, a sulfide group, a disulfide group, an epoxide group and an episulfide group, a thioester group, an alcohol group, an activated alcohol group, a phosphate group, a phosphate ester group, and a photoactivator group.

[0086] In other exemplary embodiments, the reactive group or functional group may comprise an electrophile and / or nucleophile, and in some embodiments, may form a covalent bond between them. Exemplary electrophilic and nucleophilic functional groups may comprise an activated ester, generally having the formula -COΩ, where Ω is a good leaving group (e.g., oxysuccinimidyl (-OC4H4O2), oxysulfosuccinimidyl (-OC4H3O2-SO3H), -1-oxybenzotriazoryl (-OC6HN3); or an aryloxy group or aryloxy substituted one or more times by electron-withdrawing substituents such as nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinations thereof, used to form an activated aryl ester; or an anhydride or mixed anhydride -OCOR activated by a carbodiimide. a or -OCNR a NHR b A carboxylic acid that forms, wherein R, which may be the same or differenta and R b is a C1-C6 alkyl, C1-C6 perfluoroalkyl, or C1-C6 alkoxy carboxylic acid; or, cyclohexyl, 3-dimethylaminopropyl, acyl halide group, acylnitrile, aldehyde, alkyl halide, anhydride, aryl halide, aziridine, diazoalkane, haloacetamide, halotriazine, isocyanate, isothiocyanate, maleimide, phosphoramidite, sulfonyl halide, sulfide group, disulfide group, epoxide group, and episulfide group, thioester group, activated alcohol group, phosphate group, phosphate ester group, and photoactivated group. Acyl azide can also be rearranged into isocyanate.

[0087] In some embodiments, the reactor further comprises a linker L in addition to the reactive functional moiety. The linker may be used to covalently attach the reactive functional group. If present, the linker is a single covalent bond or a series of stable bonds. The reactive functional moiety may be directly attached to or attached to a solid support, moiety, or small molecule through a series of stable bonds (if the linker is a single bond). If the linker is a series of stable covalent bonds, the linker typically comprises some non-hydrogen atoms selected from the group consisting of C, N, O, S, Si, B, and P. Additionally, the covalent bonds may comprise platinum atoms as described in U.S. Patent No. 5,714,327. Where the linker is not a single covalent bond, the linker may be any combination of stable chemical bonds, including single, double, triple, or aromatic carbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds optionally. In an exemplary embodiment, the linker comprises fewer than 15 non-hydrogen atoms and consists of a combination of ether, thioether, thiourea, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds. Typically, the linker is a combination of a single covalent bond or a single carbon-carbon bond and a carboxamide, sulfonamide, or thioether bond. The following moiety can be found in the linker: ether, thioether, carboxamide, thiourea, sulfonamide, urea, urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl, and amine moiety. Examples of L include substituted or unsubstituted polymethylene, arylene, alkylarylene, arylenealkyl, or arylthio.

[0088] Functional groups or reactive groups can be attached using any combination of linkers. If the reactive group is a maleimide or a haloacetamide, the resulting compound is particularly useful for conjugation to thiol-containing materials. If the reactive group is a hydrazide, the resulting compound is particularly useful for conjugation to peroxidized carbohydrates and glycoproteins. If the reactive group is a silyl halide, the resulting compound is particularly useful for conjugation to silica surfaces, especially when incorporated into optical fiber probes where the silica surface is subsequently used for remote ion detection or quantification.

[0089] In some non-limiting exemplary embodiments of the composition of the present disclosure, a first size exclusion support is bonded to a first moiety, and the first moiety comprises, for example, an amine-containing polymer, and a second size exclusion support is bonded to a second moiety, and the second moiety comprises, for example, dextran.

[0090] In some other non-limiting exemplary embodiments of the composition of the present disclosure, a first size exclusion support is combined with a first moiety comprising, for example, poly(ethylene glycol)diamine, polyethylenediamine, linear polyethyleneimine, or branched polyethyleneimine, and a second size exclusion support is combined with a second moiety comprising, for example, dextran.

[0091] In some other non-limiting exemplary embodiments of the composition of the present disclosure, a first size exclusion support is combined with a first moiety comprising diethylenediamine, and a second size exclusion support is combined with a second moiety, and the second moiety comprises, for example, dextran.

[0092] In some non-limiting exemplary embodiments of the composition of the present disclosure, a first size exclusion support is bonded to a first moiety, and the first moiety comprises, for example, an amine-containing polymer, and a second size exclusion support is bonded to a second moiety, and the second moiety comprises, for example, a polyethylene glycol polymer.

[0093] Equipment and devices:

[0094] In some embodiments, the present disclosure provides an apparatus and / or device and / or system for removing one or more small molecules from a sample comprising: a) a container comprising: at least one size exclusion support and at least one moiety capable of binding to one or more small molecules; and b) a reservoir located below the container. In some embodiments of the apparatus, apparatus or system of the present disclosure, the reservoir is attached to a column. In some embodiments, the reservoir is detachable from the column. The contents of the reservoir can be removed by a user. In some embodiments, the reservoir of the apparatus collects a sample with significantly reduced small molecules. In some embodiments, the reservoir of the apparatus collects a sample without small molecules.

[0095] In some embodiments of the apparatus, apparatus or system of the present disclosure, it is configured to be operable to receive gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, and combinations thereof. Structures that allow the application of the aforementioned pressures or forces include, without limitation, syringes capable of being drawn to induce positive pressure, vacuum frits for generating negative pressure, tubes or containers adaptable to commercially available centrifuges or rotary devices.

[0096] The apparatus, device, or system of the present disclosure is operable to separate, reduce, or remove an amount of one or more small molecules from a sample by applying a single sample to the apparatus, and to receive one or more forces, such as vacuum, gravity, or negative or positive pressure, applied to a sample in a container.

[0097] In some embodiments of the apparatus, apparatus or system of the present disclosure, the container is a columnar container, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate. Exemplary containers include, but are not limited to, a test tube, a spin column, a multi-well plate, a multi-well filter plate, a micro-well plate, or a micro-well filter plate.

[0098] FIG. 3 illustrates a non-limiting three-dimensional (3-D) view of an exemplary apparatus / device or system (100) according to one embodiment of the present disclosure, comprising a container (60) (e.g., a columnar tube, a test tube, or a spin column) and a container (60) having a filter, mesh, or porous surface area at the bottom end of the container (not shown). The container (60) may also include one or more frits (not shown). The container (60) includes or contains within an exemplary composition (30) (at least one size exclusion support and at least one moiety capable of bonding with one or more small molecules) and an optional lid (60') that may be used to secure the container (60) to the top end. A reservoir (70) is located at the bottom of the container (60) and is adapted or configured to receive an effluent or effluent from the container through its bottom end. The reservoir (70) may be separated from the container (60) so that a user can collect the effluent or effluent. In some embodiments, the storage (60) has a twist-off tab configuration for removal. In other embodiments, the storage (70) may be attached to the container (60) by a groove or thread that can be attached and removed by manual or mechanical means, such as by rotation, twisting-off, or attachment by a complementary fit that can be pulled.

[0099] FIGS. 4a and 4b illustrate non-limiting three-dimensional (3-D) views of an exemplary apparatus / device or system (100') according to one embodiment of the present disclosure, comprising a multi-well container (80) disposed inside each well, for example, a composition (30) (at least one size exclusion support and at least one moiety capable of being combined with one or more small molecules), and an optional lid (80') for the container. (80') may be foil, clear wrap, a tear-off seal and / or a removable lid.

[0100] The multi-well container (80) may be a multi-well plate, a multi-well plate filter, a microplate, or a microtiter plate, and comprises a flat plate having multiple wells, each well being used as a small test tube or container. Multi-well plates are provided in various formats for high-throughput use and may typically have 6, 12, 24, 48, 96, 384, 1536, 3456, 9600 or more wells arranged in a rectangular matrix or array.

[0101] A multi-well container (80) may have a reservoir (70) located below the container that is adaptable or configured to receive effluent or effluent from the container. The multi-well filter plate, container (80) may have a mesh, filter, or other type of porous bottom surface, through which effluent or effluent may flow into the reservoir (70) (not shown).

[0102] In some embodiments, this reservoir (70) is a multi-well tray for collecting leachate or effluent (e.g., see FIG. 4a). The reservoir (70) is detachable and the leachate can be collected from it by a user (see FIG. 4a and FIG. 4b). In some embodiments, the reservoir (70) is a cleaning plate or a collection plate.

[0103] FIGS. 3, FIGS. 4a, and FIGS. 4b illustrate exemplary devices only, and while this description uses these embodiments for illustrative purposes, it will be understood that other embodiments of the apparatus can be readily made by those skilled in the art as variations of the descriptions herein.

[0104] One or more small molecules that can be removed or extracted by the apparatus, apparatus or system of the present disclosure may be dyes, derivatives of dyes, biotin, biotin derivatives, crosslinking agents, reducing agents, labels, nanoparticles, radioactive ligands, mass tags, unreacted molecules and combinations thereof, intermediates and derivatives, or may include the same.

[0105] Small molecules that can be removed by the apparatus / mechanism or system of the present disclosure have a molecular weight range of < 2000 Da. This includes molecular weight ranges of approximately 100-200Da, 200-300Da, 300-400Da, 400-500Da, 500-600Da, 600-700Da, 700-800Da, 800-900Da, 900-1000Da, 1000-1100Da, 1100-1200Da, 1200-1300Da, 1300-1400Da, 1400-1500Da, 1500-1600Da, 1600-1700Da, 1700-1800Da, 1800-1900Da, and 1900-<2000Da. In some embodiments, the small molecules that can be removed by the apparatus / mechanism or system of the present disclosure are 50Da, 100Da, 150Da, 200Da, 250Da, 300Da, 350Da, 400Da, 450Da, 500Da, 550Da, 600Da, 650Da, 700Da, 750Da, 800Da, 850Da, 900Da, 950Da, 1000Da, 1050Da, 1100Da, 1150Da, 1200Da, 1250Da, 1300Da, 1350Da, 1400Da, 1450Da, 1500Da, It has a molecular weight range of 1550Da, 1600Da, 1650Da, 1700Da, 1750Da, 1800Da, 1850Da, 1900Da, 1950Da, 1975Da to about <2000Da.

[0106] In an embodiment, the system, apparatus, and device of the present disclosure include a container comprising one or more compositions of the present disclosure as described in detail in the paragraphs above and below.

[0107] One or more advantages of the systems, apparatuses, and devices of the present disclosure include one or more of the following: being economical, simple, easy to use, providing faster results, being usable for high-throughput sample preparation in a multi-well container format that may be a single-use disposable unit, and adaptable to automated and robotic sample preparation systems. Reducing the amount of small molecules from a sample using the apparatuses, devices, and systems provided herein provides rapid and high-quality biomolecules and their derivatives that can be used in downstream applications.

[0108] System

[0109] In some embodiments, the present disclosure provides a system for removing one or more small molecules from a sample, comprising: a) a container comprising a size exclusion support and one or more moieties capable of binding to one or more small molecules; and b) a reservoir located below the container. In some embodiments, the system further comprises means for causing the container and the reservoir to be subjected to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, and combinations thereof.

[0110] In some embodiments, the system of the present disclosure may include the illustrated device (100 or 100) which may be formed to be received within a centrifugal tube or any other similar rotating mechanism. In some embodiments, the system of the present disclosure may include the illustrated device (100 or 100) which may be formed to receive negative pressure (e.g., vacuum) or positive pressure (e.g., syringe, pipette).

[0111] The system of the present disclosure (not illustrated) may be a fully automated or manually operated system. In some embodiments, the system may be operated partially manually and partially automated.

[0112] The system may also include a computer system including a CPU, and hardware elements and / or software elements may be physically internal or external and may be operably connected to the hardware / software elements. The computer system may be operable to control various components of a device (100 or 100"), such as a control robot for recovering and analyzing the leachate.

[0113] The system of the present disclosure may also optionally include, but is not limited to, one or more devices operable to process eluted biomolecules, e.g., eluted derivatized proteins, e.g., conjugated antibodies or tagged proteins for fluorescence detection or immunoassay. In some embodiments, the system may include an imager or a protein or nucleic acid detector or a sequencer.

[0114] A computer system may be operable to control one or more components of the system of the present disclosure. In some embodiments, as described above, the computer system and / or its components may be physically located within or outside the device (100 or 100). The computer system may include a central processing unit, hardware and software elements operable to control and direct any automated steps of data processing of data obtained by sample processing and / or downstream data processing of data (by the device (100 or 100) and other components of the system). Accordingly, the computer system used herein may include a data analysis and control system, a data transfer system such as a read-write CD-ROM drive or DVD drive, at least one USB port, and / or at least one Ethernet port. In some embodiments, the computer system may include pre-loaded software and / or application-specific integrated circuits (ASICS) capable of enabling control of devices (100, 100) and / or other components within the system, including control of a number of components of the system, control of processing and analysis, and / or control of displaying and / or outputting results.

[0115] The system may also include, but is not limited to, additional devices or components such as a power supply, a display unit such as a monitor operable to report / view sample processing or monitor the extraction of biomolecules from a sample; a spectrophotometer; a nucleic acid extraction measuring device; a device for further processing the extracted biomolecules for further analysis; and a printer. The system of the present disclosure may be configured to fit on a laboratory benchtop.

[0116] method

[0117] In some embodiments, the present disclosure describes a method for separating a biomolecule from one or more small molecules, comprising: a) applying a sample to a container comprising a size exclusion support and at least one moiety capable of binding to at least one small molecule; and b) subjecting the container to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof, wherein the biomolecule in the sample is excluded through the size exclusion support and collected as an effluent, and at least one small molecule is separated from the sample by binding to at least one moiety.

[0118] In some embodiments of the method of the present disclosure, separation of at least one small molecule from the remainder of the sample is performed in one step. In some embodiments of the method of the present disclosure, the effluent is collected in a reservoir located below the container. In some embodiments, the small molecule may constitute an impurity or contaminant for the sample.

[0119] Various samples that may be tested by the methods of the present disclosure may be any type of biological or clinical sample having a biomolecule or a derivative thereof from which a small molecule must be separated or removed. Some exemplary, non-limiting samples include samples having protein dye conjugates, biotinylated protein samples, proteins having reducing agents such as DTT or TCEP crosslinking proteins, and protein samples having crosslinking agents. Suitable dyes for use are known to those skilled in the art and include pyrene, coumarin, cyanine, benzofuran, quinoline, quinazolinone, indole, benzazole, borapolyazindacen and xanthenes including fluorescein, rhodamine and rhodole, as well as pyrene, coumarin, cyanine, benzofuran, quinoline, quinazolinone, indole, benzazole, borapolyazindacen and fluorescein, rhodamine and rhodole, as well as xanthenes, as described in the literature (RICHARD P. HAUGLAND, MOLECμLAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (11 thOther dyes described in edition, January 2010)) are included, but not limited to, those described herein, the entirety of which is incorporated herein by reference.

[0120] The methods of the present disclosure can advantageously reduce the time required to process a sample to reduce the amount of small molecules from the sample.

[0121] Kit

[0122] The present disclosure also describes a kit for implementing the methods discussed herein and / or a composition and / or a kit containing the apparatus / device discussed herein.

[0123] In some embodiments, the present disclosure describes a kit for separating a biomolecule from one or more small molecules, comprising: an apparatus comprising: a) a container comprising at least one size exclusion resin and at least one moiety capable of binding to at least one small molecule and capturing said small molecule; and b) a reservoir located below the container, wherein the apparatus is configured to be operable by gravity flow, centrifugal force, positive pressure, negative pressure, vacuum and combinations thereof.

[0124] In some embodiments, the kit of the present disclosure comprises at least two moieties capable of binding to one or more small molecules.

[0125] In some embodiments of the kit of the present disclosure, the device is a spin column, a multi-well filter plate, or a multi-well plate. The kit may further comprise one or more buffers packaged in one or more separate containers or contained in the first container.

[0126] The kit of the present disclosure may also include one or more reagents, such as one or more washing buffers, elution buffers, filter membranes, and / or additional spin columns or multi-well plates.

[0127] The reagents and components of the kit may be contained in one or more suitable container means. The container means may generally include at least one vial, test tube, flask, bottle, syringe, or other container means in which the components may be placed and, preferably, appropriately aliquoted. If the kit contains one or more components, they may be packaged together where appropriate, or the kit will generally contain a second, third, or other additional container in which additional components may be placed separately. However, in some embodiments, specific combinations of components may be packaged together and contained in a single container means. The kit may also include means for containing any reagent container within a sealed chamber for commercial sale. Such a container may include an injection-molded or blow-molded plastic container in which the desired vial is held.

[0128] In some embodiments, the device of the kit of the present disclosure may be pre-filled with one or more reagents to process a sample and may be suitably classified into a suitable chamber. The kit or its container may have an internal compartment and a seal so that any contents therein are sterilized and prevent leakage.

[0129] Some components of the kit are provided as one and / or one or more liquid solutions. The liquid aqueous solution may be an insoluble aqueous solution, an aqueous aqueous solution, or a sterile solution. Components of the kit may also be provided as a dried powder. If reagents and / or components are provided as a dried powder, the powder may be reconstituted by the addition of a suitable solvent. It is considered that a suitable solvent may also be provided in another container means. The kit may also include a container means for containing a sterile and pharmaceutically acceptable buffer and / or other diluent.

[0130] The kit of the present disclosure may also include instructions for using the kit components and may also have instructions for using any other reagents not included in the kit. The instructions may include implementable variations.

[0131] Examples

[0132] Aspects of the present teaching may be further understood in light of the following examples, and this should not be interpreted as limiting the scope of the present teaching in any way.

[0133] Example 1. Preparation of Composition and Testing

[0134] A composition for isolating or extracting one or more small molecules from a sample comprising at least one size exclusion support and at least one moiety capable of binding to one or more small molecules was prepared and tested.

[0135] In some embodiments, an exemplary size exclusion support is modified into a moiety comprising a functional group capable of binding to small molecules by charge interaction, hydrophobic interaction, or any other interaction so that while larger biomolecules in the sample are excluded and collected, these small molecules can bind to and be removed from the sample.

[0136] In this example and the following examples, four exemplary compositions for removing small molecules were prepared and tested for their ability to remove small molecules, comprising a dye (molecular weight range of about 700 Da to 1100 Da), biotin and its derivatives (molecular weight range of about 300 Da to 1000 Da), a reducing agent (molecular weight range of about 150 Da to 300 Da), and a crosslinking agent (molecular weight range of about 300 to 600 Da). Although these exemplary small molecules and the listed molecular weight ranges were used in experimental demonstration, the present embodiment is not limited to these small molecules or molecular weight ranges, and those skilled in the art will realize that the teachings of the present invention allow for the preparation and use of compositions and devices for the removal of various types of small molecules and molecular weight ranges.

[0137] The following moiety was immobilized in various ways on a size exclusion support resin: 1. dextran with a molecular weight range of 6 kDa to 2800 kDa; 2. polyethyleneimine (PEI) (linear and branched); and 3. diethylenediamine (DEA).

[0138] Example 1A: Preparation and Testing of Chemical 1

[0139] Chemical 1: Fixes the size exclusion support to the branched polyethyleneimine moiety. :Two examples of size exclusion supports, comprising hydroxyethylcellulose resins having 7 K and 40 K size exclusion ranges, such as Thermo Scientific’s Zeba 40 K spin demineralizing column and Zeba 7 K spin demineralizing column, were modified as described below. A vicinal diol located on these size exclusion support columns was periodate oxidized to produce an aldehyde group. The resulting aldehyde was reacted with polyethyleneimine and diethylenediamine having primary amines using a Schiff base chemical reaction. Polyethyleneimine and diethylenediamine were prepared in PBS, adjusted the pH to a range of 8.0 to 8.5, and then reacted with the oxidized Zeba column. These compositions are later referred to as Zeba 7 K-PEI (PolyEthyleneImine) and Zeba 40 K-PEI.

[0140] The same chemical modification was performed on an agarose resin without size exclusion properties (Sepharose Fast Flow 4 (FF4) resin from GE). Visinal diols located on the agarose resin were periodate oxidized to generate aldehyde groups. The generated aldehydes were reacted with polyethyleneimine and diethylenediamine containing primary amines using a Schiff base chemical reaction. Polyethyleneimine and diethylenediamine were prepared in PBS, the pH was adjusted to a range of 8.0 to 8.5, and then reacted with the oxidized agarose resin. This formed the agarose-PEI described in subsequent experiments.

[0141] The following experimental steps were performed with chemically modified size exclusion supports (e.g., the Zeba column in the example described above) and non-size exclusion supports (e.g., the Sepharose resin in the example described above): 1. A bed volume of 0.5 mL of the chemically modified support was added to each spin column. 2. The spin columns containing the various chemically modified supports were spun at 1000 x g for 2 minutes to remove the stock solution. 3. a) A sample containing antibody-conjugated dye was added to the chemically modified support in the spin column at a volume of 100 μL. The column was spun at 1000 x g for 2 minutes, and the effluent was collected.

[0142] Figure 5 shows the results of experiments demonstrating the removal of exemplary small molecules (glass dyes) using two exemplary compositions in the apparatus of the present disclosure using the method according to one embodiment of the present disclosure. Small molecules embodied herein by glass dyes (DyLight™ 650) were removed using compositions of different embodiments of the present disclosure comprising size exclusion supports (at least two hydroxyethylcellulose supports having size exclusion ranges of 7 K and 40 K were each individually combined with a combination of moiety comprising polyethyleneimine (as an exemplary first moiety) and diethylenediamine (as an exemplary second moiety). The comparative efficiency of simple detergent removal resins was also tested under similar conditions.

[0143] Various lanes of the gel in Fig. 5 were loaded as follows: Lane 1 - GAM (GoatAntiMouse) DyLight™ 650 conjugate (Microcleaning - Control); Lane 2 - Detergent removal resin (Pierce TM Detergent removal resin, HiPPR TM Detergent Removal Spin Column Kit); Lane 3- Agarose-PEI (Polyethyleneimine); Lane 4- Zeba 7 K-PEI (Polyethyleneimine); Lane 5- Zeba 40 K-PEI (Polyethyleneimine).

[0144] The two bands within the box in Fig. 5 represent the protein recovered from each lane, namely the GoatAntiMouse DyLight™ 650 conjugate protein (GAM DyLight™ 650 conjugate), and the bottom band represents the glass dye DyLight™ 650 remaining in the sample. The glass or unreacted dye 650 was retained in the column and eluted after protein recovery. Lane 1 is the microcrystalline GAM 650 conjugate. The term "microcrystalline" is used herein to describe GAM DyLight™ 650 conjugates that have not passed through any resin / support. "Microcrystalline" is a control group operated on a gel to demonstrate how a sample containing glass dye that is "not" removed by the compositions, apparatus, and methods of this disclosure would appear on a gel. Lane 2 is an exemplary simple detergent removal resin (Pierce TM Detergent removal resin, HiPPR TMThis shows a GAM 650 conjugate protein passed through a detergent removal spin column kit to test whether this type of resin can remove small molecules (e.g., glass dye). The presence of the bottom band indicates that the glass dye is not completely or efficiently removed by this resin compared to other lanes (particularly lanes 4 and 5). Lane 3 indicates that the GAM 650 conjugate protein is separated from the glass dye to some extent by PEI immobilized on an agarose resin. However, Lane 3 indicates relatively low protein recovery and low removal of glass dye compared to lanes 4 and 5. Lanes 4 and 5 show the best protein recovery relatively compared to lanes 1, 2, and 3. The bottom band representing the glass dye is completely absent in lanes 4 and 5, indicating that the glass dye is efficiently removed by PEI-modified size exclusion resins (7 K and 40 K Zeba resins). However, the recovery of GAM 650 conjugates by non-size exclusion agarose resins in lanes 4 and 5 is inferior to that by size exclusion resins such as 7 K and 40 K Zeba resins. Accordingly, lanes 4 and 5 using the compositions, apparatus, and methods of the present disclosure represent the optimal removal of small molecules shown herein by the absence of free dyes and efficient conjugate-protein (large biomolecule) recovery compared to other lanes. Quantitative data for other dyes are given in the following examples.

[0145] Accordingly, in this experiment, at least two compositions of the present disclosure were analyzed: 1) a hydroxyethylcellulose size exclusion support having a 7 K size combined with a combination of moietyes comprising polyethyleneimine (as an exemplary first moiety) and diethylenediamine (as an exemplary second moiety); 2) a hydroxyethylcellulose size exclusion support having a 40 K size combined with a combination of moietyes comprising polyethyleneimine (as an exemplary first moiety) and diethylenediamine (as an exemplary second moiety); and the performance of the two compositions of the present disclosure was compared and contrasted with that of a non-size exclusion support chemically modified similarly to the compositions of the present disclosure. (i.e., compared with an agarose support combined with a moiety comprising polyethyleneimine (as an exemplary first moiety) and diethylenediamine (as an exemplary second moiety).) In addition, compared with a detergent removal resin and a "microcleaning" control. As shown in the above experiments and results (and in FIG. 5), the modified non-size exclusion support, or the detergent removal resin, could not remove small molecules as efficiently as the composition of the present disclosure comprising the modified size exclusion support.

[0146] Example 1B: Preparation of Chemical 2

[0147] Chemical 2: Fixes the dextran moiety and the size-excluded support. : In this embodiment, 7 K and 40 K hydroxyethyl cellulose resins (Zeba TM Spin desalination column, 40 K and Zeba TM A spin desalination column (7 K) was combined with a dextran moiety to form additional compositions of the present disclosure. Dextrans of different molecular weights (ranging from 6 K Da to 2.8 M Da) were immobilized on 7 K and 40 K resins.

[0148] First, dextran with a molecular weight range of 1,500,000 Da to 2,800,000 Da was immobilized on a resin as described below. Visinal diol on a hydroxycellulose-sized exclusion support was periodide oxidized to produce an aldehyde group. Ethylenediamine (EDA) or 1,5-diaminopentane (PDA) was reacted with the resulting aldehyde using a Schiff base reaction. This produced terminal amine groups on both 7 K and 40 K-sized exclusion supports. Subsequently, the dextran solution was periodide oxidized using sodium periodate to produce an aldehyde group. Then, the aldehyde group produced on the dextran was reacted with the terminal amine to produce dextran immobilized on 7 K and 40 K resins.

[0149] For comparative analysis, similar compositions and chemical modifications were prepared for agarose non-size exclusion supports. As mentioned in the above paragraph, agarose does not possess size exclusion properties. The chemical reaction is as follows:

[0150]

[0151] Example 1C: Preparation of Chemical 3

[0152] Chemical 3: The size exclusion support is fixed to the branched polyethylene glycol amine moiety. : A composition comprising a size exclusion support (Zeba, 7 K and 40 K, Thermo Scientific) and poly(ethylene glycol) bis(amine) was prepared as follows: Moiety of poly(ethylene glycol) bis(amine) (2 K Da to 20 K Da) of different molecular weights was immobilized on a size exclusion support (7 K and 40 K resin) using a Schiff base chemical reaction as described in the above paragraph.

[0153] For comparative analysis, similar chemical modifications were also performed on a size-exclusion agarose support (agarose, GE Healthcare). The chemical reaction is as follows:

[0154]

[0155] Example 1D: Testing of Chemicals 2 and 3: Chemicals 2 and 3 in a spin column device

[0156] Removal of small molecules of glass dye used

[0157] The compositions of chemical substances 2 and 3 described above were tested for small molecule removal.

[0158] All experiments for removing small molecules (unless specifically stated to use a different method / apparatus in this example and other examples) were performed using a 0.5 mL resin layer volume (various chemicals as described above) assembled in a 0.8 mL spin column to create the apparatus of the non-limiting examples of the present disclosure. The spin column was spun at 1000 x g for 2 minutes to remove the stock solution. Subsequently, the spin column was placed in a clean 2 mL centrifuge tube. A sample volume of 100 μL was added to the center of the resin, the column was spun at 1000 x g for 2 minutes, and the effluent was collected in a 2 mL tube. The results of these experiments are shown in FIG. 6.

[0159] FIG. 6 shows other exemplary small molecules, glass dyes, and DyLight using different supports of the present disclosure comprising 20k PEG diamine Zeba 7 K. TM The removal of 550 NHS esters is illustrated; Dextran-PDA Zeba 7 K (where PDA refers to 1,5-diaminopentane); and comparisons were made with a control group comprising Zeba 7 K (unmodified) and a starting sample (control), and with other chemicals comprising Zeba 7 K and dextran resin (cross-linked together). In this experiment, compared with an unmodified size exclusion resin (e.g., Lane's unmodified Zeba 7 K resin), glass dyes (e.g. DyLight TM To demonstrate the ability of the composition of the present disclosure to bind with small molecules such as ), it was performed without protein.

[0160] DyLight TM 550 NHS-ester was prepared at 1.3 mg / mL in borate buffer (this 1.3 mg / mL is equivalent to taking a 20 molar excess of dye to 10 mg of GAM protein). 500 μl of the other support and control of the present disclosure were assembled in a 0.8 ml spin column. 1.3 mg / mL of DyLight TM 100 μL of 550 NHS-Ester was added to the resin. The spin column was centrifuged at 1000 x g for 2 minutes. The effluent was collected. 10 μL of the effluent was added to 90 μL of sample buffer. Subsequently, this 10 μL was added per well on a 4-20% Trisglycine SDS gel. The gel was run for 40 minutes, and then imaged using an iBright imager (Thermo Fisher Scientific). The lanes in Fig. 6 correspond to the following support compositions used to remove the glass dye: Lane 1 - 20k PEG diamine Zeba 7 K; Lane 2 - Dextran-PDA Zeba 7 K; Lane 3 - Zeba 7 K (unmodified); Lane 4 - Zeba 7 K and dextran resin (crosslinked together); Lane 5 - Starting sample or positive control (20 molar excess of DyLight in borate buffer) TM 550 NHS-Ester).

[0161] Lane 1 of FIG. 6 shows data for removing small molecules with PEG diamine immobilized on 7 K Zeba resin. This chemical was unsuccessful in removing the glass dye. Lane 2 shows data for dextran-PDA immobilized on a 7 K size-exclusion support resin, which removed 100% of the glass dye. Lane 3, an unmodified 7 K Zeba resin (i.e., a size-exclusion resin with no added moiety—no surface chemicals), was unsuccessful in removing the glass dye. Lane 4 corresponds to a resin prepared by a different chemical for comparison with the compositional chemicals of the present disclosure. The lane 4 resin was prepared by crosslinking hydroxyethyl cellulose and dextran using a crosslinking agent, as opposed to immobilizing dextran on Zeba resin. This crosslinking chemical was not effective compared to the lane 2 chemical, which immobilized dextran on a size-exclusion support by the reductive amination method described above. As can be seen in Lane 2, the glass dye is completely removed, and the glass dye band is indicated by the missing glass dye band; the glass dye band is present in Lane 4, indicating that the glass dye is not removed by the cross-linked resin. Lane 5 represents the "micro-cleaned" sample passed as described in the above paragraph. Here, 1.3 mg / mL of DyLight TM The 550 NHS-ester did not pass through any resin. This 1.3 mg / mL solution was diluted 1:10 in sample buffer as described above and loaded onto a gel.

[0162] Example 1E: Chemicals 2 and 3 Test: Chemicals 2 and 3

[0163] Removal of small molecules of the reducing agent used

[0164] Another composition of the present disclosure was prepared as described in Chemical 3, comprising a single moiety PEG (polyethylene glycol) diamine immobilized on a size exclusion support (7 K Zeba, Thermo Scientific), and was tested for its ability to remove small molecules functioning as reducing agents as follows: 714 μl of a 70% support resin slurry was pipetted into a spin column placed in a collection reservoir tube. The spin column was spun at 1000 x g for 2 minutes to remove the stock solution. Subsequently, the spin column was placed in a clean 2 mL centrifuge tube. A 100 μl volume sample containing the reducing agent tris(2-carboxyethyl)phosphine (TCEP) at a concentration of 50 mM in PBS was added to the center of the spin column containing the composition as described above, spun at 1000 x g for 2 minutes, and the effluent was collected in the 2 mL tube. The removal of TCEP from the sample was tested by the Ellmans Assay. This assay was performed by adding the following to a 96-well plate: a) 250 μl Elman buffer; b) 10 μl Elman reagent (4 mg / ml); c) 50 μl sample (diluted 1:10); and measuring the amount of TCEP by reading the sample at 450 nm on a colorimeter (Mμltiskan, Thermo Scientific). The amount of TCEP in the sample is proportional to the intensity of the color indicated by the reading at 450 nm. Therefore, a higher reading at 450 nm corresponds to a greater amount of TCEP present in the sample. The results of this experiment are shown in Figure 7. A low value at 450 nm indicates efficient removal of TCEP.

[0165] FIG. 7 illustrates the removal of an exemplary small molecule, a reducing agent (TCEP), using a composition, apparatus, and method according to one embodiment of the present disclosure, and compares these with a control comprising unmodified Zeba 7 K. As shown in FIG. 7, the first bar corresponds to PEG diamine immobilized on a size exclusion support resin exemplified by 7 K Zeba and indicates the removal of 97% of TCEP added to the resin at 50 mM. The second bar corresponds to dextran-PDA immobilized on a size exclusion support resin exemplified by 7 K Zeba and indicates the removal of 80% of TCEP. In contrast, the third bar of FIG. 7 indicates the result of passing a 50 mM TCEP sample over a chemically unmodified size exclusion support resin exemplified by unmodified Zeba 7 K resin and indicates the removal of 41% of TCEP. The fourth bar represents the effect of another chemical comprising 7 K Zeba resin cross-linked to dextran, showing only 12% removal of TCEP. Thus, the compositions of the present disclosure prepared according to chemical 2 and chemical 3 show significantly higher removal of the small molecule reducing agent TCEP compared to other compositions.

[0166] Example 2: Removal of blended composition and small molecules in a spin column and multi-well format

[0167] From the data obtained from experiments illustrated in FIGS. 5, 6, and 7, the size exclusion support resin alone (i.e., 7 K Zeba without bonded moiety or chemicals) cannot remove small molecules containing, for example, dyes or reducing agents such as TCEP. The composition of the present disclosure, comprising a size exclusion resin and dextran, was able to remove both types of small molecules containing dyes and reducing agents. For example, the data in FIGS. 5 and 6 illustrate that the composition of the present disclosure, comprising dextran immobilized on a size exclusion support (Zeba 7 K and 40 K in FIG. 5, and Zeba 7 K in FIG. 6), removes free unreacted dyes (Dye 650, NHS 550), and that the dextran PDA in FIG. 7 removes the reducing agent TCEP. However, some compositions of the present disclosure, for example, comprising a PEG diamine moiety immobilized on a size exclusion support (Zeba 7 K), allow for the removal of significant amounts of the small molecule reducing agent TCEP (see Figs. 7 and 13) as well as DTT (see Fig. 19), but could not remove small dyes (see Fig. 6, lane 1).

[0168] Accordingly, the inventors have produced a composition capable of removing various types of small molecules by preparing a composition comprising a blend of the various compositions described herein. Some exemplary blend compositions of the present disclosure are shown in Table 1:

[0169]

[0170] Blends 1 to 5 described in Table 1, comprising a composition of dextran immobilized on a size-exclusion 7K resin and a composition of PEG immobilized on a size-exclusion 7K resin, were prepared in various proportions of each composition described in columns 2 and 3 of Table 1.

[0171] The blends of Table 1 prepared in a 1:1 ratio were tested for their ability to remove different classes of small molecules and compared with similar tests performed on the corresponding control of a non-deformed size exclusion resin (7 K Zeba resin) and a control of a microcrystalline junction protein.

[0172] The ability of the blended composition was tested in the apparatus of the present disclosure by incorporating the composition into spin columns of various sizes (0.8 mL, 2 mL, 5 mL, and 10 mL) and also onto 96-well filter plates. The storage buffer was removed by centrifuging the spin column or multi-well plate at 1000 x g for 2 minutes. An appropriate volume of sample containing small molecules was added. The column or multi-well plate was centrifuged again at 1000 x g for 2 minutes, and the eluent was collected from the effluent. Data are shown in FIGS. 8, 9, and 10 and correspond to experiments performed in the spin column format.

[0173] For Figure 8, a 700 μL volume sample of goat anti-mouse IgG conjugated with a 10 molar excess of DyLight™ 550 was applied to the center of the spin column with the composition corresponding to each lane as described below. Free dye removal was evaluated by SDS-PAGE, effluent from the imaging gel on the iBright imager, followed by quantification of dye removal by iBright analysis software. The modified 7 K 1:1 blend samples (lanes 1-2) had greater than 99% dye removal, and the unmodified 7 K Zeba resin (lane 3) had 20% dye removal compared to the "micro-cleaned" starting sample (lane 4) containing 10 mg / ml goat anti-mouse IgG with a 10 molar excess of DyLight™ 550 dye. The percentage of small molecule removal is calculated compared to that in the "micro-cleaned" control.

[0174] Figure 8 illustrates the removal of small molecule dye of DyLight™ 550 from a protein conjugate of DyLight™ 550 goat anti-mouse IgG using a 1:1 ratio of the exemplary blend compositions of Table 1. Lane 1 contains data for dye removal using a 1:1 Dextran-PDA blend; Lane 2 contains data for dye removal using a 1:1 Dextran-EDA (ethylenediamine) blend; Lane 3 contains data for dye removal using an unmodified 7 K Zeba size exclusion resin; and Lane 4 contains data for a "micro-cleaned" control of the dye DyLight™ 550 goat anti-mouse IgG. Figure 7 indicates that the blends of Lanes 1 and 2 can remove a significant amount of free dye compared to the control and Lane 3. This is illustrated in Figure 7 by the almost absence of free dye bands at the bottom of the gel.

[0175] In Fig. 9, a 2 mL volume sample of goat anti-rabbit IgG conjugated with a 10 molar excess of Alexa Fluor™ 488 was applied to the center of a spin column containing the compositions corresponding to those described below for each lane. The column was centrifuged at 1000 x g for 2 minutes, and the effluent was collected. Free dye removal from the effluent of each column was evaluated by SDS-PAGE, followed by quantification of dye removal by iBright analysis software on the effluent and imaging gel on an iBright imager. The modified 7 K 1:1 blend samples (lanes 1-2) exhibited greater than 99% dye removal compared to the "micro-cleaned" starting sample (lane 4) consisting of 10 mg / ml goat anti-rabbit IgG with a 10 molar excess of Alexa Fluor™ 488 dye, whereas the unmodified 7 K Zeba resin (lane 3) exhibited greater than 49% dye removal.

[0176] FIG. 9 illustrates the removal of small molecule dye from the Alexa Fluor™ 488 goat anti-rabbit IgG protein conjugate using the exemplary blend compositions of Table 1 in a 1:1 ratio, where Lane 1 contains data for dye removal using a 1:1 Dextran-PDA blend; Lane 2 contains data for dye removal using a 1:1 Dextran-EDA blend; Lane 3 contains data for dye removal using an unmodified 7 K Zeba size exclusion resin; and Lane 4 contains data for the dye Alexa Fluor™ 488 goat anti-rabbit IgG "micro-cleaned" control. FIG. 9 indicates that the blends of Lanes 1 and 2 remove a significant amount of free dye compared to the Lane 4 control and the Lane 3 unmodified resin. This is illustrated as having almost no free dye band at the bottom.

[0177] In Fig. 10, a 4 mL volume sample of goat anti-mouse IgG conjugated with a 10 molar excess of DyLight™ 650 was applied to the center of a spin column containing the compositions corresponding to those described below for each lane. Free dye removal was evaluated by SDS-PAGE, effluent from the imaging gel on an iBright imager, followed by quantification of dye removal by iBright analysis software. The modified 7 K 1:1 blend samples (lanes 1-2) exhibited greater than 99% dye removal compared to the "micro-cleaned" starting sample (lane 4) containing 10 mg / ml goat anti-mouse IgG with a 10 molar excess of DyLight™ 550 dye, whereas the unmodified 7 K Zeba resin (lane 3) exhibited 22% dye removal.

[0178] Figure 10 illustrates the removal of small molecule dye of DyLight™ 650 from a protein conjugate of DyLight™ 650 goat anti-mouse IgG using the exemplary blend compositions of Table 1 at a 1:1 ratio, where Lane 1 contains data for dye removal using a 1:1 Dextran-PDA blend; Lane 2 contains data for dye removal using a 1:1 Dextran-EDA blend; Lane 3 contains data for dye removal using an unmodified 7 K Zeba size exclusion resin; and Lane 4 contains data for a "micro-cleaned" control of the dye DyLight™ 650 goat anti-mouse IgG. Figure 10 indicates that the blends of Lanes 1 and 2 remove a significant amount of free dye, as shown by the almost absence of free dye bands at the bottom.

[0179] In Figs. 8, 9, and 10, the removal amount of various dyes / small molecules is expressed as a % compared to the respective amount in the "micro-washed" sample (lane 4 of each gel).

[0180] The blend of Table 1 prepared in a 1:1 ratio was also tested for its ability to remove different classes of small molecules and compared with similar tests performed on the corresponding non-size-exclusion agarose-dextran and agarose-PEG diamine resins blended in a 1:1 ratio (referred to herein as the FF4 blend in FIG. 12 (lanes 5 A and 5 B), 13, 14, 15, and 16). These compositions were incorporated into a spin column with a column volume of 0.8 ml at a bed volume of 0.5 ml to compare them with the compositions and apparatus of the present disclosure. The spin column was initially centrifuged at 1000 x g for 2 minutes to remove the storage buffer. An appropriate volume of the sample containing small molecules was added. The column was centrifuged again at 1000 x g for 2 minutes, and the eluent was collected from the flow. The data is shown, for example, in Fig. 12 (lanes 5 A and 5 B), and Figs. 13, 14, 15 and 16.

[0181] Example 3: With a composition blended in a multi-well filter plate device

[0182] Removal of glass dye small molecules

[0183] The blended composition described above was tested for small molecule removal using a multi-well filter plate device format.

[0184] All experiments for removing small molecules were performed using a 0.5 mL resin layer of a modified 7 K 1:1 resin blend (of different chemicals as variously described above) and assembled in a microwell / multiwell plate to form a mechanism of a non-limiting embodiment of the present disclosure. For these experiments, a 96-well multiwell plate containing the described chemicals was placed on top of a 96-well washing plate. Next, this assembly was placed on a 96-well plate-carrier rotor and centrifuged at 1000 xg for 2 minutes to remove the storage buffer. The plate assembly was removed from the centrifuge, and the washing plate was discarded. Subsequently, the 96-well plate was placed on top of a 96-well collection plate. Samples (20 μl, 50 μl, 100 μl) were applied to the center of the resin layer in each well. The plate assembly was centrifuged at 1000 xg for 2 minutes to collect the samples. Antibody conjugates were collected with the free dye removed. Free dye removal was evaluated by SDS-PAGE, an imaging gel on an iBright imager, followed by the quantification of dye removal using iBright analysis software. The results for the multi-well plates are shown in Fig. 11.

[0185] In Fig. 11, lanes 1 and 2 correspond to 20 μl of goat anti-rabbit IgG Alexa Fluor™ 647 conjugate applied to a modified 7 K 1:1 resin blend. Lanes 3 and 4 correspond to 50 μl of goat anti-rabbit IgG Alexa Fluor™ 647 conjugate applied to a modified 7 K 1:1 resin blend. Lanes 5 and 6 correspond to 100 μl of goat anti-rabbit IgG Alexa Fluor™ 647 conjugate applied to a modified 7 K 1:1 resin blend. Lane 7 corresponds to a "micro-cleaned" sample of goat anti-rabbit IgG Alexa Fluor™ 647 conjugate not applied to any resin blend. All samples (lanes 1–6) show greater than 99% dye removal compared to the "micro-cleaned" sample (lane 7) consisting of 10 mg / ml goat anti-rabbit IgG conjugated with a 10 molar excess of Alexa Fluor™ 647 dye. Lanes 1–2: Effluent from 20 μl samples applied to the resin layer, Lanes 3 and 4: Effluent from 50 μl samples applied to the resin layer, Lanes 5 and 6: Effluent from 100 μl samples, Lane 7.

[0186] Example 4: Blend composition for the removal of small molecule glass dyes

[0187] FIG. 12 illustrates removal data of an exemplary small molecule, two exemplary glass dyes, fluorescein, and Alexa Fluor™ 555 using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure.

[0188] The experimental steps for Fig. 12 are as follows: Dye removal protocol: 1. Pipette 1 ml of 50% resin slurry onto the spin column placed in the collection tube; 2. Spin at 1000 xg for 2 minutes; 3. Replace the collection tube and add antibody samples (GAR fluorescein conjugate, GAM Alexa 555 conjugate) to the spin column for each sample; 4. Centrifuge at 1000 xg for 2 minutes to remove free dye from the samples.

[0189] Evaluate dye removal from the gel: 1. Prepare a sample for gel operation by adding the following to a microcentrifuge tube: a) 20 μl of 2x loading buffer containing 50 mM DTT and b) 20 μl of the effluent from the antibody wash from step 4 above; 2. Heat the sample at 95°C for 8 minutes; 3. Cool the sample on ice; 4. Load 10 μl of each sample into separate wells on the gel; 5. Run the gel at 225 V for 32 minutes; 6. Remove the gel and rinse; 7. Image on an iBright imager with appropriate fluorescence.

[0190] In FIG. 12, lanes 1-6 (A or B) comprise a blended composition comprising the following: lanes 1A and 1B comprise 40 K Zeba, which is a non-deformation size exclusion resin with a molecular weight cutoff of 40 K; lanes 2A and 2B comprise 7 K Zeba, which is a non-deformation size exclusion support resin with a molecular weight cutoff of 7 K; lanes 3A and 3B comprise a 40 K blend, which is a 1:1 blend of a 40 K resin modified with dextran and a 40 K resin modified with PEG diamine; lanes 4A and 4B comprise a 7 K blend, which is a 1:1 blend of a 7 K resin modified with dextran and a 7 K resin modified with PEG diamine; Lanes 5A and 5B comprise non-size exclusion resins blended in a 1:1 ratio, comprising FF4 which is Fast Flow 4 agarose (from GE), FF4 modified with dextran, and FF4 modified with PEG diamine; lanes 6A and 6B comprise “micro-cleaned” or untreated samples (e.g., dye conjugates that do not pass through any resin). Lane A (lanes 1A-6A) comprises GAR or goat anti-rabbit IgG-fluorescein conjugate protein, and Lane B (lanes 1B-6B) comprises GAM or goat anti-mouse IgG-Alexa Flour™ 555-conjugate protein.

[0191] Lanes 6A and 6B illustrate "micro-cleaned" samples, showing two bands at the top corresponding to a reduced antibody band and a free dye band at the bottom of the gel (in Lane 6A, the free dye band corresponds to free fluorescein dye, and in Lane 6B, it corresponds to free Alexa Fluor™ 555 dye). In the treated samples (lanes 1A through 5A and lanes 1B through 5B), the presence of the bottom dye band indicates incomplete or unsuccessful removal of the free dyes (fluorescein and Alexa Fluor™ 555), as shown in lanes 1A, 1B and 2A and 2B. The presence of the free fluorescein band at the bottom of lanes 1A and 2A indicates poor or no removal of free fluorescein by the unmodified 40 K and unmodified 7 K Zeba resins, respectively. Similarly, the presence of a glass Alexa Fluor™ 555 band at the bottom of lanes 1B and 2B indicates poor removal of glass Alexa Fluor™ 555 dye by the unmodified 40 K and unmodified 7 K Zeba resins, respectively. Lanes 3A and 4A, representing the 40 K blend (1:1) and 7 K blend (1:1), indicate the absence or near absence of a glass fluorescein band at the bottom, which indicates the excellent glass dye removal characteristics of these two blends. Similarly, lanes 3B and 4B, representing the 40 K blend (1:1) and 7 K blend (1:1), indicate the absence or near absence of a glass Alexa Fluor™ 555 band at the bottom, which indicates the excellent glass dye removal characteristics of these two resins. Lanes 5A and 5B show the complete loss of GAR antibody-conjugated fluorescein (5A) and GAM antibody-conjugated Alexa Fluor™ 555 (5B), as evidenced by the absence of reduced antibody bands for 5A and 5B on the gel. In addition to the removal of free dye, there is excellent protein recovery of antibody-conjugated fluorescein and antibody-conjugated Alexa Fluor™ 555 in lanes 3A, 4A, 3B, and 4B.Bands from these lanes were quantified using iBright analysis software, and dye removal (%) and antibody-dye conjugate recovery (%) were plotted for different resins (see data in Figures 15 and 16).

[0192] Example 5: Blend composition for removing small molecule reducing agents

[0193] FIG. 13 illustrates removal data of an exemplary small molecule reducing agent using a blended composition, apparatus, kit, and method according to one embodiment of the present disclosure. The experiment was as follows: Reducing agent removal protocol: 1. Pipette 714 μl of a 70% resin slurry into a spin column in a collection tube; 2. Spin the liquid at 1000 xg for 2 minutes; 3. Replace the collection tube and add 100 μl of a TCEP sample (25 mM) containing 1 mg / ml goat anti-rabbit antibody in PBS to each spin column for each sample; 4. Centrifuge at 1000 xg for 2 minutes to remove TCEP from the sample.

[0194] Evaluation of TCEP removal by Elman assay: 1. Amount of TCEP measured by adding the following to each sample in a 96-well plate; 2. 250 μl Elman buffer; 3. 10 μl Elman reagent (4 mg / ml); 4. 50 μl sample (diluted 1:10); 5. Sample reading at 450 nm on a multiscan plate reader; Evaluation of protein recovery by 280 nm; 6. Pipette 4 μL of sample into a Nanodrop One and measure at 280 nm.

[0195] Figure 13 is a bar graph showing the removal of reducing agent TCEP using 7 K and 40 K blends (blended with dextran and PEG diamine chemicals blended in a 1:1 ratio) and compares TCEP removal with non-modified 7 K and 40 K size exclusion resins and a non-size exclusion column containing an FF4 blend. Black bars represent the percentage (%) of recovered protein. Gray bars represent the amount of removed TCEP in %. The 40 K and 7 K resins remove 7.3% and 1.8% of TCEP, respectively, while the 40 K blend and 7 K blend remove 98.5% and 88.7% of TCEP, respectively. The FF4 blend, prepared on the non-size exclusion resin, removes 42.7% of TCEP. The FF4 blend also has a poor comparative recovery of protein (36.4%) compared to the 40 K and 7 K blends, which show relatively good protein recovery of 68.9% and 72.3%, respectively.

[0196] Example 6: Blend composition for removing small molecule biotin

[0197] FIG. 14 illustrates the removal of free / unreacted biotin using the 7 K and 40 K blends of the present disclosure. These experiments are as follows:

[0198] Biotin removal protocol: 1. Pipette 1 ml of 50% resin slurry into the spin column in the collection tube; 2. Spin at 1000 xg for 2 minutes; 3. Replace the collection tube and add the biotinylated antibody sample to the sample-specific spin column; 4. Centrifuge at 1000 xg for 2 minutes to remove free biotin from the sample.

[0199] Biotin removal evaluation: 1. Amount of biotin measured by adding the following to each sample in a cuvette: a) 800 μl of PBS buffer; b) 100 μl of colorimetric HABA; c) 100 μl of sample (diluted 1:10); 2. Sample read at 500 nm on a multiscan plate reader.

[0200] Evaluation of Biotinylated Protein Recovery: 1. Pipette 10 μl of sample into a 96-well plate; 2. Pierce according to the manufacturer's instructions TM Prepare the Rapid Gold BCA protein assay working reagent; 3. Add 200 μl of working reagent to a well; 4. Incubate the plate at room temperature for 5 minutes; 5. Read the absorbance at 480 nm using a multiscan plate reader.

[0201] FIG. 14 is a bar graph showing the removal of free / unreacted biotin using 7 K and 40 K blends (as described in the above examples) compared to the removal of free biotin using unmodified 7 K and 40 K resins and the FF4 blend (as described in the above examples). Black bars represent the protein recovery rate (of GAM Ab), and gray bars represent the amount of free biotin removed as %. The unmodified 40 K and 7 K supports remove 40% and 38.75% of free biotin, respectively, while the 40 K blend and 7 K blend remove 80.31% and 72.7% of unreacted / free biotin, respectively. The FF4 blend (non-size exclusion blend) shows 89.49% biotin removal. However, the FF4 blend shows poor protein recovery, with a protein recovery of 21.4%. In contrast, 40 K and 7 K blends show excellent protein recovery.

[0202] Figures 12, 13, and 14 show the relatively good removal of small molecules, including exemplary dyes, biotin, and reducing agents, using modified 40 K and 7 K blends compared to using non-modified 40 K and 7 K size exclusion columns. Protein recovery using non-exclusion resins such as FF4 is relatively poor.

[0203] Example 7: Blend composition for small molecule removal

[0204] Figures 12, 13, and 14 show the relatively good removal of small molecules including exemplary dyes, biotin, and reducing agents using modified 40 K and 7 K blends compared to using non-modified 40 K and 7 K size exclusion columns. Protein recovery using non-exclusion resins such as FF4 is relatively poor.

[0205] In addition, additional experiments were conducted using the crosslinking agent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the results are summarized in Table 2 below.

[0206] Table 2 shows the percentage of protein recovery using the various blends described in the above examples for several different small molecules including dye (fluorescein), biotin, TCEP, and SMCC. The blend containing the non-size exclusion resin FF4 has the lowest protein recovery rate for all small molecules. Even though biotin was removed by FF4 (as shown in Example 8), the protein recovery rate is below optimal.

[0207]

[0208] Figures 15 and 16 are bar graphs from experiments showing protein recovery and Alexa Fluor™ 555 removal, with bands quantified from the gel corresponding to Figure 12 to determine the protein recovery rate (%) and free dye removal rate (%) from each tested composition. Gray bars represent the dye removal rate (%) and black bars represent the protein recovery rate (%). As demonstrated in Figures 15 and 16, the unmodified size exclusion resins 40 K Zeba and 7 K Zeba resins could not efficiently remove free fluorescein or free Alexa Fluor™ 555. In contrast, the modified 40 K and 7 K blend compositions of the present disclosure were able to remove >96% of fluorescein and >93% of Alexa Fluor™ 555. The non-size exclusion FF4 blend had poor protein recovery for both the GAR-fluorescein conjugate protein and the GAM-Alexa Fluor™ 555 conjugate protein, with protein recovery rates of 15.5% and 14.5%, respectively.

[0209] The experimental steps of Fig. 17 are as follows: 1. Prepare 1.33 ml of BS3 crosslinking agent solution in PBS; 2. Pipette 1 ml of 50% resin slurry into a spin column in a collection tube; 3. Spin the liquid at 1000 xg for 2 minutes; 4. Replace the collection tube and add the BS3 solution to each spin column; 5. Centrifuge at 1000 xg for 2 minutes to remove the BS3.

[0210] BS3 Removal Evaluation: 1. Prepare a 1:50 dilution of each effluent sample in PBS by mixing 50 μl of effluent with 450 μl of PBS; 2. Read the sample at 280 nm in a UV cary of a 500 μl cuvette.

[0211] Evaluation of BS3 cross-linked protein recovery: 1. Pipette 10 μl of sample into a 96-well plate; 2. Pierce TMPrepare the Rapid Gold BCA protein assay working reagent according to the manufacturer's instructions; 3. Add 200 μl of working reagent to a well; 4. Incubate at room temperature for 5 minutes; 5. Read the absorbance at 480 nm using a multiscan plate reader.

[0212] The experimental steps of Fig. 17 are as follows: 1. Prepare a 1.33 mM SMCC crosslinker solution in PBS; 2. Pipette 1 mL of 50% resin slurry into a spin column in a collection tube; 3. Spin the liquid at 1000 xg for 2 minutes; 4. Replace the collection tube and add the SMCC solution to each column; 4. Centrifuge at 1000 xg for 2 minutes to remove the SMCC.

[0213] SMCC Removal Evaluation: 1. Prepare a 1:50 dilution of each effluent sample in PBS by mixing 50 μl of effluent with 450 μl of PBS; 2. Read the sample at 280 nm in a UV cary of a 500 μl cuvette.

[0214] Evaluation of SMCC cross-linked protein recovery: 1. Pipette 10 μl of sample into a 96-well plate; 2. Pierce according to the manufacturer's instructions TM Prepare the Rapid Gold BCA protein assay working reagent; 3. Add 200 μl of working reagent to a well; 4. Incubate the plate at room temperature for 5 minutes; 5. Read the absorbance at 480 nm using a multiscan plate reader.

[0215] Figures 17 and 18 are bar graphs showing small molecules used as crosslinkers, including bis(sulfosuccinimidyl) subcerate (BS3) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), removed using these resins. Figure 17 shows the removal of the BS3 crosslinker, and Figure 18 shows the removal of the SMCC crosslinker. In Figure 17, the gray bars correspond to the BS3 removal rate (%), demonstrating that the unmodified 40 K and 7 K Zeba resins remove 69.1% and 63.7% of the BS3 crosslinker, respectively, while the compositions containing the 40 K blend and 7 K blend remove 85.9% and 88% of the BS3 crosslinker, respectively. The FF4 blend removes 80% of the BS3, but the protein recovery characteristics of FF4 are only about 51%. In contrast, the present compositions of the 40 K blend and 7 K blend have protein recovery rates of 80.6% and 89.7%, respectively.

[0216] In Fig. 18, the gray bars represent SMCC removal in %, demonstrating that while the unmodified 40 K and 7 K Zeba resins remove 61.6% and 53.7% of the SMCC crosslinker, respectively, the present composition blends of the 40 K and 7 K blends remove 85.9% and 89.4% of the SMCC crosslinker, respectively. The present composition blends can perform 20% better than the unmodified 7 K and 40 K. The FF4 blend removes 84.3% of the crosslinker, but the protein recovery characteristics of FF4 are relatively poor, with only 2% of the protein recovered. In contrast, the 40 K and 7 K blends show protein recovery rates of 63.1% and 83.8%, respectively.

[0217] The inventors have also indicated that the compositions of the present disclosure, comprising a blend of dextran and PDA and a blend of dextran and an EDA moiety immobilized on a size exclusion support, can remove a significantly large amount of a reducing agent, such as dithiotritol (DTT). This is illustrated in FIG. 19, where the black bars represent the removal rate (%) of DTT and the gray bars represent the protein recovery rate (%) of goat anti-rabbit IgG. The 7 K resin removed >95% of DTT with a protein recovery rate of >85%.

[0218] Example 8: For protein recovery and removal of small molecules (dye, biotin, reducing agent)

[0219] Composition of the present disclosure big Other existing products

[0220] The composition of the present disclosure has demonstrated superior performance for the removal of dyes, biotin, and reducing agents with excellent protein recovery rates compared to columns containing other existing products sold for similar applications.

[0221] FIGS. 20a and 20b illustrate the use of a spin column comprising the composition of the present disclosure or products from other suppliers sold for standard dialysis as well as similar uses, for comparison of dye removal and protein recovery. Free Alexa Fluor™ 647 dye was removed from 100 μl of a sample of 10 mg / mL goat anti-rabbit IgG labeled with a 10 molar excess of Alexa Fluor™ 647 using a spin column comprising the composition of the present disclosure or products from other suppliers. Equal volumes of samples from each flow and starting sample (lane 7) were loaded onto a gel. The samples were run on an electrophoresis gel and imaged on an iBright FL1500 imaging system (Thermo Fisher Scientific), after which iBright analysis software was used to quantify the removal of free dye.

[0222] FIG. 20a illustrates an electrophoretic gel comprising: Lane 1: a spin column comprising the composition of the present disclosure for dye and biotin removal, Lane 2: BioRad P-30, Lane 3: G-Biosciences GT-100, Lane 4: G-Biosciences GT-600, Lane 5: GE PD10, Lane 6: Thermo Scientific Slide-A-Lyzer G2 dialysis cassette, 3.5K MWCO, Lane 7: (positive control) starting sample, 10 mg / ml goat anti-rabbit IgG 10 molar excess of Alexa Fluor™ 647.

[0223] FIG. 20b illustrates graphic data representing dye removal and protein recovery for a spin column: composition of the present disclosure for small molecule removal (first set of bars), BioRad P-30 (second set of bars labeled "BR"), G-Biosciences GT-100 (third set of bars labeled "GB 1"), G-Biosciences GT-600 (fourth set of bars labeled "GB 6"), GE PD10 (fifth set of bars labeled "GE").

[0224] As can be seen in FIGS. 20a and 20b, the spin column of the present disclosure provides higher dye removal with a superior protein recovery rate compared to products from other suppliers.

[0225] FIG. 21 illustrates the use of a spin column comprising the composition of the present disclosure or a spin column comprising a product from another supplier sold for similar use, for the purpose of comparing biotin removal and protein recovery.

[0226] Biotin was removed using a spin column containing the composition of the present disclosure or a spin column containing a product from another supplier. 0.27 mM of free NHS-LC-biotin was present in a 100 μl sample. Protein recovery from goat anti-mouse (2 mg / mL) labeled with 20 X NHS-LC-biotin was performed using Pierce TM It was evaluated by the Rapid Gold BCA assay kit (Thermo Fisher Scientific Cat. No. # A 53225). Free biotin removal was quantified using the Thermo Scientific biotin quantification kit (Thermo Fisher Scientific Cat. No. # 28005).

[0227] FIG. 21 illustrates graphic data representing biotin removal and protein recovery for a spin column: composition of the present disclosure for small molecule removal (first set of bars), BioRad P-30 (second set of bar labels "BR"), G-Biosciences GT-100 (third set of bar labels "GB 1"), G-Biosciences GT-600 (fourth set of bar labels "GB 6"), GE PD10 (fifth set of bar labels "GE").

[0228] As can be seen in FIG. 21, the spin column of the present disclosure provides higher biotin removal along with higher protein recovery compared to products from other suppliers.

[0229] FIG. 22 illustrates the use of a spin column comprising the composition of the present disclosure or a spin column comprising a product from another supplier sold for a similar use, for comparison of reducing agent removal.

[0230] The reducing agent TCEP was removed from 1 mg / mL goat anti-rabbit IgG containing 25 mM TCEP in PBS using a spin column containing the composition of the present disclosure or a product from another supplier. The removal of the reducing agent was performed by applying 700 μl of the sample to a 2 mL column. Quantification of TCEP removal from the effluent was performed using the Elman assay in comparison to the starting sample.

[0231] FIG. 22 illustrates graphic data showing TCEP removal for spin columns: composition of the present disclosure for small molecule removal (first set of bars), BioRad P-30 (second set of bars labeled "BR"), G-Biosciences GT-100 (third set of bars labeled "GB 1"), G-Biosciences GT-600 (fourth set of bars labeled "GB 6"), and GE PD10 (fifth set of bars labeled "GE"). As can be seen, the TCEP removal rate was much higher in the spin columns containing the composition of the present disclosure compared to other spin columns tested.

[0232] Example 9: Composition of the present disclosure for the removal of small molecules (dyes and biotin) big dialysis

[0233] The composition of the present disclosure was used to purify small molecule impurities from proteins by comparing it with dialysis, a standard method used in the art. The composition of the present disclosure demonstrated superior performance for the removal of dyes and biotin, having a higher protein recovery rate in fractions of the time required for dialysis.

[0234] As shown in FIG. 23, free Alexa Fluor™ 647 dye was removed from a sample of 10 mg / mL goat anti-rabbit IgG (10 molar excess) labeled with Alexa Fluor™ 647 by comparing a spin column containing the composition of the present disclosure for small molecule removal with standard dialysis. Protein recovery was evaluated by A280 measurements of the starting sample and the effluent after dye removal. The sample was run on an electrophoretic gel and imaged on an iBright FL1500 imaging system (Thermo Fisher Scientific, Product # A44241), and the removal of free dye was quantified using iBright analysis software.

[0235] As can be seen in the graphic data of FIG. 23, the dye removal and protein recovery (bars of the first set) of a spin column containing the composition of the present disclosure for small molecule removal were superior to those of dialysis. Furthermore, the method using the spin column of the present disclosure takes only 15 minutes compared to the overnight time with three buffer changes for dialysis. Thus, the present composition and method provide surprisingly pure removal of proteins and small molecules in a single-step process that is significantly faster than dialysis.

[0236] Figure 24 shows data for a spin column containing the composition of the present disclosure for small molecule removal compared to standard dialysis for removing free NHS-LC-biotin. Protein recovery from goat anti-rabbit (2 mg / mL) labeled with 20 X NHS-LC-biotin was evaluated by the Rapid Gold BCA test (Thermo Fisher Scientific, Product # A53225).

[0237] As can be seen in the graphic data of FIG. 24, biotin removal and protein recovery for a spin column comprising the composition of the present disclosure (the first set of rods) for small molecule removal were superior to those for dialysis. Furthermore, the method using the spin column of the present disclosure takes only 15 minutes compared to the overnight time with three buffer changes for dialysis. Thus, the composition and method of the present disclosure provide surprisingly pure removal of proteins and small molecules in a single-step process that is significantly faster than dialysis.

[0238] Example 10: In a spin plate device for the removal of small molecules

[0239] Composition of the present disclosure

[0240] The composition of the present disclosure was placed in a spin plate device to test the removal of small molecules. In some embodiments, the composition of the present disclosure was placed in a 96-well filter plate to test high-throughput removal of small molecules from a number of samples.

[0241] As shown in Fig. 25, the composition of the present disclosure for small molecule removal was placed in a 96-well filter plate to remove free Alexa Fluor™ 647 dye from 50 μl (lanes 1 and 2) and 100 μl samples (lanes 3 and 4) of 10 mg / mL goat anti-rabbit IgG labeled with a 10 molar excess of Alexa Fluor™ 647. The samples were run on an electrophoretic gel and imaged on an iBright FL1500 imaging system (Thermo Fisher Scientific, Product # A44241), after which iBright analysis software was used to quantify the removal of free dye. Dye removal and excellent protein recovery were demonstrated.

[0242] Example 11: Composition of the present disclosure for the removal of small molecule fluorescein dye

[0243] The composition of the present disclosure was placed in a spin column to test the removal of a fluorescent dye.

[0244] As shown in Fig. 26, the composition of the present disclosure for small molecule removal was placed in a spin column to remove free fluorescein dye (Thermo Fisher Scientific Cat. No. # 46410) from 100 μl of a sample of 10 mg / mL goat anti-rabbit IgG labeled with 15 molar excess fluorescein. Equal volumes of samples from each effluent (lanes 1-3) and starting sample (lane 4) were loaded onto a gel. The samples were run on an electrophoresis gel and imaged using an iBright FL1500 imaging system (Thermo Fisher Scientific, Product # A44241), after which the removal of free dye was quantified using iBright analysis software. Dye removal and excellent protein recovery rates were demonstrated.

[0245] Example 12: Composition of the present disclosure for immunofluorescence application

[0246] The composition, apparatus, and method of the present disclosure have been found to be useful for immunofluorescence applications.

[0247] In one embodiment, an HDAC2 polyclonal antibody (Thermo Fisher Scientific, Product # PA1-861) was labeled with Alexa Fluor™ 647 (Thermo Fisher Scientific, Product # A20006) and then purified from unreacted dye using a spin column containing the composition of the present disclosure to remove small molecules. Figures 27a and 27b show the immunofluorescence analysis of HDAC2 (red) in A549 cells. Cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature, permeated with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 1% BSA in PBS. Cells were stained with the HDAC2 polyclonal antibody, Alexa Fluor™ 647 conjugate, washed using a spin column having the composition of the present disclosure (Fig. 27b), and the spin column of the present disclosure was not used for 1 hour at room temperature protected from light with unreacted dye diluted to 2.5 μg / ml in blocking buffer. By not doing It was not cleaned (Fig. 27a).

[0248] As can be seen, Fig. 27a shows a lot of background fluorescence compared to Fig. 27b, which has significantly reduced residual background.

[0249] In another embodiment, the cleaning of small molecule dyes in immunofluorescence was compared to cleaning using existing products sold for similar applications.

[0250] The PMP70 polyclonal antibody (Thermo Fisher Scientific, Product # PA1-650) was labeled with Alexa Fluor™ 647 (Thermo Fisher Scientific, Product # A20006) and purified using a spin column containing the composition of the present disclosure for removing small molecules from unreacted dyes. Immunofluorescence analysis of PMP70 (red in the original but shown in black and white herein) in A549 cells was performed as follows. Cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature, permeated with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 1% BSA in PBS. Cells were labeled with the PMP70 monoclonal antibody, Alexa Fluor TM The 647 conjugate was stained by 1) without washing (Fig. 28a), 2) washing using a conventional GE PD-10 column (Fig. 28b), and 3) washing using a spin column of the present disclosure with unreacted dye diluted to 2.5 µg / ml in blocking buffer for 1 hour at room temperature protected from light (Fig. 28c). The nucleus (blue in the original but shown in black and white in this document) was stained with Hoechst Dye at a dilution of 10,000 in blocking buffer.

[0251] As can be seen, FIG. 28a shows a lot of background immunofluorescence, reduced background fluorescence is shown in FIG. 28b, and FIG. 28c shows a significant residual background fluorescence indicating excellent washing of the dye using the spin column of the present disclosure.

[0252] In another embodiment, a ZO-1 monoclonal antibody (Thermo Fisher Scientific, Product # MA3-39100) was labeled with Alexa Fluor™ 488 (Thermo Fisher Scientific, Product # A20000) and then purified from unreacted dye using a spin column containing the composition of the present disclosure for removing small molecules. Immunofluorescence analysis of ZO-1 (original green, but shown herein in black and white) was performed on Caco-2 cells. Cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature, permeated with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 1% BSA in PBS. Cells were stained with the ZO-1 polyclonal antibody, Alexa Fluor™ 488 conjugate, washed using a spin column having the composition of the present disclosure (Fig. 29b), and the spin column of the present disclosure diluted to 5 μg / ml in blocking buffer was not used for 1 hour at room temperature protected from light. By not doing It was not cleaned (Fig. 29a).

[0253] As can be seen, Fig. 29a shows a lot of background fluorescence compared to Fig. 29b, which has significantly reduced residual background.

[0254] Example 14: The present disclosure for protein recovery and removal of small molecules

[0255] composition big ion exchange resin

[0256] All experiments for removing small molecules (in this example) were performed using a 0.5 mL resin layer volume (of various chemicals as described above and below) assembled in a 0.8 mL spin column to construct a mechanism of the non-limiting embodiment of the present disclosure. In this particular embodiment, the spin column was packed with the composition of the present disclosure (labeled as Rumba in the corresponding numerical value) or the ion-exchange resin Dowex. The spin column was spun at 1000 x g for 2 minutes to remove the stock solution. Subsequently, the spin column was placed in a clean 2 mL centrifuge tube. Volumes of samples of 50 μL, 250 μL, and 400 μL were added to the center of the resin, the column was spun at 1000 x g for 2 minutes, and the effluent was collected in a 2 mL tube. 10 μL of the effluent was added to 90 μL of sample buffer. Next, 10 μL of this was added per well on a 4-20% Trisglycine SDS gel. The gel was run for 40 minutes, and then imaged using an iBright imager (Thermo Fisher Scientific).

[0257] Two concentrations of goat anti-rabbit Alexa Fluor™ 594 conjugate ("GAR-594") were used in this experiment - 1 mg / mL (gel shown in Fig. 30a) and 10 mg / mL (gel shown in Fig. 31a).

[0258] Supports used in FIG. 30a, FIG. 30b, FIG. 31a, and FIG. 31b:

[0259] Lanes 1-3 used a dextran PEG diamine blend labeled with Roomba in FIG. 30a-31b (composition of the present disclosure).

[0260] Lanes 4-6 used Dowex (non-modified ion exchange resin).

[0261] Volumes of antibody-dye conjugates used in Figs. 30a, 30b, 31a, and 31b:

[0262] Lanes 1 and 4 - 50 μl

[0263] Lanes 2 and 5 - 250 μl

[0264] Lanes 3 and 6 - 400 μl

[0265] Lane Start - Microtable sample (10 mg / mL GAR-594 conjugate Fig. 30a or 1 mg / mL GAR-594 conjugate Fig. 31b)

[0266] The results of these experiments are shown in the gels of Figs. 30a and 31b and the bar graphs of Figs. 30b and 31b.

[0267] FIGS. 30a and 30b illustrate the removal of another exemplary small molecule, Alexa Fluor™ 594, when 10 mg / mL of dye antibody conjugate, i.e., the composition of the present disclosure, dextran PEG 20 K blend (labeled as Romba in FIGS. 30a and 30b) and pure ion exchange resin (unmodified ion exchange resin), Dowex, are added in different volumes (50 μl, 250 μl, and 400 μl) to spin columns having different supports.

[0268] The lane labeled "Start" represents the "micro-cleaned" sample passed as described in the above paragraph (this corresponds to a positive control in which unbound dye from the protein mixture is not spun to remove the dye). The upper two bands of lane "Start" correspond to reduced antibodies (light and heavy chains). The lower band corresponds to the free dye, Alexa Fluor™ 594. In FIG. 30a, lanes 1, 2, and 3 show good protein recovery when comparing the upper two bands to the "Start" lane. Additionally, the lower free dye band is missing in lanes 1-3, indicating the removal of free dye by a spin column having the resin composition of the present disclosure. In contrast, lane 4 shows a loss of protein recovery with the ion exchange resin when the conjugate is added to the unmodified ion exchange resin layer at a rate of 50 μl. This indicates that when 10 mg / mL GAR-Alexa Fluor™ 594 conjugate is added to a resin layer of 50 μl to 500 μl, the resin layer having the composition of the present disclosure has a Dextran-PEG diamine resin blend, whereas the protein yield is lost in the Dowex resin. The graph generated using iBright analysis software correlates very well with the image.

[0269] FIG. 31a illustrates the removal of small molecules of the free dye Alexa Fluor™ 594 when added at different volumes (50 μl, 250 μl, and 400 μl) of a 1 mg / mL dye antibody conjugate using different supports, namely the composition of the present disclosure, a dextran PEG 20 K blend, and a pure ion exchange resin (unmodified ion exchange resin) Dowex.

[0270] The lane labeled "Start" represents the "micro-washed" (positive control) sample processed as described in the above paragraph. The upper two bands of lane "Start" correspond to reduced antibodies (light and heavy chains). The lower band corresponds to the free dye Alexa Fluor™ 594. Lanes 1, 2, and 3 in Fig. 31a show good protein recovery when comparing the upper two bands to the "Start" lane. Additionally, the lower free dye band is missing in lanes 1-3, indicating the removal of the free dye by the composition of the present disclosure. Lanes 4, 5, and 6 show the loss of protein recovery with the ion exchange resin when 1 mg / mL antibody-dye conjugate was added to the resin layer at 50 μl, 250 μl, and 400 μl, respectively. This indicates that protein recovery with Dowex resin is poor compared to Dextran-PEG diamine blended resin at all tested volumes of GAR-Alexa Fluor™ 594 conjugates of 50 μl, 250 μl, and 400 μl at 1 mg / mL. The graph generated using iBright analysis software correlates very well with the image.

[0271] The bar graphs shown in FIGS. 30b and 31b quantify the percentage of protein recovered and removed dye when 0.05 mg to 4 mg of GAR 594 Ab-dye conjugate is loaded with > 90% dye removal, and show a protein recovery rate of > 90% with the composition of the present disclosure (shown as a rombar in FIGS. 30b and 31b).

[0272] In contrast, Dowex provides >90% protein recovery with >90% dye removal, but only at lower scales, i.e., when 2.5 mg to 4 mg of GAR 594 Ab-Dye conjugate is loaded, and at much higher concentrations of protein.

[0273] This data indicates that the composition of the present disclosure provides about 50X more flexibility, greater flexibility, and allows for protein recovery at a wider range of sample protein concentrations and volumes compared to non-modified ion exchange-only resins (e.g., Dowex).

[0274] Accordingly, the present embodiment demonstrates that the compositions of the present disclosure achieve high protein recovery as well as efficient removal of small molecules (removal of substantially all small molecules) over a wide range of low and high sample protein concentrations and high and low sample protein volumes. In contrast, the ion-exchanger Dowex is limited in that it operates only at high sample protein concentrations and high protein volumes, but fails to efficiently remove small molecules or has high protein recovery rates at low sample protein volumes and concentrations. Advantageously, the compositions, apparatus, and methods of the present disclosure provide much higher protein recovery even when sample proteins at very low concentrations are purified from small molecule contaminants.

[0275] The experiments described above were performed with Dowex or the composition of the present disclosure placed in a spin column. In other experiments (data not shown), batch processing for small molecule removal was performed in large batches for the composition of the present disclosure versus Dowex. The present method comprises the following steps of taking a large batch of dried resin of the composition of the present disclosure or Dowex: adding to a protein conjugated to a small molecule that may have free suspended small molecule contaminants (e.g., antibody-dye conjugates); mixing the dried resin with the protein conjugated to the small molecule; and collecting the conjugated protein from a filtrate in which free unbound small molecules bind to Dowex or the composition of the present disclosure. In these experiments, it is noted that when using a pure ion exchange column Dowex, a change in pH (decreased pH) is required for the ion exchange to bind the small molecules. The composition of the present disclosure did not require this additional pH change step. Therefore, additional advantages of the composition of the present disclosure are a reduced number of steps and ease of separation.

[0276] ********

[0277] Each embodiment disclosed herein may be used with or otherwise combined with any other embodiment disclosed herein. Any component of any embodiment may be used in any embodiment. Although the claimed embodiments of the present invention have been described with reference to specific embodiments, those skilled in the art will understand that various modifications may be made and equivalents may be substituted for the components without departing from the true spirit and scope of the claimed invention. Furthermore, variations may be made without departing from the essential teachings of the present invention.

Claims

Claim 1 A composition for separating one or more small molecules from a sample, comprising a first size exclusion support comprising polyacrylamide, a cellulose material, hydroxyethyl cellulose and / or a derivative thereof, and a first moiety, wherein the first moiety is an amine-containing polymer immobilized on the first size exclusion support via carbon-nitrogen bonds and can bind to the one or more small molecules, optionally the first moiety can bind to the one or more small molecules by charge interaction, hydrophilic interaction, hydrophobic interaction, affinity interaction, hydrogen bonding, or Van der Waals force. Claim 2 A composition according to claim 1, further comprising a second moiety, wherein the first moiety and the second moiety are different, and the second moiety is fixed on the first size exclusion support. Claim 3 A composition according to claim 1, further comprising a second size exclusion support, wherein the first size exclusion support and the second size exclusion support are different, and the first moiety is fixed on the second size exclusion support. Claim 4 A composition according to paragraph 3, wherein the second size exclusion support comprises a dextran polymer or agarose. Claim 5 A composition according to paragraph 2, wherein the second moiety comprises a polysaccharide, dextran, polyethylene glycol polymer, amine-containing polymer, polyamino acid, lipopolysaccharide, antibiotic, chelate group, magnetic particle, paramagnetic particle, functional group, ion-exchanger, or a combination thereof. Claim 6 A composition according to claim 1, wherein the amine-containing polymer is poly(ethylene glycol)diamine, polyethylenediamine, linear polyethyleneimine, or branched polyethyleneimine, and optionally the linear polyethyleneimine is diethylenediamine. Claim 7 A composition according to claim 5, wherein the second moiety is dextran, and optionally the molecular weight of the dextran is within the range of 6 kDa to 2800 kDa. Claim 8 A composition according to claim 1, wherein one or more small molecules comprise a dye, a derivative of a dye, biotin, a biotin derivative, a crosslinking agent, a reducing agent, a label, a nanoparticle, a radioactive ligand, a mass tag, an unreacted molecule, or a combination thereof, an intermediate, or a derivative, and optionally, one or more small molecules have a molecular weight range of < 2000 Da. Claim 9 An apparatus for separating one or more small molecules from a sample, comprising: a) a container, wherein the container comprises a composition of any one of claims 1 to 8, and optionally the container is a columnar container, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate; and b) a receptacle located below the container. Claim 10 In claim 9, a device configured to be operable to receive gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof. Claim 11 A method for separating a biomolecule from one or more small molecules, comprising: a) applying a sample containing said biomolecule to a container comprising a composition of any one of claims 1 to 8; and b) subjecting said container to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof, wherein said biomolecule in said sample is excluded through said first size exclusion support and collected as flow-through, said one or more small molecules are separated from said biomolecule by binding to said first moiety, and optionally said biomolecule excluded from said sample is 2 kDa or more. Claim 12 A method according to claim 11, wherein the first moiety is bonded to one or more small molecules by charge interaction, hydrophilic interaction, hydrophobic interaction, affinity interaction, hydrogen bonding, or van der Waals force. Claim 13 A system for separating one or more small molecules from a sample, comprising: a) a container comprising a composition of any one of claims 1 to 8; b) a storage located below the container; and c) means for causing the container and the storage to be subjected to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof. Claim 14 A kit for separating biomolecules from one or more small molecules, comprising a) a container comprising a composition of any one of claims 1 to 8 and b) a device comprising a reservoir located below the container, wherein the device is configured to be operable to receive gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof, and optionally, the device is a spin column, a multi-well filter plate, or a multi-well plate. Claim 15 In Clause 14, a kit further comprising a buffer solution. Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete

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