Components for separating molecules and methods of making and using same

Abstract

Disclosed herein are embodiments of matrices made from porous size-exclusion supports and cationic moieties for separating one or more small molecules from one or more large molecules in a sample using differences in one or more properties, such as molecular size, molecular charge, molecular isoelectric point (pI), and / or any combination of these properties, including method, system, and kit embodiments. Also disclosed herein are embodiments of methods for making the matrices and methods for using the matrices to separate small molecules from one or more large molecules in a sample.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 63 / 524,189, filed June 29, 2023, and 63 / 370,277, filed August 3, 2022, each of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to matrices, systems, methods, and kits for sample preparation, for example, separating small molecules from large molecules. [Background technology]

[0003] Sample preparation techniques for isolating biomolecules may require separation of the biomolecule from other sample and sample processing components to enable downstream analysis and processing of the biomolecule. Often, during sample preparation of biomolecules such as proteins or nucleic acids, the biomolecule must be labeled with a dye, affinity tag, radiolabel, mass tag, etc. In other examples, the biomolecule may require chemical modification, such as by reduction, oxidation, crosslinking, and / or alkylation.

[0004] One example is provided by considering bovine serum albumin (BSA), a protein isolated from bovine plasma that is commonly added to antibodies as a stabilizer. While BSA has stabilizing properties, it often interferes with downstream applications (e.g., labeling of antibodies with fluorospheres), especially when antibody levels are low. To avoid such interference, BSA must be separated from the selected antibody before labeling the antibody. Often, such separation is performed using a size-exclusion resin, requiring cumbersome fast protein liquid chromatography (FPLC) instrumentation for the purification resin. However, existing resin kits for BSA removal are often ineffective, exhibiting low antibody recovery and / or poor BSA removal properties.

[0005] Therefore, there is a need for improved purification resins, methods, and kits for purifying proteins from samples that result in proteins that are sufficiently purified for high quality downstream analysis. Summary of the Invention

[0006] The disclosed embodiments of the present disclosure advantageously provide superior separation of molecules, e.g., biomolecules, from one another. In some aspects of the present disclosure, molecules are separated from one another using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point (pI), and / or any combination of these properties. Furthermore, molecules can be separated from one another based on one or more separation matrix properties, such as, but not limited to, charge and size exclusion properties. This can advantageously reduce the time and cost associated with separating small molecules from large molecules. Molecules separated as described herein facilitate downstream processing compared to known processes.

[0007] In some embodiments, the porous size-exclusion support is produced by using enough HEC to produce a resin with a molecular weight cutoff (or "MWCO") of 40 kDa or greater. For example, some embodiments may include 60 grams to 150 grams of HEC. In some aspects, 60 grams to 130 grams of HEC are used at a 5 liter reaction scale to produce the size-exclusion support. In certain aspects disclosed herein, 60 grams to 130 grams of HEC produces 0.5 liters to 2 liters of resin bed comprising the porous size-exclusion support. In some embodiments, the porous size-exclusion support may further comprise a crosslinked porous size-exclusion support. In some embodiments, the porous size-exclusion support can be crosslinked with an epoxide-containing compound containing at least one epoxide functional group; an exemplary embodiment is crosslinked with epichlorohydrin (also referred to herein as "Epi"). For example, the porous size-exclusion support can be crosslinked with 250 milliliters to 450 milliliters of Epi.

[0008] Certain disclosed embodiments relate to matrices comprising a porous size-exclusion support having a molecular weight cutoff of 40 kDa or greater and at least one cationic moiety associated with the porous size-exclusion support, wherein the at least one cationic moiety is selected for association with small molecules having a molecular weight of less than 100 kDa, such as, but not limited to, bovine serum albumin (BSA) and / or other proteins. In some embodiments, the at least one cationic moiety associates with the small molecule through ionic interactions, hydrophilic interactions, hydrophobic interactions, affinity interactions, hydrogen bonding, and / or van der Waals forces. The cationic moiety may be, for example, an amine, diamine, polyamine, amine-containing heterocyclic compound, amine-containing aromatic compound, or amine-containing polymer. Specific examples of cationic moieties include 5,8-dimethyl-4,7,10-trioxatridecane-2,12-diamine, polyethyleneimine, diaminopentane, N,N-diethylethylenediamine, 1,2-diaminobenzene, 1,3-diaminobenzene, 1,4-diaminobenzene, and (S)-N-boc-2,3-epoxypropylamine. For certain embodiments, the cationic moiety is covalently attached to the porous size-exclusion support.

[0009] The matrix may advantageously be equilibrated with an equilibration buffer containing little or no salt (e.g., NaCl and other ionic salts), e.g., 0 mM to 5 mM salt. In some embodiments, the equilibration buffer may contain a positive charge. In other embodiments, the equilibration buffer may contain a neutral charge. In some aspects, the equilibration buffer may have a pH of 4 to 9, preferably 5 to 7. In some aspects, the equilibration buffer may have a concentration of 20 mM to 100 mM. In certain aspects disclosed herein, the equilibration buffer may contain triethylammonium bicarbonate, borate, sodium acetate, or HEPES to facilitate separation of small molecules from large molecules, e.g., to separate negatively charged small molecules from positively charged large molecules.

[0010] The present disclosure also provides, in some embodiments, a system for separating molecules of different molecular weights and / or charges and / or isoelectric points (pI) from one another. In one embodiment, the disclosed system can separate small molecules from large molecules in a sample. In one embodiment, the disclosed system can separate small molecules, e.g., 70 kDa proteins, from large molecules, e.g., 150 kDa proteins, in a sample. Such a system includes a vessel containing a matrix of the present disclosure and a receptacle configured to receive flow from the vessel. The system can be configured for gravity flow operation, centrifugal force operation, positive pressure operation, negative pressure operation, vacuum operation, or a combination thereof. The vessel can be any suitable vessel, such as a column, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate.

[0011] Also disclosed is a method for preparing a matrix comprising a porous size-exclusion support having a MWCO of 40 kDa or greater and at least one cationic moiety associated with the porous size-exclusion support. The method includes providing a porous size-exclusion support comprising hydroxyethyl cellulose having a MWCO of 40 kDa or greater, the hydroxyethyl cellulose having at least one vicinal diol. The vicinal diol is oxidized to form an aldehyde, which is then reacted with amine groups of at least 50 mg / mL of cationic moieties by reductive amination, for example, using sodium cyanoborohydride or picoline borane. The concentration of the cationic moiety used to form the resin can be varied as desired to facilitate the separation process, but is typically between 50 mg / mL and 175 mg / mL, more typically between 50 mg / mL and 160 mg / mL.

[0012] Also disclosed are methods for separating small molecules of different molecular weights and / or charges and / or pI values from one another. In one embodiment, the disclosed method can separate proteins of 70 kDa or less from at least one large molecule of greater than 100 kDa in a sample. The method includes providing a matrix comprising a porous size-exclusion support having associated therewith at least one cationic moiety, which can associate with at least one small molecule, such as, but not limited to, a negatively charged small molecule. In some embodiments, the matrix can be equilibrated with an equilibration buffer. In some aspects of the present disclosure, the at least one small molecule is then separated from at least one large molecule by applying the sample to the matrix and subjecting the matrix to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof. The at least one large molecule in the sample is excluded by the matrix and collected as flow-through in a receptacle configured to receive the flow-through. At least one small molecule (e.g., a negatively charged small molecule) is associated with at least one cationic moiety, thereby separating it from at least one large molecule in a single step. The disclosed method substantially increases the ability to separate small molecules, such as bovine serum albumin, from a sample, while also substantially increasing the recovery of large molecules, such as IgG. This further facilitates processing of the large molecules for downstream applications, such as dye labeling.

[0013] Kits for separating molecules of different molecular weights and / or charges and / or pI values from each other are also disclosed. In one embodiment, the disclosed kits can separate small molecules, e.g., 70 kDa proteins, from large molecules, e.g., 150 kDa proteins, in a sample. In another embodiment, the disclosed kits can separate negatively charged molecules from more positively charged molecules in a sample. In some embodiments, the kits can include (1) a porous size-exclusion support having at least one cationic moiety associated therewith, where the cationic moiety can associate with and capture small molecules, and (2) instructions for using the porous size-exclusion support. The kits can further include a system including a container containing an equilibration buffer and / or the porous size-exclusion support, and a receptacle configured to receive the support flow-through.

[0014] The foregoing and other objects, features, and advantages of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram illustrating one disclosed embodiment comprising a porous size-exclusion support associated with a cationic moiety, such as diaminopentane (or "PDA"). [Figure 2] FIG. 1 is a schematic diagram illustrating one disclosed embodiment comprising a porous size-exclusion support associated with a cationic moiety, such as branched polyethyleneimine (or "PEI"). [Figure 3] FIG. 1 is a schematic diagram illustrating one disclosed embodiment comprising a porous size-exclusion support associated with a cationic moiety, such as N,N diethylethylenediamine (or "DEED"). [Figure 4] FIG. 1 is a schematic diagram illustrating one disclosed embodiment of using cationic moieties associated on a porous size-exclusion support to separate small molecules from a sample containing small molecules, including but not limited to, negatively charged small molecules. [Figure 5]FIG. 1 is a schematic diagram illustrating one embodiment of the disclosed system, including a container, a receptacle, and an exemplary matrix including a porous size-exclusion carrier associated with a cationic moiety. [Figure 6] FIG. 1 is a schematic side view of one embodiment of a system according to the present disclosure, comprising a container and receptacle for processing a sample to separate at least one small molecule from at least one large molecule using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point (pI), and / or any combination of these properties. [Figure 7] FIG. 1 is a schematic perspective view of one embodiment of the disclosed system for processing a sample to separate small molecules from large molecules using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point (pI), and / or any combination of these properties, the system comprising a multi-well container and receptacle. [Figure 8] FIG. 1 is a schematic perspective view of one embodiment of the disclosed system for processing a sample to separate small molecules from large molecules using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point (pI), and / or any combination of these properties, the system comprising a multi-well container and receptacle. [Figure 9] 9 is an image of a gel comparing BSA removal and IgG recovery from a sample containing a mixture of BSA (2 mg / mL) and IgG (2 mg / mL) for Resin A, Resin B, Resin C, Resin D, Resin E, and Resin F, which are embodiments disclosed in Table 2 herein. Figure 9 shows that the BSA removal from greatest to least was achieved in the following order: Resin F, Resin D, Resin E, Resin B, then Resin A (thus indicating that in addition to PDA, MWCO also contributed to the desired BSA removal). [Figure 10]1 is a gel image comparing an embodiment of Resin 5 (described in Table 1 provided herein), an embodiment of Resin L (described in Table 2 provided herein), an embodiment of Resin F (described in Table 2 provided herein), and an embodiment of Resin D (described in Table 2 provided herein) equilibrated with different equilibration buffers (described in Table 3 provided herein) to remove BSA and recover IgG from a sample containing a mixture of BSA (10 mg / mL) and IgG (1 mg / mL). Thus, this figure demonstrates that PDA aids in removing a desirable amount of BSA, that embodiments with a MWCO of 40 kDa or greater exhibited lower BSA removal capacity than embodiments with a MWCO of 45 kDa, and that embodiments equilibrated with Tris buffer exhibited desirable IgG recovery. [Figure 11] 1 is an image of a gel showing BSA removal and IgG recovery from a sample containing a mixture of BSA (10 mg / mL) and IgG (1 mg / mL) by an embodiment of Resin G (described in Table 1 provided herein) equilibrated with 50 mM Tris pH 7 (lane 1), 50 mM TEAB pH 5 (lane 2), 50 mM TEAB pH 7 (lane 3), 50 mM sodium acetate pH 5 (lane 4), 50 mM sodium acetate pH 7 (lane 5), 50 mM HEPES pH 5 (lane 6), 50 mM HEPES pH 7 (lane 7), demonstrating desirable BSA removal and IgG recovery from the sample. [Figure 12]1 is a bar graph comparing the BSA binding capacity (300 μL of 10 mg / mL BSA) of an embodiment of Resin 5 (described in Table 1 provided herein) modified with 150 mg / mL PEI, an embodiment of Resin 3 (described in Table 1 provided herein) modified with 130 mg / mL PEI, the Melon™ Gel IgG Purification Kit, and Affi-Gel® Blue Gel (Bio-Rad). The Resin 5 embodiment exhibited a binding capacity of 3.11 mg BSA bound / mL resin, the Resin 3 embodiment exhibited a binding capacity of 2.45 mg BSA bound / mL resin, the Melon™ Gel IgG Purification Kit exhibited a binding capacity of 1.26 mg BSA bound / mL resin, and the Affi-Gel® Blue Gel (Bio-Rad) exhibited a binding capacity of 2.43 mg BSA bound / mL resin. Thus, this figure shows the desired BSA binding capacity in the 150 mg / mL PEI-modified resin 5 embodiment, the 150 mg / mL PEI-modified resin 3 embodiment, and Affi-Gel® Blue Gel (Bio-Rad), as opposed to the Melon™ Gel IgG Purification Kit. [Figure 13]1 is a bar graph comparing IgG recovery (2 mg / mL) for an embodiment of Resin 5 modified with 150 mg / mL PEI (described in Table 1 provided herein), an embodiment of Resin 3 modified with 130 mg / mL PEI (described in Table 1 provided herein), the Melon™ Gel IgG Purification Kit, and Affi-Gel® Blue Gel (Bio-Rad). The Resin 5 embodiment demonstrated 72% recovery by volume, the Resin 3 embodiment demonstrated 61% recovery by volume, the Melon™ Gel IgG Purification Kit demonstrated 80% recovery by volume, and the Affi-Gel® Blue Gel (Bio-Rad) demonstrated 25% recovery by volume. Thus, this example demonstrates desirable IgG recovery in an embodiment of Resin 5 modified with 150 mg / mL PEI, an embodiment of Resin 3 modified with 130 mg / mL PEI, and the Melon™ Gel IgG Purification Kit, unlike Affi-Gel® Blue Gel (Bio-Rad). [Figure 14A] 1 is a gel image comparing an embodiment of Resin 5 (described in Table 1 provided herein) produced with different amounts of PDA (described in Table 4 provided herein) with the Abcam BSA Removal Kit and the Melon™ Gel IgG Purification Kit to remove BSA and recover IgG from a sample containing a mixture of BSA (10 mg / mL) and GAR (1 mg / mL). An embodiment of Resin 5 modified with 150 mg / mL of PDA (also referred to as an embodiment of Resin F described in Table 2 provided herein) and an embodiment of Resin 5 modified with 75 mg / mL of PDA (also referred to as an embodiment of Resin K described in Table 2 provided herein) show higher BSA removal and higher IgG recovery than the Abcam BSA Removal Kit and the Melon™ Gel IgG Purification Kit. [Figure 14B]14B is a bar graph showing quantification of the bands from the gel of FIG. 14A using iBright Image analysis software, demonstrating that an embodiment of Resin 5 modified with 150 mg / mL PDA had 85.7% IgG recovery and 98.8% BSA removal, Resin 5 modified with 75 mg / mL showed 86.7% IgG recovery and 82.6% BSA removal, the Abcam BSA Removal Kit had 53.3% IgG recovery and 65.78% BSA removal, and the Melon™ Gel IgG Purification Kit had 78.7% IgG recovery and 8.3% BSA removal. Thus, it was demonstrated that Resin 5 modified with 150 mg / mL PDA and 75 mg / mL PDA achieved higher IgG recovery and BSA removal than the Abcam BSA Removal Kit and the Melon™ Gel IgG Purification Kit. [Figure 15] An embodiment of Resin 5 showed 86% recovery at 42,000 Da, 94% recovery at 67,000 Da, 92% recovery at 80,000 Da, and 94% recovery at 150,000 Da; an embodiment of Resin 6 showed 82% recovery at 42,000 Da, 84% recovery at 67,000 Da, 90% recovery at 80,000 Da, and 92% recovery at 150,000 Da; an embodiment of Resin 7 showed 66% recovery at 42,000 Da, 76% recovery at 67,000 Da, and 92% recovery at 80,000 Da. 1 is a bar graph showing the recovery of molecules of different sizes for establishing the MWCO of Resin 5, Resin 6, Resin 7, and Resin 8 embodiments (listed in Table 1 provided herein), showing that Resin 5 showed 84% recovery at 42,000 Da, 86% recovery at 150,000 Da, and Resin 8 showed 58% recovery at 42,000 Da, 75% recovery at 67,000 Da, 75% recovery at 80,000 Da, and 83% recovery at 150,000 Da. Thus, this figure demonstrates that decreasing the amount of HEC results in resins having a 50 kDa MWCO, an 80 kDa MWCO, and a 90 kDa MWCO (listed in Table 1 provided herein). [Figure 16A]1 is a gel image showing BSA removal and IgG recovery for Resin F embodiment, Resin G embodiment, Resin H embodiment, Resin I embodiment, and Resin J embodiment (listed in Table 1 provided herein) versus Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit from a sample containing a mixture of BSA (10 mg / mL) and GAR IgG (1 mg / mL). Thus, this figure demonstrates that Resin F embodiment and Resin G embodiment had the most desirable BSA removal and IgG recovery from the sample. [Figure 16B] iBright demonstrated that an embodiment of Resin F had 83% GAR recovery and 99% BSA removal, an embodiment of Resin G had 93% GAR recovery and 100% BSA removal, an embodiment of Resin H had 63% GAR recovery and 99% BSA removal, an embodiment of Resin I had 76% GAR recovery and 95% BSA removal, an embodiment of Resin J had 82% GAR recovery and 100% BSA removal, a Melon™ Gel IgG Purification Kit (Thermo Scientific™) had 106% GAR recovery and 82% BSA removal, Affi-Gel® Blue Gel (Bio-Rad) had 73% GAR recovery and 65% BSA removal, and an Abcam BSA Removal Kit had 125% GAR recovery and 85% BSA removal. 16B is a bar graph showing quantification of the bands of the gel of FIG. 16A using Image analysis software. Thus, this figure demonstrates higher BSA removal and IgG recovery with Resin F embodiment, Resin G embodiment, and Resin J embodiment. [Figure 17]1 is a gel image showing BSA removal and IgG recovery for Resin F embodiment, Resin G embodiment, Resin H embodiment, Resin I embodiment, and Resin J embodiment (listed in Table 2 provided herein) relative to the Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit in a sample containing a mixture of BSA (10 mg / mL) and IgG (0.1 mg / mL). Thus, this figure demonstrates that Resin F embodiment, Resin G embodiment, and Resin J embodiment performed better in mixtures with low concentrations of IgG relative to the Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit. [Figure 18] Figure 1 shows an image of fluorescently labeled GAR flow-through using NHS DyLight™ 488 (Thermo Scientific™) after removal of BSA from a mixture containing GAR (1 mg / mL) and BSA (10 mg / mL) with an embodiment of Resin F (listed in Table 2 provided herein) for the Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and the Abcam BSA Removal Kit. Thus, this figure demonstrates the desirable recovery of labeled GAR after BSA removal with an embodiment of Resin F and the Abcam BSA Removal Kit, as opposed to the Melon™ Gel IgG Purification Kit (Thermo Scientific™) and Affi-Gel® Blue Gel (Bio-Rad). [Figure 19]Figure 1 shows an image of fluorescently labeled GAR flow-through using NHS DyLight™ 488 (Thermo Scientific™) after removal of BSA from a mixture containing GAR (0.1 mg / mL) and BSA (10 mg / mL) by Resin F embodiment and Resin G embodiment (listed in Table 2 provided herein) for Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit. Thus, this figure demonstrates the desirable recovery of labeled GAR after BSA removal by Resin F embodiment and Resin G embodiment, even at low antibody concentrations such as 0.1 mg / mL, unlike Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit. [Figure 20] 1 is a gel image showing the purification of rabbit serum, mouse serum, human plasma, and human serum with an embodiment of Resin F and an embodiment of Resin G (described in Table 2 provided herein), demonstrating the desired removal of albumin and the desired recovery of IgG from the different serum species. [Figure 21A] 1 is a bar graph showing rabbit IgG (A280 amounts) for embodiments of Resin G (listed in Table 2 provided herein) equilibrated with different concentrations of Tris buffer, showing that the A280 amount of rabbit IgG for an embodiment of Resin G equilibrated with 50 mM Tris (pH 7.0) was 123, an embodiment of Resin G equilibrated with 50 mM Tris (pH 5.0) had a 124 A280 amount, an embodiment of Resin G equilibrated with 50 mM Tris (pH 7.0 + 20 μL stacker) had a 139 A280 amount, the BSA-rabbit IgG starting mixture had an 824 A280 amount, the rabbit IgG starting material had a 133 A280 amount, and BSA alone had a 634 A280 amount. This figure therefore demonstrates desirable recovery of antibody, as the A280 amount of the flow-through was similar to that of rabbit IgG. [Figure 21B] 1 is a bar graph showing the A280 amount of rabbit IgG for an embodiment of Resin G equilibrated with 50 mM Tris (pH 7.0) having a 97 A280 amount, an embodiment of Resin G equilibrated with 50 mM Tris (pH 5.0) having a 101 A280 amount, an embodiment of Resin G equilibrated with 50 mM Tris (pH 7.0 + 20 μL stacker) having a 99 A280 amount, a BSA-GAR starting mixture having a 834 A280 amount, a GAR starting mixture having a 100 A280 amount, and BSA alone having a 634 A280 amount. This figure therefore demonstrates desirable recovery of antibody, as the A280 amount of the flow-through was similar to that of the GAR. [Figure 22] Figure 10 shows images of fluorescent dye labeling of a primary antibody (GAPDH) after BSA removal from a sample containing an antibody (1 mg / mL)-BSA (10 mg / mL) mixture for an embodiment of Resin G (described in Table 2 provided herein) spun at 3,000 x G and 6,000 x G, which also labeled the antibody-BSA and free Dy650. Thus, this figure shows desirable BSA removal for both spin speeds. [Figure 23] 1 is an image of a gel obtained by loading and staining the flow-through using Coomassie stain with a Pierce Power blotter, showing BSA-free calreticulin after passage through an embodiment of Resin G (described in Table 2 provided herein), calreticulin conjugated to DyLight™ 680 (Thermo Scientific™) after BSA removal, and calreticulin as received with BSA added as a stabilizer. This figure therefore demonstrates the desirable removal of BSA from the primary antibody calreticulin prior to conjugation with DyLight™ (Thermo Scientific™) 680 by an embodiment of Resin G. [Figure 24A]1 is an image of a Western blot application using fluorescently labeled GAR after BSA purification with an embodiment of Resin H (described in Table 2 provided herein) showing BSA that was removed from GAR (left) and BSA that was not removed from GAR (right) from a sample containing a mixture of GAR (1 mg / mL) and BSA (10 mg / mL). Thus, this figure shows that BSA that was removed from GAR prior to conjugation to Dy650 exhibited much higher intensity than when BSA was not removed from GAR. [Figure 24B] 1 is a bar graph showing the fluorescence intensity of BSA removed and not removed by an embodiment of Resin H (described in Table 2 provided herein), where HeLa lysate loading for 10 μg of removed BSA has a fluorescence intensity of 13,000,000, non-removed BSA has a fluorescence intensity of 4,000,000, HeLa lysate loading for 5 μg of removed BSA has a fluorescence intensity of 9,000,000, non-removed BSA has a fluorescence intensity of 3,800,000, HeLa lysate loading for 2.5 μg of removed BSA has a fluorescence intensity of 7,800,000, non-removed BSA has a fluorescence intensity of 3,800,000, HeLa lysate loading for 1.25 μg of removed BSA has a fluorescence intensity of 4,100,000, non-removed BSA has a fluorescence intensity of 1,800,000. Thus, this figure demonstrates that BSA removed from GFAR prior to conjugation to Dy650 exhibited much higher intensity than when BSA was not removed from GAR, with a 3-fold higher fluorescence intensity observed with BSA-removed GAR Dy650. DETAILED DESCRIPTION OF THE INVENTION

[0016] I. Abbreviations BSA: bovine serum albumin DEED: Diethylethylenediamine Epi: epichlorohydrin HEC: Hydroxyethyl cellulose HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IgG: immunoglobulin G MWCO: Molecular Weight Cutoff PDA: Diaminopentane PEI: Branched polyethyleneimine TRIS: Tris(hydroxymethyl)aminomethane

[0017] II. Overview of Terms, Scope, and Definitions Both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the present disclosure.

[0018] As used herein, the use of the singular includes the plural unless specifically stated otherwise. For example, as used herein, the singular forms "a," "an," and "the" also include the plural unless the context dictates otherwise. Similarly, as used herein, singular terms also refer to the plural and vice versa unless the context dictates otherwise.

[0019] In some examples, values, procedures, or devices may be referred to as "lowest," "best," "minimum," etc. Such descriptions are intended to indicate that a selection may be made from among many possible functional alternatives, but it will be understood that such a selection need not be better than, smaller than, or otherwise preferred to other selections.

[0020] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. Unless otherwise indicated, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the present disclosure will be apparent from the following detailed description and claims.

[0021] Unless otherwise indicated, when used in the specification or claims, all numbers expressing quantities of ingredients, molecular weights, percentages, temperatures, times, and the like, should be understood to be modified by the term "about." Accordingly, unless otherwise indicated, either implicitly or explicitly, the numerical parameters set forth are approximations that may depend on the desired properties sought and / or the limits of detection under standard testing conditions / methods. Where the word "about" is recited to directly and explicitly distinguish an embodiment from the prior art discussed, the number of the embodiment is not approximate. Furthermore, not all alternatives described herein are equivalent.

[0022] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least A, B, C, AB, AC, BC, or ABC, and, in the particular context, where order is important, also includes BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations including repeats of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included.

[0023] The term "acyl halide" generally refers to -C(O)X, where X is a halogen, such as Br, F, I, or Cl.

[0024] The term "alcohol" generally refers to an organic compound containing at least one hydroxyl group. Alcohols may be monovalent (containing one -OH group), divalent (containing two -OH groups, diols, e.g., glycols), trivalent (containing three -OH groups, triols, e.g., glycerol), or polyvalent (containing more than one -OH group, polyols). The organic portion of the alcohol may be aliphatic, cycloaliphatic (alicyclic), heteroaliphatic, cycloheteroaliphatic (heterocyclic), polycyclic, aryl, or heteroaryl, and may be substituted or unsubstituted.

[0025] The term "aldehyde" generally refers to a carbonyl-containing functional group having the formula:

[0026] [ka] In the formulae, a line drawn through a bond indicates that the functional group may be attached to any other moiety, but such moiety is simply not shown.

[0027] The term "aliphatic" generally refers to substantially hydrocarbon-based compounds or their radicals (e.g., CH for hexane radical). 13 ), including alkanes, alkenes, and alkynes, including cyclic versions thereof, as well as straight-chain and branched-chain configurations, and all stereo- and positional isomers. Unless otherwise specified, an aliphatic group contains 1 to 25 carbon atoms, e.g., 1 to 15, 1 to 10, 1 to 6, or 1 to 4 carbon atoms. The term "lower aliphatic" refers to an aliphatic group containing 1 to 10 carbon atoms. The aliphatic chain can be substituted or unsubstituted. Unless explicitly referred to as "unsubstituted aliphatic," an aliphatic group can be unsubstituted or substituted. An aliphatic group can be substituted with one or more substituents (up to two substituents for each methylene carbon in the aliphatic chain, or up to one substituent for each carbon of a C=C double bond in the aliphatic chain, or up to one substituent for the carbon of the terminal methine group). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heteroalicyclic, hydroxyl, oxo, sulfonamido, sulfhydryl, thioalkoxy, or other functional groups.

[0028] The term "alkoxy" generally refers to a radical (or substituent) having the structure --OR, where R is a substituted or unsubstituted alkyl.

[0029] The term "alkyl" generally refers to a hydrocarbon group having a saturated carbon chain, which may be cyclic, branched, or unbranched.

[0030] The term "alkynyl" generally refers to an organic compound that contains at least one carbon-carbon triple bond. Alkynyl groups can be branched, straight-chain, or cyclic (e.g., cycloalkynyl).

[0031] The term "amide" generally refers to the chemical functional group -C(O)N(R')(R"), where R' and R" are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0032] The term "amino" generally refers to the chemical functional group -N(R)R', where R and R' are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0033] The term "antibody" generally refers to an immunoglobulin or immunoglobulin-like molecule (including, by way of example and not limitation, IgA (Immunoglobulin A), IgD (Immunoglobulin D), IgE (Immunoglobulin E), IgG (Immunoglobulin E), and IgM (Immunoglobulin M), combinations thereof, and similar molecules produced during the immune response in any chordate, such as a vertebrate, e.g., a mammal, such as a human, goat, rabbit, and mouse), and fragments thereof that specifically bind to a molecule of interest (or a group of closely related molecules of interest) to the substantial exclusion of binding to other molecules. An "antibody" typically includes a polypeptide ligand having at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. Immunoglobulins are composed of heavy and light chains, each of which contains a heavy chain variable (V H ) region and the light chain variable (V L ) region. H Area and V L The region is responsible for binding the antigen recognized by the immunoglobulin. Exemplary immunoglobulin fragments include, but are not limited to, proteolytic immunoglobulin fragments (e.g., F(ab')2 fragments, Fab' fragments, Fab'-SH fragments, and Fab fragments, as known in the art), recombinant immunoglobulin fragments (e.g., sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, 'F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). Other examples of antibodies include diabodies and triabodies (known in the art), and camelid antibodies. "Antibody" also includes genetically engineered molecules, such as chimeric antibodies (e.g., humanized murine antibodies), and heteroconjugate antibodies (e.g., bispecific antibodies). See Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL), Kuby, J., Immunology, 3 rdEd., W.H. Freeman & Co., New York, 1997.

[0034] The term "aromatic" generally refers to a cyclic or conjugated group containing 5 to 15 ring atoms, unless otherwise specified, having at least one ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl, indolyl, or pyrazolopyridinyl) in which at least one ring is aromatic, i.e., at least one ring, and optionally multiple fused rings, have a contiguous delocalized π-electron system. Typically, the number of out-of-plane π-electrons corresponds to Hückel's rule (4n+2). The point of attachment to the parent structure is typically through the aromatic portion of the fused ring system. For example,

[0035] [ka] However, in certain instances, the context or explicit disclosure may indicate that the point of attachment is through the non-aromatic portion of the fused ring system. For example:

[0036] [ka] An aromatic group may contain only carbon atoms in the ring, such as an aryl group, or may contain one or more ring carbon atoms and one or more ring heteroatoms (e.g., S, O, N, P, or Si) containing lone pairs of electrons, such as a heteroaryl group. The aromatic group may be substituted with one or more groups other than hydrogen, such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional groups, or organic functional groups.

[0037] The term "aryl" generally refers to an aryl group having at least 5 carbon atoms and up to 15 carbon atoms (C5-C6). 15 ), e.g., 5 to 10 carbon atoms (C5 to C 10" refers to an aromatic carbocyclic group containing a single ring or multiple fused rings, which may or may not be aromatic, provided that the point of attachment to the remainder of the compounds disclosed herein is through an atom of the aromatic carbocyclic group. Aryl groups can be substituted with one or more groups other than hydrogen, for example, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional groups.

[0038] The term "biological sample" generally refers to hematological, cytological, and histological specimens, such as cells, cell cultures, hybridomas, single-cell organisms (e.g., yeast and bacteria), 3D cell cultures (e.g., spheroids and organoids), tissues, whole organisms (e.g., flies or worms), cell-free extracts, or fluid samples containing any biological material (e.g., blood, serum, plasma, saliva, urine, cerebrospinal fluid). Biological samples can be from plants or animals (e.g., humans, mice, flies, worms, fish, frogs, fungi, etc.). Samples can refer to samples that have been processed by filtration and / or centrifugation, and can include cell culture supernatants and homogenized tissues or pulverized cells.

[0039] The term "carbamate" generally refers to -OC(O)NRR', where R and R' are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0040] The term "carbonate" generally refers to a functional group having the formula -OCOR, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0041] The term "carboxamido" generally refers to -N(R) acyl, or -C(O) amino, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0042] The term "carboxyl" generally refers to -C(O)OH.

[0043] The term "carboxylic acid" generally refers to an organic compound having the formula RCOOH, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0044] The term "cyano" generally refers to -CN.

[0045] The term "disulfide" generally refers to -SSR a In the formula, R a is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0046] The term "epoxide" refers to a compound of the general formula

[0047] [ka] In the formula, R 1 ~R 4 are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0048] The term "ester" generally refers to an ester of the formula

[0049] [ka] wherein R and R' are independently alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0050] The term "ether" generally refers to a class of organic compounds containing an ether group, i.e., an oxygen atom bonded to two aliphatic and / or aryl groups, and having the general formula R-O-R', where R and R' are independently alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0051] The term "equilibration buffer" generally refers to a buffer used to inject into a matrix according to the present disclosure to facilitate sample processing and promote affinity of the molecule of interest to the carrier.

[0052] The term "fluorophore" generally refers to a functional group or moiety of a compound that causes the compound (or a sample or composition containing the compound) to fluoresce. In some embodiments, the fluorophore can fluoresce when the compound (or a sample or composition containing the compound) is exposed to an excitation source or after the fluorophore is cleaved from the compound to which it is conjugated.

[0053] The term "functional group" generally refers to a specific group of atoms within a molecule that participates in the molecule's characteristic chemical reactions. Exemplary functional groups include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, halo (fluoro, chloro, bromo, iodo), epoxide, hydroxyl, carbonyl (ketone), aldehyde, carbonate, carboxylate, carboxyl, ether, ester, peroxy, hydroperoxy, carboxamide, amino (primary, secondary, tertiary), ammonium, imide, azide, cyanate, isocyanate, thiocyanate, nitrate, nitrite, nitrile, nitroalkyl, nitroso, pyridyl, phosphate, sulfonyl, sulfide, thiol (sulfhydryl), and disulfide.

[0054] The term "halo" generally refers to fluoro, chloro, bromo, or iodo.

[0055] The term "heteroaryl" generally refers to an aryl group containing at least one heteroatom, which may be selected from, but is not limited to, oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorus, and their oxidized forms within the ring. Heteroaryl groups can contain a single ring or multiple fused rings, which may or may not be aromatic and / or contain heteroatoms, provided that the point of attachment is through an atom of the aromatic heteroaryl group. Heteroaryl groups can be substituted with one or more groups other than hydrogen, such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional groups. In some embodiments, fluorophores may also be described herein as heteroaryl groups.

[0056] The term "hydroxyl" generally refers to the group --OH.

[0057] The term "imine" generally refers to an organic compound containing a -C=NR group, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0058] The term "immunoglobulin G (IgG)" generally refers to one of the major classes of immunoglobulins that have heavy chains known as gamma chains.

[0059] The term "isoelectric point (pI)" generally refers to the pH at which a molecule has no net charge. Polymeric molecules, such as proteins composed of amino acids, can be positive, neutral, negative, or polar in character, imparting an overall charge to the polymeric material. Molecules with low pI values have a net negative charge at neutral pH, while molecules with high pI values have a net positive charge at neutral pH.

[0060] The term "molecular weight cutoff" or "MWCO" generally refers to the minimum molecular weight of a sample component, e.g., a smaller sample component, that will be excluded by a matrix. For example, one or more small molecules, such as, but not limited to, BSA, will be excluded by the matrix, while a larger sample component, e.g., one or more large molecules, will elute faster and be recovered.

[0061] The term "multimodal" generally refers to the ability of a material or compound, such as a resin or matrix, to contribute to the separation of a first desired molecule from a second molecule, such as by providing multiple different types of interactions between the resin or matrix and the desired molecule, thereby retaining the first and / or second molecule on the resin or matrix.

[0062] The term "negatively charged small molecule" generally refers to a molecule with a low isoelectric point (pI) that carries a net negative charge at neutral pH.

[0063] The term "phosphate" generally refers to -OP(O)(ORa ) 2, wherein each R a are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other organic functional group.

[0064] The term "large positively charged molecule" generally refers to a molecule with a high isoelectric point (pI) that carries a net positive charge at neutral pH.

[0065] The term "sample" generally refers to any fluid or solution containing at least two molecules to be separated, where the first of the at least two molecules is a small molecule as defined herein and the second of the at least two molecules is a large molecule as defined herein. In some embodiments, a sample may contain small molecules, such as, but not limited to, stabilizing molecules. Stabilizing molecules may be purified proteins isolated from natural or recombinant sources, such as, for example, recombinant albumin, native albumin, human serum albumin, albumin-like stabilizers, bovine serum albumin, equivalent mammalian serum (such as, but not limited to, rabbit serum and mouse serum), ovalbumin, glycerol, and / or gelatin. A sample may also contain one or more molecules derived from a biological sample.

[0066] The terms "separation," "extraction," "extracted," "removal," "reduce" or "reducing the amount," or "purification" generally refer to the removal or isolation of a substance, e.g., a small molecule such as BSA, or a large molecule or biomolecule such as IgG, from a mixture containing small and / or large molecules. The extracted substance, or the sample from which it is extracted, has significantly reduced amounts of components present in the sample prior to separation compared to before extraction, and the extracted substance may be substantially reduced, substantially removed, substantially concentrated, substantially pure, or pure (free of any contaminants).

[0067] This disclosure uses the terms "small molecule" and "large molecule" to refer to species that are separate from one another. A "small molecule" or "smaller molecule" generally refers to any molecule with a molecular weight less than 100 kDa, while a "large molecule" is a molecule with a molecular weight equal to or greater than 100 kDa. Small molecules (such as, but not limited to, biomolecules) may be used to process, derivatize, conjugate, crosslink, label, tag, or chemically or biologically modify larger molecules for further analysis. Small molecules may be stabilizing molecules, such as purified proteins isolated from natural or recombinant sources. For example, recombinant albumin, native albumin, human serum albumin, albumin-like stabilizers, bovine serum albumin, equivalent mammalian serum, ovalbumin, and / or gelatin. Derivatization includes labeling a molecule with a label, such as a dye, affinity tag, radiolabel, mass tag, metal, or the like. Derivatization also includes chemical modification of a molecule by reduction, oxidation, methylation, biological or biochemical modification of a biomolecule, and the like. Derivatives of biomolecules include, but are not limited to, tagged proteins or nucleic acids; labeled biomolecules labeled with various labels, including, but not limited to, dyes, fluorescent dyes, radioactive labels, affinity labels, mass tags, and metals; conjugated biomolecules, including conjugated antibodies; biomolecules conjugated to nanoparticles; metals, such as gold, conjugated to nanoparticles; dyes or labels, such as biotin, conjugated to toxins; and chemical derivatives of biomolecules, including, but not limited to, reduced proteins, oxidized proteins, methylated nucleic acids, and proteins with sulfhydryl modifications. Large molecules and / or biomolecules include, but are not limited to, proteins, glycoproteins, and antibodies. In one example, the small molecule is BSA, which has a molecular weight of 67,000 Da. In one example, the large molecule is IgG, which has a molecular weight of 150 kDa. In a specific disclosed example, the sample contains a mixture of BSA and IgG.

[0068] The term "size-exclusion support" generally refers to an inert, porous solid having a porosity that determines the size of molecules that can be included or excluded from entering the pores. In some disclosed embodiments, the pores of the porous size-exclusion support have a molecular size cut-off (MWCO) of 40 kDa or greater.

[0069] The term "sulfonamide" generally refers to the group -SOamyl or -N(R)sulfonyl, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0070] The term "sulfonate" generally refers to -SO3 - where the negative charge of the sulfonate group is M + Counterions may be balanced with positive counterions such as M + is K + , Na + , Li + Alkaline ions such as + N(R b ) 4, wherein R b is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group; ammonium ion; or, e.g., [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 The ion may be an alkaline earth ion such as

[0071] The term "sulfonyl" generally refers to a functional group having the general formula:

[0072] [ka] wherein R represents the remainder of the molecule to which the sulfonyl group is attached, and R' is selected from hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0073] The term "thioester" generally refers to a functional group having the general formula:

[0074] [ka] wherein R represents the remainder of the molecule to which the thioester group is attached, and R' is selected from alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group.

[0075] The term "thioether" generally refers to a functional group having the general formula: RS-R', where R and R' are independently selected from alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (e.g., optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional group. Thioethers are similar to ethers, except that thioethers contain a sulfur atom instead of the oxygen atom of an ether.

[0076] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for all purposes. In the event that one or more of the incorporated literature and similar materials defines or uses a term in a manner that contradicts the definition of that term in this application, the definition provided hereby controls. While the present teachings have been described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art in light of the present teachings.

[0077] III. Matrix Certain disclosed embodiments of the present disclosure relate to matrices for separating molecules, such as biomolecules, from one another. In some embodiments of the present disclosure, molecules are separated from one another using differences in one or more properties, such as molecular size, molecular charge, isoelectric point (pI), and / or a combination of these properties. In certain embodiments, molecules can be separated from one another based on one or more separation matrix properties, such as charge on the matrix and size-exclusion properties of the matrix. The matrix may include a porous size-exclusion support, at least one cationic moiety, and may further be equilibrated with an equilibration buffer. Each of these components is described in more detail below.

[0078] In some embodiments, a sample solution containing at least one small molecule and at least one large molecule is applied to a matrix, where the large molecule elutes faster than the small molecule, which is captured by the matrix. The matrices of the present disclosure provide unexpectedly rapid, economical, and efficient separation of small molecules from large molecules.

[0079] One embodiment of the present disclosure describes a matrix for separating, extracting, removing, and / or reducing the amount of one or more small molecules from one or more large molecules. In some embodiments, the small molecules can be, but are not limited to, proteins, globular proteins, serum albumin proteins, polypeptides, etc.

[0080] In certain disclosed embodiments, one or more small molecules can be separated from one or more large molecules using one or more properties, including, but not limited to, the isoelectric point of the molecule. In some embodiments, the one or more small molecules can be one or more negatively charged small molecules. In certain disclosed embodiments, the one or more negatively charged small molecules can have an isoelectric point (pI) value in the range of 4.5 to 5.5. In some embodiments, the one or more negatively charged small molecules can have a pI value of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, and / or 5.5. In one exemplary embodiment, the one or more negatively charged small molecules have a pI value of 4.9.

[0081] In some embodiments, the matrix separates, extracts, removes, and / or reduces the amount of one or more small molecules from one or more large molecules based on, but not limited to, the size of the molecules. In some aspects of the present disclosure, the one or more small molecules may have a molecular weight in the range of less than 100 kDa, less than 80 kDa, and / or less than 70 kDa. In some embodiments, the one or more small molecules can have a molecular weight of 50 kDa to 80 kDa, e.g., 50 kDa, 51 kDa, 52 kDa, 53 kDa, 54 kDa, 55 kDa, 56 kDa, 57 kDa, 58 kDa, 59 kDa, 60 kDa, 61 kDa, 62 kDa, 63 kDa, 64 kDa, 65 kDa, 66 kDa, 65 kDa, 66 kDa, 67 kDa, 68 kDa, 69 kDa, 70 kDa, 71 kDa, 72 kDa, 73 kDa, 74 kDa, 75 kDa, 76 kDa, 77 kDa, 78 kDa, 79 kDa, and / or 80 kDa. In some exemplary embodiments, the one or more small molecules is BSA, which has a molecular weight of 67,000 Da (67 kDa).

[0082] One embodiment of the present disclosure describes a matrix for separating, extracting, removing, and / or reducing the amount of one or more small molecules from one or more large molecules. In some embodiments, the one or more large molecules may be, but are not limited to, a glycoprotein, a phosphoprotein, an antibody, or an immunoglobulin.

[0083] In certain disclosed embodiments, one or more small molecules can be separated from one or more large molecules using one or more properties, including, but not limited to, the isoelectric point of the molecules. In some embodiments, the one or more large molecules can be one or more positively charged large molecules. In certain disclosed embodiments, the one or more positively charged large molecules can have an isoelectric point (pI) value in the range of 8.0 to 11.5. In some embodiments, the one or more positively charged large molecules can have a pI value of 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, and / or 11.5. In one exemplary embodiment, the one or more positively charged large molecules have a pI value of 11.0.

[0084] In some embodiments, the matrix separates, extracts, removes, and / or reduces the amount of one or more small molecules from one or more large molecules based on, but not limited to, the size of the molecules. In some embodiments, the one or more large molecules may have a molecular weight of 100 kDa or greater, and in certain embodiments, a molecular weight of 150 kDa or greater. For example, 100 kDa, 101 kDa, 102 kDa, 103 kDa, 104 kDa, 105 kDa, 106 kDa, 107 kDa, 108 kDa, 109 kDa, 110 kDa, 110 kDa, 111 kDa, 112 kDa, 113 kDa, 114 kDa, 115 kDa, 116 kDa, 117 kDa, 118 kDa, etc. a, 119kDa, 120kDa, 121kDa, 122kDa, 123kDa, 124kDa, 125kDa, 126kDa, 127kDa, 128kDa , 129kDa, 130kDa, 131kDa, 132kDa, 133kDa, 134kDa, 135kDa, 136kDa, 137kDa, 138kDa, 1 39kDa, 140kDa, 141kDa, 142kDa, 143kDa, 144kDa, 145kDa, 146kDa, 147kDa, 148kDa, 14 9kDa, 150kDa, 151kDa, 152kDa, 153kDa, 154kDa, 155kDa, 156kDa, 157kDa, 158kDa, 159k Da, 160 kDa, 161 kDa, 162 kDa, 163 kDa, 164 kDa, 165 kDa, 166 kDa, 167 kDa, 168 kDa, 169 kDa, 170 kDa, 180 kDa, 220 kDa, 250 kDa, 300 kDa, 350 kDa, 450 kDa, 550 kDa, 750 kDa, 900 kDa. In one exemplary embodiment, the large molecule is an IgG having a molecular weight of 150,000 Da (150 kDa).

[0085] A. Size Exclusion Support The matrix according to the present disclosure may include a porous size-exclusion carrier. The porous size-exclusion carrier may include spherical beads made of a gel or a gel-like material having pores. The pore size range of the porous size-exclusion carrier determines the size of molecules that can be included in or excluded from the size-exclusion carrier. Without being bound by a single theory, it is currently believed that when a sample solution passes through the size-exclusion carrier, at least one small molecule in the sample enters a pore in the size-exclusion carrier and is forced to follow a circuitous path before exiting the size-exclusion carrier. Meanwhile, large molecules take a relatively direct path through the size-exclusion carrier. Therefore, without being bound by a particular theory of operation, it is currently believed that the difference in flow rates between small and large molecules allows faster-flowing large molecules to be separated from slower-flowing small molecules as the sample moves through the size-exclusion carrier.

[0086] Some exemplary size-exclusion supports are made from agarose, polyacrylamide, cellulosic materials, and / or derivatives thereof. In some embodiments, the porous size-exclusion support may comprise an agarose support, a polyacrylamide support, a cellulosic material support, or a derivative thereof. In some exemplary embodiments, the porous size-exclusion support comprises HEC.

[0087] In some embodiments, the porous size-exclusion support is produced by using enough HEC to produce a resin with a MWCO of 40 kDa or greater.

[0088] In certain disclosed embodiments, the porous size exclusion carrier is made from a range of 50 grams (g) to 250 grams (g) of HEC, including, but not limited to, 50 g of HEC to 150 g of HEC. In some embodiments, the porous size exclusion carrier is made from 50 g, 51 g, 52 g, 53 g, 54 g, 55 g, 56 g, 57 g, 58 g, 59 g, 60 g, 61 g, 62 g, 63 g, 64 g, 65 g, 66 g, 67 g, 68 g, 69 g, 70 g, 71 g, 72 g, 73 g, 74 g, 75 g, 76 g, 77 g, 78 g, 79 g, 80 g, 81 g, 82 g, 83 g, 84 g, 85 g, 86 g, 87 g, 88 g, 89 g, 90 g, 91 g, 92 g, 93 g, 94 g, 95 g, 96 g, 97 g, 98 g, 99 g, 100 g, 101 g, 102 g, 103 g, 104 g, 105 g, 106 g, 107 g, 108 g, 109 g, 110 g, 111 g, 112 g, 113 g, 114 g, 115 g, 116 g, 117 g, 118 g, 119 g, 120 g, 121 g, 122 g, 123 g, 124 g, 125 g, 126 g, 127 g, 128 g, 129 g, 130 g, 13 9g, 90g, 91g, 92g, 93g, 94g, 95g, 96g, 97g, 98g, 99g, 100g, 101g, 102g, 103g, 104g, 105g, 106g, 107g, 10 8g, 109g, 110g, 111g, 112g, 113g, 114g, 115g, 116g, 117g, 118g, 119g, 120g, 121g, 122g, 123g, 124g, 12 5g, 126g, 127g, 128g, 129g, 130g, 131g, 132g, 133g, 134g, 135g, 136g, 137g, 138g, 139g, 140g, 141g, 1 42g, 143g, 144g, 145g, 146g, 147g, 148g, 149g, 150g, 160g, 170g, 180g, 190g, 200g, 201g, 202g, 203g, 2 The porous size-exclusion carrier can be produced using 0.4g, 205g, 206g, 207g, 208g, 209g, 210g, 211g, 212g, 213g, 214g, 215g, 216g, 217g, 218g, 219g, 220g, 221g, 222g, 223g, 224g, 225g, 226g, 227g, 228g, 229g, 230g, 240g, or 250g of HEC. In one exemplary embodiment, 80g of HEC was used to produce the porous size-exclusion carrier. In another exemplary embodiment, 90g of HEC was used to produce the porous size-exclusion carrier. In another exemplary embodiment, 108g of HEC was used to produce the porous size-exclusion carrier. In another exemplary embodiment, 129g of HEC was used to produce the porous size-exclusion carrier. In another exemplary embodiment, 147 g of HEC was used to prepare the porous size-exclusion support.In another exemplary embodiment, 216 g of HEC was used to prepare the porous size-exclusion support.

[0089] In some aspects of the present disclosure, the porous size-exclusion support is produced using 60 g to 130 g of HEC at a reaction scale of greater than 0 liters (L) to 10 liters (L), for example, but not limited to, greater than 0 L to 5 L, to produce the size-exclusion support. In some embodiments, a 0.5 L to 2 L resin bed comprising the porous size-exclusion support is produced from 60 g to 130 g of HEC. In certain disclosed aspects of the present disclosure, a 0.5 L to 1.5 L resin bed comprising the porous size-exclusion support is produced from 80 g to 130 g of HEC used at a 5 L reaction scale.

[0090] In some aspects of the present disclosure, the size-exclusion support may be crosslinked with a crosslinker, such as, but not limited to, an epoxide-containing compound containing at least one epoxide functional group, e.g., 1, 2, 3, or 4 epoxide groups. In some embodiments, the epoxide-containing compound may contain one or more halo functional groups, aliphatic functional groups, heteroaliphatic functional groups, or combinations thereof. In some embodiments, the heteroaliphatic functional group may include a polyethylene glycol (or "PEG") spacer arm. The epoxide-containing compound may have Formula I:

[0091] [ka] In the formula, R is C1 to C 10 In one preferred embodiment, the epoxide-containing compound is:

[0092] [ka] In another preferred embodiment, the epoxide-containing compound can be 1,4-butane diglycidyl ether.

[0093] In some embodiments, the porous size-exclusion support can be crosslinked with a crosslinker in the range of 250 milliliters (mL) to 700 milliliters (mL), for example, 250 mL, 251 mL, 252 mL, 253 mL, 254 mL, 255 mL, 256 mL, 257 mL, 258 mL, 259 mL, 260 mL, 261 mL, 262 mL, 263 mL, 264 mL, 265 mL, 266 mL, 267 mL, 268 mL, 269 mL, 270 mL, 271 mL, 272 mL, 273 mL, 274 mL, 275 mL, 276 mL, 277 mL, 278 mL, 279 mL, 300 mL, 301 mL, 302 mL, 303 mL, 304 mL, 305 mL, 306 mL, 307 mL, 308 mL, 309 mL, 310 mL, 311 mL, 312 mL, 313 mL, 314 mL, 315 mL, 316 mL, 317 mL, 318 mL, 319 mL, 320 mL, 321 mL, 322 mL, 323 mL, 324 mL, 325 mL, 326 mL, 327 mL, 328 mL, 329 mL, 330 mL, 331 mL, 332 mL, 333 mL, 334 mL, 335 mL, 336 mL, 337 mL, 338 mL, 339 mL, 340 mL, 341 mL, 342 mL, 343 mL, 344 mL, 345 mL, 346 mL, 9mL, 280mL, 281mL, 282mL, 283mL, 284mL, 285mL, 286mL, 287mL, 288mL, 289mL, 290mL, 291mL, 292mL, 293mL, 294mL, 295mL, 296mL, 297mL, 928mL, 299mL, 300mL, 301mL, 302mL, 303mL, 304mL, 305mL, 306mL, 307mL, 308mL, 309mL, 310mL, 311mL, 312mL, 313mL, 314mL, 315mL, 316mL, 317mL, 318mL, 319mL, 320mL , 321mL, 322mL, 323mL, 324mL, 325mL, 326mL, 327mL, 328mL, 329mL, 330mL, 331mL, 332mL, 333mL, 334mL, 335mL, 336mL, 337mL, 338mL, 339mL, 340mL, 341 mL, 342mL, 343mL, 344mL, 345mL, 346mL, 347mL, 348mL, 349mL, 355mL, 356mL, 357mL, 358mL, 359mL, 360mL, 361mL, 362mL, 363mL, 364mL, 365mL, 366mL, 36 7mL, 368mL, 369mL, 370mL, 371mL, 372mL, 373mL, 374mL, 375mL, 376mL, 377mL, 378mL, 379mL, 380mL, 381mL, 382mL, 383mL, 384mL, 385mL, 386mL, 387mL, 3 88mL, 389mL, 390mL, 391mL, 392mL, 393mL, 394mL, 395mL, 396mL, 397mL, 398mL, 399mL, 400mL, 401mL, 402mL, 403mL, 404mL, 405mL, 406mL, 407mL, 408mL,409 mL, 410 mL, 411 mL, 412 mL, 413 mL, 414 mL, 415 mL, 416 mL, 417 mL, 418 mL, 419 mL, 420 mL, 421 mL, 422 mL, 423 mL, 424 mL, 425 mL, 426 mL, 427 mL, 428 mL, 429 mL, 430 mL, 440 mL, 450 mL, 460 mL, 470 mL, 480 mL, 500 mL, 520 mL, 540 mL, 560 mL, 580 mL, 600 mL, 620 mL, 640 mL, 660 mL, 680 mL, or 700 mL of crosslinker can be used, and in certain embodiments, the above ranges / amounts of Epi are used as the crosslinker.

[0094] In one exemplary embodiment, 265 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 303 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 375 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 397 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 410 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 441 mL of Epi was used to crosslink the porous size-exclusion support. In yet another exemplary embodiment, 662 mL of Epi was used to crosslink the porous size-exclusion support.

[0095] In some embodiments, the porous size-exclusion support can be crosslinked by providing 250 mL to 700 mL of Epi at a reaction scale of greater than 0 L to 5 L. In some specific aspects of the present disclosure, a resin bed of greater than 0 L to 2 L containing the porous size-exclusion support can be produced from a 5 L reaction scale of 250 mL to 400 mL of Epi.

[0096] In some embodiments, the porous size-exclusion carrier has a MWCO of 40 kDa or greater, and therefore the molecules to be excluded have a molecular weight of 40 kDa or greater. The pore size of the porous size-exclusion carrier may have a MWCO size for excluding molecules from the pores of 40 kDa or greater to 150 kDa, more typically 50 kDa to 150 kDa, and even more typically 40 kDa or greater to 60 kDa, for example, but not limited to, 40 kDa, 41 kDa, 42 kDa, 43 kDa, 44 kDa, 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa, 51 kDa, 52 kDa, 53 kDa, 54 kDa, 55 kDa, 56 kDa, and the like. In one exemplary embodiment, the porous size exclusion support has a MWCO of 40 kDa or greater. In another exemplary embodiment, the porous size exclusion carrier has a MWCO of 45 kDa. In another exemplary embodiment, the porous size exclusion carrier has a MWCO of 50 kDa. In another exemplary embodiment, the porous size exclusion carrier has a MWCO of 80 kDa. In another exemplary embodiment, the porous size exclusion carrier has a MWCO of 90 kDa.

[0097] B. Cationic moiety Matrices according to the present disclosure may include a porous size-exclusion carrier and a cationic moiety. In some aspects of the present disclosure, small molecules, such as, but not limited to, negatively charged molecules, can be separated from large molecules, such as, but not limited to, positively charged molecules, based on one or more matrix properties. In some embodiments, the one or more matrix properties may be the charge and / or size-exclusion properties of the matrix.

[0098] In some embodiments, the cationic moieties of the matrix of the present disclosure are amines, imines, or combinations thereof. In some aspects of the present disclosure, such cationic moieties may include amine-containing polymers; alkylamines, particularly lower alkylamines (e.g., pentylamine); amine-containing heterocyclic compounds; amine-containing aromatic compounds; or other amine / diamine compounds.

[0099] In an exemplary embodiment, prior to association with the porous size-exclusion support, the amine-containing polymer is a branched polyethyleneimine (PEI) having the basic structure shown below, which becomes charged to provide cationic moieties upon exposure to appropriate conditions, such as, but not limited to, an equilibration buffer.

[0100] [ka]

[0101] In another exemplary embodiment, prior to association with the porous size-exclusion support, the amine-containing polymer is a diamine containing one or more polyethylene glycol groups, such as 5,8-dimethyl-4,7,10-trioxatridecane-2,12-diamine (also known commercially as Jeffamine), having the basic structure shown below: Upon exposure to appropriate conditions, such as, but not limited to, an equilibration buffer, the diamine becomes charged to provide cationic moieties.

[0102] [ka]

[0103] In another exemplary embodiment, prior to association with the porous size-exclusion support, the alkyldiamine is selected from the group consisting of the structure NH(CH 25 Diaminopentane (PDA) (also shown below) has an NH2 that becomes charged to provide a cationic moiety when exposed to appropriate conditions, such as, but not limited to, an equilibration buffer.

[0104] [ka]

[0105] In another exemplary embodiment, prior to association with the porous size-exclusion support, the diamine is N,N diethylethylenediamine (DEED) having the structure shown below, which becomes charged to provide cationic moieties upon exposure to appropriate conditions, such as, but not limited to, an equilibration buffer.

[0106] [ka]

[0107] In another aspect of the present disclosure, the amine-containing aromatic compound is a C3-C 15 and at least one amine-containing compound. In certain disclosed embodiments of the present disclosure, the amine-containing compound may have the general formula II, or an enantiomer, diastereomer, tautomer, salt, solvate, and / or isotopically substituted derivative thereof:

[0108] [ka] wherein, with respect to Formula II, J, Q, T, X, Y, and Z are the same or different, and each of J, Q, T, X, Y, and Z is independently nitrogen or CR c Selected from R c Each occurrence is independently selected from hydrogen, halo, aliphatic, heteroaliphatic, or amino. c In some embodiments, prior to association with the porous size-exclusion support, each of J, Q, T, X, Y, and Z is CR c and there are at least two R c The group contains an amino group, and the remaining R cThe group is hydrogen. In certain embodiments, the compound of Formula II can be selected from, but is not limited to, 1,2-diaminobenzene having the structure shown below, 1,3-diaminobenzene having the structure shown below, and / or 1,4-diaminobenzene having the structure shown below. Upon exposure to appropriate conditions, such as, but not limited to, an equilibration buffer, each of these compounds becomes charged to provide a cationic moiety.

[0109] [ka]

[0110] In certain disclosed embodiments, an amine-containing heterocyclic compound can be associated with a size-exclusion support containing HEC. In one example, an epoxyamine compound can be associated with a size-exclusion support containing HEC. In some embodiments, the amine-containing can be an epoxyamine, such as, but not limited to, (S)-N-boc-2,3-epoxypropylamine, having the following basic structure, which becomes charged to provide a cationic moiety when exposed to appropriate conditions, such as, but not limited to, an equilibration buffer.

[0111] [ka]

[0112] In some embodiments, the matrix of the present disclosure comprises at least one cationic moiety associated with a porous size-exclusion carrier. In some embodiments, either the porous size-exclusion carrier or the cationic moiety may comprise a reactive functional group. For example, functional groups on the porous size-exclusion carrier may be used to interact with and associate with one or more cationic moieties to form a matrix.

[0113] In some embodiments, the matrix comprises at least one cationic moiety immobilized on at least one size-exclusion support. In some embodiments, the cationic moiety may be "immobilized" by being covalently attached to the size-exclusion support through the formation of a covalent bond. In some embodiments, the covalent bond may be formed using alkylation or by forming an amide or amine bond between the porous size-exclusion support and the cationic moiety. In exemplary embodiments, immobilization according to the present disclosure is achieved by first oxidizing the hydroxyl groups of HEC to aldehyde groups using any suitable oxidizing agent, and in some instances, periodate (IO). 4- or IO6 5- The aldehyde group generated by oxidation can react with the terminal amine on the cationic moiety to covalently attach the cationic moiety to the HEC support, and the resulting intermediate can be reduced to form an amine by using a reducing agent (such as, but not limited to, sodium cyanoborohydride or picoline borane).

[0114] In some embodiments, the epoxyamine can be immobilized on a size-exclusion support. The epoxyamine can be associated with and / or immobilized on a size-exclusion support comprising HEC by reacting with the hydroxyl group provided by HEC. In some aspects of the present disclosure, the epoxyamine compound is a protected amine compound, and the protected amine compound is deprotected after being associated with or immobilized on the size-exclusion support.

[0115] In some embodiments, the cationic moiety is associated with one or more small molecules, such as, but not limited to, negatively charged small molecules. For example, the negatively charged small molecule can be associated with the cationic moiety through interactions and / or bonds, such as, but not limited to, ionic interactions, hydrophilic interactions, hydrophobic interactions, affinity interactions, hydrogen bonding, van der Waals forces, and / or covalent bonds.

[0116] In certain disclosed embodiments, functional groups on the cationic moieties can react with functional groups on the porous size-exclusion support and / or small molecules (e.g., but not limited to, negatively charged small molecules) to separate or extract from at least one large molecule. The functional groups can include, but are not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amide, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, and / or any other functional group for associating and / or interacting with the porous size-exclusion support and / or small molecule.

[0117] In another embodiment, the functional group is R, which represents a reactive functional moiety. x or a reactive functional moiety R attached to either the porous size exclusion support or moiety by a covalent bond L x Represents (-LR x ), or, The reactive group serves as a site of association, attachment and / or interaction with a moiety or small molecule, and the reactive group chemically reacts with a suitable reactive group or functional group on the porous size-exclusion support, moiety, or small molecule. In exemplary embodiments, the reactive group or functional group can 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 imidoester, 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 / or a photoactivatable group.

[0118] In another exemplary embodiment, the reactive group or functional group may comprise an electrophilic and / or nucleophilic species, which in some embodiments may form a covalent bond therebetween. Exemplary electrophilic and nucleophilic functional groups include aryloxy groups, or aryloxy groups substituted one or more times with electron-withdrawing substituents such as nitro, fluoro, chloro, cyano, trifluoromethyl, or combinations thereof, which are used to form activated aryl esters; or -OCOR groups, which are activated by carbodiimides to form anhydrides or mixed anhydrides. a or -OCNR a NHR b wherein R a and R b may be the same or different, and are C1-C6 alkyl, C r or cyclohexyl, 3-dimethylaminopropyl, acyl halide, acyl nitrile, aldehyde, alkyl halide, anhydride, aryl halide, aziridine, diazoalkane, haloacetamide, halotriazine, isocyanate, isothiocyanate, maleimide, phosphoramidite, sulfonyl halide, sulfide, disulfide, epoxide, and episulfide groups, thioester, activated alcohol, phosphate, phosphate ester, and photoactivatable group. Acyl azides can also rearrange to isocyanates.

[0119] In some embodiments, the reactive group further comprises a linker L in addition to the reactive functional group. The linker can be used to covalently bond the reactive functional group. When present, the linker is a single covalent bond or a series of stable bonds. The reactive functional group moiety can be directly attached to a solid support, moiety, or small molecule through a series of stable bonds (when the linker is a single bond). When the linker is a series of stable covalent bonds, the linker typically incorporates several non-hydrogen atoms selected from C, N, O, S, Si, B, and P. Additionally, the covalent bond can incorporate a platinum atom, as described in U.S. Pat. No. 5,714,327. When the linker is not a single covalent bond, it can optionally 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. In exemplary embodiments, the linker contains fewer than 15 non-hydrogen atoms and is composed of a combination of ether, thioether, thiourea, amine, ester, carboxamide, sulfonamide, hydrazide, aromatic, and / or heteroaromatic bonds. Typically, the linker is a single covalent bond or a single carbon-carbon bond combined with a carboxamide, sulfonamide, or thioether bond. In some embodiments, the following moieties may be found in the linker: ether, thioether, carboxamide, thiourea, sulfonamide, urea, urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl, and amine moieties. Examples of L include substituted or unsubstituted polymethylene, arylene, alkylarylene, aryl, or arylthio.

[0120] Any combination of linkers can be used to attach functional or reactive groups. When the reactive group is maleimide or haloacetamide, the resulting compound is particularly useful for conjugation to thiol-containing substances. When the reactive group is hydrazide, the resulting compound is particularly useful for conjugation to periodate-oxidized carbohydrates and glycoproteins.

[0121] In some embodiments, the cationic moiety can include open and / or unreacted amines. For example, a matrix including a size-exclusion carrier and a cationic moiety (e.g., but not limited to, PDA) can include open / unreacted amines. In certain disclosed embodiments, the open / unreacted amines on the PDA can be reacted with polysaccharide-derived molecules having a molecular weight range of 0 to 2,000,000. In one exemplary embodiment, the open and / or unreacted amines on the cationic moiety PDA are reacted with dextran having a molecular weight of 1,000,000.

[0122] In some embodiments, the concentration of the cationic moiety added to the matrix can range from 50 mg / mL to 175 mg / mL. In some embodiments, the concentration of the cationic moiety is 50 mg / mL to 100 mg / mL, 50 mg / mL to 75 mg / mL, 50 mg / mL to 150 mg / mL, 100 mg / mL to 155 mg / mL, or 130 mg / mL to 175 mg / mL. In one exemplary embodiment, the concentration of the cationic moiety can be 50 mg / mL. In another exemplary embodiment, the concentration of the cationic moiety can be 75 mg / mL. In another exemplary embodiment, the concentration of the cationic moiety can be 150 mg / mL.

[0123] 1 shows a non-limiting exemplary embodiment in which a porous size-exclusion support 10 is produced using 129 grams of HEC and 265 milliliters of Epi to create a 1.5 liter resin bed, where the HEC is modified with cationic PDA moieties 20 to form matrix 30. Matrix 30 has a MWCO of 50 kDa and an overall positive charge.

[0124] 2 shows another non-limiting exemplary embodiment of a porous size-exclusion support 10 produced using 129 grams of HEC and 265 milliliters of Epi to make a 1.5 liter resin bed, where the HEC is modified with branched PEI cationic moieties 40 to form matrix 50. Matrix 50 has a MWCO of 50 kDa and an overall positive charge.

[0125] 3 shows another non-limiting exemplary embodiment of a porous size-exclusion support 10 produced using 129 grams of HEC and 265 milliliters of Epi to make a 1.5 liter resin bed, where the HEC is modified with DEED cationic moieties 60 to form a matrix 70. The matrix 70 has a MWCO of 50 kDa and an overall positive charge.

[0126] C. Equilibration buffer In certain aspects of the present disclosure, a buffer can be provided to increase the binding capacity of the matrix for smaller sample components while also decreasing the binding capacity of the matrix for larger sample components. For example, without being bound by a single theory of operation, the buffer increases the binding capacity of cationic moieties for small molecules in a sample containing small molecules, such as, but not limited to, small negatively charged molecules, and decreases the binding capacity of cationic moieties for large molecules, such as, but not limited to, large positively charged molecules. In certain disclosed embodiments, an additional buffer can be used to improve recovery of larger sample components.

[0127] In some embodiments, the buffer is an equilibration buffer. In another embodiment of the present disclosure, the equilibration buffer is substantially free of or free of salts, such as, but not limited to, NaCl. An equilibration buffer containing substantially no salt generally refers to greater than 0 mM salt to 10 mM salt, preferably greater than 0 mM salt to 5 mM salt. In some aspects, the equilibration buffer can have a pH of 4 to 9. In another embodiment, the equilibration buffer can have a positive charge. In certain disclosed embodiments, the equilibration buffer can contain a neutral charge.

[0128] In some embodiments, the positively charged equilibration buffer may include, but is not limited to, carbonate buffer, bicarbonate buffer, phosphate buffer, citric acid / citrate buffer, and combinations thereof. For example, the buffer may include Tris buffer, which is a buffer containing 2-amino-2-(hydroxymethyl)propane-1,3-diol, also known as tris(hydroxymethyl)aminomethane, and triethylammonium bicarbonate.

[0129] In some aspects of the present disclosure, the equilibration buffer can have a pH of 4 to 9, e.g., a pH of 5 to 8, e.g., a pH of 5 to 7. In certain aspects of the present disclosure, the equilibration buffer has a pH of 5, a pH of 7, and / or a pH of 8.5. In one exemplary embodiment, the equilibration buffer is sodium acetate having a pH of 5. In another exemplary embodiment, the equilibration buffer is sodium acetate having a pH of 7. In another exemplary embodiment, the equilibration buffer is HEPES having a pH of 5. In another exemplary embodiment, the equilibration buffer is HEPES having a pH of 7. In another exemplary aspect, the equilibration buffer is borate having a pH of 5. In yet another exemplary embodiment, the equilibration buffer is borate having a pH of 8.5.

[0130] In some embodiments, the equilibration buffer is a salt-free (eg, sodium chloride-free) charged buffer.

[0131] In some embodiments, the equilibration buffer is used at a concentration of 1 mM to 100 mM, e.g., 10 mM to 100 mM, 20 mM to 100 mM, 40 mM to 100 mM, or 50 mM to 100 mM. In an exemplary embodiment, the equilibration buffer concentration is 50 mM.

[0132] 4 shows a process for separating small molecules 90 from a sample containing large molecules 100 using a matrix 80 having bound cationic moieties 82. Small molecules 90 associate with cationic moieties 82 to form composition 110. A positively charged buffer can be used to facilitate separation of small molecules (e.g., negatively charged molecules such as BSA) from large molecules (e.g., positively charged molecules such as IgG).

[0133] IV. System The present disclosure also relates to embodiments of systems comprising one or more of the disclosed matrices and further comprising a container. Such systems offer one or more advantages, including, but not limited to, economic feasibility; simplicity and ease of use; faster results compared to previous products in the art; suitability as a single-use disposable unit; suitability for high-throughput sample preparation in multi-well container formats; and suitability for automated and robotic sample preparation systems. Reducing the concentration of small molecules in a sample using the systems provided herein provides rapid recovery of biomolecules and excellent purity of the biomolecules and their derivatives for downstream applications.

[0134] In some embodiments, the present disclosure provides a system for removing one or more small molecules from a sample using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point (pI), and / or any combination of these properties. The system can include (i) a container having at least one size-exclusion carrier and at least one cationic moiety capable of associating with one or more small molecules, and (ii) a receptacle configured to receive flow-through from the container. Furthermore, molecules can be separated from one another based on one or more separation matrix properties, such as the charge and size-exclusion properties of the size-exclusion carrier associated with the at least one cationic moiety. In some disclosed system embodiments, the receptacle is attached to a column. In some embodiments, the receptacle is removable from the column. The contents of the receptacle can be used or removed by a user as desired. In some embodiments, the receptacle collects a sample containing substantially reduced small molecules. In some embodiments, the receptacle collects a sample free of small molecules.

[0135] The system may be operatively configured to operate by gravity flow. Alternatively, an externally actively applied pressure or force, such as centrifugal force, positive pressure, negative pressure, vacuum, and combinations thereof, may be applied to facilitate flow. Structures that enable the application of the above pressures or forces include, but are not limited to, syringes that can be pulled to create positive pressure, vacuum frits to generate negative pressure, and / or tubes or containers that are compatible with commercially available centrifuges or rotating devices. In some embodiments, the system may be configured for use with or within a centrifuge tube or any other equivalent rotating device.

[0136] In some embodiments of the disclosed system, the vessel is a columnar vessel, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate. Exemplary vessels include, but are not limited to, a test tube, a spin column, a multi-well plate, a multi-well filter plate, a microwell plate, or a microwell filter plate.

[0137] 5 shows an exemplary separation system 200 including a container 240 and a sample flowing through the container. The sample includes small molecules 210 and large molecules 230. The container 240 contains a matrix 220 configured to separate the small molecules 210 from the large molecules 230 using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, isoelectric point (pI), and / or a combination of these properties. The system 200 also includes a receptacle 250 for collecting the large molecules 230.

[0138] 6 illustrates an exemplary separation system 300 according to one embodiment of the present disclosure. System 300 includes a vessel 310 (such as a cylindrical tube, test tube, or spin column), a matrix 320 contained in vessel 310, and a receptacle 330 positioned below vessel 310 and adapted or configured to receive fluid flowing from vessel 310 through its bottom end 340. In some embodiments, vessel 310 may also include one or more frits (not shown). In some embodiments, system 300 may include an optional lid 350 that can be used to secure vessel 310 at its top end 360. In some embodiments, receptacle 330 can be detachable from vessel 310, allowing a user to collect the flow-through. In some embodiments, receptacle 330 has a twist-off tab configuration for removal. In other embodiments, receptacle 330 can be threadedly coupled to vessel 310 or attached using a complementary fitting that can be pulled apart, or the like.

[0139] FIG. 7 is a perspective view of an exemplary system 400 according to one embodiment of the present disclosure, including a multiwell container 410. The multiwell container 410 includes walls defining a plurality of wells 412. The walls may include edges 420, sides 430, and a top 440 that define the plurality of wells. The multiwell container 410 contains a matrix including at least one size-exclusion carrier and at least one cationic moiety according to the present disclosure that can associate with one or more small molecules using properties such as, but not limited to, size, charge, isoelectric point (pI), and / or any combination of these properties. As shown in FIG. 7, the multiwell container can include an optional container lid 450. The lid 450 can be any suitable removable / detachable structure, such as foil, clear wrap, or a peel-off seal. The multiwell container 410 can be a multiwell plate, a multiwell plate filter, a microplate, or a microtiter plate, and includes a flat plate with multiple wells 412, each of which serves as a small test tube or vessel. Multiwell plates come in a variety of formats for high throughput applications and can contain 6, 12, 24, 48, 96, 384, 1536, 3456, 9600 or more wells arranged in a rectangular matrix or array.

[0140] 8 shows an exemplary system 500 according to one embodiment, comprising a multi-well vessel 510. The system 500 further comprises a receptacle 550 positioned below the vessel 510 and adapted or configured to receive fluid flowing from the vessel. The vessel 510 comprises an end 520, a top 540, and a side 530. In some embodiments, the receptacle 550 may comprise a multi-well tray for collecting flow-through. The receptacle 550 may be removable and can be collected by a user. In some embodiments, the receptacle 550 is a wash plate or a collection plate.

[0141] Systems according to the present disclosure may be fully automated or may be manually operated systems, and in some embodiments, the system may be partially manually and partially automated.

[0142] The system may also include a computer system comprising a CPU, hardware elements, and / or software elements. A suitable computer system may be operable to control various components of the system, such as a control robot for collecting and analyzing the flow-through. In some embodiments, the computer system and / or its components may be physically present within system 300, 400, or 500 or may be external. As used herein, a computer system 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 preloaded software and / or application specific integrated circuits (ASICS) for controlling the disclosed systems, such as systems 300, 400, 500, and / or other components of the system, including sample processing and analysis, display, and / or export of results.

[0143] Embodiments of the disclosed systems may also optionally comprise one or more devices or components operable to further process the flow-through, in some embodiments, the flow-through may be eluted molecules, such as, but not limited to, eluted larger sample components, e.g., one or more large molecules.

[0144] The system may also include additional devices or components such as a power source, a display unit such as a monitor operable to display sample processing and / or monitor extraction of biomolecules from the sample, a spectrophotometer, a device for measuring nucleic acid extraction, a device for further processing the extracted biomolecules for further analysis, a printer, etc. The system of the present disclosure may be configured to fit on a laboratory benchtop.

[0145] V. Manufacturing method Embodiments of the present disclosure also relate to methods for producing matrices or systems according to the present disclosure. In some embodiments, the method of production comprises immobilizing cationic moieties on a porous size-exclusion support.

[0146] Porous size-exclusion supports can include spherical beads made of gel or gel-like materials with pores. Some exemplary size-exclusion supports include agarose, polyacrylamide, cellulosic materials (e.g., hydroxyethyl cellulose), and / or derivatives thereof.

[0147] In some embodiments, the porous size-exclusion carrier can be crosslinked with at least one crosslinker. The pore size range of the porous size-exclusion carrier determines the size of molecules that can be included in or excluded from the porous size-exclusion carrier. Without being bound by this theory, it is currently believed that when a sample solution passes through a porous size-exclusion carrier, molecules with a molecular weight less than or substantially equal to the MWCO are forced to follow a circuitous path before exiting the porous size-exclusion carrier. Meanwhile, large molecules take a relatively direct path through the porous size-exclusion carrier. Therefore, the difference in flow rate between small and large molecules allows faster-flowing large molecules to be separated from slower-flowing small molecules as the sample moves through the size-exclusion carrier.

[0148] Disclosed embodiments of the porous size-exclusion support may include a product formed by reacting 50 grams (g) to 250 grams (g) of HEC. In some embodiments, the disclosed size-exclusion support may include a range of 80 grams to 130 grams of HEC per 1.5 L of resin bed. In one exemplary embodiment, 80 g of HEC was used to prepare the porous size-exclusion support. In another exemplary embodiment, 90 g of HEC was used to prepare the porous size-exclusion support. In another exemplary embodiment, 108 g of HEC was used to prepare the porous size-exclusion support. In another exemplary embodiment, 129 g of HEC was used to prepare the porous size-exclusion support. In another exemplary embodiment, 147 g of HEC was used to prepare the porous size-exclusion support. In another exemplary embodiment, 216 g of HEC was used to prepare the porous size-exclusion support.

[0149] In some embodiments, the porous size-exclusion support is produced by crosslinking HEC with a crosslinking agent. For example, HEC can be crosslinked with an epoxide, such as, but not limited to, epichlorohydrin (Epi). In some embodiments, the porous size-exclusion support can be crosslinked with 250 mL to 450 mL of Epi. The support can be produced using 250 mL to 450 mL of epichlorohydrin for a 1.5 L resin bed. In one exemplary embodiment, 265 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 303 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 375 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 397 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 410 mL of Epi was used to crosslink the porous size-exclusion support. In another exemplary embodiment, 441 mL of Epi was used to crosslink the porous size-exclusion support.

[0150] In some embodiments, the cationic moiety is immobilized to the porous exclusion support by the formation of a covalent bond. In some embodiments, the covalent bond can be formed by a reaction such as, but not limited to, amidation, alkylation, amination, or other covalent bond-forming reactions. In some such embodiments, functional groups (e.g., hydroxyl groups) on the size-exclusion support are oxidized, for example, by using periodate, to generate aldehydes. These generated aldehyde groups can react with terminal amines on the cationic moiety to form imine intermediates, which can then be chemically reduced to form amines.

[0151] In some embodiments, the size-exclusion carrier is oxidized by adding an oxidizing agent such as 15-35 mg / mL periodate. In some embodiments, the size-exclusion carrier is oxidized by adding 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, or 35 mg / mL periodate. In some embodiments, the size-exclusion carrier is oxidized using sodium periodate.

[0152] In some embodiments, 5-15 mg / mL of reducing agent is added. In some embodiments, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, or 15 mg / mL of reducing agent is added. In an exemplary embodiment, the reducing agent is sodium cyanoborohydride. In another exemplary embodiment, the reducing agent is picoline borane.

[0153] In some embodiments, the cationic moiety associated with the size carrier may comprise an amine group. In some embodiments, the cationic moiety comprises an amine-containing polymer, a pentylamine group, a diamine, or an imine group. In an exemplary embodiment, the amine-containing polymer is branched PEI. In another exemplary embodiment, the diamine is PDA. In another embodiment, the diamine is DEED.

[0154] In some embodiments, the concentration of the cationic moiety used to modify the support material can range from 50 mg / mL to 175 mg / mL. In some embodiments, the concentration of the cationic moiety can include 50 mg / mL to 170 mg / mL, 60 mg / mL to 165 mg / mL, 65 mg / mL to 160 mg / mL, 70 mg / mL to 155 mg / mL, 75 mg / mL to 150 mg / mL, 80 mg / mL to 145 mg / mL, 85 mg / mL to 140 mg / mL, 90 mg / mL to 135 mg / mL, or 95 mg / mL to 130 mg / mL. In one exemplary embodiment, the concentration of the cationic moiety is 50 mg / mL. In another exemplary embodiment, the concentration of the cationic moiety is 75 mg / mL. In yet another exemplary embodiment, the concentration of the cationic moiety can be 150 mg / mL.

[0155] In an exemplary embodiment, sodium metaperiodate is dissolved in water and mixed with a porous size-exclusion support matrix bed containing HEC to oxidize vicinal diols present in the HEC to aldehyde groups. The mixture is allowed to react for a suitable time, such as 2 to 4 hours, at a temperature ranging from 15°C to 30°C. In an exemplary embodiment, the mixture is allowed to react for at least 4 hours at room temperature with constant overhead stirring. In some embodiments, during the reductive amination step (i.e., the step in which the amine groups on the cationic moieties react with the aldehyde groups on the size-exclusion support matrix bed and reduce the intermediate imine to an amine), the cationic moieties are prepared at a pH of 8.0 to 8.5 and added to the slurry. A suitable reducing agent, such as sodium cyanoborohydride, is added to the mixture, and the reaction is allowed to proceed for a suitable time, such as 8 to 12 hours, with stirring at 20°C to 30°C, followed by washing with water and NaCl.

[0156] VI.How to use Certain disclosed embodiments relate to a method for separating at least one large molecule from at least one small molecule using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point (pI), and / or any combination of these properties. The method may include applying a sample to a porous size-exclusion support containing at least one cationic moiety capable of binding or associating with a small molecule, and subjecting the container to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof. Large molecules in the sample can be excluded by the porous size-exclusion support and collected as flow-through. A small molecule can interact with the cationic moiety, thereby being separated from the large molecule in the sample using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point (pI), and / or any combination of these properties.

[0157] In certain aspects of the present disclosure, the equilibration buffers disclosed herein can also be provided to increase the binding capacity of the matrix for smaller sample components while decreasing the binding capacity of the matrix for larger sample components.

[0158] Currently known methods for removing small molecules, such as, but not limited to, BSA, from large molecules, such as, but not limited to, antibodies, in a sample require an initial step, such as a pre-salting step, to remove salt from the antibody solution. Without being bound by theory of operation, salt generates counterions that reduce the rate of BSA removal in subsequent steps. The present disclosure enables the separation of small molecules, such as, but not limited to, BSA, from large molecules, such as, but not limited to, antibodies, in a single step. Thus, embodiments of the present disclosure do not require a pre-desalting step to achieve the desired BSA removal and antibody recovery, such as, but not limited to, IgG. Thus, in some embodiments, separation of small molecules from the remainder of the sample is performed in a single step.

[0159] In some embodiments, the flow-through is collected in a receptacle located below the vessel. In some embodiments, small molecules may constitute sample impurities or contaminants.

[0160] Samples that can be processed by the methods of the present disclosure can be any type of biological or clinical sample containing biomolecules or their derivatives from which small molecules must be separated or removed. Some exemplary, non-limiting samples include samples with small molecules such as BSA and large molecules such as IgG.

[0161] The methods of the present disclosure advantageously reduce the time required to process a sample and / or increase the amount of small molecules removed from the sample.

[0162] VII. Kit The present disclosure also describes kits for carrying out the methods discussed herein and / or kits including the matrices and / or kits including the systems discussed herein.

[0163] In some embodiments, the present disclosure describes a kit for separating large molecules from small molecules using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point, and / or any combination of these properties. The kit can include a system including: (i) a container that holds a size-exclusion carrier comprising a cationic moiety associated with the carrier, where the cationic moiety can also associate with small molecules; and (ii) a receptacle located below the container, where the device is configured to operate for gravity flow or can be operated by applying centrifugal force, positive pressure, negative pressure, vacuum, and combinations thereof.

[0164] In some embodiments of the kits of the present disclosure, the device is a spin column, a multi-well filter plate, or a multi-well plate. The kit may further include one or more equilibration buffers packaged in one or more separate containers or contained in the first container.

[0165] In some embodiments, the equilibration buffer does not contain salt (e.g., an ionic salt such as NaCl). In other aspects disclosed herein, the equilibration buffer may contain a small amount of salt. In some aspects, the equilibration buffer may have a pH of 4 to 9. In other embodiments, the equilibration buffer may have a positive charge. In certain disclosed embodiments, the equilibration buffer may contain a neutral charge.

[0166] In some embodiments, the equilibration buffer may include, but is not limited to, a carbonate buffer, a bicarbonate buffer, a phosphate buffer, or a citric acid / citrate buffer. In an exemplary embodiment, the buffer comprises a Tris buffer. In another exemplary embodiment, the buffer comprises a TEAB buffer.

[0167] In some aspects of the present disclosure, the equilibration buffer can have a pH of 4 to 9, e.g., a pH of 5 to 8, e.g., a pH of 5 to 7. In certain aspects, the equilibration buffer has a pH of 5, a pH of 7, and / or a pH of 8.5. In one exemplary embodiment, the equilibration buffer is sodium acetate having a pH of 5. In another exemplary embodiment, the equilibration buffer is sodium acetate having a pH of 7. In another exemplary embodiment, the equilibration buffer is HEPES having a pH of 5. In another exemplary embodiment, the equilibration buffer is HEPES having a pH of 7. In another exemplary aspect, the equilibration buffer is borate having a pH of 5. In yet another exemplary embodiment, the equilibration buffer is borate having a pH of 8.5.

[0168] In some embodiments, the positively charged equilibration buffer is present at a concentration of 1 mM to 100 mM, 2 mM to 75 mM, 5 mM to 50 mM, 10 mM to 25 mM, or 5 to 25 mM. In some embodiments, the positively charged equilibration buffer is present at a concentration of 1 mM to 100 mM, 2 mM to 75 mM, 5 mM to 50 mM, 10 mM to 25 mM, or 5 to 25 mM Tris buffer. In an exemplary embodiment, the equilibration buffer concentration can be 50 mM.

[0169] Kits of the present disclosure may also include one or more reagents such as one or more wash buffers, elution buffers, filter membranes and / or additional spin columns or multi-well plates.

[0170] The reagents and components of the kit may be contained in one or more suitable containers. Containers may generally include at least one vial, test tube, flask, bottle, syringe, or other container into which the components are placed, preferably suitably aliquoted. When multiple components are present in the kit, they may be packaged together if suitable, or the kit will typically include a second, third, or other additional container into which additional components may be individually placed. However, in some embodiments, a particular combination of components may be packaged together in a single container means. The kit may also include components for housing any reagent containers in close confinement for commercial sale. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are retained.

[0171] In some embodiments, the components of the disclosed kits may be pre-filled with one or more reagents for processing samples and suitably aliquoted into appropriate chambers. The kit or its container may have a seal to keep the internal compartment and any contents therein sterile and hermetic.

[0172] Some components of the kit may be provided in one or more liquid solutions. The liquid solutions may be non-aqueous, aqueous, or sterile. Kit components may also be provided as dry powders. When reagents and / or components are provided as dry powders, the powder may be reconstituted by the addition of a suitable solvent. It is envisioned that a suitable solvent may also be provided in another container means. The kit may also include container means for containing a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0173] Kits of the present disclosure may also include instructions for using the kit components, and may also include instructions for the use of other reagents not included in the kit. The instructions may include variations that may be performed.

[0174] VIII. Overview of Some Embodiments Disclosed herein are embodiments of a matrix comprising a porous size-exclusion carrier and at least one cationic moiety associated with the porous size-exclusion carrier, wherein the matrix is capable of separating one or more molecules in a sample by using the molecular weight of the one or more molecules, the charge of the one or more molecules, the isoelectric point (pI) of the one or more molecules, or a combination thereof.

[0175] In any or all embodiments, the porous size-exclusion support has a molecular weight cutoff of 40 kDa or greater.

[0176] In any or all embodiments, at least one cationic moiety is covalently attached to the porous size-exclusion support.

[0177] In any or all embodiments, at least one cationic moiety comprises an amine, a diamine, a polyamine, or an amine-containing polymer.

[0178] In any or all embodiments, the amine-containing polymer is polyethyleneimine.

[0179] In any or all embodiments, the diamine is diaminopentane, N,N-diethylethylenediamine, or a combination thereof.

[0180] In any or all embodiments, the porous size-exclusion support comprises hydroxyethyl cellulose.

[0181] Any or all of the embodiments further include epichlorohydrin.

[0182] Also disclosed herein are embodiments of a system for separating one or more molecules in a sample using the molecular weight of the one or more molecules, the charge of the one or more molecules, the isoelectric point (pI) of the one or more molecules, or a combination thereof, the system including a container containing a matrix of the above embodiments and a receptacle positioned to receive a flow from the container.

[0183] In any or all embodiments, the system may be configured for gravity flow operation, centrifugal force operation, positive pressure operation, negative pressure operation, vacuum operation, or a combination thereof.

[0184] In any or all embodiments, the vessel is a column, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate.

[0185] Also disclosed herein is a method for making a multimodal resin, the method comprising: providing a porous size-exclusion support comprising hydroxyethyl cellulose having a MWCO of 40 kDa or greater, the hydroxyethyl cellulose having at least one vicinal diol; oxidizing the at least one vicinal diol to an aldehyde; and reacting the aldehyde with an amine group of a cationic moiety via reductive amination.

[0186] In any or all embodiments, the porous size-exclusion carrier is manufactured using an amount of hydroxyethyl cellulose ranging from 50 g to 250 g.

[0187] In any or all embodiments, the method further comprises providing a cross-linking agent to cross-link the porous size-exclusion support.

[0188] In any or all embodiments, the crosslinking agent is epichlorohydrin.

[0189] In any or all embodiments, epichlorohydrin is used in an amount ranging from 250 mL to 450 mL.

[0190] In any or all embodiments, the cationic moiety is diaminopentane, polyethyleneimine, or N,N-diethylethylenediamine.

[0191] In any or all embodiments, the cationic moiety has a concentration range of 50 mg / mL to 175 mg / mL.

[0192] Also disclosed herein is a method for separating one or more molecules in a sample, the method comprising: providing a matrix comprising a porous size-exclusion support having at least one cationic moiety associated therewith, wherein the matrix is capable of separating one or more molecules using the molecular weight of the one or more molecules, the charge of the one or more molecules, the isoelectric point (pI) of the one or more molecules, or a combination thereof; equilibrating the matrix with an equilibration buffer; and separating the one or more molecules by applying a sample to the matrix and subjecting the matrix to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof, wherein the one or more molecules in the sample are excluded by the matrix and collected as flow-through, and the one or more molecules associate with the at least one cationic moiety and are thereby separated from the sample in a single step.

[0193] In any or all embodiments, the flow-through is collected in a receptacle located below the vessel.

[0194] In any or all embodiments, the equilibration buffer is a positively charged buffer, a neutrally charged buffer, a low salt buffer, or a salt-free buffer.

[0195] In any or all embodiments, the equilibration buffer is sodium acetate having a pH of 5 or 7, HEPES having a pH of 5 or 7, or borate having a pH of 5 or 8.5.

[0196] In any or all embodiments, the positively charged buffer is Tris buffer or triethylammonium bicarbonate.

[0197] In any or all embodiments, a first molecule of the two or more molecules comprises at least one small molecule having a molecular weight range of less than 100 kDa, and a second molecule of the one or more molecules comprises at least one large molecule having a molecular weight range of 100 kDa or greater.

[0198] In any or all embodiments, the one or more molecules is bovine serum albumin.

[0199] In any or all embodiments, the one or more molecules is an antibody.

[0200] In any or all embodiments, the antibody is an IgG.

[0201] In any or all embodiments, one or more molecules having an isoelectric point value in the range of 4.5 to 5.5 are separated from one or more molecules having an isoelectric point value of 8.0 to 11.5.

[0202] In any or all embodiments, one or more molecules are more negatively charged than the other molecules, which have positively charged molecules.

[0203] In any or all embodiments, the at least one cationic moiety associates with one or more molecules through ionic interactions, hydrophilic interactions, hydrophobic interactions, affinity interactions, hydrogen bonding, or van der Waals forces.

[0204] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged large molecule, the size exclusion support includes HEC and Epi, the cationic moiety is diaminopentane, and greater than 80% of the at least one negatively charged molecule is separated and greater than 80% of the positively charged large molecules are recovered as flow-through.

[0205] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged large molecule, the size exclusion support includes HEC and Epi, the cationic moiety is diaminopentane, the cationic moiety is modified with dextran, and more than 80% of the at least one negatively charged molecule is separated and more than 80% of the positively charged large molecules are recovered as flow-through.

[0206] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged large molecule, the size exclusion support includes HEC and Epi, and the cationic moiety is N,N-diethylethylenediamine, and greater than 50% of the at least one negatively charged molecule is separated and greater than 90% of the positively charged large molecules are recovered as flow-through.

[0207] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged large molecule, the size exclusion support includes HEC and Epi, the cationic moiety is diaminopentane, the equilibration buffer is a Tris buffer, and more than 75% of the at least one negatively charged molecule is separated and more than 80% of the positively charged large molecules are recovered as flow-through.

[0208] In any or all embodiments, when the negatively charged molecule is bovine serum albumin and the cationic moiety is diaminopentane, the cationic moiety has a binding capacity for bovine serum albumin of 1.6 mg / mL and the equilibration buffer is Tris buffer.

[0209] In any or all embodiments, when the negatively charged molecule is bovine serum albumin and the cationic moiety is polyethyleneimine, the cationic moiety has a binding capacity for bovine serum albumin of 3.11 mg / mL, and the equilibration buffer is Tris buffer.

[0210] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged large molecule, the size exclusion carrier includes HEC, the cationic moiety is polyethyleneimine, the equilibration buffer is a Tris buffer, and more than 70% of the positively charged large molecules are recovered as flow-through.

[0211] Also disclosed herein are kit embodiments for separating positively charged large molecules from one or more negatively charged small molecules in a sample, the kit including a porous size-exclusion support having associated therewith at least one cationic moiety capable of associating with and capturing at least one negatively charged small molecule, and instructions for using the porous size-exclusion support.

[0212] In any or all embodiments, the kit further comprises an equilibration buffer.

[0213] In any or all embodiments, the equilibration buffer is Tris buffer or triethylammonium bicarbonate.

[0214] In any or all embodiments, the kit further includes a system including a container containing the porous size-exclusion carrier and a receptacle positioned to receive the flow-through of the carrier.

[0215] In any or all embodiments, the system of the kit is configured to operate by gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, and combinations thereof.

[0216] In any or all embodiments, the container of the kit is a spin column, a multi-well filter plate, or a multi-well plate.

[0217] IX. Working Example Aspects of the present teachings can be further understood in light of the following examples. Matrices comprising at least one size-exclusion carrier and at least one cationic moiety were prepared and tested for separating and / or extracting one or more small molecules from a sample that can associate with the small molecules using differences in one or more properties, such as, but not limited to, molecular size, molecular charge, molecular isoelectric point, and / or any combination of these properties. In some embodiments, exemplary size-exclusion carriers were modified with cationic moieties that include functional groups that can associate with negatively charged small molecules through ionic, hydrophobic, or any other interaction, thereby removing the negatively charged small molecules from the sample, while allowing at least one positively charged large molecule in the sample to be excluded and recovered.

[0218] In the following example, a high concentration of BSA (67,000 Da) was separated from IgG (150,000 Da) in a sample containing a BSA-IgG mixture. The Pierce™ Rapid Gold BCA Protein Kit was used to measure the respective concentrations of BSA and IgG as the proteins were spun through the resin. Quantitative iBright analysis of the SDS-Page results provided accurate estimates of BSA and IgG recovery when the IgG-BSA mixture was spun through the resin. Furthermore, the following example addresses the issue of BSA directly competing with IgG during antibody labeling without adversely affecting the original amount of IgG, demonstrating superior performance over currently available resins in separating BSA from IgG.

[0219] Additionally, in some of the following examples, BSA removal and IgG purification kits were used. For example, kits containing Melon Gel resin, which is used for binding and removing serum proteins, such as the Abcam BSA Removal Kit (ab173231) commercially available from Abcam and the Melon™ Gel IgG Purification Kit commercially available from Thermo Scientific™, were used in the following examples. Additionally, cross-linked agarose beads coupled to Cibacron Blue F3GA dye, such as Affi-Gel® Blue Gel commercially available from Bio-Rad, were used in the following examples.

[0220] Although these exemplary small molecules and listed molecular weight ranges were used in the experimental demonstrations, one skilled in the art will understand that the present embodiments are not limited to any of these small molecules or molecular weight ranges, and that the teachings herein will enable one skilled in the art to prepare and use matrices and systems for removing a variety of small molecules and molecular weight ranges.

[0221] Example 1 Preparative Chemistry: A porous size-exclusion support comprising HEC cross-linked with Epi in the presence of a non-polar phase-containing solvent and surfactant (i.e., non-ionic detergent) was modified according to Scheme 1 to produce the embodiments in Table 1.

[0222] [ka]

[0223] [Table 1]

[0224] Next, the vicinal diols located on these size-exclusion support columns were oxidized using periodate to generate aldehyde groups. PDA, branched PEI, or DEED was prepared in PBS, the pH was adjusted to 8.0–8.5, and reacted with the oxidation column according to the following procedure. According to Scheme 2, different size-exclusion supports (see Table 1) with different MWCOs were reacted with the cationic reagents PDA and DEED to generate the multimodal resins shown in Table 2.

[0225] [Table 2]

[0226] [ka]

[0227] Scheme 2 includes the following steps: (1) 100 mL of size-exclusion base matrix resin was prepared; (2) 2.3 grams of sodium metaperiodate was dissolved in water to prepare 100 mL of 23 mg sodium metaperiodate / mL resin; (3) 100 mL of 23 mg sodium metaperiodate per mL of resin was added to a 100 mL bed of size-exclusion matrix resin; (4) The reaction was allowed to proceed at room temperature with constant overhead stirring for 4 hours, thereby oxidizing the vicinal diols present in HEC to aldehyde groups; (5) A 100 mL volume of 50–150 mg / mL HEC was prepared. (6) adding the prepared reagent to the resin slurry, whereby the amine provided by the reagent reacts with the aldehyde group formed in the periodate oxidation (step 4), followed by reductive amination by adding 10 mg sodium cyanoborohydride / mL resin; (7) allowing the reaction to proceed for 8-12 hours at room temperature with constant overhead stirring; and (8) washing the resin with 2x bed volume water, 2x bed volume NaCl, and 2x bed volume water.

[0228] Method for evaluating the resin on a BSA-IgG mixture: (1) spin a 562 μL resin bed at 3,000 × G for 1 minute and remove the storage solution; (2) equilibrate the resin with buffer (3 × 300 μL); (3) spin at 3,000 × G for 1 minute and remove the buffer; (4) add 100 μL of a BSA-rabbit IgG mixture (BSA 10 mg / mL and IgG 1 mg / mL) to the resin; (5) spin at 3,000 × G for 2 minutes and collect the flow-through containing unbound IgG and unbound BSA; (6) prepare a 1:10 dilution of the flow-through and start by adding 10 μL / well to a 4-20% Tris-glycine gel; (7) run the gel at 200 V for 45-50 minutes; (8) stain / destain the gel using a Pierce Power stainer.

[0229] Example 2 Resin MWCO Effect on BSA Removal and Antibody Recovery: In this example, the MWCO effects of Resin A Embodiment, Resin B Embodiment, Resin D Embodiment, Resin E Embodiment, and Resin F Embodiment from Table 2 were compared to determine the effect of increasing the MWCO on the removal of BSA from a BSA (2 mg / mL) and IgG (2 mg / mL) mixture.

[0230] Figure 9 is an image of a gel showing the removal of BSA from the BSA / IgG described above and in Table 2. As shown by Figure 9, the BSA removal properties, as indicated by the disappearance of the lower bands in the gel, increased in the order of Resin F embodiment (45 kDa MWCO) > Resin D embodiment (40 kDa MWCO) > Resin E embodiment (30 kDa MWCO) > Resin B embodiment (7 kDa MWCO) > Resin A embodiment (2 kDa MWCO), respectively. Thus, the MWCO had a significant effect on the removal of BSA from the samples, in addition to the PDA-modified embodiment.

[0231] Example 3 Effect of PDA modification and equilibration buffer: In this example, an embodiment of Resin 5 from Table 1, an embodiment of Resin L from Table 2, an embodiment of Resin F from Table 2, and an embodiment of Resin D from Table 2 were evaluated for their ability to remove BSA and recover IgG from a BSA-IgG mixture using different buffers in the equilibration as shown in Table 3.

[0232] Melon Gel Purification Buffer (MGPB): A low phosphate buffer containing no sodium chloride. Without being bound by theory, the low negative charge of this buffer was chosen to increase the BSA binding capacity for positively charged embodiments of the resin.

[0233] Tris buffer: a positively charged buffer without a negative charge. While not wishing to be bound by this theory, the absence of a negative charge may increase the BSA binding capacity, similar to that of MGPB. However, it also repels the positively charged amine groups on IgG, thereby increasing IgG recovery.

[0234] FIG. 10 is an image of a gel showing BSA removal and IgG recovery according to Table 3.

[0235] [Table 3]

[0236] As shown in Figure 10 and Table 3, lanes 1A and 1B show no BSA removal, while the PDA-modified resin embodiment removed more BSA from the BSA-IgG mixture. Furthermore, the PDA concentration (50 mg / mL vs. 150 mg / mL) had little effect on BSA removal, and the 40 kDa MWCO resin embodiment in lanes 4A and 4B showed lower BSA removal than the 45 kDa MWCO resin embodiment in lanes 3A and 3B. Furthermore, the buffer used for equilibration demonstrated that the resin embodiment equilibrated with MGPB showed lower IgG recovery than the resin embodiment equilibrated with 50 mM Tris.

[0237] Example 4 In this example, the ability of an embodiment of Resin G according to the table using different equilibration buffers to remove BSA and recover IgG from a sample containing a mixture of BSA (10 mg / mL) and IgG (1 mg / mL) was compared.

[0238] FIG. 11 is an image of a gel showing the removal of BSA and recovery of IgG in a sample containing a mixture of BSA (10 mg / mL) and IgG (1 mg / mL) by an embodiment of Resin G (described in Table 1 provided herein) equilibrated with 50 mM Tris pH 7 (lane 1), 50 mM TEAB pH 5 (lane 2), 50 mM TEAB pH 7 (lane 3), 50 mM sodium acetate pH 5 (lane 4), 50 mM sodium acetate pH 7 (lane 5), 50 mM HEPES pH 5 (lane 6), 50 mM HEPES pH 7 (lane 7), 1 mg / mL rabbit IgG / 10 mg / mL BSA (lane 8), 1 mg / mL rabbit IgG (lane 9), 10 mg / mL BSA.

[0239] This example demonstrates desirable BSA removal and IgG recovery with different equilibration buffers having a pH between 5 and 7. Furthermore, equilibration buffers with a neutral charge, such as sodium acetate, showed desirable BSA removal and IgG recovery.

[0240] Example 5 In this example, the BSA binding capacity (300 μL of 10 mg / mL) and IgG recovery (2 mg / mL) of an embodiment of Resin 5 from Table 1 modified with 150 mg / mL PEI were compared to an embodiment of Resin 3 from Table 1 modified with 150 mg / mL PEI, Affi-Gel® Blue Gel (Bio-Rad), and Melon™ Gel IgG Purification Kit (Thermo Scientific™) in a sample containing 300 μL of 10 mg / mL.

[0241] Figure 12 is a bar graph showing mg BSA bound / mL resin. As shown in Figure 12, an embodiment of Resin 5 (described in Table 1 provided herein) had a binding capacity of 3.11 mg BSA bound / mL resin, an embodiment of Resin 3 (described in Table 1 provided herein) had a binding capacity of 2.45 mg BSA bound / mL resin, the Melon™ Gel IgG Purification Kit had a binding capacity of 1.26 mg BSA bound / mL resin, and the Affi-Gel® Blue Gel (Bio-Rad) had a binding capacity of 2.43 mg BSA bound / mL resin.

[0242] 13 is a bar graph showing IgG recovery (2 mg / mL) where an embodiment of Resin 5 (listed in Table 1 provided herein) showed 72% recovery by volume, an embodiment of Resin 3 (listed in Table 1 provided herein) showed 61% recovery by volume, the Melon™ Gel IgG Purification Kit showed 80% recovery by volume, and Affi-Gel® Blue Gel (Bio-Rad) showed 25% recovery by volume.

[0243] Thus, this example demonstrates that desirable BSA binding capacities were achieved when PEI was used as the modification reagent, with BSA binding capacities comparable to Affi-Gel® Blue Gel (Bio-Rad) and IgG recovery comparable to the Melon™ Gel IgG Purification Kit.

[0244] Example 6 In this example, an embodiment of Resin 5 from Table 1 was modified with different concentrations of PDA to produce an embodiment of Resin F and an embodiment of Resin K, as described in Table 2, which were compared to the commercially available Abcam BSA Removal Kit and Melon™ Gel IgG Purification Kit (Thermo Scientific™) for their ability to remove BSA and recover IgG from samples containing a BSA-IgG mixture. Table 4 presents the data from FIG. 14A, an image of a gel showing the BSA removal and IgG recovery capabilities of an embodiment of Resin 5 modified with different concentrations of PDA to produce an embodiment of Resin F and an embodiment of Resin K relative to the Abcam BSA Removal Kit and the Melon™ Gel IgG Purification Kit (Thermo Scientific™). As shown in FIG. 14A, lane 5 is a mixture of 10 mg / mL BSA and 1 mg / mL GAR. Lane 6 was 1 mg / mL GAR, lane 7 was 10 mg / mL BSA, all lanes were calibrated and samples were loaded onto the gel at 10 μL / well.

[0245] [Table 4]

[0246] FIG. 14A shows the superior BSA-removed IgG recovery characteristics in lanes 1 and 2 when compared to the commercially available Abcam BSA Removal Kit and Melon™ Gel IgG Purification Kit (Thermo Scientific™) in lanes 3 and 4, respectively.

[0247] Figure 14B is a bar graph obtained from quantitating the bands in Figure 14A using IBright image analysis software, showing that an embodiment of Resin F (an embodiment of Resin 5 in Table 1 modified with 150 mg / mL PDA) had 85.7% IgG recovery and 98.8% BSA removal, Resin K (an embodiment of Resin 5 in Table 1 modified with 75 mg / mL PDA) showed 86.7% IgG recovery and 82.6% BSA removal, the Abcam BSA Removal Kit had 53.3% IgG recovery and 65.78% BSA removal, and the Melon™ Gel IgG Purification Kit (Thermo Scientific™) had 78.7% IgG recovery and 8.3% BSA removal. Thus, an embodiment of Resin F and an embodiment of Resin K achieved higher IgG recovery and BSA removal than the Abcam BSA Removal Kit and the Melon™ Gel IgG Purification Kit (Thermo Scientific™).

[0248] Example 7 Increased MWCO: In this example, a higher MWCO resin with a MWCO of 45,000 Da was produced with PDA modification and measured for its ability to remove BSA and recover IgG from a sample containing BSA-IgG. The higher MWCO was achieved for Resin 5 embodiment, Resin 6 embodiment, Resin 7 embodiment, and Resin 8 embodiment, prepared according to Table 1.

[0249] 15 is a bar graph showing recoveries at 42,000 Da, 67,000 Da, 80,000 Da, and 150,000 Da for an embodiment of Resin 5, an embodiment of Resin 6, an embodiment of Resin 7, and an embodiment of Resin 8. The embodiment of Resin 5 exhibited 86% recovery at 42,000 Da, 94% recovery at 67,000 Da, 92% recovery at 80,000 Da, and 94% recovery at 150,000 Da. The embodiment of Resin 6 exhibited 82% recovery at 42,000 Da, 84% recovery at 67,000 Da, 90% recovery at 80,000 Da, and 92% recovery at 150,000 Da. An embodiment of Resin 7 showed 66% recovery at 42,000 Da, 76% recovery at 67,000 Da, 84% recovery at 80,000 Da, and 86% recovery at 150,000 Da. An embodiment of Resin 8 showed 58% recovery at 42,000 Da, 75% recovery at 67,000 Da, 75% recovery at 80,000 Da, and 83% recovery at 150,000 Da.

[0250] This example demonstrates that by decreasing the amount of HEC, resins of 50K MWCO, 80K MWCO, and 90K MWCO were produced (see Table 1).

[0251] Example 8 In this example, Resin F embodiment, Resin G embodiment, Resin H embodiment, Resin I embodiment, and Resin J were prepared according to Table 2 and tested for their ability to remove BSA and recover GAR from samples containing BSA-GAR mixtures and compared to commercially available Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit.

[0252] Figure 16A is an image of a gel showing BSA removal and antibody recovery, and Table 5 is the legend for Figure 16A.

[0253] [Table 5]

[0254] As shown in Figure 16A, Resin F embodiment (45K MWCO) and Resin G embodiment (50K MWCO) showed the greatest BSA removal and IgG recovery from a sample containing a mixture of BSA (10 mg / mL) and GAR IgG (1 mg / mL) when compared to the Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® (Bio-Rad), and Abcam BSA Removal Kit. Resin H embodiment (80K MWCO) and Resin I embodiment (90K MWCO), both modified with 75 mg / mL PDA, performed better at removing BSA and recovering IgG when compared to the Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit. However, they did not perform as well as Resin F embodiment and Resin G embodiment at recovering IgG. Resin J was produced by reacting an embodiment of Resin 6 in Table 1 with PDA, followed by further reaction of the open amine end of the PDA with dextran having a molecular weight of 1,000,000. However, this did not improve performance compared to Resins F and G.

[0255] FIG. 16B is a bar graph further showing GAR (1 mg / mL) recovery and BSA (10 mg / mL) removal. As shown in FIG. 16B, an embodiment of Resin F exhibited 83% GAR recovery and 99% BSA removal, an embodiment of Resin G exhibited 93% GAR recovery and 100% BSA removal, an embodiment of Resin H exhibited 63% GAR recovery and 99% BSA removal, an embodiment of Resin I exhibited 76% GAR recovery and 95% BSA removal, an embodiment of Resin J exhibited 82% GAR recovery and 100% BSA removal, the Melon™ Gel IgG Purification Kit (Thermo Scientific™) exhibited 106% GAR recovery and 82% BSA removal, Affi-Gel® Blue Gel (Bio-Rad) exhibited 73% GAR recovery and 65% BSA removal, and the Abcam BSA Removal Kit exhibited 125% GAR recovery and 85% BSA removal. Furthermore, the artificially high IgG recovery shown by the data from the Abcam BSA Removal Kit and Melon™ Gel IgG Purification Kit (Thermo Scientific™) can be explained by insufficient BSA removal resulting in smear in the gel, which leads to overlapping bands with IgG. Thus, this example demonstrated higher BSA removal and superior IgG recovery with Resin F embodiment, Resin G embodiment, and Resin J embodiment when compared to the commercially available resin and Resin H embodiment and Resin I embodiment.

[0256] Example 9 In this example, Resin F embodiment, Resin G embodiment, Resin H embodiment, Resin I embodiment, and Resin J embodiment were prepared according to Table 2 and compared to Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit for their ability to remove BSA and IgG from a sample containing a mixture of BSA (10 mg / mL) and GAR IgG (0.1 mg / mL).

[0257] FIG. 17 is an image of a gel showing BSA removal and IgG recovery, and Table 6 is the legend for FIG.

[0258] [Table 6]

[0259] As shown in FIG. 17 , Resin F and Resin G embodiments demonstrated the greatest BSA removal and IgG recovery from a mixture containing BSA (10 mg / mL) and GAR IgG (0.1 mg / mL) when compared with the Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit. Furthermore, Resin H performed better than the Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit. However, it did not perform as well as Resin F and Resin G embodiments in recovering IgG. Resin I embodiment demonstrated lower BSA removal capacity when the sample contained a 0.1 mg / mL GAR and 10 mg / mL BSA mixture. Resin J embodiment did not perform better than Resin F or Resin G. The Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit performed poorly in removing BSA and recovering GAR IgG in samples containing a low GAR-BSA mixture (0.1 mg / mL GAR and 10 mg / mL BSA). Lane 11 shows BSA (10 mg / mL) alone, which represents the smear obtained by running only BSA.

[0260] Example 10 In this example, a mixture of GAR (1 mg / mL) and BSA (10 mg / mL) was processed through an embodiment of Resin F from Table 2, Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit. The resulting flow-through was then labeled with NHS DyLight™ 488 (Thermo Scientific™), and free dye was purified using Pierce™ dye and biotin removal resin.

[0261] FIG. 18 shows the fluorescent dye labeling to BSA that was removed from the antibody prior to the labeling reaction, and Table 7 shows the legend corresponding to FIG.

[0262] [Table 7]

[0263] Figure 18 shows the efficiency of labeling to GAR when BSA is removed, as demonstrated by the resin F embodiment for the Melon™ Gel IgG Purification Kit (Thermo Scientific™) and Affi-Gel® Blue Gel (Bio-Rad), which contained label due to unremoved BSA. The Abcam BSA Removal Kit showed comparable results to the resin F embodiment.

[0264] Figure 19 is an image of fluorescent dye labeling for BSA removed from antibody prior to labeling reaction. A mixture of GAR (0.1 mg / mL) and BSA (10 mg / mL) was passed through an embodiment of Resin F from Table 2, an embodiment of Resin G from Table 2, a Melon™ Gel IgG Purification Kit (Thermo Scientific™), Affi-Gel® Blue Gel (Bio-Rad), and a BSA Removal Kit. The resulting flow-through was then labeled with NHS DyLight™ 650 (Thermo Scientific™), and the free dye was purified using Pierce™ dye and biotin removal resin. Table 8 is the legend for Figure 19.

[0265] [Table 8]

[0266] Figure 19 demonstrates the efficiency of labeling to GAR when BSA is removed, as demonstrated by embodiments of Resin F and Resin G, when compared with the Melon™ Gel IgG Purification Kit, Affi-Gel® Blue Gel (Bio-Rad), and Abcam BSA Removal Kit, and includes labeling efficiency due to unremoved BSA. Thus, Figure 19 shows that even at low antibody concentrations, such as 0.1 mg / mL, embodiments of Resin F and Resin G were able to recover the antibody after BSA was purified and successfully dye-label it.

[0267] Example 11 Removal of serum from serum samples: In this example, rabbit serum, mouse serum, human plasma, and human serum were purified using an embodiment of Resin F and an embodiment of Resin G from Table 2. Figure 20 is an image of a gel showing the results of an embodiment of Resin F and an embodiment of Resin G on a starting mixture, where lanes 1, 5, and 9 contain rabbit serum, lanes 2, 6, 10 contain mouse serum, lanes 3, 7, 11 contain human plasma, and lanes 4, 8, and 12 contain human serum, and Table 9 shows the data from Figure 20.

[0268] [Table 9]

[0269] As shown in Figure 20, an embodiment of Resin F and an embodiment of Resin G demonstrated excellent ability to remove albumin from different serum species and good ability to recover IgG.

[0270] Example 12 Effect of pH of equilibration buffer: In this example, the effect of equilibration buffer Tris 50 mM on the BSA removal and IgG recovery capacity of an embodiment of Resin G according to Table 2. The starting and flow-through volumes were measured at A280 using a nanodrop. The A280 volume was calculated by measuring the A280 on the nanodrop and taking into account the volume of sample added and collected. The Nanodrop was used as a control to determine the 280 reading of BSA at 10 mg / mL.

[0271] 21A is a bar graph showing the A280 amount of rabbit IgG. As shown in FIG. 21A, an embodiment of Resin G equilibrated with 50 mM Tris (pH 7.0) had an A280 amount of 123, an embodiment of Resin G equilibrated with 50 mM Tris (pH 5.0) had an A280 amount of 124, an embodiment of Resin G equilibrated with 50 mM Tris (pH 7.0 + 20 μL stacker) had an A280 amount of 139, the BSA-rabbit IgG starting mixture had an A280 amount of 824, the rabbit IgG starting material had an A280 amount of 133, and BSA alone had an A280 amount of 634.

[0272] Figure 21B is a bar graph showing the A280 content of GAR. As shown in Figure 21B, an embodiment of Resin G equilibrated with 50 mM Tris (pH 7.0) had an A280 content of 97, an embodiment of Resin G equilibrated with 50 mM Tris (pH 5.0) had an A280 content of 101, an embodiment of Resin G equilibrated with 50 mM Tris (pH 7.0 + 20 μL stacker) had an A280 content of 99, the BSA-GAR starting mixture had an A280 content of 834, the GAR starting material had an A280 content of 100, and BSA alone had an A280 content of 634.

[0273] This example demonstrates that the A280 amounts of the flow-through were similar to those of GAR and rabbit IgG, indicating a desirable recovery of antibody after purification with an embodiment of Resin G.

[0274] Example 13 Labeling of primary antibody: In this example, the primary antibody GAPDH was labeled with a fluorescent dye after removing the BSA from the BSA-antibody mixture. Figure 22 is an image of a gel showing the removal of BSA from the primary antibody GAPDH before conjugation with DyLight™ 650 (Thermo Scientific™), and Table 10 shows the data from Figure 22.

[0275] [Table 10]

[0276] The resin was spun at 3000×G and 6000×G. 1 mg / mL GAPDH was spiked with 10 mg / mL BSA and passed through an embodiment of Resin G from Table 2. The flow-through was collected, and the resin was washed with 50 mM Tris to remove any GAPDH that may have been bound to the resin. The flow-through from the two elutions was collected, pooled, and then conjugated with NHS DyLight™ 650 (Thermo Scientific™) and purified using Pierce™ dye and biotin removal resin. GAPDH containing BSA was also labeled, as was free Dy650 added as a control lane.

[0277] This example demonstrates successful removal of BSA from the primary antibody GAPDH prior to conjugation with DyLight™ 650 (Thermo Scientific™), thus demonstrating complete removal of BSA at spin speeds of 3,000×G and 6,000×G.

[0278] Example 14 In this example, the ability of an embodiment of Resin G from Table 2 to remove BSA from the primary antibody calreticulin prior to conjugation with DyLight™ 680 NHS (Thermo Scientific™) was tested. 1 mg / mL BSA was added as a stabilizer to obtain 1 mg / mL of calreticulin antibody. This antibody was passed through an embodiment of Resin G, the flow-through was collected, and the resin was washed three times with 50 mM Tris to remove any primary antibody that may have bound to the resin. The flow-through was collected, pooled, and concentrated before being conjugated with DyLight™ 680 NHS (Thermo Scientific™) and purified using Pierce™ dye and biotin removal resin.

[0279] Figure 23 is an image of a gel obtained by loading the flow-through and stained using Coomassie stain using a Pierce Power blotter. Table 11 shows the data in Figure 23.

[0280] [Table 11]

[0281] As shown in Figure 23, lane 1 shows BSA-free calreticulin after passing through an embodiment of Resin G. Lane 2 shows calreticulin conjugated to DyLight™ 680 (Thermo Scientific™) after BSA has been removed. Lane 3 is calreticulin with BSA still added as a stabilizer. With this in mind, this example demonstrates the successful removal of BSA from primary antibody calreticulin prior to conjugation with DyLight™ 680 (Thermo Scientific™) by an embodiment of Resin G.

[0282] Example 15 Western Blot Application: In this example, Western blot applications using fluorescently labeled GAR after BSA purification were tested. 1 mg / mL goat anti-rabbit (GAR) was spiked with 10 mg / mL BSA. Resin 7 embodiment from Table 1 was modified with 150 mg / mL PDA and used to remove BSA. GAR was then conjugated to DyLight™ 650 (Thermo Scientific™). GAR containing BSA was also conjugated to DyLight™ 650 (Thermo Scientific™) as a control. HeLa lysate was loaded onto the gel at 10 μg, 2.5 μg, and 1.25 μg, and then transferred to a nitrocellulose membrane. The membrane was blocked and then incubated with HSP90 primary antibody. The membrane was washed and then incubated with GAR Dy650, washed, and then scanned with an IBright imager using fluorescence mode. GAR conjugated with Dy650 without removing BSA was used as a comparative control.

[0283] Figure 24A is an image of a gel showing BSA removed from GAR (left) and not removed from GAR (right), and Figure 24B is a bar graph showing the fluorescence intensity of BSA removed and not removed from HeLa lysate loading. As shown in Figure 24B, HeLa lysate loading at 10 μg of stripped BSA had a fluorescence intensity of 13,000,000, and unstripped BSA had a fluorescence intensity of 4,000,000; HeLa lysate loading at 5 μg of stripped BSA had a fluorescence intensity of 9,000,000, and unstripped BSA had a fluorescence intensity of 3,800,000; HeLa lysate loading at 2.5 μg of stripped BSA had a fluorescence intensity of 7,800,000, and unstripped BSA had a fluorescence intensity of 3,800,000; and HeLa lysate loading at 1.25 μg of stripped BSA had a fluorescence intensity of 4,100,000, and unstripped BSA had a fluorescence intensity of 1,800,000.

[0284] This example demonstrates that BSA removed from GAR prior to conjugation to Dy650 exhibits much higher intensity on the blot and quantification graph than when BSA was not removed from GAR. Furthermore, the observed fluorescence intensity increased threefold with GAR Dy650 from which BSA was removed. Therefore, if the target is low in abundance in the cell lysate or the performance of the antibody containing BSA is poor, performance can be improved by removing the BSA using an embodiment of Resin 7 modified with 150 mg / mL PDA.

[0285] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Variations, modifications, and substitutions to these disclosed embodiments will be apparent to those skilled in the art without departing from the present disclosure. It is understood that all such various alternatives to the embodiments described herein may be employed in practicing the present disclosure. The following claims define the scope of the present disclosure.

Claims

1. A matrix, a porous size exclusion support; and at least one cationic moiety associated with the porous size-exclusion support, wherein the matrix is capable of separating one or more molecules in a sample by using the molecular weight of the one or more molecules, the charge of the one or more molecules, the isoelectric point (pI) of the one or more molecules, or a combination thereof.

2. The matrix of claim 1 , wherein the porous size-exclusion carrier has a molecular weight cutoff of 40 kDa or greater.

3. 3. The matrix of claim 1 or claim 2, wherein at least one cationic moiety is covalently attached to the porous size-exclusion support.

4. The matrix of any one of claims 1 to 3, wherein the at least one cationic moiety comprises an amine, a diamine, a polyamine, or an amine-containing polymer.

5. 5. The matrix of claim 4, wherein the at least one cationic moiety is polyethyleneimine, diaminopentane, 5,8-dimethyl-4,7,10-trioxatridecane-2,12-diamine, 1,2-diaminobenzene, 1,3-diaminobenzene, 1,4-diaminobenzene, or N,N-diethylethylenediamine.

6. The matrix of any one of claims 1 to 5, wherein the porous size-exclusion carrier comprises hydroxyethyl cellulose.

7. The matrix of any one of claims 1 to 6, wherein the porous size-exclusion support is cross-linked with epichlorohydrin.

8. 1. A system for separating one or more molecules in a sample using the molecular weight of the one or more molecules, the system comprising: A container comprising a matrix according to any one of claims 1 to 7; a receptacle positioned to receive a flow-through from the vessel; The system is configured for gravity flow operation, centrifugal force operation, positive pressure operation, negative pressure operation, vacuum operation, or a combination thereof, and the vessel is a column vessel, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate.

9. 1. A method for making a multimodal resin, comprising: providing a porous size-exclusion carrier comprising hydroxyethyl cellulose having a MWCO of 40 kDa or greater, the hydroxyethyl cellulose having at least one vicinal diol; oxidizing said at least one vicinal diol to an aldehyde; reacting the aldehyde with an amine group of a cationic moiety by reductive amination.

10. 10. The method of claim 9, wherein the porous size-exclusion carrier is produced using hydroxyethyl cellulose in an amount ranging from 50 grams to 250 grams.

11. 11. The method of claim 9 or claim 10, further comprising providing a cross-linking agent for cross-linking the porous size-exclusion support.

12. 12. The method of claim 11, wherein the cross-linking agent is epichlorohydrin and is used in an amount ranging from 250 milliliters to 450 milliliters.

13. 13. The method of any one of claims 9 to 12, wherein the cationic moiety is polyethyleneimine, diaminopentane, 5,8-dimethyl-4,7,10-trioxatridecane-2,12-diamine, (S)-N-boc-2,3-epoxypropylamine, 1,2-diaminobenzene, 1,3-diaminobenzene, 1,4-diaminobenzene, or N,N-diethylethylenediamine.

14. The method of any one of claims 9 to 13, wherein the cationic moiety is used at a concentration ranging from 50 mg / mL to 175 mg / mL.

15. 1. A method for separating one or more molecules in a sample, comprising: Providing a matrix according to any one of claims 1 to 7; equilibrating the matrix with an equilibration buffer; applying the sample to the matrix and separating the one or more molecules by subjecting the matrix to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof; The one or more molecules of the sample are excluded by the matrix and collected as flow-through, wherein the one or more molecules are associated with the at least one cationic moiety.

16. 16. The method of claim 15, wherein the one or more molecules are removed without a desalting step.

17. 17. The method of claim 15 or claim 16, wherein the flow-through is collected in a receptacle located below the vessel.

18. The method of any one of claims 15 to 17, wherein the equilibration buffer is positively charged or neutrally charged.

19. 19. The method of claim 18, wherein the positively charged buffer is Tris buffer or triethylammonium bicarbonate.

20. The method of any one of claims 15 to 17, wherein the equilibration buffer comprises between 0 mM and 5 mM salt.

21. The method according to any one of claims 15 to 17, wherein the equilibration buffer has a pH between 4 and 9.

22. 22. The method of claim 21, wherein the equilibration buffer comprises sodium acetate, HEPES, or borate.

23. the one or more molecules at least one small molecule having a molecular weight range of less than 100 kDa; and at least one large molecule having a molecular weight range of 100 kDa or greater.

24. 24. The method of any one of claims 15 to 23, wherein the matrix separates one or more molecules having an isoelectric point value in the range of 4.5 to 5.5 from one or more molecules having an isoelectric point value in the range of 8.0 to 11.

5.

25. 25. The method of any one of claims 15 to 24, wherein the matrix separates the one or more negatively charged molecules from one or more positively charged molecules.

26. 26. The method of any one of claims 15 to 25, wherein the at least one cationic moiety associates with the one or more molecules through ionic interactions, hydrophilic interactions, hydrophobic interactions, affinity interactions, hydrogen bonds, or van der Waals forces.

27. 1. A kit for separating a positively charged large molecule from one or more negatively charged small molecules in a sample, said kit comprising: a porous size-exclusion support having associated therewith at least one cationic moiety, said cationic moiety being capable of associating with and capturing said at least one negatively charged small molecule; and instructions for using said porous size-exclusion carrier.

28. 28. The kit of claim 27, further comprising an equilibration buffer, a container containing the porous size-exclusion support, a receptacle positioned to receive the flow-through of the support, or any combination thereof.

29. 29. The kit of claim 27 or claim 28, wherein the equilibration buffer comprises Tris, triethylammonium bicarbonate, sodium acetate, HEPES, or borate.

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