Components for separating molecules and methods of making and using same

Abstract

Disclosed herein are embodiments of matrices made from a porous size exclusion carrier and a cationic moiety for separating one or more small molecules from one or more macromolecules in a sample using differences in one or more properties, such as the size of the molecules, the charge of the molecules, the isoelectric point (pI) of the molecules, and / or any combination of these properties, methods, systems, and kit embodiments are included. Also disclosed herein are embodiments of methods of making the matrices and using the matrices to separate small molecules from one or more macromolecules in a sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 524,189, filed on June 29, 2023, and U.S. Provisional Application No. 63 / 370,277, filed on August 3, 2022, each of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to matrices, systems, methods, and kits for sample preparation, such as separation of small molecules from macromolecules. Background Art

[0004] Sample preparation techniques used to isolate biomolecules may require separation of the biomolecules from other sample components and from sample processing components to enable downstream analysis and processing of the biomolecules. During sample preparation of biomolecules such as proteins or nucleic acids, it is often necessary to label the biomolecules with dyes, affinity tags, radioactive labels, mass spectrometry tags, etc. In other cases, the biomolecules may need to be chemically modified, such as by reduction, oxidation, cross-linking, and / or alkylation.

[0005] An example is provided by considering bovine serum albumin (BSA), a protein isolated from bovine plasma that is often added to antibodies as a stabilizer. Although BSA has stabilizing properties, it often interferes with downstream applications (e.g., labeling antibodies with fluorescent microspheres), especially in the presence of low antibody levels. In order to avoid such interference, BSA should be separated from the antibody before labeling the selected antibody. Such separations are usually performed using size exclusion resins, which involve cumbersome fast protein liquid chromatography (FPLC) setups to purify the resin; however, existing resin kits for BSA removal are often ineffective, have low antibody recovery, and / or have low BSA removal properties.

[0006] Therefore, there is a need for improved purification resins, methods, and kits for purifying proteins from samples such that the proteins are sufficiently pure for high quality downstream analysis. Summary of the invention

[0007] Embodiments disclosed in the present disclosure advantageously provide excellent separation of molecules (such as biomolecules) from each other. In some aspects of the present disclosure, molecules are separated from each other using one or more characteristics, such as but not limited to the size of the molecule, the charge of the molecule, the isoelectric point (pI) of the molecule and / or the difference of any combination of these characteristics. In addition, molecules can be separated from each other based on one or more separation matrix characteristics, such as but not limited to charge and size exclusion characteristics. This can advantageously reduce the time and expense associated with the separation of small molecules and larger molecules. As described herein, the molecules separated are conducive to downstream processing relative to known methods.

[0008] In some embodiments, a porous size exclusion support is produced by using enough HEC to produce a resin having a molecular weight cutoff (or "MWCO") greater than or equal to 40 kDa. For example, some embodiments may include 60 grams of HEC to 150 grams of HEC. In some aspects, 60 grams to 130 grams of HEC are used to produce a size exclusion support in a 5 liter reaction scale. In specific aspects disclosed herein, a 0.5 liter to 2 liter resin bed comprising a porous size exclusion support is produced from 60 grams of HEC to 130 grams of HEC. In some embodiments, the porous size exclusion support may also include a cross-linked porous size exclusion support. In some embodiments, the porous size exclusion support may be cross-linked with an epoxide-containing compound comprising at least one epoxide functional group, wherein an exemplary embodiment is cross-linked with epichlorohydrin (also referred to herein as "Epi"). For example, a porous size exclusion support may be cross-linked with 250 milliliters of Epi to 450 milliliters of Epi.

[0009] Certain disclosed embodiments relate to a matrix comprising a porous size exclusion support having a molecular weight cutoff greater than or equal to 40 kDa 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 a small molecule having a molecular weight 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 can be, for example, an amine, a diamine, a polyamine, an amine-containing heterocyclic compound, an amine-containing aromatic compound, or an amine-containing polymer. Specific examples of cationic moieties include 5,8-dimethyl-4,7,10-trioxatridecane-2,12-diamine, polyethyleneimine, pentamethylenediamine, N,N-diethylethylenediamine, 1,2-phenylenediamine, 1,3-phenylenediamine, 1,4-phenylenediamine, and (S)-N-boc-2,3-epoxypropylamine. For certain embodiments, the cationic moiety is covalently bound to the porous size exclusion support.

[0010] The matrix can be advantageously balanced with a balancing buffer, which contains little to no salt (e.g., NaCl and other ionic salts), such as 0 mM salt to 5 mM salt. In some embodiments, the balancing buffer may contain a positive charge. In another embodiment, the balancing buffer may contain a neutral charge. In some aspects, the balancing buffer may have a pH of 4 to a pH of 9, preferably a pH of 5 to a pH of 7. In some aspects, the balancing buffer may have a concentration of 20 mM to 100 mM. In particular aspects disclosed herein, the balancing buffer may include triethylammonium bicarbonate, borate, sodium acetate or HEPES to facilitate separation of small molecules from macromolecules. For example, negatively charged small molecules are separated from positively charged macromolecules.

[0011] In some embodiments, the disclosure also provides a system for separating molecules with different molecular weights and / or charge and / or isoelectric point (pI) values ​​from each other. In one embodiment, the system of the disclosure can separate small molecules from macromolecules in a sample. In one embodiment, the system of the disclosure can separate small molecules (such as 70kDa proteins) from a sample from macromolecules (such as 150kDa proteins). Such systems include containers comprising matrix of the disclosure and receivers positioned to receive streams from the container. 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 container can be any suitable container, such as a columnar container, a pipe, a porous tube, a porous plate or a porous filter plate.

[0012] Also disclosed is a method for preparing a matrix comprising a porous size exclusion support having a MWCO greater than or equal to 40 kDa and at least one cationic moiety associated with the porous size exclusion support. The method includes providing a porous size exclusion support comprising a hydroxyethyl cellulose having a MWCO greater than or equal to 40 kDa or greater, the hydroxyethyl cellulose having at least one vicinal diol. The vicinal diol is oxidized to form an aldehyde, and the aldehyde is then reacted with an amine group of the cationic moiety at least greater than 50 mg / mL via a reductive amination reaction using, for example, 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 within a concentration of 50 mg / mL to 175 mg / mL, more typically 50 mg / mL to 160 mg / mL.

[0013] A method for separating small molecules with different molecular weights and / or charges and / or pI values ​​from each other is also disclosed. In one embodiment, the method of the present disclosure can separate proteins less than or equal to 70kDa from at least one macromolecule greater than 100kDa in a sample. The method includes providing a matrix comprising a porous size exclusion carrier, the porous size exclusion carrier having at least one cationic portion associated therewith, wherein the cationic portion 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 balanced with a balancing buffer. In some aspects of the present disclosure, the sample is then applied to the matrix to separate at least one small molecule from at least one macromolecule by subjecting the matrix to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum or a combination thereof. At least one macromolecule in the sample is excluded by the matrix and is collected as a flow-through in a receiver positioned to receive the flow-through. At least one small molecule (e.g., a negatively charged small molecule) associates with at least one cationic portion and is thereby separated from at least one macromolecule in a single step. The disclosed method substantially improves the ability to separate small molecules (such as bovine serum albumin) from a sample, and also substantially improves the recovery of large molecules (such as IgG). This in turn facilitates the processing of large molecules in downstream applications such as dye labeling.

[0014] Also disclosed is a kit for separating molecules with different molecular weights and / or charges and / or pI values ​​from each other. In one embodiment, the kit of the present disclosure can separate small molecules (such as proteins of 70kDa) from macromolecules (such as proteins as large as 150kDa) from samples. In another embodiment, the kit of the present disclosure can separate negatively charged molecules from more positively charged molecules in a sample. In some embodiments, the kit may include (1) a porous size exclusion carrier having at least one cationic portion associated therewith, wherein the cationic portion is capable of associating with a small molecule and capturing the small molecule, and (2) instructions for using the porous size exclusion carrier. The kit may also include an equilibrium buffer and / or a system comprising a container for accommodating the porous size exclusion carrier and a receiver positioned to receive a flow through the carrier.

[0015] The foregoing and other objects, features and advantages of the present disclosure will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram illustrating one disclosed embodiment that includes associating a porous size exclusion support with a cationic moiety such as pentamethylenediamine (or "PDA").

[0017] Figure 2is a schematic diagram illustrating one disclosed embodiment that includes associating a porous size exclusion support with a cationic moiety such as a branched polyethyleneimine (or "PEI").

[0018] Figure 3 is a schematic diagram illustrating one disclosed embodiment that includes associating a porous size exclusion support with a cationic moiety such as N,N-diethylethylenediamine (or "DEED").

[0019] Figure 4 is a schematic diagram illustrating one disclosed embodiment of using cationic moieties associated to a porous size exclusion support to separate small molecules from a sample containing small molecules, such as but not limited to negatively charged small molecules.

[0020] Figure 5 is a schematic diagram illustrating one embodiment of the disclosed system including a container, a receptacle, and an exemplary matrix comprising a porous size exclusion support associated with a cationic moiety.

[0021] Figure 6 is a schematic side view of one embodiment of a system according to the present disclosure, the system comprising a container and a receiver 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, the size of the molecules, the charge of the molecules, the isoelectric point (pI) of the molecules, and / or any combination of these properties.

[0022] Figure 7 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, the size of the molecules, the charge of the molecules, the isoelectric point (pI) of the molecules, and / or any combination of these properties, and wherein the system includes a porous container and a receiver.

[0023] Figure 8 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, the size of the molecules, the charge of the molecules, the isoelectric point (pI) of the molecules, and / or any combination of these properties, and wherein the system includes a porous container and a receiver.

[0024] Fig. 9 is a gel image comparing the removal of BSA and recovery of IgG from a sample comprising a mixture of BSA (2 mg / mL) and IgG (2 mg / mL) for the following embodiments disclosed in Table 2 herein: Resin A, Resin B, Resin C, Resin D, Resin E, and Resin F; wherein Fig. 9It was shown that greatest to least BSA removal was achieved in the following order (and thus showing that MWCO, in addition to PDA, also contributed to the desired BSA removal): Resin F, Resin D, Resin E, Resin B, then Resin A.

[0025] Fig.10 is a gel image comparing Resin 5 embodiments (as described in Table 1 provided herein), Resin L embodiments (as described in Table 2 provided herein), Resin F embodiments (as described in Table 2 provided herein), and Resin D embodiments (as described in Table 2 provided herein) equilibrated with different equilibration buffers (as described in Table 3 provided herein) to remove BSA and recover IgG from a sample comprising a mixture of BSA (10 mg / mL) and IgG (1 mg / mL); therefore, this figure demonstrates that PDA facilitates the removal of a desired amount of BSA, that embodiments having a MWCO greater than or equal to 40 kDa exhibit a lower ability to remove BSA than the 45 kDa MWCO embodiment, and that embodiments equilibrated with Tris buffer show desirable IgG recovery.

[0026] Fig.11 is a gel image showing BSA removal and IgG recovery from a sample comprising a mixture of BSA (10 mg / mL) and IgG (1 mg / mL) by an embodiment of Resin G (as 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), showing the desired BSA removal and IgG recovery from the sample.

[0027] Fig.12 The comparison was made between the resin 5 embodiment modified with 150 mg / mL PEI (as described in Table 1 provided herein), the resin 3 embodiment modified with 130 mg / mL PEI (as described in Table 1 provided herein), the Melon TM Gel IgG Purification Kit and Bar graph of BSA binding capacity (300 μL of 10 mg / mL BSA) of Blue Gel (Bio-Rad); Resin 5 embodiment exhibited a binding capacity of 3.11 mg bound BSA / mL resin, Resin 3 embodiment exhibited a binding capacity of 2.45 mg bound BSA / mL resin, Melon T The Gel IgG Purification Kit exhibits a binding capacity of 1.26 mg bound BSA / mL resin, and Blue Gel (Bio-Rad) exhibited a binding capacity of 2.43 mg bound BSA / mL resin; thus, the figure shows the binding capacity of Resin 5 embodiment modified with 150 mg / mL PEI, Resin 3 embodiment modified with 150 mg / mL PEI, and Blue Gel (Bio-Rad) and Melon TM Gel IgG purification kits vary in expected BSA binding capacity.

[0028] Fig.13 The comparison was made between the resin 5 embodiment modified with 150 mg / mL PEI (as described in Table 1 provided herein), the resin 3 embodiment modified with 130 mg / mL PEI (as described in Table 1 provided herein), the Melon TM Gel IgG Purification Kit and Histogram of IgG recovery (2 mg / mL) from Blue Gel (Bio-Rad); Resin 5 embodiment showed 72% volume recovery, Resin 3 embodiment showed 61% volume recovery, Melon TM The gel IgG purification kit exhibited 80% volume recovery, and Blue Gel (Bio-Rad) showed 25% volume recovery; therefore, this example shows that the resin 5 embodiment modified with 150 mg / mL PEI, the resin 3 embodiment modified with 130 mg / mL PEI and the Melon TM Gel IgG purification kit is different from Desired IgG recovery by Blue Gel (Bio-Rad).

[0029] Fig.14A The present invention is to compare the resin 5 embodiment (as described in Table 1 provided herein) produced with different amounts of PDA (as described in Table 4 provided herein) with the Abcam BSA removal kit and Melon TM Gel image of the IgG purification kit removing BSA and recovering IgG from a sample containing a mixture of BSA (10 mg / mL) and GAR (1 mg / mL); Resin 5 embodiment modified with 150 mg / mL PDA (also referred to herein as Resin F embodiment as described in Table 2 provided herein) and Resin 5 embodiment modified with 75 mg / mL PDA (also referred to herein as Resin K embodiment as described in Table 2 provided herein) show improved performance over the Abcam BSA Removal Kit and Melon TM Gel IgG Purification Kit Higher BSA removal and higher IgG recovery.

[0030] Fig. 14BThe iBright image analysis software is used to analyze the Fig.14A , which demonstrates that the resin 5 embodiment modified with 150 mg / mL PDA has 85.7% IgG recovery and 98.8% BSA removal; the 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 TM The 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 better results than the Abcam BSA Removal Kit and Melon TM Gel IgG Purification Kit for higher IgG recovery and BSA removal.

[0031] Fig.15 is a bar graph showing the recovery percentage of molecules of different sizes for establishing the MWCO of Resin 5 embodiment, Resin 6 embodiment, Resin 7 embodiment, and Resin 8 embodiment (as described in Table 1 provided herein), which shows that Resin 5 embodiment exhibits 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; Resin 6 embodiment exhibits 82% recovery at 42,000 Da, 84% recovery at 67,000 Da, 90% recovery at 80,000 Da, and 91% recovery at 150,000 Da. 00Da, and 92% recovery at 150,000Da; Resin 7 embodiment showed 66% recovery at 42,000Da, 76% recovery at 67,000Da, 84% recovery at 80,000Da, and 86% recovery at 150,000Da; Resin 8 embodiment showed 58% recovery at 42,000Da, 75% recovery at 67,000Da, 75% recovery at 80,000Da, and 83% recovery at 150,000Da; Therefore, this figure demonstrates that by reducing the amount of HEC, resins with 50kDa MWCO, 80kDa MWCO, and 90kDa MWCO are produced (as described in Table 1 provided herein).

[0032] Fig.16A is a gel image showing the relative performance of Resin F Embodiment, Resin G Embodiment, Resin H Embodiment, Resin I Embodiment, and Resin J Embodiment (as described in Table 1 provided herein) to Melon TMGel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA removal kits from a sample containing a mixture of BSA (10 mg / mL) and GAR IgG (1 mg / mL); therefore, this figure demonstrates that the Resin F embodiment and the Resin G embodiment have the most ideal BSA removal and IgG recovery from the sample.

[0033] Fig. 16B The iBright image analysis software is used to analyze the Fig.16A 93% GAR recovery and 100% BSA removal; Resin H embodiment has 63% GAR recovery and 99% BSA removal; Resin I embodiment has 76% GAR recovery and 95% BSA removal; Resin J embodiment has 82% GAR recovery and 100% BSA removal; Melon TM Gel IgG Purification Kit (Thermo Scientific TM ) with 106% GAR recovery and 82% BSA removal; Blue Gel (Bio-Rad) had 73% GAR recovery and 65% BSA removal; Abcam BSA removal kit had 125% GAR recovery and 85% BSA removal; thus, this figure demonstrates higher BSA removal and IgG recovery for Resin F embodiment, Resin G embodiment, and Resin J embodiment.

[0034] Fig.17 is a gel image showing the performance of Resin F Embodiment, Resin G Embodiment, Resin H Embodiment, Resin I Embodiment, and Resin J Embodiment (as described in Table 2 provided herein) relative to Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA removal kit BSA removal and IgG recovery; therefore, this figure demonstrates that relative to Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA Removal Kit, Resin F Embodiment, Resin G Embodiment and Resin J Embodiment performed better in mixtures with low concentrations of IgG.

[0035] Fig.18 is relative to Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA Removal Kit, after removing BSA from a mixture containing GAR (1 mg / mL) and BSA (10 mg / mL) using Resin F embodiment (as described in Table 2 provided herein), NHSDyLight TM 488(Thermo Scientific TM ) was performed on the image of the fluorescently labeled GAR flowthrough; thus, this figure demonstrates that, unlike Melon TM Gel IgG Purification Kit (Thermo Scientific TM )and Blue Gel (Bio-Rad), expected ability to recover labeled GAR after removal of BSA by Resin F embodiment and Abcam BSA removal kit.

[0036] Fig.19 is relative to Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA Removal Kit, after removing BSA from a mixture containing GAR (0.1 mg / mL) and BSA (10 mg / mL) using Resin F embodiment and Resin G embodiment (as described in Table 2 provided herein), NHSDyLight TM 488(Thermo Scientific TM ) was performed on the image of the fluorescently labeled GAR flowthrough; thus, this figure demonstrates that, unlike Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA removal kits, the desired ability to recover labeled GAR after removal of BSA by Resin F embodiment and Resin G embodiment at even low antibody concentrations (such as 0.1 mg / mL).

[0037] Fig. 20 are gel images showing the clearance of rabbit serum, mouse serum, human plasma, and human serum by Resin F embodiment and Resin G embodiment (as described in Table 2 provided herein) and demonstrating the desired albumin removal and desired IgG recovery from the different serum species.

[0038] Fig.21A is a bar graph showing rabbit IgG (A280 amount) of Resin G embodiments equilibrated with different concentrations of Tris buffer (as described in Table 2 provided herein), which shows that the A280 amount of rabbit IgG of Resin G embodiments equilibrated with 50 mM Tris (pH 7.0) has an A280 amount of 123; the Resin G embodiment equilibrated with 50 mM Tris (pH 5.0) has an A280 amount of 124; the Resin G embodiment equilibrated with 50 mM Tris (pH 7.0 + stacker 20 μL) has an A280 amount of 139; the BSA-rabbit IgG starting mixture has an A280 amount of 824; the rabbit IgG starting material has an A280 amount of 133; and BSA has only an A280 amount of 634; therefore, this graph demonstrates ideal antibody recovery because the flow-through A280 amount is similar to the A280 amount of rabbit IgG.

[0039] Fig. 21B is a bar graph showing GAR (A280 amount) of Resin G embodiments equilibrated with different concentrations of Tris buffer (as described in Table 2 provided herein), which shows that the A280 amount of rabbit IgG of Resin G embodiments equilibrated with 50mM Tris (pH7.0) has an A280 amount of 97; Resin G embodiments equilibrated with 50mM Tris (pH 5.0) have an A280 amount of 101; Resin G embodiments equilibrated with 50mM Tris (pH 7.0 + stacking agent 20μL) have an A280 amount of 99; BSA-GAR starting mixture has an A280 amount of 834; GAR starting material has an A280 amount of 100; and BSA has only an A280 amount of 634; therefore, this graph demonstrates ideal antibody recovery because the flow-through A280 amount is similar to the A280 amount of GAR.

[0040] Fig. 22is an image showing the primary antibody (GAPDH) labeled with a fluorescent dye after removal of BSA from a sample comprising a mixture of antibody (1 mg / mL)-BSA (10 mg / mL) after spinning at 3,000XG and at 6,000XG for the Resin G embodiment (as described in Table 2 provided herein), with antibody-BSA and free Dy 650 also labeled; thus, the figure shows the expected BSA removal for both spin speeds.

[0041] Fig.23 is an image of a gel obtained by loading and staining the flow-through using a Pierce Power blotting apparatus using Coomassie stain, showing calreticulin without BSA after passage through a resin G embodiment (as described in Table 2 provided herein), conjugated to DyLight TM 680 (Thermo Scientific TM ) and calreticulin received with BSA added as a stabilizer; thus, this figure demonstrates that when the primary anti-calreticulin is combined with DyLight TM (Thermo Scientific TM )680 Prior to conjugation, the desired BSA was removed from the primary anti-calreticulin by the resin G embodiment.

[0042] Fig.24A are images of a Western blot application using fluorescently labeled GAR after BSA removal with the Resin H embodiment (as described in Table 2 provided herein), showing removal of BSA from GAR (left) and without removal of BSA from GAR (right) from a sample comprising a mixture of GAR (1 mg / mL) and BSA (10 mg / mL); thus, the figure shows that removal of BSA from GAR prior to conjugation to Dy 650 shows much higher intensity than when BSA is not removed from GAR.

[0043] Fig. 24B2.5 μg of HeLa lysate with BSA removed had a fluorescence intensity of 7,800,000, and the BSA was not removed had a fluorescence intensity of 3,800,000; and 1.25 μg of HeLa lysate with BSA removed had a fluorescence intensity of 4,100,000, and the BSA was not removed had a fluorescence intensity of 1,800,000; Thus, this figure demonstrates that, in comparison with Dy Removal of BSA from GFAR before 650 conjugation showed much higher intensity than when BSA was not removed from GAR, and three times the fluorescence intensity was observed in GAR Dy 650 with BSA removed. DETAILED DESCRIPTION

[0044] I. Abbreviations

[0045] BSA: bovine serum albumin

[0046] DEED: Diethylethylenediamine

[0047] Epi: Epichlorohydrin

[0048] HEC: Hydroxyethyl Cellulose

[0049] HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

[0050] IgG: Immunoglobulin G

[0051] MWCO: Molecular Weight Cut-off

[0052] PDA: Pentylene diamine

[0053] PEI: branched polyethyleneimine

[0054] TRIS: Tris(hydroxymethyl)aminomethane

[0055] II. Overview of Terms, Scope and Definitions

[0056] 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.

[0057] As used herein, the use of the singular includes the plural unless specifically stated otherwise. For example, the singular forms "a", "an", and "the" used in the specification also include plural aspects unless the context dictates otherwise. Similarly, any singular term used in the specification also contemplates the plural, and vice versa, unless the context dictates otherwise.

[0058] In some instances, values, procedures, or devices may be referred to as "lowest," "optimal," "minimum," etc. It should be understood that such descriptions are intended to indicate that choices may be made among a number of functional alternatives used, and that such choices are not necessarily better, lesser, or otherwise preferred than other choices.

[0059] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. Unless otherwise indicated, materials, methods and examples are only illustrative and are not intended to be limiting. Other features of the present invention are apparent from the following detailed description and claims.

[0060] Unless otherwise indicated, all numerals expressing the amount of components, molecular weight, percentage, temperature, time, etc. as used in this specification or claims are understood to be modified by the term "about". Therefore, unless otherwise implicitly or explicitly indicated, the numerical parameters are approximate values, which may depend on the desired properties sought and / or the detection limits under standard test conditions / methods. When the embodiments are directly and clearly distinguished from the discussed prior art, unless the word "about" is narrated, the embodiment numbers are not approximate. In addition, not all alternatives narrated herein are equivalents.

[0061] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items 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 if order is important in the particular context, BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc.

[0062] The term "acyl halide" generally refers to -C(O)X, where X is a halogen such as Br, F, I or Cl.

[0063] The term "alcohol" generally refers to an organic compound containing at least one hydroxyl group. The alcohol can be a monohydric alcohol (containing one -OH group), a dihydric alcohol (containing two -OH groups; diols, such as ethylene glycol), a trihydric alcohol (containing three -OH; triols, such as glycerol) groups, or a polyhydric alcohol (containing two or more -OH groups; polyols). The organic portion of the alcohol can be aliphatic, cycloaliphatic (alicyclic), heteroaliphatic, cycloheteroaliphatic (heterocyclic), polycyclic, aryl, or heteroaryl, and can be substituted or unsubstituted.

[0064] The term "aldehyde" generally refers to a carbonyl-bearing functional group having the formula:

[0065]

[0066] Where a line is drawn through a bond it indicates that the functional group may be attached to any other moiety, but such moieties are simply not represented.

[0067] The term "aliphatic" generally refers to a substantially hydrocarbon-based compound or group thereof (e.g., for a hexane group, CH 13 ), including alkanes, alkenes, alkynes, including their cyclic forms, and also including straight and branched arrangements, as well as all stereoisomers and positional isomers. Unless otherwise specifically stated, aliphatic groups contain 1 to 25 carbon atoms; for example, 1 to 15, 1 to 10, 1 to 6, or 1 to 4 carbon atoms. The term "lower aliphatic" refers to aliphatic groups containing 1 to 10 carbon atoms. An aliphatic chain may be substituted or unsubstituted. Unless explicitly stated as "unsubstituted aliphatic", an aliphatic group may be unsubstituted or substituted. An aliphatic group may 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 a terminal methine). Exemplary substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxy, oxo, sulfonamide, thiol, thioalkoxy, or other functional groups.

[0068] The term "alkoxy" generally refers to a group (or substituent) having the structure -OR, where R is a substituted or unsubstituted alkyl group.

[0069] The term "alkyl" generally refers to a hydrocarbon group having a saturated carbon chain. The chain may be cyclic, branched or unbranched.

[0070] The term "alkynyl" generally refers to an organic compound having at least one carbon-carbon triple bond. An alkynyl group can be branched, straight-chain or cyclic (eg, cycloalkynyl).

[0071] 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 (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfano, or other functional groups.

[0072] 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 (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional groups.

[0073] The term "antibody" generally refers to immunoglobulins or immunoglobulin-like molecules (including, for example, but not limited to, IgA (immunoglobulin A), IgD (immunoglobulin D), IgE (immunoglobulin E), IgG (immunoglobulin E) and IgM (immunoglobulin M), combinations thereof, and similar molecules produced during an immune response in any chordate (such as a vertebrate), e.g., a mammal (such as a human, goat, rabbit, and mouse)) and fragments thereof, which specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules. An "antibody" generally comprises a polypeptide ligand having at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigenic epitope. Immunoglobulins are composed of heavy and light chains, each of which has a variable region, called a variable heavy (V H ) area and variable lightness (V L ) area. H Area and V LThe two domains are together responsible for binding the antigen recognized by the immunoglobulin. Exemplary immunoglobulin fragments include, but are not limited to, proteolytic immunoglobulin fragments [such as F(ab′)2 fragments, Fab′ fragments, Fab′-SH fragments, and Fab fragments known in the art], recombinant immunoglobulin fragments (such as 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 (as known in the art) and camelid antibodies. "Antibodies" also include genetically engineered molecules, such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Edition, WH Freeman & Co., New York, 1997.

[0074] The term "aromatic" generally refers to a cyclic or conjugated group containing 5 to 15 ring atoms, unless otherwise specified, having at least one single ring (e.g., phenyl) or multiple fused rings in which at least one ring is aromatic (e.g., naphthyl, indolyl, or pyrazolopyridinyl); that is, at least one ring and the optional multiple fused rings have a continuous delocalized π electron system. Typically, the number of out-of-plane π electrons corresponds to Huckel's rule (4n+2). The point of attachment to the parent structure is usually through the aromatic portion of the fused ring system. For example, However, in certain instances, context or explicit disclosure may indicate that the point of attachment is through the non-aromatic portion of the fused ring system. For example, The aromatic group may contain only carbon atoms in the ring, such as in an aryl group, or it may contain one or more ring carbon atoms and one or more ring heteroatoms containing a lone pair of electrons (e.g., S, O, N, P, or Si), such as in a heteroaryl group. The aromatic group may be substituted with one or more non-hydrogen groups, such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional groups, or organic functional groups.

[0075] The term "aryl" generally refers to a group containing at least 5 carbon atoms and up to 15 carbon atoms (C5-C 15 ), such as 5 to 10 carbon atoms (C5-C 10) is an aromatic carbocyclic group having a single ring or multiple fused rings, which may or may not be aromatic, provided that the point of attachment to the rest of the compounds disclosed herein is through an atom of the aromatic carbocyclic group. The aryl group may be substituted with one or more non-hydrogen groups, such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl or other functional groups.

[0076] The term "biological sample" 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 including any biological material (e.g., blood, serum, plasma, sputum, 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 supernatants of cell cultures and homogenized tissues or disrupted cells.

[0077] The term "carbamate" generally refers to -OC(O)NRR', where R and R' are independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional groups.

[0078] The term "carbonate" generally refers to a functional group having the formula -OCOR, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional group.

[0079] The term "carboxamide" generally refers to -N(R)acyl or -C(O)amino, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional groups.

[0080] The term "carboxy" generally refers to -C(O)OH.

[0081] The term "carboxylic acid" generally refers to an organic compound of the formula RCOOH, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional group.

[0082] The term "cyano" generally refers to -CN.

[0083] The term "disulfide" generally refers to -SSR a , where R a is selected from hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl or other functional groups.

[0084] The term "epoxide" generally refers to a cyclic ether having a 3-membered ring having the following general formula: Where R 1 To R 4 and R is independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional groups.

[0085] The term "ester" generally refers to a compound having the formula: wherein R and R' are independently alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional groups.

[0086] The term "ether" generally refers to a class of organic compounds containing an ether group, which is an oxygen atom attached to two aliphatic and / or aryl groups and has the general formula ROR', wherein R and R' are independently alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl or other functional groups.

[0087] The term "equilibration buffer" generally refers to a buffer used to infuse a matrix according to the present disclosure to facilitate processing of the sample and to facilitate affinity of the molecule of interest to the support.

[0088] The term "fluorophore" generally refers to a functional group or moiety of a compound that causes a compound (or a sample or composition comprising the compound) to fluoresce. In some embodiments, a fluorophore can fluoresce when the compound (or a sample or composition comprising the compound) is exposed to an excitation source or after cleavage from a compound conjugated to the fluorophore.

[0089] The term "functional group" generally refers to a specific group of atoms in a molecule that is responsible for the characteristic chemical reactions of the molecule. Exemplary functional groups include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, halo (fluorine, chlorine, bromine, iodine), epoxide, hydroxyl, carbonyl (ketone), aldehyde, carbonate, carboxylate, carboxyl, ether, ester, peroxy, peroxyhydroxyl, carboxamide, amino (primary, secondary, tertiary), ammonium, imide, azide, cyanate, isocyanate, thiocyanate, nitrate, nitrite, nitrile, nitroalkyl, nitroso, pyridyl, phosphate, sulfonyl, sulfide, thiol (sulfhydryl), disulfide.

[0090] The term "halo" generally refers to fluoro, chloro, bromo or iodo.

[0091] The term "heteroaryl" generally refers to an aryl group containing at least one heteroatom in the ring, which heteroatom can be selected from, but not limited to, oxygen, nitrogen, sulfur, silicon, boron, selenium, phosphorus, and their oxidized forms. The heteroaryl group can contain a single ring or multiple condensed rings, wherein the condensed rings may or may not be aromatic and / or may or may not contain heteroatoms, provided that the point of attachment is through the atom of the aromatic heteroaryl group. The heteroaryl group can be substituted by one or more non-hydrogen groups, such as alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, halogenated aliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, halogenated aryl, alkylsulfanyl or other functional groups. In some embodiments, the fluorophore may also be described herein as a heteroaryl group.

[0092] The term "hydroxy" refers to the group -OH.

[0093] 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 (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional group.

[0094] The term "immunoglobulin G (IgG)" generally refers to one of the major classes of immunoglobulins having a heavy chain called the gamma chain.

[0095] The term "isoelectric point (pi)" generally refers to the pH at which a molecule carries no net electrical charge. Polymeric molecules, such as proteins composed of amino acids, can be positively charged, neutral, negatively charged, or polar in nature, giving the polymeric material an overall charge. Molecules with low pi values ​​carry a net negative charge at neutral pH, while molecules with high pi values ​​carry a net positive charge at neutral pH.

[0096] The term "molecular weight cutoff" or "MWCO" generally refers to the lowest molecular weight of sample components (such as smaller sample components) that are excluded by a matrix. For example, one or more small molecules (such as, but not limited to, BSA) are excluded by the matrix, while larger sample components (such as one or more macromolecules) elute faster and are recovered.

[0097] The term "multimodal" generally refers to the ability of a material or compound (such as a resin or matrix) to provide multiple different types of interactions between the resin or matrix and the desired molecule to facilitate separation of a first desired molecule from a second molecule (such as by retaining the first and / or second molecule in the resin or matrix).

[0098] The term "negatively charged small molecule" generally refers to a molecule with a low isoelectric point (pi), which carries a net negative charge at neutral pH.

[0099] The term "phosphate" generally refers to -OP(O)(OR a )2, where each R a and R is independently hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other organic functional groups.

[0100] The term "positively charged macromolecule" generally refers to a molecule with a high isoelectric point (pi), which carries a net positive charge at neutral pH.

[0101] The term "sample" generally refers to any fluid or solution containing at least two molecules that need to be separated, wherein 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 macromolecule as defined herein. In some embodiments, the sample may contain small molecules, such as but not limited to stabilizing molecules. Stabilizing molecules can be purified proteins isolated from natural or recombinant sources. For example, recombinant albumin, natural albumin, human serum albumin, albumin-like stabilizers, bovine serum albumin, similar mammalian serum (such as but not limited to rabbit serum and mouse serum), ovalbumin, glycerol and / or gelatin. The sample may also contain one or more molecules derived from a biological sample.

[0102] The terms "separate", "extract", "extracted", "remove", "reduce" or "reduce the amount thereof" or "purify" generally refer to removing or isolating a substance, e.g., a small molecule (such as BSA) or a macromolecule or biomolecule (such as IgG), from a mixture containing small molecules and / or macromolecules. The extracted substance or the sample from which the substance is extracted has significantly reduced amounts of components present in the sample before separation compared to before extraction, and the extracted substance can be substantially reduced, substantially removed, substantially concentrated, substantially pure or pure (free of any contaminants).

[0103] The present disclosure uses the terms "small molecule" and "macromolecule", which refer to substances to be separated from each other. "Small molecule" or "smaller molecule" generally refers to any molecule with a molecular weight less than 100 kDa, and "macromolecule" is a molecule with a molecular weight greater than or equal to 100 kDa. Small molecules (such as but not limited to biomolecules) can be used to treat, derivatize, conjugate, cross-link, label, tag, chemically or biologically modify macromolecules for further analysis. Small molecules can be stable molecules, such as but not limited to purified proteins isolated from natural or recombinant sources. For example, recombinant albumin, native albumin, human serum albumin, albumin-like stabilizers, bovine serum albumin, similar mammalian serum, ovalbumin and / or gelatin. Derivatization includes labeling the molecule with a marker such as a dye, an affinity tag, a radioactive label, a mass spectrometry tag, a metal, etc. Derivatization also includes chemically modifying the molecule by reduction, oxidation, methylation, chemical or biochemical modification of the biomolecule, etc. Derivatives of biomolecules include, but are not limited to: tagged proteins or nucleic acids; labeled biomolecules labeled with a variety of markers, such as, but not limited to, dyes, fluorescent dyes, radioactive markers, affinity markers, mass spectrometry tags, metals, etc.; conjugated biomolecules, including conjugated antibodies; biomolecules conjugated to nanoparticles; metals, such as gold conjugated to nanoparticles; dyes or markers, such as biotin conjugated to toxins; chemical derivatives of biomolecules, such as, but not limited to, reduced proteins, oxidized proteins, methylated nucleic acids, and proteins with sulfhydryl-modified proteins. Macromolecules and / or biomolecules include, for example, but not limited to, proteins, glycoproteins, and antibodies. In one example, the small molecule is BSA with a molecular weight of 67,000Da. In one example, the macromolecule is IgG with a molecular weight of 150kDa. In certain disclosed examples, the sample comprises a mixture of BSA and IgG.

[0104] 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 cut-off size (MWCO) of 40 kDa or greater.

[0105] The term "sulfonamide" generally refers to the group -SO2amide or -N(R)sulfonyl, where R is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional groups.

[0106] The term "sulfonate" generally refers to -SO3 - , where the negative charge of the sulfonic acid group can be balanced by a positive counterion, such as M + The counter ion, M + Can be an alkali ion such as K + 、Na + , Li + ; ammonium ions, such as + N(R b )4, where R b is hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional group; or an alkaline earth ion such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 or[Ba 2+ ] 0.5 .

[0107] The term "sulfonyl" generally refers to a functional group having the following general formula:

[0108]

[0109] wherein R represents the remainder of the molecule to which the sulfonyl group is bound, and R′ is selected from hydrogen, alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl or other functional groups.

[0110] The term "thioester" generally refers to a functional group having the following general formula:

[0111]

[0112] wherein R represents the remainder of the molecule to which the thioester group is bound, and R′ is selected from alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl, or other functional groups.

[0113] The term "thioether" generally refers to a functional group having the general formula RSR', where R and R' are independently selected from alkyl, heteroalkyl, haloalkyl, aliphatic, heteroaliphatic, haloaliphatic, aryl (such as optionally substituted phenyl or benzyl), heteroaryl, haloaryl, alkylsulfanyl or other functional groups. Thioethers are similar to ethers except that thioethers contain a sulfur atom in place of the oxygen atom of an ether.

[0114] All documents 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 form of these documents and similar materials, are expressly incorporated herein by reference as a whole for any purpose. In the case where one or more of the incorporated documents and similar materials define or use the term in a manner inconsistent with the definition of the term in this application, the definition provided in this specification shall prevail. Although the present teaching content is described in conjunction with various embodiments, it is not intended that the present teaching be limited to such embodiments. On the contrary, the present teaching encompasses various substitutions, modifications and equivalents, as will be understood by those of ordinary skill in the art based on the present teaching.

[0115] III. Matrix

[0116] Some disclosed embodiments of the present disclosure relate to a matrix for separating molecules (such as biomolecules) from each other. In some embodiments of the present disclosure, molecules are separated from each other using one or more characteristics, such as the size of the molecule, the charge of the molecule, the isoelectric point (pI) of the molecule and / or the difference of the combination of these characteristics. In certain embodiments, molecules can be separated from each other based on one or more separation matrix characteristics, such as the charge on the matrix and the size exclusion characteristics of the matrix. The matrix can include a porous size exclusion carrier, at least one cationic part, and can be further balanced with a balance buffer. Each of these components is described in more detail below.

[0117] In some embodiments, a sample solution comprising at least one small molecule and at least one macromolecule is applied to the matrix, wherein the macromolecule elutes faster than the small molecules captured by the matrix. The matrix disclosed herein provides unexpectedly rapid, economical and efficient separation of small molecules from macromolecules.

[0118] 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 macromolecules. In some embodiments, the small molecule can be, but is not limited to, a protein, a globular protein, a spherical protein, a serum albumin, a polypeptide, and the like.

[0119] In some disclosed embodiments, one or more characteristics (including but not limited to isoelectric point) of the molecule can be used to separate one or more small molecules from one or more macromolecules. In some embodiments, one or more small molecules can be one or more negatively charged small molecules. In a specifically disclosed embodiment, 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, 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 an exemplary embodiment, one or more negatively charged small molecules have a pI value of 4.9.

[0120] In some embodiments, the matrix separates, extracts, removes and / or reduces the amount of one or more small molecules from one or more macromolecules based on, but not limited to, the size of the molecule. In some aspects of the present disclosure, one or more small molecules can 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, one or more small molecules can have a molecular weight of 50 kDa to 80 kDa, such as 50 kDa, 51 kDa, 52 kDa, 53 kDa, 54 kDa, 55 kDa, 56 kDa, 57 kD, 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, one or more small molecules are BSA having a molecular weight of 67,000 Da (67 kDa).

[0121] 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 macromolecules. In some embodiments, the one or more macromolecules may be, but are not limited to, glycoproteins, phosphoproteins, antibodies or immunoglobulins.

[0122] In specific disclosed embodiments, one or more properties of the molecule, including but not limited to isoelectric point, can be used to separate one or more small molecules from one or more macromolecules. In some embodiments, the one or more macromolecules can be one or more positively charged macromolecules. In specific disclosed embodiments, the one or more positively charged macromolecules 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 macromolecules 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 an exemplary embodiment, the one or more positively charged macromolecules have a pI value of 11.0.

[0123] In some embodiments, the matrix separates, extracts, removes and / or reduces the amount of one or more small molecules from one or more macromolecules based on, but not limited to, the size of the molecule. In some embodiments, the one or more macromolecules may have a molecular weight greater than or equal to 100 kDa, and in certain embodiments, a molecular weight greater than or equal to 150 kDa. 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, 119 kDa, 120 kDa, 121 kDa, 122 kDa, 123 kDa, 124 kDa, 125 kDa, 126 kDa, 127 kDa, 128 kDa, 129 kDa, 130 kDa, 131 kDa, 132 kDa, 133 kDa, 134 kDa, 135 kDa, 136 kDa, 137 kDa, 138 kDa, 139 kDa, 140 kDa, 141 kDa, 142 kDa, 143 kDa, 144 kDa 9kDa, 140kDa, 141kDa, 142kDa, 143kDa, 144kDa, 145kDa, 146kDa, 147kDa, 148kDa, 149 kDa, 150kDa, 151kDa, 152kDa, 153kDa, 154kDa, 155kDa, 156kDa, 157kDa, 158kDa, 159k Da, 160kDa, 161kDa, 162kDa, 163kDa, 164kDa, 165kDa, 166kDa, 167kDa, 168kDa, 169kD a, 170kDa, 180kDa, 220kDa, 250kDa, 300kDa, 350kDa, 450kDa, 550kDa, 750kDa, 900kDa. In an exemplary embodiment, the macromolecule is an IgG having a molecular weight of 150,000 Da (150 kDa).

[0124] A. Size Exclusion Support

[0125] According to the matrix disclosed herein, a porous size exclusion carrier may be included. The porous size exclusion carrier may include spherical beads made of a gel or gel-like material having holes. The pore size range of the porous size exclusion carrier determines the size of the molecules that can be included or excluded from entering the size exclusion carrier. Without being bound by a single theory, it is currently believed that when a sample solution passes through a size exclusion carrier, at least one small molecule in the sample enters the pores of the size exclusion carrier and is forced to follow a circuitous path before leaving the size exclusion carrier later. On the other hand, larger molecules take a relatively direct path through the size exclusion carrier. Therefore, without being bound by a specific operating theory, it is currently believed that when a sample travels through a size exclusion carrier, the flow rate difference between small molecules and large molecules allows the separation of faster flowing large molecules and slower flowing small molecules.

[0126] Some exemplary size exclusion carriers are made of agarose, polyacrylamide, cellulose materials and / or their derivatives. In some embodiments, the porous size exclusion carrier can include an agarose carrier, a polyacrylamide carrier, a cellulose material carrier or their derivatives. In some exemplary embodiments, the porous size exclusion carrier comprises HEC.

[0127] In some embodiments, a porous size exclusion support is produced by using sufficient HEC to produce a resin having a MWCO greater than or equal to 40 kDa.

[0128] In a specific disclosed embodiment, the porous size exclusion support is produced from 50 grams (g) to 250 grams (g) of HEC, such as but not limited to 50 g HEC to 150 g HEC. In some embodiments, 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 g, 89g, 90g, 91g, 92g, 93g, 94g, 95g, 96g, 97g, 98g, 99g, 100g, 101g, 102g, 103g, 104g, 105g, 106g, 10 7g, 108g, 109g, 110g, 111g, 112g, 113g, 114g, 115g, 116g, 117g, 118g, 119g, 120g, 121g, 122g, 123g, 1 24g, 125g, 126g, 127g, 128g, 129g, 130g, 131g, 132g, 133g, 134g, 135g, 136g, 137g, 138g, 139g, 140g , 141g, 142g, 143g, 144g, 145g, 146g, 147g, 148g, 149g, 150g, 160g, 170g, 180g, 190g, 200g, 201g, 202 In one exemplary embodiment, 80 g of HEC is used to produce a porous size exclusion carrier. In another exemplary embodiment, 90 g of HEC is used to produce a porous size exclusion carrier. In another exemplary embodiment, 108 g of HEC is used to produce a porous size exclusion carrier. In another exemplary embodiment, 129 g of HEC was used to produce the porous size exclusion support. In another exemplary embodiment, 147 g of HEC was used to produce the porous size exclusion support. In another exemplary embodiment, 216 g of HEC was used to produce the porous size exclusion support.

[0129] In some aspects of the present disclosure, the porous size exclusion support is produced with 60 g HEC to 130 g HEC at a reaction scale of greater than 0 liter (L) to 10 liters (L), such as but not limited to greater than 0 L to 5 L, to produce the size exclusion support. In some embodiments, 0.5 L to 2 L of a resin bed comprising a porous size exclusion support is produced from 60 g HEC to 130 g HEC. In specific disclosed aspects of the present disclosure, 0.5 L to 1.5 L of a resin bed comprising a porous size exclusion support is produced from 80 g HEC to 130 g HEC used in a 5 L reaction scale.

[0130] In some aspects of the present disclosure, the size exclusion support can be cross-linked with a cross-linking agent such as, but not limited to, an epoxide-containing compound comprising at least one epoxide functional group (such as 1, 2, 3, or 4 epoxide groups). In some embodiments, the epoxide-containing compound can include one or more halogenated functional groups, aliphatic functional groups, heteroaliphatic functional groups, or combinations thereof. In some embodiments, the heteroaliphatic functional group can include a polyethylene glycol (or "PEG") spacer arm. The epoxide-containing compound can have Formula I,

[0131]

[0132] Where R is C1-C 10 alkyl; and LG may be a halogenated functional group or a glycidol moiety. In a preferred embodiment, the epoxide-containing compound may be epichlorohydrin (Epi) having the following structure: In another preferred embodiment, the epoxide-containing compound may be 1,4-butane diglycidyl ether.

[0133] In some embodiments, the porous size exclusion support can be cross-linked with a cross-linking agent 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, 280 mL, 281 mL, 282 mL, 283 mL, 284 mL, 285 mL, 286 mL, 287 mL, 288 mL, 289 mL, 290 mL, 291 mL, 292 mL, 293 mL, 294 mL, 295 mL, 296 mL, 297 mL, 298 mL, 299 mL 8mL, 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, 341mL, 342mL, 343mL, 344mL, 345mL, 346mL, 347 mL, 348mL, 349mL, 355mL, 356mL, 357mL, 358mL, 359mL, 360mL, 361mL, 362mL, 363mL, 364mL, 365mL, 366mL, 367mL, 368mL, 369mL, 370mL, 371mL, 37 2mL, 373mL, 374mL, 375mL, 376mL, 377mL, 378mL, 379mL, 380mL, 381mL, 382mL, 383mL, 384mL, 385mL, 386mL, 387mL, 388mL, 389mL, 390mL, 391mL, 3 92mL, 393mL, 394mL, 395mL, 396mL, 397mL, 398mL, 399mL, 400mL, 401mL, 402mL, 403mL, 404mL, 405mL, 406mL, 407mL, 408mL, 409mL, 410mL, 411mL,600mL, 620mL, 640mL, 660mL, 680mL or 700mL of a cross-linking agent, wherein specific embodiments use the above ranges / amounts of Epi as a cross-linking agent.

[0134] In one exemplary embodiment, 265 mL of Epi is used to crosslink the porous size exclusion carrier. In another exemplary embodiment, 303 mL of Epi is used to crosslink the porous size exclusion carrier. In another exemplary embodiment, 375 mL of Epi is used to crosslink the porous size exclusion carrier. In another exemplary embodiment, 397 mL of Epi is used to crosslink the porous size exclusion carrier. In another exemplary embodiment, 410 mL of Epi is used to crosslink the porous size exclusion carrier. In another exemplary embodiment, 441 mL of Epi is used to crosslink the porous size exclusion carrier. In yet another exemplary embodiment, 662 mL of Epi is used to crosslink the porous size exclusion carrier.

[0135] In some embodiments, the porous size exclusion support can be cross-linked at a reaction scale of greater than 0 L to 5 L by providing 250 mL Epi to 700 mL Epi. In some specific aspects of the present disclosure, a resin bed comprising a porous size exclusion support of greater than 0 L to 2 L can be produced from 250 mL Epi to 400 mL Epi at a 5 L reaction scale.

[0136] In some embodiments, the porous size exclusion support has a MWCO greater than or equal to 40 kDa, so the excluded molecules have a molecular weight greater than or equal to 40 kDa. The pore size of the porous size exclusion support can have a MWCO size for excluding from the pore molecules greater than or equal to 40 kDa to 150 kDa, more typically 50 kDa to 150 kDa, and even more typically greater than or equal to 40 kDa to 60 kDa, including in between, such as but not limited to greater than or equal 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 In one exemplary embodiment, the porous size exclusion carrier has a MWCO of greater than or equal to 40 kDa. 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 support has a MWCO of 80 kDa.In another exemplary embodiment, the porous size exclusion support has a MWCO of 90 kDa.

[0137] B. Cationic part

[0138] The matrix according to the present disclosure can include a porous size exclusion support and a cationic portion. In some aspects of the present disclosure, small molecules (such as but not limited to negatively charged molecules) can be separated from macromolecules (such as but not limited to positively charged molecules) based on one or more matrix properties. In some embodiments, one or more matrix properties can be the charge and / or size exclusion properties of the matrix.

[0139] In some embodiments, the cationic portion of the matrix of the present disclosure is an amine, an imine, or a combination thereof. In some aspects of the present disclosure, such cationic portions may include amine-containing polymers; alkylamines, particularly lower alkylamines (e.g., amylamine); amine-containing heterocyclic compounds; amine-containing aromatic compounds; or other amine / diamine compounds.

[0140] In an exemplary embodiment, prior to association with the porous size exclusion support, the amine-containing polymer is a branched polyethyleneimine (PEI) having a base structure as shown below, which becomes charged to provide a cationic moiety when exposed to appropriate conditions such as but not limited to an equilibration buffer.

[0141]

[0142] In another exemplary embodiment, 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 a basic structure as shown below, prior to association with the porous size exclusion support. When exposed to appropriate conditions (such as, but not limited to, an equilibration buffer), the diamine becomes charged to provide a cationic moiety.

[0143]

[0144] In another exemplary embodiment, the alkyl diamine is pentamethylenediamine (PDA) having the structure NH2(CH2)5NH2 (also shown below), which becomes charged to provide a cationic moiety when exposed to appropriate conditions (such as but not limited to an equilibration buffer) prior to binding to the porous size exclusion support.

[0145]

[0146] In another exemplary embodiment, prior to binding to the porous size exclusion support, the diamine is N,N diethylethylenediamine (DEED) having the structure shown below, which becomes charged to provide a cationic moiety when exposed to appropriate conditions such as but not limited to an equilibration buffer.

[0147]

[0148] In another aspect of the present disclosure, the amine-containing aromatic compound may include at least one C3 to C 15 In the specific disclosed aspects of the present disclosure, the amine-containing compound may have the general formula II, or enantiomers, diastereomers, tautomers, salts, solvates and / or isotope-substituted derivatives thereof.

[0149]

[0150] wherein, with respect to Formula II, J, Q, T, X, Y and Z are the same or different, and J, Q, T, X, Y and Z are each independently selected from nitrogen or CR c , where R cis independently selected at each occurrence 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 at least two R c The group contains an amino group and the remaining R c The group is hydrogen. In a specific embodiment, the compound of formula II can be selected from, but not limited to, 1,2-phenylenediamine having the structure shown below, 1,3-phenylenediamine having the structure shown below, and / or 1,4-phenylenediamine having the structure shown below. When exposed to suitable conditions (such as, but not limited to, an equilibrium buffer), each of these compounds becomes charged to provide a cationic moiety.

[0151]

[0152] In specific disclosed embodiments, an amine-containing heterocyclic compound can be associated with a size exclusion support comprising HEC. In one example, an epoxyamine compound can be associated with a size exclusion support comprising HEC. In some embodiments, the amine-containing compound 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 suitable conditions, such as, but not limited to, an equilibration buffer.

[0153]

[0154] In some embodiments, the matrix of the present disclosure comprises at least one cationic moiety associated with a porous size exclusion support. In some embodiments, the porous size exclusion support or the cationic moiety may comprise a reactive functional group. For example, the functional group on the porous size exclusion support may be used to interact and associate with one or more cationic moieties to form a matrix.

[0155] In some embodiments, the matrix comprises at least one cationic moiety immobilized to at least one size exclusion support. In some embodiments, the cationic moiety can be "immobilized" by covalently binding to the size exclusion support via formation of a covalent bond. In some embodiments, the covalent bond can 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 performed by first using any suitable oxidizing agent (some examples use periodate (IO 4- or IO6 5-)) is achieved by oxidizing the hydroxyl groups of HEC to aldehyde groups. The aldehyde groups generated by oxidation can react with the terminal amine on the cationic moiety to covalently bind the cationic moiety to the HEC support, and the resulting intermediate can be reduced to form an amine using a reducing agent (such as, but not limited to, sodium cyanoborohydride or methylpyridine borane).

[0156] In some embodiments, the epoxyamine can be fixed to the size exclusion support. The epoxyamine can be associated with and / or fixed to the size exclusion support comprising HEC by reacting with the hydroxyl groups provided by HEC. In some aspects of the present disclosure, the epoxyamine compound is a protected amine compound, wherein the protected amine compound is deprotected after being associated with or fixed to the size exclusion support.

[0157] 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 molecules 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.

[0158] In specific disclosed embodiments, the functional groups on the cationic moiety can react with functional groups on the porous size exclusion support and / or small molecules (such as but not limited to negatively charged small molecules) to separate or extract from at least one macromolecule. The functional groups can include, but are not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide and / or any other functional group suitable for associating and / or interacting the cationic moiety with the porous size exclusion support and / or small molecule.

[0159] In another embodiment, the functional group comprises R representing a reactive functional moiety. x ; or represents a reactive functional portion R attached to the porous size exclusion support or portion via a covalent bond L x (-LR x) represents at least one reactive group. The reactive group serves as a site for association, attachment and / or interaction with a part or a small molecule, wherein the reactive group reacts chemically with a suitable reactive group or functional group, a part or a small molecule on a porous size exclusion carrier. In an exemplary embodiment, the reactive group or functional group can be an activated ester of acrylamide, a carboxylic acid, an acyl halide, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a borate, a thioboronic acid group (thioboronate group), a carboxylic acid, a diazoalkane, a haloacetamide, a halogenated triazine, a hydrazine, a hydrazide, an imino ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a sulfonyl halide, a thiol group, a sulfide group, a disulfide group, an epoxy group, an epithio group, a thioester group, an alcohol group, an activated alcohol group, a phosphoric acid group, a phosphate group and / or a photoactivated group.

[0160] In another exemplary embodiment, the reactive groups or functional groups may include electrophiles and / or nucleophiles, and in some embodiments, covalent bonds may be generated between them. Exemplary electrophiles and nucleophiles functional groups may be aryloxy groups or aryloxy groups substituted one or more times with electron withdrawing substituents such as nitro, fluorine, chlorine, cyano, trifluoromethyl, or combinations thereof to form activated aryl esters; or activated by carbodiimide to form anhydrides or mixed anhydrides-OCOR a or -OCNR a NHR b Carboxylic acid, where R a and R b can be the same or different, and are C1-C6 alkyl, C r C6 perfluoroalkyl or C1-C6 alkoxy; or cyclohexyl, 3-dimethylaminopropyl, acyl halide, acyl nitrile, aldehyde, alkyl halide, acid anhydride, aryl halide, aziridine, diazoalkane, haloacetamide, halogenated triazine, isocyanate, isothiocyanate, maleimide, phosphoramidite, sulfonyl halide, sulfide, disulfide, epoxy and epithio, thioester, activated alcohol, phosphoric acid, phosphate ester and photoactivated group. Acyl azide can also be rearranged as isocyanate.

[0161] In some embodiments, in addition to the reactive functional part, the reactive group further includes a joint L. The joint can be used for covalently linking reactive functional groups. When present, the joint is a single covalent bond or a series of stable bonds. The reactive functional part can be directly attached (wherein the joint is a single bond) to a solid support, part or to a small molecule through a series of stable bonds. When the joint is a series of stable covalent bonds, the joint generally includes several non-hydrogen atoms selected from C, N, O, S, Si, B and P. In addition, the covalent bond can include platinum atoms such as described in U.S. Patent No. 5,714,327. When the joint is not a single covalent bond, the joint can be any combination of stable chemical bonds, and the stable chemical bonds optionally include single, double, three or aromatic carbon-carbon bonds, and 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 joint comprises less 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 joint is a combination of a single covalent bond or a single carbon-to-carbon bond and carboxamide, sulfonamide or thioether bonds. In some embodiments, the following parts may be present in the joint: ether, thioether, carboxamide, thiourea, sulfonamide, urea, urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl and amine moiety. The example of L comprises substituted or unsubstituted polymethylene, arylidene, alkylarylidene, aryl or aryl thiol.

[0162] Any combination of linkers can be used to attach functional groups or reactive groups. When the reactive group is a maleimide or haloacetamide, the resulting compound can be particularly useful for conjugation with thiol-containing species. In the case where the reactive group is a hydrazide, the resulting compound can be particularly useful for conjugation with periodate-oxidized carbohydrates and glycoproteins.

[0163] In some embodiments, the cationic moiety can include open and / or unreacted amines. For example, a matrix comprising a size exclusion carrier and a cationic moiety (such as, but not limited to, PDA) can include open / unreacted amines. In a specific disclosed embodiment, the open / unreacted amines on the PDA can react with polysaccharide-derived molecules having a molecular weight range of 0 to 2,000,000. In an exemplary embodiment, the open and / or unreacted amines on the cationic moiety PDA react with a dextran having a molecular weight of 1,000,000.

[0164] In some embodiments, the concentration of the cationic moiety added to the matrix can be in the range of 50mg / mL to 175mg / mL. In some embodiments, the concentration of the cationic moiety is 50mg / mL to 100mg / mL, 50mg / mL to 75mg / mL, 50mg / mL to 150mg / mL, 100mg / mL to 155mg / mL, 130mg / mL to 175mg / mL. In an exemplary embodiment, the concentration of the cationic moiety can be 50mg / mL. In another exemplary embodiment, the concentration of the cationic moiety can be 75mg / mL. In another exemplary embodiment, the concentration of the cationic moiety can be 150mg / mL.

[0165] Figure 1 A non-limiting exemplary embodiment is depicted in which a porous size exclusion support 10 is produced with 129 grams of HEC and 265 milliliters of Epi to prepare a 1.5 liter resin bed, and in which the HEC is modified with a cationic PDA moiety 20 to form a matrix 30. The matrix 30 has a MWCO of 50 kDa and an overall positive charge.

[0166] Figure 2 Another non-limiting exemplary embodiment of a porous size exclusion support 10 is depicted, produced with 129 grams of HEC and 265 milliliters of Epi to prepare a 1.5 liter resin bed, and wherein the HEC is modified with branched PEI cationic moieties 40 to form a matrix 50. The matrix 50 has a MWCO of 50 kDa and an overall positive charge.

[0167] Figure 3 Another non-limiting exemplary embodiment of a porous size exclusion support 10 is depicted, produced with 129 grams of HEC and 265 milliliters of Epi to prepare a 1.5 liter resin bed, and wherein 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.

[0168] C. Equilibration Buffer

[0169] In certain aspects of the present disclosure, a buffer may be provided such that it increases the binding capacity of the matrix to smaller sample components while also reducing the binding capacity of the matrix to larger sample components. For example, and without being bound by a single theory of operation, the buffer increases the binding capacity of the cationic moiety to small molecules (such as, but not limited to, negatively charged small molecules) in a sample containing small molecules; and reduces the binding capacity of the cationic moiety to macromolecules (such as, but not limited to, positively charged macromolecules). In specific disclosed embodiments, additional buffers may be used to improve the recovery of larger sample components.

[0170] In some embodiments, the buffer is an equilibrium buffer. In another embodiment of the present disclosure, the equilibrium buffer contains substantially no or no salt, such as but not limited to NaCl. The equilibrium buffer comprising substantially no amount of salt generally refers to a salt greater than 0mM salt to 10mM salt, preferably greater than 0mM salt to 5mM salt. In some aspects, the equilibrium buffer can have a pH of 4 to 9, and in another embodiment, the equilibrium buffer can have a positive charge. In a specifically disclosed embodiment, the balance can include a neutral charge.

[0171] In some embodiments, the balancing buffer with positive charge 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, i.e., a buffer solution comprising 2-amino-2-(hydroxymethyl)propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane); and triethylammonium bicarbonate.

[0172] In some aspects of the present disclosure, the balancing buffer may have a pH of 4 to 9, such as a pH of 5 to 8, for example a pH of 5 to 7. In specific aspects of the present disclosure, the balancing buffer has a pH of 5, a pH of 7, and / or a pH of 8.5. In an exemplary embodiment, the balancing buffer is sodium acetate having a pH of 5. In another exemplary embodiment, the balancing buffer is sodium acetate having a pH of 7. In another exemplary embodiment, the balancing buffer is HEPES having a pH of 5. In another exemplary embodiment, the balancing buffer is HEPES having a pH of 7. In another exemplary aspect, the balancing buffer is borate having a pH of 5. In yet another exemplary embodiment, the balancing buffer is borate having a pH of 8.5.

[0173] In some embodiments, the equilibration buffer is a salt-free (eg, sodium chloride-free) charging buffer.

[0174] In some embodiments, the equilibration buffer is used at a concentration of 1 mM to 100 mM, such as 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.

[0175] Figure 4 A method is shown for separating small molecules 90 from a sample containing macromolecules 100 using a matrix 80 having cationic moieties 82 associated therewith. Small molecules 90 associate with cationic moieties 82 to form composition 110. Positively charged buffers can be used to facilitate the separation of small molecules (e.g., negatively charged molecules such as BSA) from macromolecules (e.g., positively charged molecules such as IgG).

[0176] IV. System

[0177] The present disclosure also relates to embodiments of systems comprising one or more disclosed matrices and further comprising containers. Such systems provide one or more advantages, including, but not limited to: economic feasibility; simplicity and ease of use; providing faster results relative to conventional products in the art; adaptability as a single-use disposable unit; adaptability for high-throughput sample preparation in a porous container format; and adaptability for automated and robotic sample preparation systems. Reducing the concentration of small molecules in a sample using the system provided herein provides rapid recovery of biomolecules, as well as excellent purity of biomolecules and their derivatives that can be used for downstream applications.

[0178] 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 characteristics, such as, but not limited to, the size of the molecule, the charge of the molecule, the isoelectric point (pI) of the molecule, and / or any combination of these characteristics. The system may include: (i) a container having at least one size exclusion carrier and at least one cationic moiety that can be associated with one or more small molecules; and (ii) a receiver positioned to receive a flow from the container. In addition, molecules can be separated from each other based on one or more separation matrix characteristics, such as the charge and size exclusion characteristics of the size exclusion carrier associated with at least one cationic moiety. In some embodiments of the disclosed system, the receiver is attached to a column. In some embodiments, the receiver is detachable from the column. The contents of the receiver can be used or removed by a user as needed. In some embodiments, the receiver collects a sample containing significantly reduced small molecules. In some embodiments, the receiver collects a sample without small molecules.

[0179] The system can be operably configured to operate by gravity flow. Alternatively, external, actively applied pressure or force, such as centrifugal force, positive pressure, negative pressure, vacuum, and combinations thereof, can be applied to promote flow. Structures that allow the application of the above pressure or force include, but are not limited to: a syringe that can be pumped to generate positive pressure; a vacuum frit for generating negative pressure; and / or a tube or container suitable for use in a commercially available centrifuge or rotating device. In some embodiments, the system can be configured for use with or in a centrifuge tube or any other similar rotating instrument.

[0180] In some embodiments of the disclosed systems, the container is a columnar container, a tube, a porous tube, a porous plate, or a porous filter plate. Exemplary containers include, but are not limited to, test tubes, spin columns, porous plates, porous filter plates, microplates, or microporous filter plates.

[0181] Figure 5An exemplary separation system 200 is depicted that includes a container 240 and a sample flowing through the container. The sample contains small molecules 210 and macromolecules 230. The container 240 contains a matrix 220 that is configured to separate the small molecules 210 from the macromolecules 230 using differences in one or more properties, such as, but not limited to, the size of the molecules, the charge of the molecules, the isoelectric point (pI) of the molecules, and / or a combination of these properties. The system 200 also includes a receiver 250 for recovering the macromolecules 230.

[0182] Figure 6 An exemplary separation system 300 according to one embodiment of the present disclosure is depicted. The system 300 includes: a container 310 (such as a cylindrical tube, a test tube, or a spin column); a matrix 320 contained by the container 310; a receiver 330 located below the container 310, which is adapted or configured to receive a fluid flowing through the container 310 through the bottom end 340 of the container. In some embodiments, the container 310 may also include one or more frits (not depicted). In some embodiments, the system 300 may include an optional cover 350 that can be used to secure the container 310 at the top end 360. In some embodiments, the receiver 330 can be detachable from the container 310 so that a user can collect the flow-through. In some embodiments, the receiver 330 has a twist-off tab configuration for removal. In other embodiments, the receiver 330 can be threadedly coupled to the container 310, or attached using complementary mating pieces that can be pulled apart.

[0183] Figure 7 4 is a perspective view of an exemplary system 400 including a porous container 410 according to one embodiment of the present disclosure. The porous container 410 includes a wall defining a plurality of pores 412. The wall may include an end 420, a side 430, and a top 440 defining a plurality of pores. The porous container 410 contains a matrix comprising at least one size exclusion support 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. Figure 7 Shown, porous container can comprise optional container cover 450.Lid 450 can be any suitable removable / detachable structure, such as foil, transparent wrapper or tear-off seal.Porous container 410 can be porous plate, porous plate filter, microplate or microtiter plate, comprise the flat plate that comprises a plurality of holes 412, wherein each hole is used as little test tube or container.For high throughput purposes, porous plate has various forms and can comprise 6,12,24,48,96,384,1536,3456,9600 or more holes that are arranged as rectangular matrix or array.

[0184] Figure 8An exemplary system 500 including a porous container 510 according to one embodiment is depicted. The system 500 also includes a receiver 550 located below the container 510, which is adapted or configured to receive fluid flowing out of the container. The container 510 includes an end 520, a top 540, and a side 530. In some embodiments, the receiver 550 may include a porous plate to collect the flow-through liquid. The receiver 550 may be removable and can be collected by a user. In some embodiments, the receiver 550 is a wash plate or a collection plate.

[0185] The system according to the present disclosure may be fully automated or may be a manually operated system. In some embodiments, the system may be partially manually operated and partially operated by automation.

[0186] System can also include computer system, and this computer system includes CPU, hardware element and / or software element.Suitable computer system is operable to control various parts of system, such as to extract circulation liquid and analyze circulation liquid control robot.In some embodiments, computer system and / or its parts can be physically resident in system 300,400 or 500 or can be resident outside.Computer system used herein can include data analysis and control system, data transmission system such as read-write CD ROM drive or DVD drive, at least one USB port and / or at least one Ethernet port.In some embodiments, computer system can include preloaded software and / or application specific integrated circuit (ASICS) to control disclosed system, such as system 300,400,500 and / or other parts of system, including sample handling and analysis, display and / or output result.

[0187] The disclosed system embodiments may also optionally include 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, such as one or more macromolecules.

[0188] The system may also include additional devices or components, such as, but not limited to, a power supply, a display unit, such as a monitor operable to observe sample processing and / or monitor the extraction of biomolecules from the sample; a spectrophotometer; a device to measure nucleic acid extraction; a device to further process the extracted biomolecules for further analysis; a printer, etc. The system of the present disclosure may be configured to be mounted on a laboratory bench top.

[0189] V. Preparation Method

[0190] Embodiments of the present disclosure also relate to methods for preparing a matrix or system according to the present disclosure. In some embodiments, the method of preparation comprises immobilizing a cationic moiety to a porous size exclusion support.

[0191] The porous size exclusion support may comprise spherical beads made of a gel or gel-like material having pores.Some exemplary size exclusion supports comprise agarose, polyacrylamide, a cellulosic material (eg, hydroxyethyl cellulose), and / or derivatives thereof.

[0192] In some embodiments, the porous size exclusion carrier can be cross-linked with at least one cross-linking agent. The pore size range of the porous size exclusion carrier determines the size of the molecule that can be included or excluded from entering the porous size exclusion carrier. Not bound by this theory, it is currently believed that when the sample solution passes through the porous size exclusion carrier, the molecule with a molecular weight less than or substantially equal to MWCO is forced to follow a circuitous path before leaving the porous size exclusion carrier later. On the other hand, macromolecules take a relatively direct path through the porous size exclusion carrier. Therefore, when the sample travels through the size exclusion carrier, the flow rate difference between small molecules and macromolecules allows the macromolecules that flow faster to be separated from the small molecules that flow slower.

[0193] Embodiments of the disclosed porous size exclusion carrier may include a product formed by reacting 50 grams (g) to 250 grams (g) of HEC. In some embodiments, for a 1.5L resin bed, the disclosed size exclusion carrier may include HEC in the range of 80 grams to 130 grams of HEC. In an exemplary embodiment, 80g of HEC is used to produce the porous size exclusion carrier. In another exemplary embodiment, 90g of HEC is used to produce the porous size exclusion carrier. In another exemplary embodiment, 108g of HEC is used to produce the porous size exclusion carrier. In another exemplary embodiment, 129g of HEC is used to produce the porous size exclusion carrier. In another exemplary embodiment, 147g of HEC is used to produce the porous size exclusion carrier. In another exemplary embodiment, 216g of HEC is used to produce the porous size exclusion carrier.

[0194] In some embodiments, a porous size exclusion carrier is produced by cross-linking HEC with a cross-linking agent. For example, HEC can be cross-linked with epoxides such as, but not limited to, epichlorohydrin (Epi). In some embodiments, a porous size exclusion carrier can be cross-linked with Epi in the range of 250mL to 450mL. For a 1.5L resin bed, the carrier can be produced by epichlorohydrin in the range of 250mL to 450mL. In an exemplary embodiment, 265mL of Epi is used to cross-link a porous size exclusion carrier. In another exemplary embodiment, 303mL of Epi is used to cross-link a porous size exclusion carrier. In another exemplary embodiment, 375mL of Epi is used to cross-link a porous size exclusion carrier. In another exemplary embodiment, 397mL of Epi is used to cross-link a porous size exclusion carrier. In another exemplary embodiment, 410mL of Epi is used to cross-link a porous size exclusion carrier. In another exemplary embodiment, 441mL of Epi is used to cross-link a porous size exclusion carrier.

[0195] In some embodiments, the cationic moiety is fixed to the porous exclusion carrier by forming a covalent bond. In some embodiments, the covalent bond can be formed by reaction, such as but not limited to amidation, alkylation, amination or other reactions forming covalent bonds. In some such embodiments, the functional group (e.g., hydroxyl group) of the size exclusion carrier is oxidized (such as by using periodate) to produce an aldehyde. The aldehyde groups produced can react with the terminal amine on the cationic moiety to form an imine intermediate, which can be chemically reduced to form an amine.

[0196] In some embodiments, 15mg / mL to 35mg / mL of an oxidant (such as periodate) is added to oxidize the size exclusion carrier. In some embodiments, 15mg / mL, 16mg / mL, 17mg / mL, 18mg / mL, 19mg / mL, 20mg / mL, 21mg / mL, 22mg / mL, 23mg / mL, 24mg / mL, 25mg / mL, 26mg / mL, 27mg / mL, 28mg / mL, 29mg / mL, 30mg / mL, 31mg / mL, 32mg / mL, 33mg / mL, 34mg / mL, 35mg / mL of periodate are added to oxidize the size exclusion carrier. In some embodiments, sodium periodate is used to oxidize the size exclusion carrier.

[0197] In some embodiments, 5 mg / mL to 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, 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.

[0198] 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.

[0199] In some embodiments, the concentration of the cationic moiety for modifying the support material can be in the range of 50mg / mL to 175mg / mL. In some embodiments, the concentration of the cationic moiety can include 50mg / mL to 170mg / mL, 60mg / mL to 165mg / mL, 65mg / mL to 160mg / mL, 70mg / mL to 155mg / mL, 75mg / mL to 150mg / mL, 80mg / mL to 145mg / mL, 85mg / mL to 140mg / mL, 90mg / mL to 135mg / mL or 95mg / mL to 130mg / mL. In an exemplary embodiment, the concentration of the cationic moiety is 50mg / mL. In another exemplary embodiment, the concentration of the cationic moiety is 75mg / mL. In another exemplary embodiment, the concentration of the cationic moiety can be 150mg / mL.

[0200] In an exemplary embodiment, sodium metaperiodate is dissolved in water and mixed with a porous size exclusion support matrix bed containing HEC to oxidize the vicinal diol present in the HEC to an aldehyde group. The mixture is reacted at a temperature ranging from 15°C to 30°C for a suitable period of time, such as 2 hours to 4 hours. In an exemplary embodiment, the mixture is reacted at room temperature for at least four hours under constant overhead stirring. In some embodiments, during the reductive amination step (i.e., reacting the amine groups on the cationic part with the aldehyde groups on the size exclusion support matrix bed and reducing the intermediate imine to an amine), the cationic part is 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 at 20°C 30°C under stirring for a suitable period of time, such as 8 hours to 12 hours, and washed with water and NaCl.

[0201] VI. How to use

[0202] Certain disclosed embodiments relate to a method of separating at least one macromolecule from at least one small molecule using a difference in one or more properties, such as, but not limited to, the size of the molecule, the charge of the molecule, the isoelectric point (pi) of the molecule, and / or any combination of these properties. The method may include: applying a sample to a porous size exclusion carrier, the porous size exclusion carrier comprising at least one cationic moiety that can bind or associate with the small molecule; and subjecting the container to gravity flow, centrifugal force, positive pressure, negative pressure, vacuum, or a combination thereof. The macromolecules in the sample can be excluded by the porous size exclusion carrier and collected as a flow-through. The one small molecule can interact with the cationic moiety, thereby separating from the macromolecules in the sample using a difference in one or more properties, such as, but not limited to, the size of the molecule, the charge of the molecule, the isoelectric point (pi) of the molecule, and / or any combination of these properties.

[0203] In certain aspects of the present disclosure, the equilibration buffer disclosed herein can be provided such that it increases the binding capacity of the matrix for smaller sample components while also decreasing the binding capacity of the matrix for larger sample components.

[0204] Currently known methods for removing small molecules (such as but not limited to BSA) from larger molecules (such as but not limited to antibodies) in samples require initial steps, such as pre-salting steps to remove salt from antibody solutions. Without being bound by operating theory, salts produce counterions and will reduce the removal of BSA in subsequent steps. The present disclosure allows the separation of small molecules (such as but not limited to BSA) to be removed from larger molecules (such as but not limited to antibodies) in one step. Therefore, embodiments of the present disclosure do not require a pre-desalting step to achieve desired BSA removal and antibody recovery, such as but not limited to IgG. Therefore, in some embodiments, separation of small molecules from the remainder of the sample is performed in one step.

[0205] In some embodiments, the flow-through is collected in a receiver positioned below the container. In some embodiments, small molecules may constitute sample impurities or contaminants.

[0206] The sample that can be processed by the method of the present disclosure can be any type of biological or clinical sample containing biomolecules or derivatives thereof from which small molecules must be separated or removed. Some exemplary non-limiting samples include samples with small molecule BSA and large molecule IgG.

[0207] 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 a sample.

[0208] VII. Kit

[0209] The present disclosure also describes kits for practicing the methods discussed herein and / or kits containing matrices and / or kits containing the systems discussed herein.

[0210] In some embodiments, the present disclosure describes a kit for separating macromolecules from small molecules using differences in one or more properties, such as, but not limited to, the size of the molecule, the charge of the molecule, the isoelectric point of the molecule, and / or any combination of these properties. The kit may include: a system comprising (i) a container containing a size exclusion carrier, the size exclusion carrier comprising a cationic moiety associated with the carrier, wherein the cationic moiety may also be associated with a small molecule; and (ii) a receiver located below the container, wherein the device is operably configured for gravity flow, or may be operated by applying centrifugal force, positive pressure, negative pressure, vacuum, and combinations thereof.

[0211] In some embodiments of the kits of the present disclosure, the device is a spin column, a multiwell filter plate, or a multiwell plate.The kit may also include one or more equilibration buffers packaged in one or more separate containers or contained in the first container.

[0212] In some embodiments, the equilibrium buffer does not contain salt (e.g., ionic salts such as NaCl). In other aspects disclosed herein, the equilibrium buffer may contain a small amount of salt. In some aspects, the equilibrium buffer may have a pH of 4 to 9, and in another embodiment, the equilibrium buffer may have a positive charge. In a specifically disclosed embodiment, the equilibrium may contain a neutral charge.

[0213] In some embodiments, the equilibration buffer may include, but is not limited to, carbonate buffer, bicarbonate buffer, phosphate buffer, citric acid / citrate buffer. In an exemplary embodiment, the buffer comprises a Tris buffer. In another exemplary embodiment, the buffer comprises a TEAB buffer.

[0214] In some aspects of the present disclosure, the balancing buffer may have a pH of 4 to 9, such as a pH of 5 to 8, such as a pH of 5 to 7. In specific aspects, the balancing buffer has a pH of 5, a pH of 7, and / or a pH of 8.5. In one exemplary embodiment, the balancing buffer is sodium acetate having a pH of 5. In another exemplary embodiment, the balancing buffer is sodium acetate having a pH of 7. In another exemplary embodiment, the balancing buffer is HEPES having a pH of 5. In another exemplary embodiment, the balancing buffer is HEPES having a pH of 7. In another exemplary aspect, the balancing buffer is borate having a pH of 5. In yet another exemplary embodiment, the balancing buffer is borate having a pH of 8.5.

[0215] In some embodiments, the positively charged equilibration buffer is present at a concentration of 1mM to 100mM, 2mM to 75mM, 5mM to 50mM, 10mM to 25mM, 5mM to 25mM. In some embodiments, the positively charged equilibration buffer is present at a concentration of 1mM to 100mM, 2mM to 75mM, 5mM to 50mM, 10mM to 25mM, 5mM to 25mM Tris buffer. In an exemplary embodiment, the equilibration buffer concentration can be 50mM.

[0216] The 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 multiwell plates.

[0217] The reagent and component of test kit can be included in one or more suitable containers.Container can comprise at least one vial, test tube, flask, bottle, syringe or other container that component can be placed into and preferably suitably aliquoted generally.When there is more than one component in test kit, if suitable, described component can be packaged together, or test kit can contain second container, the third container or other additional container that can be placed separately usually with additional component.However, in some embodiments, some combination of component can be packaged together, be included in a container utensil.Test kit can also comprise for accommodating any reagent container for commercial sale in closed restriction mode.Such container can comprise injection or blow-molded plastic container that expects vial is retained therein.

[0218] In some embodiments, the components of the kit of the present disclosure may be pre-filled with one or more reagents for processing a sample and may be appropriately aliquoted into appropriate chambers. The kit or its container may have a seal for maintaining the internal compartment and any contents therein sterile and spill-proof.

[0219] Some components of the test kit are provided in the form of one and / or more liquid solutions. The liquid solution can be a non-aqueous solution, an aqueous solution, and can be a sterile solution. The components of the test kit can also be provided in the form of a dry powder. When the reagents and / or components are provided in the form of a dry powder, the powder can be reconstituted by adding a suitable solvent. It is contemplated that a suitable solvent can also be provided in another container apparatus. The test kit can also include a container apparatus for holding a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0220] The kits of the present disclosure may also include instructions for employing the kit components, and may also have instructions for using any other reagents not included in the kit. The instructions may include variations that may be implemented.

[0221] VIII. Overview of Several Implementation Schemes

[0222] Disclosed herein are embodiments of a matrix comprising: 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.

[0223] In any or all embodiments, the porous size exclusion support has a molecular weight cut-off greater than or equal to 40 kDa.

[0224] In any or all embodiments, the at least one cationic moiety is covalently bound to the porous size exclusion support.

[0225] In any or all embodiments, the at least one cationic moiety comprises an amine, a diamine, a polyamine, or an amine-containing polymer.

[0226] In any or all embodiments, the amine-containing polymer is polyethyleneimine.

[0227] In any or all embodiments, the diamine is pentamethylenediamine, N,N-diethylethylenediamine, or a combination thereof.

[0228] In any or all embodiments, the porous size exclusion support comprises hydroxyethyl cellulose.

[0229] In any or all embodiments, epichlorohydrin is also included.

[0230] Also disclosed herein are embodiments of a system for separating one or more molecules in a sample using the molecular weight of one or more molecules, the charge of one or more molecules, the isoelectric point (pI) of one or more molecules, or a combination thereof, the system comprising a container comprising the matrix of the above embodiments and a receiver positioned to receive a flow from the container.

[0231] In any or all embodiments, the system is configured for gravity flow operation, centrifugal force operation, positive pressure operation, negative pressure operation, vacuum operation, or a combination thereof.

[0232] In any or all embodiments, the container is a columnar container, a tube, a porous tube, a porous plate, or a porous filter plate.

[0233] Also disclosed herein is a method for preparing a multimodal resin, the method comprising: providing a porous size exclusion support, the porous size exclusion support comprising hydroxyethyl cellulose having a MWCO greater than or equal to 40 kDa, 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 portion via reductive amination.

[0234] In any or all embodiments, hydroxyethyl cellulose is used in an amount ranging from 50 g to 250 g to produce the porous size exclusion support.

[0235] In any or all embodiments, the method further comprises providing a cross-linking agent to cross-link the porous size exclusion support.

[0236] In any or all embodiments, the cross-linking agent is epichlorohydrin.

[0237] In any or all embodiments, epichlorohydrin is used in an amount ranging from 250 mL to 450 mL.

[0238] In any or all embodiments, the cationic moiety is pentamethylenediamine, polyethyleneimine, or N,N-diethylethylenediamine.

[0239] In any or all embodiments, the cationic moiety has a concentration range of 50 mg / mL to 175 mg / mL.

[0240] 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 can separate the 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 applying the sample to the matrix to separate the one or more molecules by 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 a flow-through, and wherein the one or more molecules are associated with the at least one cationic moiety and thereby separated from the sample in a single step.

[0241] In any or all embodiments, the flow-through is collected in a receiver positioned below the container.

[0242] 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.

[0243] In any or all embodiments, the equilibration buffer is sodium acetate having a pH of 5 or a pH of 7, HEPES having a pH of 5 or a pH of 7, or borate having a pH of 5 or a pH of 8.5.

[0244] In any or all embodiments, the positively charged buffer is Tris buffer or triethylammonium bicarbonate.

[0245] In any or all embodiments, the first of the two or more molecules includes: at least one small molecule having a molecular weight range of less than 100 kDa; and the second of the one or more molecules includes at least one macromolecule having a molecular weight range of greater than or equal to 100 kDa.

[0246] In any or all embodiments, the one or more molecules is bovine serum albumin.

[0247] In any or all embodiments, one or more molecules are antibodies.

[0248] In any or all embodiments, the antibody is IgG.

[0249] In any or all embodiments, one or more molecules having an isoelectric point value 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.

[0250] In any or all embodiments, one or more molecules are more negatively charged molecules than other molecules having positively charged molecules.

[0251] In any or all embodiments, at least one cationic moiety is associated with one or more molecules through ionic interactions, hydrophilic interactions, hydrophobic interactions, affinitive interactions, hydrogen bonding, or van der Waals forces.

[0252] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged macromolecule, and the size exclusion support comprises HEC and Epi, and the cationic moiety is pentamethylenediamine; wherein greater than 80% of the at least one negatively charged molecule is separated and greater than 80% of the positively charged macromolecule is recovered as a flow-through.

[0253] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged macromolecule, and the size exclusion support comprises HEC and Epi, the cationic portion is pentamethylenediamine, and the cationic portion is modified with dextran; wherein greater than 80% of the at least one negatively charged molecule is separated and greater than 80% of the positively charged macromolecule is recovered as a flow-through.

[0254] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged macromolecule, and the size exclusion support comprises HEC and Epi, and the cationic moiety is N,N-diethylethylenediamine; wherein greater than 50% of the at least one negatively charged molecule is separated and greater than 90% of the positively charged macromolecule is recovered as a flow-through.

[0255] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged macromolecule, and the size exclusion support comprises HEC and Epi, and the cationic moiety is pentamethylenediamine; the equilibration buffer is Tris buffer; and wherein greater than 75% of the at least one negatively charged molecule is separated and greater than 80% of the positively charged macromolecule is recovered as flow-through.

[0256] In any or all embodiments, the negatively charged molecule is bovine serum albumin and when the cationic moiety is pentamethylenediamine and the equilibration buffer is Tris buffer, the cationic moiety has a binding capacity for bovine serum albumin of 1.6 mg / mL.

[0257] In any or all embodiments, the negatively charged molecule is bovine serum albumin and when the cationic moiety is polyethyleneimine and the equilibration buffer is Tris buffer, the cationic moiety has a binding capacity for bovine serum albumin of 3.11 mg / mL.

[0258] In any or all embodiments, the one or more molecules include at least one negatively charged small molecule and at least one positively charged macromolecule, and the size exclusion support comprises HEC, and the cationic portion is polyethyleneimine; the equilibration buffer is Tris buffer; and wherein greater than 70% of the positively charged macromolecules are recovered as flow-through.

[0259] Also disclosed herein is an embodiment of a kit for separating positively charged macromolecules from one or more negatively charged small molecules in a sample, the kit comprising: a porous size exclusion support having at least one cationic portion associated therewith, wherein the cationic portion is capable of associating with and capturing at least one negatively charged small molecule; and instructions for using the porous size exclusion support.

[0260] In any or all embodiments, the kit further comprises an equilibration buffer.

[0261] In any or all embodiments, the equilibration buffer is Tris buffer or triethylammonium bicarbonate.

[0262] In any or all embodiments, the kit further comprises a system comprising: a container containing a porous size exclusion support; and a receiver positioned to receive a flow through the support.

[0263] 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.

[0264] In any or all embodiments, the container of the kit is a spin column, a multiwell filter plate, or a multiwell plate.

[0265] IX. Practical Example

[0266] Aspects of the present teachings may be further understood according to the following examples. A matrix comprising at least one size exclusion carrier and at least one cationic moiety for separating and / or extracting one or more small molecules from a sample is prepared and tested, and the matrix can associate with one or more small molecules using differences in one or more properties (such as, but not limited to, the size of the molecule, the charge of the molecule, the isoelectric point of the molecule, and / or any combination of these properties). In some embodiments, an exemplary size exclusion carrier is modified with a cationic moiety comprising a functional group that can associate with a negatively charged small molecule by ionic interaction, hydrophilic interaction, or any other interaction, and the cationic moiety can associate with a negatively charged small molecule and thereby remove the negatively charged small molecule from the sample while allowing at least one positively charged macromolecule in the sample to be excluded and collected.

[0267] 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. TM The Rapid Gold BCA Protein Kit was used to measure the respective concentrations of BSA and IgG as the proteins were spun through separately. Quantitative iBright analysis of SDS-Page results provided an accurate estimate of the recovery of BSA and IgG as the IgG-BSA mixture was spun through the resin. In addition, the following examples address the issue of BSA directly competing with IgG during antibody labeling without adversely affecting the original amount of IgG, and demonstrate superior performance relative to existing commercial resins in separating BSA from IgG.

[0268] In addition, in some of the following examples, BSA removal and IgG purification kits were used. For example, the Abcam BSA removal kit (ab173231) commercially available from Abcam and a kit containing Melon gel resin for binding and removal of serum proteins, such as the kit from Thermo Scientific TMMelon TM In addition, cross-linked agarose beads conjugated to Cibacron Blue F3GA dye, such as those commercially available from Bio-Rad, were used in the following examples. Blue Gel.

[0269] Although these exemplary small molecules and listed molecular weight ranges are used for experimental demonstration, one of ordinary skill in the art will recognize that the present embodiments are not limited to these small molecules or molecular weight ranges, and that the teachings herein enable one of ordinary skill in the art to prepare and use matrices and systems for the removal of a variety of small molecules and molecular weight ranges.

[0270] Example 1

[0271] Preparative Chemistry: A porous size exclusion support comprising HEC cross-linked with Epi in the presence of a solvent containing a non-polar phase and a surfactant (ie, non-ionic detergent) was modified according to Scheme 1 to produce the embodiments of Table 1.

[0272]

[0273]

[0274] Next, the vicinal diols positioned on these size exclusion support columns were oxidized using periodate to generate aldehyde groups. PDA, branched PEI or DEED were prepared in PBS, the pH was adjusted to a range of 8.0 to 8.5, and reacted with the oxidized columns according to the following process. Different size exclusion supports with different MWCO (see Table 1) were reacted with cationic reagents PDA and DEED to produce the multimodal resins of Table 2 according to Scheme 2.

[0275]

[0276]

[0277] Scheme 2 includes the following steps: (1) preparing 100 mL of size exclusion base matrix resin; (2) dissolving 2.3 g of sodium metaperiodate in water to prepare 100 mL of 23 mg sodium metaperiodate / mL resin; (3) adding 100 mL of 23 mg sodium metaperiodate / mL resin to 100 mL of size exclusion base resin bed; (4) allowing the reaction to proceed at room temperature for 4 hours with constant overhead stirring to oxidize the vicinal diols present in HEC to aldehyde groups; (5) preparing a 100 mL volume of 50 mg / mL to 150 mg / mL HEC in PBS. / mL concentration of cationic moiety, such as but not limited to PDA, PEI or DEED, and adjusting the pH of the solution to a range of 8.0 to 8.5; (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), and adding 10 mg of sodium cyanoborohydride / mL of resin for reductive amination; (7) allowing the reaction to proceed at room temperature for 8 to 12 hours with constant overhead stirring; and (8) washing the resin with 2 bed volumes of water, 2 bed volumes of NaCl, and 2 bed volumes of water.

[0278] Method used to evaluate the effect of the resin on the BSA-IgG mixture: (1) Spin 562 μL of the resin bed at 3000X G for 1 minute to remove the storage solution; (2) Equilibrate the resin with buffer (3X 300 μL); (3) Spin at 3000X G for 1 minute to remove the buffer; (4) Add 100 μL of BSA-rabbit IgG mixture (BSA 10 mg / mL and IgG 1 mg / mL) to the resin; (5) Spin at 3000X G for 2 minutes to collect the flow-through containing unbound IgG and unbound BSA; (6) Prepare a 1:10 dilution of the flow-through and the starting solution; add 10 μL / well to a 4% to 20% Tris glycine gel; (7) Run the gel at 200 V for 45 minutes to 50 minutes; (8) Stain / de-stain the gel using a Pierce Power stainer.

[0279] Example 2

[0280] Effect of Resin MWCO 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 of Table 2 were compared to determine the effect of increasing MWCO on the removal of BSA from a mixture of BSA (2 mg / mL) and IgG (2 mg / mL).

[0281] Fig. 9 Figure 2 is a gel image showing the removal of BSA from the BSA / IgG described above and in Table 2. Fig. 9As shown, the BSA removal properties indicated by the disappearance of the lower band in the gel increased in the following order: Resin F embodiment (45kDa MWCO) > Resin D embodiment (40kDa MWCO) > Resin E embodiment (30kDa MWCO) > Resin B embodiment (7kDa MWCO) > Resin A embodiment (2kDa MWCO). Therefore, except for the PDA-modified embodiment, MWCO has a significant effect on the removal of BSA from the sample.

[0282] Example 3

[0283] Effect of PDA modification and equilibration buffer: In this example, Resin 5 embodiment of Table 1, Resin L embodiment of Table 2, Resin F embodiment of Table 2, and Resin D embodiment of Table 2 were used, wherein different buffers were used for equilibration as shown in Table 3, and their ability to remove BSA and recover IgG from a BSA-IgG mixture was evaluated.

[0284] Melon Gel Purification Buffer (MGPB): A low phosphate concentration buffer without sodium chloride. Without being bound by this theory, the low negative charge of this buffer was chosen to increase the binding capacity of BSA to the resin embodiments with a positive charge.

[0285] Tris buffer: A positively charged buffer without a negative charge. Without being bound by theory, the lack of negative charge would allow for increased BSA binding similar to MGPB; however, it would also repel the positively charged amine groups on IgG, thereby increasing IgG recovery.

[0286] Fig.10 is a gel image showing BSA removal and IgG recovery according to Table 3.

[0287]

[0288] like Fig.10 As shown in Table 3, lanes 1A and 1B show no BSA removal, however, the PDA modified resin embodiment removed more BSA from the BSA-IgG mixture. In addition, the concentration of PDA (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 a lower ability to remove BSA than the 45 kDa MWCO resin embodiment in lanes 3A and 3B. In addition, 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.

[0289] Example 4

[0290] In this example, the ability of the Resin G embodiments according to the Table to remove BSA and recover IgG from a sample comprising a mixture of BSA (10 mg / mL) and IgG (1 mg / mL) using different equilibration buffers was compared.

[0291] Fig.11 is a gel image showing BSA removal and IgG recovery from a sample comprising a mixture of BSA (10 mg / mL) and IgG (1 mg / mL) by an embodiment of Resin G 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 (as described in Table 1 provided herein).

[0292] This example demonstrates the desired BSA removal and IgG recovery using different equilibration buffers having a pH between 5 and 7. In addition, equilibration buffers having a neutral charge, such as sodium acetate, exhibited the desired BSA removal and IgG recovery.

[0293] Example 5

[0294] In this example, the BSA binding capacity (300 μL of 10 mg / mL) and IgG recovery (2 mg / mL) of the resin 5 embodiment of Table 1 modified with 150 mg / mL PEI were compared with the resin 3 embodiment of Table 1 modified with 150 mg / mL PEI in a sample containing 300 μL of 10 mg / mL. Blue Gel (Bio-Rad) and Melon TM Gel IgG Purification Kit (Thermo Scientific TM ) for comparison.

[0295] Fig.12 is a bar graph showing mg bound BSA / mL resin. Fig.12 As shown, the resin 5 embodiment (as described in Table 1 provided herein) has a binding capacity of 3.11 mg bound BSA / mL resin, the resin 3 embodiment (as described in Table 1 provided herein) has a binding capacity of 2.45 mg bound BSA / mL resin, and the Melon TMThe Gel IgG Purification Kit has a binding capacity of 1.26 mg bound BSA / mL resin, and Blue Gel (Bio-Rad) has a binding capacity of 2.43 mg bound BSA / mL resin.

[0296] Fig.13 is a bar graph showing IgG recovery (2 mg / mL). Accordingly, Resin 5 embodiment (as described in Table 1 provided herein) exhibited 72% volume recovery, Resin 3 embodiment (as described in Table 1 provided herein) exhibited 61% volume recovery, Melon TM The gel IgG purification kit showed 80% volume recovery, and Blue Gel (Bio-Rad) showed 25% volume recovery.

[0297] Therefore, this example demonstrates that the desired BSA binding capacity is achieved when PEI is used as a modifying agent, with similar Blue Gel (Bio-Rad) has a BSA binding capacity comparable to that of Melon TM Gel IgG purification kit for comparable IgG recovery.

[0298] Example 6

[0299] In this example, the resin 5 embodiment of Table 1 was modified with different concentrations of PDA to produce the resin F embodiment and the resin K embodiment as described in Table 2, which were combined with the commercially available Abcam BSA removal kit and Melon TM Gel IgG Purification Kit (Thermo Scientific TM ) were compared for their ability to remove BSA and recover IgG from samples containing a BSA-IgG mixture. Table 4 presents Fig.14A , which is a gel image showing the performance of Resin 5 Embodiment modified with different concentrations of PDA to produce Resin F Embodiment and Resin K Embodiment relative to the Abcam BSA Removal Kit and Melon TM Gel IgG Purification Kit (Thermo Scientific TM )'s BSA removal capacity and IgG recovery capacity. Fig.14A As shown, lane 5 is BSA 10 mg / mL and GAR 1 mg / mL mixture; lane 6 is 1 mg / mL GAR, and lane 7 is BSA 10 mg / mL; and all lanes were normalized and samples were loaded onto the gel at 10 μL / well.

[0300]

[0301] Fig.14A When compared with the commercially available Abcam BSA removal kit and Melon TM Gel IgG Purification Kit (Thermo Scientific TM ) when compared, excellent BSA removal IgG recovery properties in lanes 1 and 2.

[0302] Fig. 14B IBright image analysis software was used to analyze Fig.14A The bar graph obtained by quantifying the bands of . Accordingly, Resin F embodiment (Resin 5 embodiment of Table 1 modified with 150 mg / mL PDA) had 85.7% IgG recovery and 98.8% BSA removal; Resin K (Resin 5 embodiment of Table 1 modified with 75 mg / mL PDA) showed 86.7% IgG recovery and 82.6% BSA removal; Abcam BSA removal kit had 53.3% IgG recovery and 65.78% BSA removal; and Melon TM Gel IgG Purification Kit (Thermo Scientific TM ) with 78.7% IgG recovery and 8.3% BSA removal. TM Gel IgG purification kit (ThermoScientific TM ), Resin F embodiment and Resin K embodiment achieved higher IgG recovery and BSA removal compared to the above embodiments.

[0303] Example 7

[0304] Increase MWCO: In this example, higher MWCO resins with a MWCO of 45,000Da were produced and PDA modified and their ability to remove BSA and recover IgG from samples containing BSA-IgG was measured. Higher MWCO was achieved in Resin 5 embodiment, Resin 6 embodiment, Resin 7 embodiment and Resin 8 embodiment prepared according to Table 1.

[0305] Fig.15: is a bar graph showing the recovery percentages of Resin 5 embodiment, Resin 6 embodiment, Resin 7 embodiment and Resin 8 embodiment at 42,000 Da, 67,000 Da, 80,000 Da and 150,000 Da. Accordingly, Resin 5 embodiment 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. Resin 6 embodiment 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. Resin 7 embodiment 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. Resin 8 embodiment 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.

[0306] This example demonstrates that by decreasing the amount of HEC, resins with 50K MWCO, 80K MWCO, and 90K MWCO are produced (see Table 1).

[0307] Example 8

[0308] In this example, Resin F embodiment, Resin G embodiment, Resin H embodiment, Resin I embodiment and Resin J were produced according to Table 2 and tested for their ability to remove BSA and recover GAR from a sample containing a BSA-GAR mixture and compared with commercially available Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA removal kit were compared.

[0309] Fig.16A is a gel image showing BSA removal and antibody recovery, and Table 5 is Fig.16A Legend for .

[0310]

[0311] like Fig.16A As shown, when compared with Melon TMGel IgG Purification Kit (Thermo Scientific TM ), When compared to the Abcam BSA Removal Kit (Bio-Rad), the Resin F embodiment (45K MWCO) and the 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). TM Gel IgG Purification Kit (Thermo Scientific TM ), BlueGel (Bio-Rad) and Abcam BSA removal kit, resin H embodiment (80K MWCO) and resin I embodiment (90K MWCO) modified with 75 mg / mL PDA performed better in removing BSA and recovering IgG; however, they did not perform as well as resin F embodiment and resin G embodiment in recovering IgG. Resin J was produced by reacting resin 6 embodiment of Table 1 with PDA and then further reacting 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.

[0312] Fig. 16B The bar graph further demonstrates the recovery of GAR (1 mg / mL) and the removal of BSA (10 mg / mL). Fig. 16B As shown, the resin F embodiment showed 83% GAR recovery and 99% BSA removal; the resin G embodiment showed 93% GAR recovery and 100% BSA removal; the resin H embodiment showed 63% GAR recovery and 99% BSA removal; the resin I embodiment showed 76% GAR recovery and 95% BSA removal; the resin J embodiment showed 82% GAR recovery and 100% BSA removal; the Melon TM Gel IgG Purification Kit (Thermo Scientific TM ) showed 106% recovery of GAR and 82% removal of BSA; Blue Gel (Bio-Rad) showed 73% GAR recovery and 65% BSA removal; and Abcam BSA removal kit showed 125% GAR recovery and 85% BSA removal. TM Gel IgG Purification Kit (Thermo Scientific TM) The artificially high IgG recovery shown in the data can be explained by poor BSA removal producing a smear in the gel, which results in band overlap with IgG. Thus, this example demonstrates higher BSA removal and superior IgG recovery for Resin F Embodiment, Resin G Embodiment, and Resin J Embodiment when compared to commercially available resins and Resin H Embodiment and Resin I Embodiment.

[0313] Example 9

[0314] In this example, Resin F embodiment, Resin G embodiment, Resin H embodiment, Resin I embodiment and Resin J embodiment were produced according to Table 2 and compared with Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA Removal Kit were compared 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).

[0315] Fig.17 is a gel image showing BSA removal and IgG recovery, and Table 6 is Fig.17 Legend for .

[0316]

[0317]

[0318] like Fig.17 As shown, when compared with Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), When compared to the Blue Gel (Bio-Rad) and Abcam BSA Removal Kit, Resin F embodiment and Resin G embodiment showed the best BSA removal and IgG recovery from a mixture containing BSA (10 mg / mL) and GAR IgG (0.1 mg / mL). In addition, Resin H performed better than Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA removal kit; however, did not perform as well as the Resin F embodiment and the Resin G embodiment in recovering IgG. When the sample contained a mixture of 0.1 mg / mL GAR and 10 mg / mL BSA, the Resin I embodiment showed lower ability to remove BSA. The Resin J embodiment did not perform as well as the Resin F embodiment or the Resin G embodiment. Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA removal kits 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 alone (10 mg / mL), which shows the smear obtained by running only BSA.

[0319] Example 10

[0320] In this example, a mixture of GAR (1 mg / mL) and BSA (10 mg / mL) was passed through a resin F embodiment according to Table 2, Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and Abcam BSA removal kit were used for processing. TM 488 (ThermoScientific TM ) and the resulting flow-through was labeled using Pierce TM Dye and biotin removal resins remove free dye.

[0321] Fig.18 The fluorescent dye label of BSA is removed from the antibody before the labeling reaction, and Table 7 shows the corresponding Fig.18 Legend for .

[0322]

[0323]

[0324] Fig.18 The labeling efficiency of GAR is shown when BSA is removed, as shown by the resin F embodiment relative to Melon TM Gel IgG Purification Kit (Thermo Scientific TM )and Blue Gel (Bio-Rad) (where the labeling is due to unremoved BSA). The Abcam BSA removal kit demonstrated comparable results to the Resin F embodiment.

[0325] Fig.19 is an image of the fluorescent dye labeling of BSA removed from the antibody before the labeling reaction. A mixture of GAR (0.1 mg / mL) and BSA (10 mg / mL) was passed through the resin F embodiment according to Table 2, the resin G embodiment according to Table 2, the Melon TM Gel IgG Purification Kit (Thermo Scientific TM ), Blue Gel (Bio-Rad) and BSA removal kit. Then use NHS DyLight TM 650 (Thermo Scientific TM ) and the resulting flow-through was labeled using Pierce TM Dye and biotin removal resins remove free dye. Table 8 is Fig.19 Legend for .

[0326]

[0327] Fig.19 Demonstrating the labeling efficiency of GAR when BSA is removed, as shown by TM Gel IgG purification kit, This was demonstrated by the resin F embodiment and the resin G embodiment when compared with the Blue Gel (Bio-Rad), Abcam BSA removal kit (where the labeling efficiency is due to the non-removed BSA). Fig.19 It was demonstrated that even at low antibody concentrations such as 0.1 mg / mL, the Resin F embodiment and the Resin G embodiment were able to recover the antibody after BSA clearance and were able to successfully dye-label it.

[0328] Embodiment 11

[0329] Removal of serum from serum samples: In this example, the Resin F embodiment and the Resin G embodiment according to Table 2 were used to remove rabbit serum, mouse serum, human plasma, and human serum. Fig. 20 is a gel image showing the results of the Resin F embodiment and the Resin G embodiment relative to the starting mixture, wherein 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 Fig. 20 data.

[0330]

[0331] like Fig. 20 As shown, Resin F embodiment and Resin G embodiment demonstrated excellent albumin removal capability and good ability to recover IgG from different serum species.

[0332] Example 12

[0333] Effect of Equilibrium Buffer pH: In this example, the effect of equilibrium buffer Tris 50 mM on the ability of the resin G embodiment according to Table 2 to remove BSA and recover IgG. The starting amount and the flow rate were measured at A280 using nanodrop. The A280 amount was calculated by measuring A280 on the nanodrop and taking into account the volume of the sample added and recovered. Using Nanodrop as a control, the A280 measurement of 10 mg / mL BSA was determined.

[0334] Fig.21A is a bar graph showing the A280 amount of rabbit IgG. Fig.21A As shown, the resin G embodiment equilibrated with 50mM Tris (pH7.0) has an A 280 amount of 123A; the resin G embodiment equilibrated with 50mM Tris (pH 5.0) has an A 280 amount of 124A; the resin G embodiment equilibrated with 50mM Tris (pH 7.0+stacking agent 20μL) has an A 280 amount of 139A; the BSA-rabbit IgG starting mixture has an A 280 amount of 824A; the rabbit IgG starting material has an A 280 amount of 133A; and BSA alone has an A 280 amount of 634A.

[0335] Fig. 21B is a bar graph showing the A 280 amount of GAR. Fig. 21B As shown, the resin G embodiment equilibrated with 50 mM Tris (pH 7.0) has a 97A 280 amount; the resin G embodiment equilibrated with 50 mM Tris (pH 5.0) has a 101A 280 amount; the resin G embodiment equilibrated with 50 mM Tris (pH 7.0 + stacking agent 20 μL) has a 99A 280 amount; the BSA-GAR starting mixture has a 834A 280 amount; the GAR starting material has a 100A 280 amount; and BSA alone has a 634A 280 amount.

[0336] This example demonstrates that the amount of A280 in the flow-through is similar to that from GAR and rabbit IgG, indicating desirable antibody recovery after cleanup with the Resin G embodiment.

[0337] Embodiment 13

[0338] Labeling of the primary antibody: In this example, the primary antibody GAPDH was labeled with a fluorescent dye after removal of BSA from the BSA-antibody mixture. Fig. 22 Is displayed on the DyLight TM 650 (Thermo Scientific TM ) Gel image of removal of BSA from primary anti-GAPDH before conjugation, and Table 10 shows Fig. 22 data.

[0339]

[0340] The resin was spun at 3000X G and 6000X G, and 10 mg / mL BSA was spiked into 1 mg / mL GAPDH and passed through the resin G embodiment according to Table 2. The flow-through was collected and the resin was washed with 50 mM Tris to collect GAPDH that may have bound to the resin. The flow-through from both elutions was collected, combined, and then washed with NHSDyLight TM 650(ThermoScientific TM ) and conjugated using Pierce TM The dye and biotin removal resins were used for cleanup. GAPDH with BSA was also labeled, and free Dy650 was added as a control lane.

[0341] This example demonstrates that in TM 650 (Thermo Scientific TM ) BSA was successfully removed from the primary antibody GAPDH prior to conjugation and thus complete removal of BSA was demonstrated at spin speeds of 3,000XG and 6,000XG.

[0342] Embodiment 14

[0343] In this example, the effects of combining a primary antibody to calreticulin with DyLight TM 680NHS(ThermoScientific TM ) Before conjugation, the ability of BSA to be removed from the primary anti-calreticulin according to the resin G embodiment of Table 2 was measured. 1 mg / mL BSA was added as a stabilizer to obtain 1 mg / mL calreticulin antibody. The antibody was passed through the resin G embodiment and the flow-through was collected, and the resin was washed three times with 50 mM Tris to collect the primary antibody that may have bound to the resin. The flow-through was collected, combined, concentrated, and then mixed with DyLight TM 680NHS (Thermo ScientificTM ) and conjugated using Pierce TM Clearing was performed with dye and biotin removal resin.

[0344] Fig.23 The gel images were obtained by loading the flow-through and staining with Coomassie stain using the Pierce Power blotting apparatus. Table 11 shows Fig.23 data.

[0345]

[0346] like Fig.23 As shown, lane 1 shows calreticulin without BSA after passing through the resin G embodiment. Lane 2 shows calreticulin conjugated to DyLight TM 680 (Thermo Scientific TM Lane 3 is the calreticulin protein received with BSA added as a stabilizer. In view of this, this example demonstrates that when the primary anti-calreticulin protein is combined with DyLight TM 680 (Thermo Scientific TM ) BSA was successfully removed from the primary anti-calreticulin by the resin G embodiment prior to conjugation.

[0347] Embodiment 15

[0348] Western Blot Application: In this example, the Western Blot application using fluorescently labeled GAR after BSA removal was tested. 10 mg / mL BSA was doped into 1 mg / mL goat anti-rabbit (GAR). Resin 7 according to Table 1 was modified with 150 mg / mL PDA for BSA removal. GAR was then conjugated to DyLight TM 650 (Thermo Scientific TM ). GAR containing BSA was also conjugated to DyLight TM 650 (Thermo Scientific TM ), 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 the HSP 90 primary antibody. The membrane was washed and then incubated with the HSP 90 primary antibody. The membrane was washed and then incubated with GAR Dy 650, washed, and then scanned on an IBright imager using the fluorescence mode. GAR conjugated with Dy 650 without BSA removal was used as a comparative control.

[0349] Fig.24AAre images of gels showing removal of BSA from GAR (left) and without removal of BSA from GAR (right). Fig. 24B is a bar graph showing the fluorescence intensity of HeLa lysate loaded with and without BSA removal. Fig. 24B As shown, 10 μg of HeLa lysate from which BSA was removed had a fluorescence intensity of 13,000,000 and that without BSA removal had a fluorescence intensity of 4,000,000; 5 μg of HeLa lysate from which BSA was removed had a fluorescence intensity of 9,000,000 and that without BSA removal had a fluorescence intensity of 3,800,000; 2.5 μg of HeLa lysate from which BSA was removed had a fluorescence intensity of 7,800,000 and that without BSA removal had a fluorescence intensity of 3,800,000; 1.25 μg of HeLa lysate from which BSA was removed had a fluorescence intensity of 4,100,000 and that without BSA removal had a fluorescence intensity of 1,800,000.

[0350] This example demonstrates that removal of BSA from GAR prior to conjugation of GAR with Dy 650 shows much higher intensity on the blot and in the quantitative plot than when BSA is not removed from GAR. In addition, the observed fluorescence intensity increases by a factor of three with GARDy650 with BSA removed. Thus, for low abundance targets in cell lysates or for poorly performing antibodies with BSA, performance can be enhanced by removing BSA with the resin 7 embodiment modified with 150 mg / mL PDA.

[0351] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those of ordinary skill in the art that such embodiments are provided by way of example only. Modifications, changes, and substitutions to these disclosed embodiments will be apparent to those of ordinary skill in the art without departing from the present disclosure. It should be 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 comprising: Porous size exclusion supports; at least one cationic moiety associated with the porous size exclusion support; and 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 support has a molecular weight cut-off greater than or equal to 40 kDa.

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

4. The matrix according to 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. The matrix of claim 4, wherein the at least one cationic moiety is polyethyleneimine, pentamethylenediamine, 5,8-dimethyl-4,7,10-trioxatridecan-2,12-diamine, 1,2-phenylenediamine, 1,3-phenylenediamine, 1,4-phenylenediamine or N,N-diethylethylenediamine.

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

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

8. 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; as well as a receiver positioned to receive flow-through from the container, wherein the system is configured for gravity flow operation, centrifugal force operation, positive pressure operation, negative pressure operation, vacuum operation, or a combination thereof; and wherein the container is a columnar container, a tube, a porous tube, a porous plate, or a porous filter plate.

9. A method for preparing a multimodal resin, the method comprising: providing a porous size exclusion support comprising hydroxyethyl cellulose having a MWCO greater than or equal to 40 kDa, the hydroxyethyl cellulose having at least one vicinal diol; oxidizing the at least one vicinal diol to an aldehyde; as well as The aldehyde is reacted with the amine group of the cationic moiety via reductive amination.

10. The method according to claim 9, wherein an amount of hydroxyethyl cellulose ranging from 50 grams to 250 grams is used to produce the porous size exclusion support.

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

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

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

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

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

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

17. A method according to claim 15 or claim 16, wherein the flow-through is collected in a receiver positioned below the container.

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

19. The method according to 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 0 mM salt to 5 mM salt.

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

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

23. The method of any one of claims 15 to 22, wherein the one or more molecules comprise: at least one small molecule having a molecular weight range of less than 100 kDa; as well as At least one macromolecule having a molecular weight range greater than or equal to 100 kDa.

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

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

27. A kit for separating positively charged macromolecules from one or more negatively charged small molecules in a sample, the kit comprising: a porous size exclusion support having at least one cationic moiety associated therewith, wherein the cationic moiety is capable of associating with and capturing the at least one negatively charged small molecule; as well as Instructions for using the porous size exclusion support.

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

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

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