Multi-mode chromatography resins containing anionic and cationic ligands

Abstract

The present invention relates to a multi-mode chromatography resin comprising anionic and cationic ligands on each resin particle, and to a method for synthesizing said multi-mode chromatography resin by contacting a base with a quaternary and / or tertiary amine on the surface of a substrate. The multi-mode resin can be tuned by varying the ratio of cations to anions. The multi-mode chromatography resin can be used for purifying proteins.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 464,263, filed May 5, 2023, the disclosure of which is incorporated herein by reference in its entirety (including all figures, tables and amino acid or nucleic acid sequences). Background of the Invention

[0004] Protein purification has always been challenging. Protein production typically involves synthesizing target proteins using engineered bacteria or eukaryotic cell lines. It is well known that cell culture methods used for protein production produce proteins containing varying amounts of impurities. Protein purification usually involves attempting to remove impurities, including, for example, cells, cell debris, DNA, RNA, endotoxins, peptides, viruses, and components of the cell culture medium. Controlling these impurities can affect several properties of the purified protein, including, for example, protein stability and efficacy.

[0005] Despite the availability of various techniques for protein purification, separating proteins from impurities remains challenging. Therefore, there is still a need for compositions and methods for protein purification that exhibit high binding affinity to the target protein and limited nonspecific binding. Summary of the Invention

[0006] This document provides chromatographic media comprising a substrate (e.g., beads or other particulate substrate) containing two ligands, preferably one ligand being an anionic group and the second ligand being a cationic group. Chromatographic media (optionally contained within a column) are also provided, comprising a substrate (e.g., multiple beads or other substrates containing anionic and cationic ligands) including both anionic and cationic ligands.

[0007] A method for preparing the substrate comprising anionic and cationic ligands is also provided. In some embodiments, the method includes incubating the substrate with an alkali to form a substrate containing a combination of cations and anions, the substrate comprising a quaternary amine, a tertiary amine, or a combination thereof. In some embodiments, the substrate comprising anionic and cationic ligands can be prepared by incubating with an alkali at a specific temperature and concentration for a specific time to achieve a specific cation-to-anion ratio. In some embodiments, the incubation temperature can be from about 15°C to about 150°C; the concentration of the alkali can be from about 0.1N to about 10N; and the incubation time can be from about 1 second to about 3 months. Figure 1 One implementation of the method is shown.

[0008] Methods of performing chromatography are also provided. In certain embodiments, the methods comprise: contacting a sample comprising a target molecule (e.g., a protein) with a substrate comprising anionic and cationic ligands as described herein under conditions such that the target is captured by the substrate comprising anionic and cationic ligands; and collecting the target molecule from an eluate of the substrate comprising anionic and cationic ligands.

[0009] In some embodiments, the bulk substrate comprising anionic and cationic ligands can limit the amount of contaminants or impurities that bind to the substrate. The ability of the substrate comprising anionic and cationic ligands to specifically bind to a target protein can be determined by the ratio of cationic (e.g., quaternary amines and tertiary amines) to anionic (e.g., carboxymethyl groups). In some embodiments, the protein is an antibody or another therapeutic protein. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 A mechanism is shown by changing the resin, where some amine ligands are removed and carboxylic acid groups are substituted for some of the amine ligands.

[0011] Figure 2A Titrations curves are shown for chromatography resins with different Q and CM ligand distributions. Figure 2B A mixture of MP-HQ resin and Macro Prep-CM (MP-CM) resin (measured ratio of 17% CM) is shown. Figure 2A Binding and elution curves for iodide on each of the resins described in Example 1.

[0012] Figure 3 Conversion of quaternary amines on the surface of MP-HQ resin to carboxymethyl groups using NaOH is shown.

[0013] Figure 4 Performance testing of each resin treated with NaOH is shown, which shows a trend of decreasing elution times as the ratio of carboxymethyl (CM) groups to quaternary amines increases.

[0014] Figure 5 Performance comparison of a resin containing 19% CM groups to a mixture of MP-HQ resin and Macro Prep-CM (MP-CM) resin (measured ratio of 17% CM) is provided. The resin containing 19% CM, where quaternary amines and CM ligands are mixed on a single resin, appears to have better protein separation than the mixture of MP-HQ resin and MP-CM resin.

[0015] Figure 6 Binding affinity of the substrate comprising anionic and cationic ligands is shown to vary depending on whether the quaternary amines and carboxymethyl groups are distributed within a single resin (19% CM) or the column contains a mixture of two resins (Q and CM mixed (17%)).

[0016] Figure 7 Scanning electron microscope (SEM) images of both MP-HQ resin (top row) and MP-HQ resin treated with sodium hydroxide for 48 hours (bottom row) are shown, which show no significant difference in shape or structure.

[0017] Figure 8 Different microenvironments of a substrate where both quaternary amine and carboxymethyl groups are distributed on a single resin and a mixture of two different substrates, one of which contains a quaternary amine resin and the other has a carboxymethyl resin. DETAILED DESCRIPTION

[0018] The present disclosure relates to substrates comprising anionic and cationic ligands, methods of making substrates comprising anionic and cationic ligands, and methods of using substrates comprising anionic and cationic ligands.

[0019] DEFINITIONS

[0020] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. To the extent the term "includes" is used in the summary and / or detailed description of specific embodiments and / or claims, such term is intended to be interpreted as "including but not limited to." The transitional terms / phrases "comprise", "comprises", "comprising", "consist", "consists", "consisting", "consisting of", and any grammatical variants thereof, are used interchangeably.

[0021] The phrase "consisting essentially of or "consisting essentially of means that the recited embodiments encompass embodiments that contain the specified materials or steps, and further encompass embodiments that contain materials or steps that do not materially affect the basic and novel characteristics of the recited embodiments.

[0022] The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend on the context in which the number is used, i.e. the limit of the measuring system or the setting of the range finder. In the context of polynucleotide lengths, these polynucleotides contain the stated number of bases or base pairs, with a variation of 0-10% (X ± 10%) around that value. In the context of compositions in which the term "about" or "approximately" is used in connection with an amount of an ingredient, these compositions comprise the stated amount of the ingredient with a variation (error range) of 0-10% (X ± 10%) around that stated value. In the context of pH, the term "about" or "approximately" is intended to include values of ± 0.2 units of the stated pH.

[0023] In the present disclosure, ranges are used as shorthand for describing a range of values inclusively. Whenever a numerical range is given, it should be understood that any suitable value within the range can be selected as the upper value or the lower value of the range. For example, the range of 0.1-1.0 should be interpreted to include not only the specifically enumerated endpoints of 0.1 and 1.0, but also to include all the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.1-1.0, as well as all intermediate ranges of values, e.g., 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. Consideration is given to values within a range having at least two significant digits, e.g., the range of 5-10 represents all values between 5.0 and 10.0, as well as between 5.00 and 10.00, inclusive of the endpoint values.

[0024] The term "protein" can be used to refer to an amino acid polymer, or a group of two or more interacting or associated amino acid polymers. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers (including those containing modified residues) and non-naturally occurring amino acid polymers. The term "protein" includes therapeutic proteins, including but not limited to enzymes, growth regulators, clotting factors, cytokines, antibodies, hormones, transcription factors, and phosphoproteins.

[0025] The term "antibody" refers to an immunoglobulin or a fragment form thereof. The term includes, but is not limited to, polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG, and IgM classes, derived from human or other mammalian cell lines, including natural forms or genetically modified forms, such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. "Antibody" encompasses complex forms, including but not limited to fusion proteins containing an immunoglobulin portion. "Antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, Fc, and other combinations, whether or not they retain antigen-binding function.

[0026] The term "substrate comprising anionic and cationic ligands" as referred to within this disclosure is intended to mean a particulate substrate comprising two types of ligands, one comprising anionic groups and the other comprising cationic groups. The phrase "substrate comprising anionic groups and cationic groups" can be used interchangeably with the phrase "substrate comprising anionic and cationic groups" and is equivalent. The particulate substrate includes spherical or oblong particles having a diameter between about 1 pm and about 1000 pm and can be monodisperse or polydisperse. For example, beads (also referred to as substrates) can be defined as small size beads (< about 50 pm), medium size beads (about 50 to about 100 pm), or large size beads (greater than about 100 pm). Of course, the beads can have other shapes such as shard-like shapes. When spherical beads are used as the substrate, the size distribution of the spherical beads can be monodisperse or polydisperse. In other embodiments, the substrate comprises a shape selected from the group consisting of: cube, tetragon, prism, pyramid, platonic solid, torus, cone, cylinder, sphere, and mixtures thereof. In any of these embodiments, at least one dimension (length, width, diameter, and / or height) of the beads is between about 1 pm and about 1000 pm; between about 5 pm and about 750 pm; between about 5 pm and about 600 pm; between about 5 pm and about 500 pm; between about 5 pm and about 400 pm; between about 5 pm and about 250 pm; between about 5 pm and about 150 pm; or between about 5 pm and about 100 pm.

[0027] The term "sample" refers to any composition / constituency containing a target molecule that needs to be purified. The term "contaminant" or "impurity" refers to any substance to be removed from the sample. In some embodiments, the sample is a composition comprising contaminating substances from a cell culture.

[0028] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items. For example, the phrase "A, B, and / or C" includes separate A, separate B, separate C, A with B, A with C, B with C, and A, B, and C. Furthermore, "or" is used in its inclusive and not its exclusive sense, e.g., when used in the phrase "at least one of A, B, and C," the term "or" means one, some, or all of A, B, and C. Conjunctive language such as the phrase "at least one of X, Y, and Z," is to be understood to express the possibility of one, two, or all of X, Y, and Z, i.e., X, Y, and Z individually or in any combination. Unless otherwise expressly specified, conjunctive language, such as the phrase "at least one of X, Y, and Z," is to be understood in the context as expressing the possibility of one, some, or all of X, Y, and Z, i.e., X, Y, and Z individually or in any combination.

[0029] The term "substrate" refers to a solid particulate substrate, such as a bead, particle, or other particulate substrate. Such substrates include ligands, for example, anionic ligands and cationic ligands (also referred to herein as anionic groups and / or cationic groups). The substrate can be porous (microporous or macroporous) or non-porous. The substrate can have a quaternary amine, a tertiary amine, a sulfate, or a combination thereof on its surface. The substrate can be treated with a base to change the ligands on the surface. The substrate can also include a linker coupled to the substrate. The particulate substrate can be formed from methacrylate, polystyrene, agarose, polyacrylamide, or any other suitable substrate. The substrate particles can have any shape, such as spherical or beetle-shaped, and can also be rigid or malleable. In certain embodiments, rigid substrates are preferred. The substrates can be polydisperse or monodisperse with respect to size dispersion.

[0030] Producing substrates comprising anionic and cationic ligands

[0031] In general, chromatographic substrates comprising anionic groups and cationic groups can be produced by the following general method. First, a desired chromatographic support substrate is selected based on its chemical and mechanical properties, which has, for example, quaternary amines, tertiary amines, and / or sulfates on the surface of the support substrate. Thus, in some embodiments, the substrate comprises only quaternary amines. In other embodiments, the substrate comprises only tertiary amine groups. In other embodiments, the substrate comprises only sulfate groups. In yet other embodiments, the substrate comprises a mixture of quaternary amine groups and tertiary amine groups. In still other embodiments, the substrate comprises a mixture of quaternary amine groups and sulfate groups, or a mixture of sulfate groups and tertiary amine groups. In various additional embodiments, any commercially available substrate comprising quaternary amines, tertiary amines, or a combination thereof can be used to make the substrates disclosed herein, for example:

[0032] Functional group - N + Macro-Prep® High Q support with functional group - N(CH3)3 (Bio-Rad Laboratories, Inc., Hercules, CA);

[0033] Functional group - N + Macro-Prep® DEAE resin with functional group - N(C2H5)2 (Bio-Rad Laboratories, Inc.);

[0034] AG® resin (Bio-Rad Laboratories, Inc.) including functional group - CH2N + AG® 1 and AG® MP-1 with functional group - CH2N(CH3)3 (Bio-Rad Laboratories, Inc.); + AG® 2 with functional group - CH2N(CH3)2C2H4OH (Bio-Rad Laboratories, Inc.); +AG® 3-X4 and AG® 4-X4, AG® 50W and AG® MP-50 containing strong sulfonic acid functional groups, AG® 50W and AG® 1 resins containing functional groups -CH2N + (CH3)3 and -CH2COO - AG® 11A8, and AG® 501-X8 and AG® MP-501-X8(D) which have a mixture of AG® 50W and AG® 1 resins.

[0035] Bio-Rex resins (Bio-Rad Laboratories, Inc.) including Bio-Rex 5 containing functional groups -N(CH3)2, Bio-Rex MSZ 1 resin which is a mixture of AG® 50W and AG® 1 resins, and Bio-Rex 70 resin containing carboxylic acid exchange groups;

[0036] Nuvia™ resins (Bio-Rad Laboratories, Inc.) including Nuvia™ HP-Q containing functional groups -N + (CH3)3, Nuvia™ HR-S containing functional groups -SO3 - , Nuvia™ S containing functional groups -SO3 - , Nuvia™ Q containing functional groups -N(CH3)3 + , and Nuvia™ cPrime containing hydrophobic cation exchange functional groups;

[0037] UNOsphere™ resins (Bio-Rad Laboratories, Inc.) including UNOsphere™ S resin containing functional groups -SO3 - , and UNOsphere™ Q containing functional groups -N + (CH3)3; and

[0038] ENrich Q (Bio-Rad Laboratories, Inc.) containing quaternary ammonium functional groups.

[0039] After selecting the desired chromatographic support, the chromatographic support matrix containing, for example, sulfate, quaternary amine, tertiary amine, or combinations thereof, is treated with a base. The chromatographic support matrix can then be washed to remove any residual base.

[0040] In certain embodiments, the substrate is a resin bead having a surface containing a quaternary amine, a sulfate, and / or a tertiary amine, preferably the substrate is ionically charged. Thus, in some embodiments, the substrate contains only quaternary amines. In other embodiments, the substrate contains only tertiary amine groups. In other embodiments, the substrate contains only sulfate amine groups. In yet other embodiments, the substrate contains a mixture of quaternary amine groups and tertiary amine groups and / or sulfate groups. The resin bead can be made from a variety of polymers that can be used, including, but not limited to, polystyrene (e.g., polystyrene sulfonate), cross-linked polystyrene, divinylbenzene, cellulose, agarose, polymethacrylate, polyacrylamide, dextran, or any combination thereof. In certain embodiments, the quaternary amine can be, for example, cetyltrimethylammonium, chlorhexidine, quaternary ammonium methacrylate, or benzalkonium chloride. In certain embodiments, the tertiary amine can be, for example, benzyl dimethyl amine, pyridine, tertiary ammonium methacrylate, or imidazole.

[0041] In certain embodiments, the base is, for example, sodium hydroxide, lithium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide, tetramethylammonium hydroxide, guanidine, rubidium hydroxide, or any combination thereof. In certain embodiments, the concentration of the base applied to the substrate containing quaternary and / or tertiary amines is about 0.1 N to about 10 N, about 2.5 N to about 7.5 N, or about 4.5 N. In certain embodiments, the substrate can be treated with the base at a temperature of about 15 °C to about 150 °C, about 20 °C to about 125 °C, about 25 °C to about 100 °C, about 30 °C to about 75 °C, about 35 °C to about 50 °C, or about 40 °C for about 1 second to about 3 months, about 1 minute to about 10 weeks, about 2 minutes to about 6 weeks, about 5 minutes to about 4 weeks, about 10 minutes to about 2 weeks, about 15 minutes to about 1 week, about 30 minutes to about 3 days, about 1 hour to about 48 hours, or about 6 hours to about 24 hours.

[0042] In certain embodiments, after the substrate is treated with the base, the substrate can be washed with, for example, deionized water. In certain embodiments, the deionized water can remove any free base from the substrate. In certain embodiments, the substrate can be washed with deionized water at a temperature of about 15 °C to about 150 °C, about 20 °C to about 125 °C, about 25 °C to about 100 °C, about 30 °C to about 75 °C, about 35 °C to about 50 °C, or about 40 °C for about 1 second to about 24 hours, about 1 minute to about 120 minutes, about 2 minutes to about 30 minutes, about 5 minutes to about 15 minutes, or about 10 minutes.

[0043] Substrates of various diameters and / or dimensions can also be used, including small-sized particles or beads (<50 pm in at least one dimension, e.g., length, width, diameter, and / or height), medium-sized particles or beads (about 50 to about 100 pm in at least one dimension, e.g., length, width, diameter, and / or height), and large-sized particles or beads (> about 100 pm in at least one dimension, e.g., length, width, diameter, and / or height).

[0044] In certain embodiments, the resulting substrate comprises cationic groups and anionic groups, and the ratio of cationic groups (e.g., sulfate, quaternary and / or tertiary amine) to anionic groups (e.g., carboxymethyl groups) can be less than or equal to about 50:1, about 25:1, about 15.5:1, about 2.4:1, about 1.2:1, about 1:1, or about 0.7:1. Other embodiments provide a ratio of cationic groups to anionic groups represented by X:1, where X is any value between 0.2 and 50, increasing in increments of 0.1 or 0.05. Still other embodiments provide a ratio of cationic groups to anionic groups represented by X:1, where X is any value between 0.2 and 50, increasing in increments of 0.1 or 0.05. Still other embodiments provide a ratio of cationic groups to anionic groups ranging from 0.5:1 to 50:1.

[0045] In certain embodiments, the amount of anionic groups attached to the substrate can be about 5% to about 95%, about 5% to about 65%, about 7% to about 63%, about 10% to about 43%, or about 19% relative to the total ionic capacity of the substrate. The amount of anionic groups attached to the substrate relative to the amount of anionic groups is such that the total amount of anionic groups and cationic groups on the surface is 100%. Thus, the amount of cations attached to the surface relative to the total ionic capacity of the substrate can be about 5% to about 95%, about 35% to about 95%, about 37% to about 93%, about 67% to about 90%, or about 81%. It is understood that the total ionic capacity is all (100%) of the anionic groups and cationic groups present on the substrate.

[0046] In other embodiments, the amount of anionic groups attached to the surface of the substrate is about X%, where X is any integer between 1 and 99, and the amount of cationic groups attached to the surface of the substrate is about Y%, where Y is any integer between 1 and 99, and the sum of the selected X and Y values is 100 (100%).

[0047] In certain embodiments, the total ion capacity can be about 100 μeq / mL to about 1000 μeq / mL, about 250 μeq / mL to about 750 μeq / mL, about 350 μeq / mL to about 500 μeq / mL, about 399 μeq / mL to about 499 μeq / mL, about 438 μeq / mL to about 487 μeq / mL, or about 475 μeq / mL. In certain embodiments, the cationic capacity can be about 10 μeq / mL to about 1000 μeq / mL, about 50 μeq / mL to about 750 μeq / mL, about 100 μeq / mL to about 500 μeq / mL, about 146 μeq / mL to about 454 μeq / mL, about 250 μeq / mL to about 449 μeq / mL, or about 384 μeq / mL. In certain embodiments, the anionic capacity can be about 1 μeq / mL to about 1000 μeq / mL, about 10 μeq / mL to about 750 μeq / mL, about 15 μeq / mL to about 500 μeq / mL, about 33 μeq / mL to about 253 μeq / mL, about 50 μeq / mL to about 188 μeq / mL, or about 91 μeq / mL.

[0048] A substrate comprising anionic groups (ligands) and cationic groups (ligands) can be produced by contacting a solid particulate substrate comprising a quaternary amine, sulfate, and / or tertiary amine with a base. Methods of producing a substrate comprising anionic and cationic ligands, the method comprising, for example:

[0049] a) contacting a solid substrate comprising a quaternary amine, sulfate, and / or tertiary amine with a base (e.g., 4.5 N sodium hydroxide, at 40 °C for about 1 second to about 48 hours); and

[0050] b) optionally, washing the solid substrate contacted with the base (e.g., at about 18 °C to about 25 °C, using a sodium hydroxide solution with sodium chloride, for about 1 second to about 1 hour).

[0051] c) optionally, washing the solid substrate contacted with the sodium hydroxide solution with deionized water, at about 18 °C to about 25 °C, for about 1 second to about 1 hour.

[0052] Methods of use

[0053] Proteins can be purified using the substrate comprising anionic and cationic ligands using any conventional means known to those of skill in the art. Examples of proteins include, but are not limited to, antibodies, enzymes, growth regulators, clotting factors, transcription factors, and phosphoproteins. In many such conventional procedures, the substrate comprising anionic and cationic ligands is equilibrated at the pH to be used for binding the target molecule (e.g., an antibody or non-antibody protein) using a buffer ("equilibration buffer") prior to use. All features that will affect the binding environment, including ionic strength and conductivity, as appropriate, can be equilibrated.

[0054] In some embodiments, the substrate comprising anionic and cationic ligands described herein can be used in a "bind-elute" mode in order to purify one target molecule, or two or more target molecules, from a sample. In some embodiments, once the target molecule is bound to the substrate comprising anionic and cationic ligands, the target molecule can be eluted using a change in the pH and / or salt content of the elution buffer.

[0055] In some embodiments, once the substrate comprising anionic and cationic ligands is equilibrated, a sample comprising a target molecule is loaded onto the substrate comprising anionic and cationic ligands ("loading step"). The sample can optionally be diluted or equilibrated into the equilibration buffer, and the target molecule can be allowed to bind to the substrate comprising anionic and cationic ligands. Once bound to the substrate comprising anionic and cationic ligands, the substrate comprising anionic and cationic ligands can be washed with a "wash solution" to remove contaminants that are not bound to the substrate comprising anionic and cationic ligands.

[0056] Non-limiting examples of solutions suitable for use with the disclosed chromatography using substrates comprising anionic and cationic ligands (e.g., wash solutions, equilibration solutions) include water (e.g., deionized water) or a buffer (e.g., Bis-Tris at a pH of about 7). Other non-limiting examples of suitable buffers include phosphate buffers (e.g., monosodium, disodium, and / or trisodium phosphate buffers), ammonium phosphate, basic calcium phosphate, and potassium phosphate (monobasic and / or dibasic) buffers. Other buffers include acetate buffers (e.g., sodium acetate), acetic acid, malonic acid, succinate buffers, imidazole buffers, arginine buffers, glycine buffers, HEPES (2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid), BICINE (N,N-bis(2-hydroxyethyl)glycine), Bis-tris (bis-trimethane), Bis-tris propane, TRIS (tris(hydroxymethyl)aminomethane), MES (2-morpholinoethanesulfonic acid monohydrate, or ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffers. Buffers are typically prepared at a concentration of about 10 mM to about 100 mM or about 20-50 mM. Optionally, these buffers can further comprise a salt. Examples of salts that can be used for this purpose are alkali and alkaline earth metal halides, notably sodium and potassium halides, and as specific examples NaCl or KCl. Other salts include: sulfates, acetates, bromides, perchlorates, iodides, thiocyanates, and suitable cations such as ammonium, alkali and alkaline earth metals.

[0057] In some embodiments, the substrate comprising anionic and cationic ligands can be washed with a wash buffer (such as the equilibration buffer) to remove any unbound proteins or substances that can be present in the source solution. The bound proteins (e.g., antibodies or non-antibody proteins, as desired) can then be eluted using an elution buffer. The target proteins can be eluted using isocratic elution, where the buffer conditions or salt conditions are changed in a step elution, or gradient elution using buffers at constant pH and salt or phosphate ion gradients, for example.

[0058] In other embodiments, the binding and washing steps are performed in the presence of at least one salt in the sample and the wash solution. Examples of salts that can be used for this purpose are alkali and alkaline earth metal halides, notably sodium and potassium halides, and as a specific example, sodium chloride. The salt concentration can vary; in most cases, a suitable concentration can be in the range of about 10 mM to about 2 M, about 10 mM to about 1.5 M, about 10 mM to about 1 M, about 10 mM to about 750 mM, about 10 mM to about 500 mM, about 10 mM to about 250 mM, about 20 mM to about 150 mM, about 20 mM, or about 150 mM. In some cases, a salt gradient can be used to elute the bound protein. In other embodiments, step elution can be utilized, in which the amount of salt contained within the buffer is changed (e.g., increased) and passed through the column to elute the bound protein. In some embodiments, the concentration of salt ranges between X mM and 1000 mM, where X is any integer between 1 and 999; between X mM and 750 mM, where X is any integer between 1 and 749; between X mM and 500 mM, where X is any integer between 1 and 499; between X mM and 250 mM, where X is any integer between 1 and 249; between X mM and 100 mM, where X is any integer between 1 and 99; between X mM and 50 mM, where X is any integer between 1 and 49.

[0059] The substrates comprising anionic and cationic ligands can be used in any conventional configuration, including packed columns and fluidized or expanded bed columns, and can be used in any conventional process, including batch mode for loading, washing, and elution, as well as continuous or flow-through mode. The use of packed flow-through columns is particularly convenient for preparative scale extractions and analytical scale extractions. Thus, the columns can range in diameter from about 1 mm to about 1 m, and in height from about 1 cm to about 30 cm or more.

[0060] The chromatographic steps described herein can be performed in conventional purification configurations, including but not limited to packed columns and fluidized or expanded bed columns, and can be performed by any conventional chromatographic process, including batch mode for loading, washing, and elution, as well as continuous or flow-through mode. In some embodiments, the media is packed in a column ranging in diameter from less than 0.5 centimeters to greater than 1 meter, and in column height from less than 1 centimeter to greater than 30 centimeters. In other embodiments, the substrates comprising anionic and cationic ligands are disposed in a spin column.

[0061] In other embodiments, the substrate comprising anionic and cationic ligands is disposed in a chromatography column, a sample is applied to the top of the column, and gravity forces the sample, wash buffer, and / or elution buffer through the column. In other embodiments, the column containing the substrate comprising anionic and cationic ligands can be run from top to bottom or bottom to top with or without pressure, and the direction of fluid flow in the column can be reversed during the process. In some cases, it can be advantageous to reverse the liquid flow while maintaining the packed configuration of the packed bed. The methods described herein can be used to purify a variety of types of target molecules, including naturally occurring proteins, antibodies, and recombinant proteins.

[0062] The output of the chromatography column containing the substrate comprising anionic and cationic ligands can be monitored for the presence of the target molecule or other components of the sample as desired to determine a fraction that contains the target molecule and is free of contaminants, or at least has a reduced amount of contaminants, compared to the original sample. In some embodiments, at least 90%, 95%, 99% of the contaminants in the sample are removed in the resulting purified target molecule fraction. Exemplary methods for measuring the output include monitoring a characteristic absorbance wavelength of the target molecule (e.g., 254 nm). The term "fraction" is used to refer to a portion of the chromatography output and is not intended to limit the manner in which the output is collected or whether the output is collected in portions or continuously.

[0063] Any antibody preparation can be used in the present application, including unpurified or partially purified antibodies from natural, synthetic, and / or recombinant sources. Unpurified antibody preparations can be from a variety of sources, such as plasma, serum, ascites fluid, milk, plant extracts, bacterial lysates, yeast lysates, or conditioned cell culture media. Partially purified preparations can be from unpurified preparations that have been processed by at least one chromatography, precipitation, other fractionation step, or any combination thereof. In some embodiments, the antibodies have not been purified by protein affinity prior to purification.

[0064] In certain embodiments, the substrate comprising anionic and cationic ligands can be used to purify non-antibody proteins, including therapeutic proteins. Examples of therapeutic proteins include, but are not limited to, enzymes, growth regulators, clotting factors, transcription factors, and phosphoproteins.

[0065] In certain embodiments, the amount of anion and cation and the surface of the substrate comprising anionic and cationic ligands can be varied based on the properties of the protein to be purified. In certain embodiments, the reaction of a base with a quaternary and / or tertiary amine does not change the overall shape or underlying structure of the substrate.

[0066] All patents, patent applications, provisional applications, and publications, including all tables and figures, referred to in this disclosure are hereby incorporated by reference in their entirety for all purposes to the extent that they are not inconsistent with the explicit teachings of this specification.

[0067] Additional disclosure and claimable subject matter

[0068] 1. A substrate comprising anionic and cationic ligands, said substrate prepared by a method comprising:

[0069] a) contacting a solution comprising a base with a substrate, said substrate comprising sulfate, quaternary amine, and / or tertiary amine on a surface thereof.

[0070] 2. The substrate comprising anionic and cationic ligands of embodiment 1, said method further comprising:

[0071] b) washing the substrate contacted with the base using a wash solution.

[0072] 3. The substrate comprising anionic and cationic ligands of embodiment 1, wherein said substrate comprises methacrylate, polystyrene, agarose, divinylbenzene, polyacrylamide, cellulose, dextran, or a mixture thereof.

[0073] 4. The substrate comprising anionic and cationic ligands of any one of embodiments 1-3, wherein said base is sodium hydroxide, lithium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide, tetramethylammonium hydroxide, guanidine, rubidium hydroxide, or any combination thereof.

[0074] 5. The substrate comprising anionic and cationic ligands of any one of embodiments 1-4, wherein the concentration of the base is about 0.1 N to about 10 N.

[0075] 6. The substrate comprising anionic and cationic ligands of embodiment 5, wherein the concentration of the base is about 4.5 N.

[0076] 7. The substrate comprising anionic and cationic ligands of any one of embodiments 1-6, wherein step a) occurs at a temperature of about 15 °C to about 150 °C.

[0077] 8. The substrate comprising anionic and cationic ligands of embodiment 7, wherein said step a) occurs at a temperature of 40 °C.

[0078] 9. The substrate comprising anionic and cationic ligands of any one of embodiments 1-8, wherein step a) occurs for about 1 second to about 3 months.

[0079] 10. The substrate comprising anionic and cationic ligands of embodiment 9, wherein step a) occurs for about 1 second to about 48 hours.

[0080] 11. The anionic and cationic ligand-containing substrate of any preceding embodiment, wherein the substrate comprises: a cube, a tetragon, a prism, a pyramid, a platonic solid, a torus, a cone, a cylinder, a sphere, and mixtures thereof; at least one dimension (length, width, diameter, and / or height) between about 1 pm and about 1000 pm; between about 5 pm and about 750 pm; between about 5 pm and about 600 pm; between about 5 pm and about 500 pm; between about 5 pm and about 400 pm; between about 5 pm and about 250 pm; between about 5 pm and about 150 pm; or between about 5 pm and about 100 pm.

[0081] 12. The anionic and cationic ligand-containing substrate of embodiment 11, wherein the substrate is a sphere having a diameter of between about 1 pm and about 1000 pm; less than about 50 pm, between about 50 pm and about 100 pm; or greater than 100 pm and less than about 1000 pm.

[0082] 13. The anionic and cationic ligand-containing substrate of any one of embodiments 1-12, wherein a portion of sulfate, quaternary amine, and / or tertiary amine on the substrate is converted to an anionic group by treating the substrate with a solution comprising a base.

[0083] 14. The anionic and cationic ligand-containing substrate of embodiment 13, wherein the anionic group is a carboxymethyl group.

[0084] 15. The anionic and cationic ligand-containing substrate of embodiment 13 or 14, wherein the ratio of cationic groups to anionic groups of the anionic and cationic ligand- containing substrate is less than or equal to about 50: 1, about 25: 1, about 15.5: 1, about 2.4: 1, about 1.2: 1, about 1: 1, about 0.7: 1, about 0.5: 1, about 0.3: 1, or about 0.2: 1.

[0085] 16. The anionic and cationic ligand-containing substrate of embodiment 13 or 14, wherein the anionic and cationic ligand-containing substrate has:

[0086] a) an amount of anionic groups (ligands) on the surface that is between about 5% and about 95%, between about 7% and about 63%, between about 10% and about 43%, or about 19% of the total ionic capacity of the substrate, relative to the total ionic capacity of the substrate, the total ionic capacity being all (100%) anionic and cationic groups present on the anionic and cationic group-containing substrate; or

[0087] b) the amount of anionic groups (ligands) attached to the surface of the substrate is about X%, wherein X is any integer between 1 and 99, and the amount of cationic groups (ligands) attached to the surface of the substrate is about Y%, wherein Y is any integer between 1 and 99, and the sum of the selected values of X and Y is 100 (100%).

[0088] 17. A chromatography column comprising a plurality of substrates comprising anionic and cationic ligands according to embodiments 1-16.

[0089] 18. A method of performing chromatography, the method comprising:

[0090] contacting a sample comprising a target molecule with a plurality of substrates comprising anionic and cationic ligands according to any one of embodiments 1-16 or a chromatography column according to embodiment 17 under conditions such that the target molecule is captured by the plurality of substrates comprising anionic and cationic ligands;

[0091] optionally washing the plurality of substrates comprising anionic and cationic ligands with a solution; and

[0092] eluting and collecting the target molecule.

[0093] 19. The method of embodiment 18, wherein the target molecule is a protein.

[0094] 20. The method of embodiment 18, wherein the solution comprises Bis-Tris.

[0095] 21. A method of making a substrate comprising anionic and cationic ligands, the method comprising:

[0096] a) contacting a solution comprising a base with a substrate, the substrate comprising sulfate, quaternary amine, and / or tertiary amine on a surface thereof.

[0097] 22. The method of embodiment 21, further comprising washing the substrate contacted with the base with a wash solution, and optionally, recovering the substrate.

[0098] 23. The method of embodiment 21, wherein the substrate comprises methacrylate, polystyrene, agarose, divinylbenzene, polyacrylamide, cellulose, dextran, or a mixture thereof.

[0099] 24. The method of any one of embodiments 21-23, wherein the base is sodium hydroxide, lithium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide, tetramethylammonium hydroxide, guanidine, rubidium hydroxide, or any combination thereof.

[0100] 25. The method of any of embodiments 21-24, wherein the concentration of the base is about 0.1 N to about 10 N.

[0101] 26. The method of embodiment 25, wherein the concentration of the base is about 4.5 N.

[0102] 27. The method of any of embodiments 21-26, wherein step a) occurs at a temperature of about 15 °C to about 150 °C.

[0103] 28. The method of embodiment 27, wherein step a) occurs at a temperature of 40 °C.

[0104] 29. The method of any of embodiments 21-28, wherein step a) occurs for about 1 second to about 3 months.

[0105] 30. The method of embodiment 29, wherein step a) occurs for about 1 second to about 48 months.

[0106] 31. The method of any preceding embodiment, wherein the substrate comprises: a cube, a tetragon, a prism, a pyramid, a platonic solid, a torus, a cone, a cylinder, a sphere, and mixtures thereof; at least one dimension (length, width, diameter, and / or height) between about 1 pm and about 1000 pm; between about 5 pm and about 750 pm; between about 5 pm and about 600 pm; between about 5 pm and about 500 pm; between about 5 pm and about 400 pm; between about 5 pm and about 250 pm; between about 5 pm and about 150 pm; or between about 5 pm and about 100 pm.

[0107] 32. The method of embodiment 31, wherein the substrate is a sphere having a diameter of: between about 1 pm and about 1000 pm; less than about 50 pm, between about 50 pm and about 100 pm; or greater than 100 pm and less than about 1000 pm.

[0108] 33. The method of any of embodiments 21-32, wherein a portion of the sulfate, quaternary amine, and / or tertiary amine on the substrate is converted to an anionic group by treating the substrate with a solution comprising a base.

[0109] 34. The method of embodiment 33, wherein the anionic group is a carboxymethyl group.

[0110] 35. The method of embodiments 33 or 34, wherein the ratio of cationic groups to anionic groups of the substrate comprising anionic and cationic ligands is less than or equal to about 50: 1, about 25: 1, about 15.5: 1, about 2.4: 1, about 1.2: 1, about 1: 1, about 0.7: 1, about 0.5: 1, about 0.3: 1, or about 0.2: 1.

[0111] 36. The method of embodiments 33 or 34, wherein the substrate comprising anionic and cationic ligands has:

[0112] a) the amount of anionic groups (ligands) on the surface is about 5% to about 95%, about 7% to about 63%, about 10% to about 43%, or about 19% of the total ionic capacity of the substrate, the total ionic capacity being all (100%) anionic and cationic groups present on the substrate comprising anionic and cationic groups, relative to the total ionic capacity of the substrate; or

[0113] b) the amount of anionic groups (ligands) attached to the surface of the substrate is about X%, where X is any integer between 1 and 99, and the amount of cationic groups (ligands) attached to the surface of the substrate is about Y%, where Y is any integer between 1 and 99, the sum of the selected values of X and Y being 100 (100%).

[0114] Example 1 - Synthesis of a resin containing cationic and anionic ligands

[0115] The Macro-Prep High Q from Bio-Rad Laboratories was selected as the strong anion exchange resin substrate. The Macro-Prep High Q was placed in a bottle containing 4.5 N sodium hydroxide. The bottle was heated to 40 °C and agitated for different incubation times, achieved by a shaking water bath, a belt heater, and an orbital shaker or reactor, etc. After treatment, the NaOH was removed and the resin was rinsed with water. The polymer resin was packed into a column and interfaced to a liquid chromatography system. The flow rate was adjusted so that the contact time varied from 1 second to 48 hours, and a solution containing 4.5 N sodium hydroxide was flowed through the column at an ambient temperature of 40 °C. After the sodium hydroxide was flowed through the column, deionized water was flowed through the column at about 18 °C to 25 °C for about 1 second to about 1 hour.

[0116] Example 2 - Purification of a protein

[0117] This example demonstrates the chromatographic performance of Macro-Prep HQ (material from Example 1) treated with sodium hydroxide at 40°C for different times. The sodium hydroxide treated Macro-Prep HQ was packed into a chromatographic column. The conditions were as follows

[0118] Column: 1 mL (0.56 x 1.0 cm)

[0119] Flow rate: 2 mL / min

[0120] Sample: Lysozyme (0.5 mg / mL) Figure 6

[0121] Bovine Serum Albumin (BSA) (0.5 mg / mL) Figure 6

[0122] 1 mL Anion Exchange Standard (Myoglobin (pi ~ 7.2), Conalbumin (pi ~ 6.85), Ovalbumin (pi ~ 5.19), and Soybean Trypsin Inhibitor (pi ~ 4.95) resuspended in 50 mM Bis-Tris pH 7.0, 20 mL) Figure 4 and 5

[0123] Equilibration: Buffer A: 50 mM Bis-Tris, pH 7.0

[0124] Buffer B: 50 mM Bis-Tris, 1 M sodium chloride, pH 7.0

[0125] (Step 1) 100% B, 15 column volumes;

[0126] (Step 2) 100% A, 45 column volumes;

[0127] Post load: Buffer B: 50 mM Bis-Tris, 1 M sodium chloride, pH 7.0

[0128] (Step 1) 100% A, 5 column volumes;

[0129] Elution: Buffer A: 50 mM Bis-Tris, pH 7.0

[0130] Buffer B: 50 mM Bis-Tris, 1 M sodium chloride, pH 7.0

[0131] (Step 1) 0-100% B, 20 column volumes;

[0132] (Step 2) 100% B, 10 column volumes;

[0133] ​​​Regeneration: Buffer B: 50 mM Bis-Tris, 1 M sodium chloride, pH 7.0

[0134] (Step 1) 100% B, 5 column volumes.

Claims

1. A substrate comprising anionic and cationic ligands, said substrate prepared by a method comprising: a) contacting a solution comprising a base with a substrate, said substrate comprising sulfate, quaternary amine, and / or tertiary amine on a surface thereof.

2. The substrate comprising anionic and cationic ligands of claim 1, said method further comprising: b) washing said substrate contacted with a base using a wash solution.

3. The substrate comprising anionic and cationic ligands of claim 1, wherein said substrate comprises methacrylate, polystyrene, agarose, divinylbenzene, polyacrylamide, cellulose, dextran, or a mixture thereof.

4. The substrate comprising anionic and cationic ligands of any one of claims 1-3, wherein said base is sodium hydroxide, lithium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide, tetramethylammonium hydroxide, guanidine, rubidium hydroxide, or any combination thereof.

5. The substrate comprising anionic and cationic ligands of any one of claims 1-3, wherein said base has a concentration of about 0.1 N to about 10 N.

6. The substrate comprising anionic and cationic ligands of claim 5, wherein said base has a concentration of about 4.5 N.

7. The substrate comprising anionic and cationic ligands of any one of claims 1-3, wherein step a) occurs at a temperature of about 15 °C to about 150 °C.

8. The substrate comprising anionic and cationic ligands of claim 7, wherein said step a) occurs at a temperature of 40 °C.

9. The substrate comprising anionic and cationic ligands of any one of claims 1-3, wherein step a) occurs for about 1 second to about 3 months.

10. The substrate comprising anionic and cationic ligands of claim 9, wherein step a) occurs for about 1 second to about 48 hours.

11. The substrate comprising anionic and cationic ligands of any one of claims 1-3, wherein a portion of the sulfate, quaternary amine, and / or tertiary amine on said substrate is converted to anionic groups by treating said substrate with a solution comprising a base.

12. The substrate comprising anionic and cationic ligands of claim 11, wherein said anionic groups are carboxymethyl groups.

13. The substrate comprising anionic and cationic ligands of claim 11, wherein the ratio of cationic groups to anionic groups of said substrate comprising anionic and cationic ligands is less than or equal to about 50: 1, about 25: 1, about 15.5: 1, about 2.4: 1, about 1.2: 1, about 1: 1, about 0.7: 1, about 0.5: 1, about 0.3: 1, or about 0.2:

1.

14. The substrate comprising anionic and cationic ligands of claim 11, wherein said substrate comprising anionic and cationic ligands has: a) the amount of anionic groups (ligands) on the surface relative to the total ionic capacity of the substrate is about 5% to about 95%, about 7% to about 63%, about 10% to about 43%, or about 19% of the total ionic capacity of the substrate, the total ionic capacity being all (100%) anionic and cationic groups present on the substrate comprising anionic and cationic groups; or b) the amount of anionic groups (ligands) attached to the surface of the substrate is about X%, where X is any integer between 1 and 99, and the amount of cationic groups (ligands) attached to the surface of the substrate is about Y%, where Y is any integer between 1 and 99, the sum of the selected values of X and Y being 100 (100%).

15. A chromatography column comprising a plurality of substrates comprising anionic and cationic ligands according to claims 1-3.

16. A method of performing chromatography, the method comprising: contacting a sample comprising a target molecule with a chromatography column according to claim 15 under conditions such that the target molecule is captured by a plurality of the substrates comprising anionic and cationic ligands; optionally washing the plurality of the substrates comprising anionic and cationic ligands with a solution; and eluting and collecting the target molecule.

17. The method of claim 16, wherein the solution comprises Bis-Tris.

18. A method of preparing a substrate comprising anionic and cationic ligands, the method comprising: a) contacting a solution comprising a base with a substrate, the substrate comprising sulfate, quaternary amine, and / or tertiary amine on its surface.

19. The method of claim 18, wherein the substrate comprises methacrylate, polystyrene, agarose, divinylbenzene, polyacrylamide, cellulose, dextran, or a mixture thereof.

20. The method of any one of claims 18-19, wherein the base is sodium hydroxide, lithium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide, tetramethylammonium hydroxide, guanidine, rubidium hydroxide, or any combination thereof.

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