Removal of polymers from solution by using macrocycles and / or hydrophobic particles

Abstract

The present invention provides a method for removing free polymers present in a solution containing a hydrophobic protein, comprising the steps of: (a) contacting a solution containing free polymer not bound to a hydrophobic protein with macrocycles and / or hydrophobic particles, so that the free polymer binds to the macrocycles and / or hydrophobic particles, thereby obtaining a complex of the polymer and the macrocycles and / or hydrophobic particles; and (b) removing the complex of the polymer and the macrocycle and / or hydrophobic protein from the solution. Further described are the use of macrocycles and / or hydrophobic particles in this method, compositions of macrocycles for this method, and kits containing macrocycles and / or hydrophobic particles for use in this method.

Description

[Technical Field]

[0001] The present invention relates to methods for removing free polymers from solutions using macrocycles such as cyclodextrins, calixarenes, cucurbiturils or pillararenes, and / or hydrophobic particles such as butyl agarose, the use of these materials in such methods, compositions containing materials adapted for the methods, and kits containing macrocycles and / or hydrophobic particles for removing free polymers from solutions. [Background technology]

[0002] Solubilizing, stabilizing, and purifying membrane proteins from their native membrane environment is a well-established procedure. It depends on many parameters, most of which can be optimized for higher efficiency during the purification process. A key step in the purification process is the effective solubilization of the target protein from the cell membrane, which is achieved by using polymers.

[0003] To effectively solubilize the maximum amount of membrane proteins, the polymer must be used in excess. However, not all of the polymer used during the solubilization step is utilized, resulting in free polymer in the purified solubilization supernatant. Due to their chemical properties (nonspecific interactions of the polymer, interference with downstream processes), free polymers pose disadvantages. On the one hand, protein binding efficiency during affinity chromatography is affected. Free copolymers significantly interfere with the protein binding steps to many different resins, resulting in significant losses during protein-resin binding. Furthermore, downstream processing is similarly affected. Due to their chemical properties, free polymers significantly interfere with several downstream processes, such as mass spectrometry and enzyme kinetic analysis or SDS-PAGE. Summary of the Invention

[0004] Therefore, the technical problem underlying the present invention is to provide methods, uses, compositions and kits that allow for the removal of free polymer from purified solubilized supernatants, so that the above-mentioned disadvantages can be avoided. This object is solved by the subject matter defined in the independent claims. Preferred embodiments are defined in the dependent claims.

[0005] It has been found that free polymers present in a solution containing a hydrophobic protein can be removed by a method comprising the following steps: (a) contacting a solution containing free polymer that is not bound to a hydrophobic protein with macrocycles and / or hydrophobic particles such that the free polymer binds to the macrocycles and / or hydrophobic particles to form complexes of the polymer with the macrocycles and / or hydrophobic particles; and (b) Removing the complexes of polymer and macrocycles and / or hydrophobic particles from the solution.

[0006] As understood by the present invention, the term "hydrophobic protein" refers to integral membrane proteins, peripheral membrane proteins, or proteins with exposed hydrophobic regions, such as GTPases, ATG proteins, proteins containing ENTH / ANTH or Bar domains, and their interacting proteins. A review can be found in Mikhail A Zhukovsky, Angela Filograna, Alberto Luini, Daniela Corda, Carmen Valente; Protein Amphipathic Helix Insertion: A Mechanism to Induce Membrane Fission; Front Cell Dev Biol. 2019 Dec 10; 7:291. doi:10.3389 / fcell.2019.00291. eCollection 2019.

[0007] The free polymer to be removed by the present invention can be derived from the solubilization, stabilization, and, if necessary, purification of membrane proteins from their native membrane environment. This is a well-established procedure. Those skilled in the art understand the methods and materials for its implementation. Briefly, one step of this process is the effective solubilization of target proteins from cell membranes, which is achieved by using specific polymers known in the art. To effectively solubilize the maximum amount of membrane protein, the polymer used must be used in excess. However, because not all of the polymer used during the solubilization process is utilized, free polymers will be generated in the purified solubilization supernatant.

[0008] The polymer used in this step can be a homopolymer or a copolymer, as described in more detail below.

[0009] The term "remove" as used herein means that the amount of free polymer is reduced after carrying out the method according to the invention compared to the amount of free polymer before carrying out the method according to the invention.

[0010] The method according to the present invention allows for the rapid, labor-free, and highly efficient removal of polymers from a solution. Furthermore, the removal of the polymers prevents them from interfering with downstream processing, such as mass spectrometry and enzyme kinetic analysis of the remaining components of the solution, e.g., membrane proteins. Polymer removal of 95% or even more is possible.

[0011] Below, we present a general protocol for the purification of membrane proteins stabilized in copolymers (e.g., AASTY (copolymer of styrene and acrylic acid), Ultrasolute Amphipol (polyacrylic acid partially linked to amides via cycloalkylamines or cycloalkylalkylamines)).

[0012] As mentioned above, solubilization, stabilization, and purification of membrane proteins from their native membrane environment depend on many parameters. Most parameters can be optimized for higher efficiency during the purification process. These parameters include buffer conditions (e.g., salt, pH), polymer selection, protein-to-solubilizing agent ratio, temperature, and time. First, cells are lysed and centrifuged using the following parameters: add a protease inhibitor (PI) to the buffer, readjust the pH, and then disrupt the cells (e.g., sonicate or French press). Centrifuge at 9,000 rcf for 30 minutes at 4°C, discard the pellet (cell debris), collect the supernatant, centrifuge at 100,000 rcf for 1 hour at 4°C, discard the supernatant, and homogenize the pellet. Next, membrane proteins are solubilized. The polymer forms synthetic nanodiscs around the protein, thereby maintaining the natural phospholipid environment and preserving the protein's natural, and therefore functional, properties in a convenient one-step process (solubilization and stabilization). On the other hand, the surfactant forms micelles around the hydrophobic belt, thereby removing lipids from the environment. In the natural state, the unique lipid environment must be preserved.

[0013] In one embodiment, the hydrophobic protein is a membrane protein, particularly a membrane-bound or integral membrane protein. Examples of membrane proteins can be selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transporter proteins such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-bound ATPases, and SLC transporters. That is, as a starting material for the method according to the present invention, a solution of free polymers is used, which is derived from the solubilization, stabilization, and purification of the above-mentioned membrane proteins from their natural environment using polymers.

[0014] As understood by the present invention, a macrocycle is a molecule containing a ring of at least 12 atoms / ions. Furthermore, as understood by the present invention, a hydrophobic particle is a particle consisting of a solid phase and a surface bearing hydrophobic groups. According to Wikipedia, hydrophobic materials are water-repellent. Examples of these groups include linear and branched alkyl, phenyl, methylphenyl, ethylphenyl, aromatic groups such as styrene, polyacrylates and methacrylates, fatty acid esters, and polypropylene glycol. It is noted that hydrophobic particles are distinct from hydrophobic proteins; i.e., hydrophobic proteins are not considered hydrophobic particles. Hydrophobic particles can be hydrophobic resins derived from materials such as polystyrene, polyacrylate, and polymethacrylate, and can be composites of one or more hydrophobic substances with hydrophilic materials such as silica, metal oxides, polysaccharides such as dextran, or agarose. In one embodiment, the macrocycle is selected from the group consisting of cyclodextrins, calixarenes, cucurbiturils, and pillararenes. An example of a hydrophobic particle is butyl agarose.

[0015] In a further embodiment, the cyclodextrin is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, δ-cyclodextrin, or a mixture of at least two of the foregoing cyclodextrins. The cyclodextrin can reversibly bind to free copolymers in solution, which can be achieved by concentration-dependent stepwise binding. Because polymers may react differently to cyclodextrins depending on their unique chemical properties, routine testing can be performed to select the appropriate cyclodextrin for polymer removal.

[0016] In one embodiment, the macrocycles are provided on magnetic or agarose beads, which shows significant effects on polymer binding.

[0017] The magnetic beads that can be used contain a magnetic core made of magnetite covered with another material. Magnetic beads are usually ferrimagnetic (or ferromagnetic) or superparamagnetic. Ferrimagnetic / ferromagnetic cores are usually large (over 30 nm) and exhibit a strong magnetic moment. They retain this magnetic moment even after the magnetic field is removed. This effect is called "remanence." A strong magnetic field results in rapid separation of the beads in the magnetic field. At the same time, they can exhibit self-magnetism and adhere to metal surfaces. Superparamagnetic magnetic cores are smaller (5-30 nm) and have a weaker magnetic moment. The beads lose their magnetism when the external magnetic field is removed. At the same time, they are easier to use on metal surfaces.

[0018] Alternative embodiments include functionalized membranes and so-called monolithic columns. For example, cellulose membranes can be chemically modified with cyclodextrins, hydrophobic polymers, linear and branched alkyl, or aromatic molecules to prepare functionalized membranes that suppress unbound copolymers. The chemistry used for this modification is nearly identical to that described for agarose and magnetic beads (see below).

[0019] Porous monolithic columns were developed by Frechet and Svec, who polymerized styrene and (meth)acrylates in the presence of porogens. Silica monoliths are solid, continuous blocks of porous material with a bimodal pore size distribution (macropores and mesopores). Further details are presented in Unger KK, Skudas R, Schulte MM. Particle-packed columns and monolithic columns in high-performance liquid chromatography—comparison and critical appraisal. J Chromatogr A 2008;1184:393-415.

[0020] The synthesis of cyclodextrin-modified agarose and magnetic beads can be carried out by, but not limited to, the following method: - Reaction of epoxy-activated agarose or mag beads with cyclodextrin (variant A). This synthetic variant is described in Xiangling He, Tianwei Tan, Bingze Xu, Jan-Christer Janson, "Separation and purification of puerarin using cyclodextrin-coupled agarose gel media," Journal of Chromatography A, 1022 (2004) 77-82. - NHS-activated agarose or Mag beads are reacted with aminocyclodextrin (Variant B). This synthetic variant is described in Trung Nguyen, Neel S. Joshi, and Matthew B. Francis, An Affinity-Based Method for the Purification of Fluorescently-Labeled Biomolecules, Bioconjugate Chem. 2006, 17, 869-872.

[0021] Both synthetic variants result in particles modified with cyclodextrins, which effectively remove macromolecules according to the present patent application. In particular, magnetic beads with the following properties can yield very favorable results: medium-sized beads (20-40 µm) with a ferrimagnetic core and agarose coating: high polymer binding, low nonspecific binding, and efficient separation; and large or extra-large magnetic agarose beads (70-120 µm or up to 1000 µm).

[0022] In one embodiment, the cyclodextrin is provided in a cross-linked form. These cyclodextrin particles also allow for the removal of copolymers. Cross-linking of cyclodextrins can be carried out using reagents such as epichlorohydrin, diepoxide, and carbonyldiimidazole of divinylsulfone. Examples of the preparation of cross-linked carbohydrates can be found in Xiangling He, Tianwei Tan, Bingze Xu, and Jan-Christer Janson, "Separation and purification of puerarin using cyclodextrin-coupled agarose gel media," Journal of Chromatography A, 1022 (2004) 77-82.

[0023] Other materials that can be used are calixarenes, cucurbiturils, pillararenes in cross-linked or solid phase bound form. Additionally, 6% to 12% agarose, cross-linked or dextran conjugated to other polymer particles, and copolymers of methylenebisacrylamide and dextran, which can be unmodified or modified with hydrophobic groups such as linear and branched alkyl, alkenyl, alkynyl, and aromatic groups, are effective in reducing the polymer concentration in protein solutions.

[0024] Organic polymer particles, such as Biobeads SM-2 (polystyrene particles, Bio-Rad Inc., CA, USA), Macroprep methyl or butyl HIC resin (polymethacrylate particles, Bio-Rad Inc.), allow for polymer reduction.

[0025] Calixarenes are phenolic molecules linked by methylene groups or other functional groups. For example, in Aseyev's work (Wiktorowicz, H. Tenhu and V. Aseyev, Polym. Chem., 2013, 4, 2898), they are functionalized with tetraethylene glycol or alkyl groups to adjust the polarity of the molecule. These molecules can be attached to solid phases using standard methods known to those skilled in the art, for example, as described for cyclodextrins.

[0026] Cucurbiturils were first synthesized by Behrend in 1905 through the acid-catalyzed condensation reaction of urea, glyoxal, or formaldehyde. They can noncovalently interact with various sizes of positively charged and neutral guests via hydrogen bonding, charge-dipole, and hydrophobic / hydrophilic effects to form supramolecular host-guest complexes. Cucurbituril [6] can form inclusion complexes with hydrophobic neutral guests (tetrahydrofuran and benzene), protonated amines, and p-methylbenzylamine, while cucurbituril [7] can interact with naphthalene, protonated adamantanamine, and carborane, respectively. Cucurbituril[8], with its large cavity, is involved in complexation with large guest molecules (cyclen, cyclam, and their metal complexes). The synthesis of supramolecular hydrogels formed from CB[8]-encapsulated hyperbranched polymers (HBP-CB[8]) and linear hydroxyethyl cellulose-functionalized naphthalene is described in the paper "Adv. Funct. Mater., 2018, 28, 1702994" by C.S.Y. Tan, J. Liu, A.S. Groombridge, S.J.B. Arrow, C.A.D. Reiss, and O.A.S. Cherman; this procedure can also be used to prepare cucurbituril-modified particles.

[0027] Pillararene compounds have a pillar-like structure due to the methylene bridge at the para position of the functionalized aromatic ring. This structure makes them very effective in binding electron-withdrawing or neutral guests. Their good solubility in both organic and aqueous solutions makes them suitable for a wide range of applications.

[0028] For example, these molecules (T. Ogoshi, S. Kanai, S. Fujinami, TA Yamagishi and Y. Nakamoto, J. Am. Chem. Soc., 2008, 130, 5022-5023), prepared by the reaction of 1,4-dimethoxybenzene with paraformaldehyde and a Lewis acid, can be used to selectively bind copolymers from solution.

[0029] Agarose, dextran polymers, dextran-functionalized agarose, copolymers of methylene bisacrylamide and dextran, etc., can be modified with hydrophobic groups such as butyl, phenyl, or octyl by reacting the polysaccharide with butyl, phenyl, or octyl glycidyl esters under anhydrous conditions in the presence of a Lewis catalyst such as boron trifluoride diethyl ether complex. This procedure is described by Feng Qing-zheng; Meng Qing-qiang; Wang Jia-xing; Ma Guang-hui; Ma Run-yu; and Su Zhi-guo, "Preparation of Butyl-agarose Chromatography Media with Controlled Ligand Density through Direct Coupling Reaction," The Chinese Journal of Process Engineering, 2006, 6(6):959-963. Butyl, phenyl, and octyl agarose can be purchased from Cube Biotech, Monheim, Germany.

[0030] Suitable particle, membrane, and monolith columns for copolymer removal can be applied to medium-pressure chromatography gravity-flow columns, FPLC columns, and HPLC columns, which can be used in automated chromatography systems such as Akta (Cytiva) or BioLogic™ low-pressure liquid chromatography systems (Bio-Rad).

[0031] In one embodiment, the macrocycles and / or hydrophobic particles are washed with a solvent that is the solvent for the polymer solution before performing step (a). In yet another embodiment, steps (a) and (b) are repeated at least once, e.g., 1 to 3 times, for a total of 1 to 4 times. Repeating steps (a) and (b) can remove more free protein than performing them only once, allowing for tailoring of removal based on downstream processing needs.

[0032] As noted above, the polymer can be a homopolymer or a copolymer. In one embodiment, the polymer can have hydrophobic groups such as COOH, maleimide, OH, amine, ammonium salt, zwitterion such as phosphocholine, and polymerized styrene groups, polymerized diisobutylene groups, or linear C1-C16 (such as methyl and ethyl) aliphatic groups, branched C1-C16 (such as isopropyl or t-butyl) aliphatic groups, cyclic C5-C12 aliphatic or aromatic groups.

[0033] The molecular weight of the polymer used in accordance with the method of the present invention can be, for example, 1900 to 20000, 2000 to 18000, 2000 to 15000, 4000 to 16000, 4000 to 13000, or 5000 to 14000. The molecular weight can be measured by gel permeation chromatography or mass spectrometry.

[0034] Examples of polymers include, but are not limited to, styrene / maleic acid copolymers sold under the trade name "SMA," styrene / maleic acid copolymer derivatives such as SMA 200 and 300, and styrene / maleimide copolymers such as SMA 502. These materials can also be functionalized at the COOH groups with amines such as ethanolamine or ethylenediamine to form amides, or with alcohols such as glycerol to form esters. Polymers can also be functionalized with polyethylene glycol to form esters and aminated polyethylene glycol to form amides.

[0035] The polymer can be, for example, a diisobutylidene / maleic acid copolymer, such as DIBMA10 and DIBMA12 from Cube Biotech, and derivatives of diisobutylidene / maleic acid copolymers, such as DIBMA Gly, DIBMA Glu, Glyco DIBMA, and diisobutylidene / maleimide copolymers. DIBMA copolymers can be functionalized with the same molecules as SMA.

[0036] Further polymers may be copolymers of styrene and acrylic acid, in particular those with molecular weights of 5,500 and 11,000 and acrylic acid / styrene ratios of 45% / 55% to 55% / 45%, sold under the name "AASTY".

[0037] Modified polymers of polyacrylic acid can be used, in which 10-90% of the carboxylic acid groups can be modified to amides with cyclohexylamine, 2-cyclohexylethylamine, or in some embodiments with substances such as DCC (dicyclohexylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide), or PyBOP, HBTU, TBTU, etc. These substances are sold under the name "Amphipol Ultrasolve." In addition to the polymer disclosure above, further polymer descriptions are provided below.

[0038] Polymers with hydrophilic and hydrophobic functional groups: Examples of hydrophilic functional groups can be, but are not limited to, acrylic and methacrylic acid, maleic acid, carboxylic acid groups in general, amides with α,ω-alkylenediamines, ω-hydroxyalkylamines and ω-aminoallylthiols, trimethylammonio-alkylamines, amides from aminoglycerol TRIS or bis-tris, maltosamine, glucosamine, mannosamine and other amino-functionalized carbohydrates, amides with taurine.

[0039] Also included are esters of carboxylic acid groups with polyethylene glycols, diols, triols, polyols, and carbohydrates. Other examples may be maleimides with the nitrogen atom functionalized with an alkyl chain bearing an alcohol, thiol, amine, ammonium salt, and the like.

[0040] Alternatively, zwitterionic molecules consisting of ammonium and phosphate groups can be attached to the carboxyl groups, as described in U.S. Patent Application Publication Nos. 2020281855A1 or 2021171673A1.

[0041] Examples of hydrophobic groups include, but are not limited to, polymerized styrene and derivatives such as methylstyrene, diisobutylene, and linear and branched alkanes such as 2-propyl, hexyl, octyl, or decyl, which are linked to the carboxyl group through an ester or amide functional group. Also suitable are maleimide groups with alkyl or aryl groups on the amino functional group.

[0042] Examples of the synthesis of styrene-maleic acid copolymers can be found in Shintaro Sugai, Nobumichi Ohno, Conformational transitions of the hydrophobic polyacids, Biophysical Chemistry, Volume 11, Issues 3-4, June 1980, Pages 387-395.

[0043] The use of SMA to form complexes with lipids is described in WO 2006 / 129127 and references therein. SMA can be purchased from Orbiscope or Cube Biotech as SMALP140, SMALP200, or SMALP300.

[0044] The synthesis of copolymers from diisobutylene and maleic anhydride is described in U.S. Patent No. 4,250,289 by BASF. The hydrolysis of anhydride copolymers to diisobutylene-co-maleic acid is described by Lee, Nature Protocols Vol. 11, No. 7, 2016, pp. 1149-1162, and is described for SMA copolymers but can be applied successfully to DIBMA.

[0045] The synthesis of a DIBMA polymer with glucosamine functionalization on 50% of all carboxyl groups can be found in Bartholomaus Danielczak, Marie Rasche, Julia Lenz, Eugenio Perez Patallo, Sophie Weyrauch, Florian Mahler, Michael Tope Agbadaola, Annette Meister, Jonathan Oyebamiji Babalola, Carolyn Vargas, Cenek Kolar and Sandro Keller, A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs, DOI: 10.1039 / D1NR03811G (Paper) Nanoscale, 2022, 14, 1855-1867. DIBMA can be purchased from Cube Biotech as DIBMA10 and DIBMA12.

[0046] The preparation of poly(acrylic acid-co-styrene) copolymers is described in WO 2020 / 257637 and Simon Harrisson, Francesca Ercole, Benjamin W. Muir, “Living spontaneous gradient copolymers of acrylic acid and styrene: one-pot synthesis of pH-responsive amphiphiles,” Polym. Chem., 2010, 1, 326-332.

[0047] In some cases, the copolymer is a copolymer of styrene and acrylic acid, or a copolymer of styrene and an acrylic acid derivative. Any copolymer derivative may be used in the copolymer. Examples of derivatives include acrylates, methacrylates, acrylic esters, acrylamides, and N-substituted acrylamides. In certain cases, the acrylic esters or acrylamides are substituted with zwitterionic species, as described in U.S. Patent Application Publication No. 20190062469A1, the disclosure of which is incorporated herein by reference. In certain embodiments, the copolymer comprises an acrylic acid or acrylic acid derivative content of 20% to 80%, 30% to 70%, 35 to 65%, or 40 to 60%.

[0048] The synthesis of amphipols is described in WO 115083 and Marconnet, A., Michon, B., Le Bon, C., Giusti, F., Tribet, C., & Zoonens, M. (2020). Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers. Biomacromolecules. doi:10.1021 / acs.biomac.0c00929.

[0049] Additional polyacrylates modified with alkyl groups such as pentyl, hexyl, and tert-butyl are described in U.S. Patent Application Publication No. 2020 / 0383918.

[0050] A polymethacrylate containing approximately 0.52% butyl methacrylate (BMA) in the copolymer and approximately 0.48% methyl acryoloxycholine (MAC) in the copolymer, with a degree of polymerization (DP) of approximately 39.00, is sold by Avanti Polar Lipids under the trade name Polymethacrylate Copolymer (N-C4-52-6.9). Other polymethacrylates are described in Yasuhara K, Arakida J, Ravula T, Ramadugu SK, Sahoo B, Kikuchi JI, Ramamoorthy A. 2017. Spontaneous Lipid Nanodisc Formation by Amphiphilic Polymethacrylate Copolymers. J Am Chem Soc. 139(51):18657-18663.

[0051] Polyacrylate polymers modified with alkanes such as n-butyl, t-butyl, pentyl, neopentyl, and hexyl are described in Nathaniel Z. Hardin, Thirupathi Ravula, Giacomo Di Mauro, Ayyalusamy Ramamoorthy, Hydrophobic Functionalization of Polyacrylic Acid as a Versatile Platform for the Development of Polymer Lipid Nanodiscs, Small. 2019 March;15(9):e1804813. doi:10.1002 / smll.201804813, and U.S. Patent Application Publication No. 2020383918A1.

[0052] Alternatively, U.S. Patent Application Publication No. 2022093587A mentions linear polysaccharides functionalized with hydrophobic groups and having a degree of polymerization of less than 100. An example of a linear carbohydrate is inulin, and examples of the hydrophobic group are alkyl, alkenyl, alkynyl, cycloalkyl, or heteroalkyl having 1 to 3 heteroatoms. The hydrophobic group is attached to the carbohydrate via an ether, ester, or amide group.

[0053] In one embodiment, step (a) of the method according to the invention is carried out for 1 minute or less. Longer incubation times do not provide a significant enhancement effect. That is, a very rapid method for removing polymer from solution is provided by the method according to the invention.

[0054] In the following, a general description of the method according to the invention is given in order to illustrate it in more detail: To remove up to 50% of the free polymer from the solution, an equal volume of Mag Beads slurry (25%, v / v) can be used.

[0055] One step reduction: Prepare one sample tube containing, for example, 50 μl of Mag Beads slurry. Remove the storage buffer. Wash with sample buffer, for example, twice, and remove the sample buffer. Add, for example, 50 μl of sample to this first sample tube, incubate the mixture, for example, for about 1 minute, and remove the sample.

[0056] A four-step removal process can result in up to a 95% reduction in free copolymer. For example, prepare four sample tubes containing 50 μl of Mag Beads slurry. Remove the storage buffer. Wash with sample buffer, for example, twice, and remove the sample buffer. For example, add 50 μl of sample to the first sample tube, incubate the mixture for, for example, 1 minute, remove the sample and add it to the next sample tube, and continue incubating for, for example, about 1 minute. This is repeated two more times.

[0057] The present invention further provides the use of a cyclodextrin in the method according to the invention. The cyclodextrin used, and details of the method, are described above, to which reference is made in its entirety.

[0058] The present invention further provides compositions comprising cyclodextrins bound to a carrier for carrying out the above-described practice, and kits for carrying out the method, for which reference is made to the above detailed description for a description of the materials and process steps of the method. The present invention will now be further described with reference to the following examples and figures, which should not be construed as limiting the invention thereto. [Brief explanation of the drawings]

[0059] [Figure 1] FIG. 1 shows the removal of free polymer using cyclodextrin bound to magnetic beads. [Figure 2] FIG. 10 shows further removal of free polymer using cyclodextrin bound to magnetic beads. [Figure 3] FIG. 10 shows further removal of free polymer using cyclodextrin bound to magnetic beads. [Figure 4] FIG. 10 shows further removal of free polymer using cyclodextrin bound to magnetic beads. [Figure 5] FIG. 1 shows the removal of free polymer using cyclodextrin bound to agarose beads. [Figure 6A] ~ [Figure 6C] FIG. 1 shows a comparison of the reduction of free copolymer using cyclodextrin-bound agarose and size exclusion chromatography. [Figure 7] FIG. 1 shows the results of polymer removal from cell lysates containing complexed and solubilized membrane proteins. DETAILED DESCRIPTION OF THE INVENTION

[0060] Example 1: Removal of free copolymer from small volume samples using CD (cyclodextrin)-bound magnetic beads: An equal amount of magnetic beads (Magbeads from Cube biotech GmbH) is used as a slurry (25%) in order to remove up to 50% by weight of free copolymer from the solution.

[0061] 1 step reduction: Prepare one sample tube with 50 μl of Mag Beads slurry each. Remove storage buffer. Wash twice with sample buffer. Remove sample buffer. Add 50 μl of sample to the first sample tube, incubate for 1 minute, then remove sample. Using a four-step removal process, free copolymer is reduced by up to 95%.

[0062] 4-step reduction: Prepare four sample tubes each with 50 μl of Mag Beads slurry. Remove storage buffer. Wash twice with sample buffer. Remove sample buffer. Add 50 μl of sample to the first sample tube, incubate for 1 minute, remove sample and add to the next sample tube and incubate for 1 minute. Repeat this two more times. Use sample in the assay of choice.

[0063] The details of the experiments shown in FIGS. 1 to 4 and the results obtained will be described below. Figure 1: In the experiment related to Figure 1, 50 μl of a 2.5% SMA200 solution was incubated with different volumes of CDα-coupled magnetic beads for 1 minute, and a reduction capacity of up to 95% was obtained (using a four-step removal process). The four-step removal process results in higher polymer reduction compared to a one-step removal process using a similar volume of magnetic beads. The 2.5% SMA200 solution was set to 100% (alone). (CDα = cyclodextrin alpha-coupled magnetic beads; 50 μl of 25% slurry = 12.5 μl of pure CDα).

[0064] Figure 2: In the experiment related to Figure 2, 50 μl of a 1.25% AASTY11-45 solution was incubated for 1 minute with the same volume (50 μl of 25% slurry) of different CD-conjugated magnetic beads (alone = no magnetic beads, gamma = CDγ-conjugated magnetic beads, gamma 15222 = CDγ-conjugated magnetic beads, alpha = CDα-conjugated magnetic beads, beta = CDβ-conjugated magnetic beads). A reduction capacity of up to 60% was obtained (using a one-step removal process). AASTY was the only polymer tested that showed significantly enhanced binding to CDα. The 1.25% AASTY11-45 solution was set to 100% (50 μl of 25% slurry = 12.5 μl of pure CDα volume).

[0065] Figure 3: In the experiments related to Figure 3, 50 μl of a 1.25% DIBMA12 solution was incubated for 1 minute with the same volume of different CD-bound magnetic beads (50 μl of 25% slurry) (alone = no magnetic beads, gamma = CDγ-bound magnetic beads, gamma 15222 = CDγ-bound magnetic beads, alpha = CDα-bound magnetic beads), and a reduction capacity of up to 60% was obtained (using a one-step removal process). DIBMA12 does not show significantly enhanced binding to CDα compared to CDγ. The 1.25% DIBMA12 solution was set to 100% (50 μl of 25% slurry = 12.5 μl of pure CDα volume).

[0066] Figure 4: In the experiments shown in Figure 4, 50 μl of different concentrations of AASTY11-45 solution (2.5%, 1.25%, 0.5%, 0.25%) were incubated with the same volume (50 μl of 25% slurry) of CDγ-conjugated magnetic beads for 1 minute (alone = no magnetic beads). Using a similar volume of CDγ-conjugated magnetic beads does not necessarily result in enhanced reduction capacity using a one-step removal process. A 10-fold diluted (0.25%) AASTY11-45 sample resulted in a 65.4% reduction, while the 2.5% AASTY11-45 sample using a similar volume of CDγ-magnetic beads resulted in a 46% reduction, and the 1.25% AASTY11-45 sample resulted in only a 27.7% reduction. Individual AASTY11-45 solutions were set as 100% (from left to right: 2.5%, 1.25%, 0.5%, 0.25%). (50 μl of 25% slurry = 12.5 μl volume of pure CDα).

[0067] Example 2: Removal of free copolymer from large volume samples using CD-bound agarose: To remove up to 90% of the free copolymer from the solution, use an equal volume of agarose (twice the volume of a 50% slurry, e.g., 5 ml of copolymer solution and 10 ml of agarose slurry). A larger agarose bed ensures more efficient removal of the free copolymer.

[0068] 1 step reduction: Prepare a column packed with agarose slurry (e.g., for 5 ml of copolymer solution, pack 10 ml of 50% agarose slurry into a 1 cm diameter column). Allow the stock solution to drain by gravity flow. Wash the agarose with 5 column volumes (CV) of sample buffer. Add the sample and allow it to drain by gravity. Collect the sample and use it in the assay of choice.

[0069] In the experiment shown in Figure 5, 5 ml of a 5% / 2.5% DIBMA10 solution was loaded onto a column packed with different volumes of CDγ-conjugated agarose beads, and a reduction capacity of up to 90% was obtained (using 5 ml of pure agarose beads with a bed height of 10 cm). The bed volume and column diameter play an important role in the binding capacity of DIBMA10. The 5% DIBMA10 was set to 100%.

[0070] Comparative Example: Removal by Superose 6 Comparison of free copolymer reduction using cyclodextrin-bound agarose and size exclusion chromatography.

[0071] When a 2.5% Ultrasolute amphipol solution was passed through cyclodextrin-coupled agarose beads and washed twice with equal volumes (Figure 6A), a 90% reduction in the amount of detectable Ultrasolute amphipol in the flow-through fraction (FT) was observed. The first wash (W1) showed 30% detectable Ultrasolute amphipol, and the second wash (W2) showed 13% detectable Ultrasolute amphipol. The copolymer binds to the cyclodextrin matrix during its migration through the resin and is then washed off in multiple steps. This is not the case when a 2.5% Ultrasolute amphipol solution is passed through a Superose 6 10 / 300 GL column (Cytiva) (Figure 6B). The copolymer elutes broadly over a 7.5 ml volume. This equates to a size distribution ranging from approximately 700 kDa to approximately 17 kDa (see Figure 6C), indicating that extraction of free copolymer by size exclusion chromatography is not possible, as most proteins also elute in a similar manner. On the other hand, passing the copolymer in a solubilized membrane solution through a cyclodextrin matrix is ​​a simple, rapid and reliable method for extracting the free copolymer from solution.

[0072] Example 3: Polymer removal protocol from cell lysates containing complexed and solubilized membrane proteins. A general description of this protocol is as follows: The solution may contain up to 5% surfactant and amphiphilic polymer. The distilled water, buffer, and protein solutions used can be forced through the column by gravity or by manual or automated pressure application. 1. Prepare a spin column, dropping tube, or equivalent column equipped with a frit and fill it with 2 mL (50% suspension) of a partially or completely hydrophobic functionalized resin (e.g., cyclodextrin agarose or butyl agarose). 2. To equilibrate, wash the compacted agarose with 3x resin volumes of distilled water and 3x resin volumes of protein buffer (e.g.: 150 mM NaCl, 20 mM HEPES, pH 7.5). 3. Load 500 µL of cell lysate containing the desired protein-Nanodisc complex and excess polymer onto the column, ensuring the solution completely enters the resin. Discard the flow-through fraction. 4. To elute the solubilized membrane proteins, add 1.5 mL of protein buffer and collect the flow-through fraction containing the protein-Nanodisc complex of interest.

[0073] Figure 7 describes the results of polymer removal from a cell lysate containing complexed and solubilized membrane proteins. The solution can contain up to 5% AASTY copolymer. Spent distilled water, buffer, and protein solution can be allowed to flow through the column by gravity or can be slowly forced through the column by manual or automated pressure application. 1. Prepare a spin column, dropping tube, or equivalent column equipped with a frit and fill it with 2 mL (50% suspension) of a partially or completely hydrophobic functionalized resin (e.g., cyclodextrin agarose or butyl agarose). 2. To equilibrate, wash the compacted agarose with 3x resin volumes of distilled water and 3x resin volumes of protein buffer (e.g.: 150 mM NaCl, 20 mM HEPES, pH 7.5). 3. Load 500 µL of cell lysate containing the desired protein-Nanodisc complex and excess polymer onto the column, ensuring the solution completely enters the resin. Discard the flow-through fraction. 4. To elute the solubilized membrane proteins, add 1.5 mL of protein buffer and collect the flow-through fraction containing the protein-Nanodisc complex of interest.

Claims

1. 1. A method for removing free polymers present in a solution containing a hydrophobic protein, comprising: (a) contacting the solution containing the free polymer not bound to the hydrophobic protein with macrocycles and / or hydrophobic particles, so that the free polymer binds to the macrocycles and / or hydrophobic particles, resulting in a complex between the polymer and the macrocycles and / or hydrophobic particles; and (b) removing said complexes of polymer and said macrocycles and / or hydrophobic particles from said solution.

2. The method of claim 1 , wherein the hydrophobic protein is a membrane protein.

3. The method of claim 2, wherein the membrane protein is a membrane-associated protein or an integral membrane protein.

4. The method of claim 2 or 3, wherein the membrane protein is selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transporter proteins such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-bound ATPases, and SLC transporters.

5. 5. The method according to claim 1, wherein the macrocycle is selected from the group consisting of cyclodextrins, calixarenes, cucurbiturils and pillararenes, and the hydrophobic particle is butyl agarose.

6. 6. The method of claim 5, wherein the cyclodextrin is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and δ-cyclodextrin or a mixture of at least two of said cyclodextrins.

7. 7. The method of claims 5 and 6, wherein the macrocycle is provided on a magnetic bead or an agarose bead, or as a particle comprising a cross-linked cyclodextrin.

8. The method of any one of claims 1 to 7, wherein the macrocycles and / or hydrophobic particles are washed with a solvent that is a solvent for the solution of the polymer before step (a) is carried out.

9. The method of any one of claims 1 to 8, wherein steps (a) and (b) are repeated at least once.

10. The method according to any one of claims 1 to 9, wherein the polymer is a polymer capable of solubilizing and stabilizing membrane proteins and GPCRs.

11. 11. The method of claim 10, wherein the polymer has hydrophilic groups such as COOH, maleimide, OH, amines, ammonium salts, zwitterions such as phosphocholine, and hydrophobic groups such as polymerized styrene groups, polymerized diisobutylene groups, or linear C1-C16 aliphatic groups, branched C1-C16 aliphatic groups, cyclic C5-C12 aliphatic groups, or C5-C12 aromatic groups.

12. The method according to claims 10 and 11, wherein the molecular weight of the polymer is from 1,900 to 20,000.

13. 13. The method of any one of claims 10 to 12, wherein the polymer is selected from the group consisting of diisobutylene / maleic acid copolymer, styrene-maleic acid copolymer, (acrylic acid-co-styrene) copolymer, and polyacrylic acid partially linked to an amide by a cycloalkylamine or cycloalkylalkylamine.

14. The method of any one of claims 1 to 13, wherein step (a) is carried out for 1 minute or less.

15. Use of macrocycles and / or hydrophobic particles in the method according to any one of claims 1 to 14.

16. A composition comprising a macrocycle bound to a support for carrying out the method of any one of claims 1 to 14.

17. A kit for carrying out the method according to any one of claims 1 to 14, comprising: macrocycles and / or hydrophobic particles, and - instructions for carrying out said method for removing said polymer.

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