WO2017046625A1 - Nouveaux chélateurs pour la purification par affinité de protéines recombinantes - Google Patents

Nouveaux chélateurs pour la purification par affinité de protéines recombinantes Download PDF

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WO2017046625A1
WO2017046625A1 PCT/IB2015/002003 IB2015002003W WO2017046625A1 WO 2017046625 A1 WO2017046625 A1 WO 2017046625A1 IB 2015002003 W IB2015002003 W IB 2015002003W WO 2017046625 A1 WO2017046625 A1 WO 2017046625A1
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solid phase
aminopolycarboxylic acid
immobilized
acid compound
linker
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Cube Biotech Gmbh
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3265—Non-macromolecular compounds with an organic functional group containing a metal, e.g. a metal affinity ligand
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J45/00—Ion-exchange in which a complex or a chelate is formed; Use of material as complex or chelate forming ion-exchangers; Treatment of material for improving the complex or chelate forming ion-exchange properties
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • the present invention relates to a method of preparing a solid-phase bound multimeric chelator where the chelator is coupled via one carboxylic group.
  • the chelator consists of aminopolycarboxylic acid, connected to other aminocarboxylic acids via di-or trifunctional linkers. Examples for this invention have the formulas:
  • Z is a solid phase
  • S is an optional spacer molecule, able to bind both to the solid phase and the chelator
  • APA1 is a tetradentate aminopolycarboxylic acid
  • K is a bifunctional linker molecule
  • L is a trifunctional linker molecule
  • APA2 is a pentadentate aminopolycarboxylic acid
  • n is 1 - 50.
  • the invention relates to a method for purifying his-tagged recombinant polypeptides and proteins, phosphoproteins and metal-coordinating proteins with solid phase-bound di-, tri- and tetra- and polymeric chelators.
  • These chelators are polycarboxylates, such as EDTA, DTPA, and the like, and they are connected via linkers, forming chains. Coupling groups to the linker are selected from ester-, thioester- or amide functionalities.
  • the chelator chains are bound to a solid phase via an ester-, thioester- or amide group of one terminal chelator.
  • the solid-phase bound chelator chains, loaded with metal ions provide a strong metal binding, high stability against chelators and reduct- ants, high affinity and an extremely high binding capacity.
  • the present invention relates to a method of preparing a solid-phase bound multimeric chelator where the chelator is coupled via one carboxylic group.
  • the chelator consists of aminopolycarboxylic acid, connected to other aminocarboxylic acids via di-or trifunctional linkers. Examples for this invention have the formulas:
  • Z is a solid phase
  • S is an optional spacer molecule, able to bind both to the solid phase and the chelator
  • APA1 is a tetradentate aminopolycarboxylic acid
  • K is a bifunctional linker molecule
  • L is a trifunctional linker molecule
  • APA2 is a pentadentate aminopolycarboxylic acid
  • n is 1 - 50.
  • the invention relates to a method for purifying his-tagged recombinant polypeptides and proteins, phosphoproteins and metal-coordinating proteins with solid phase-bound di-, tri- and tetra- and polymeric chelators.
  • These chelators are polycarboxylates, such as EDTA, DTPA, and the like, and they are connected via linkers, forming chains. Coupling groups to the linker are selected from ester-, thioester- or amide functionalities.
  • the chelator chains are bound to a solid phase via an ester-, thioester- or amide group of one terminal chelator.
  • the solid-phase bound chelator chains, loaded with metal ions provide a strong metal binding, high stability against chelators and reduct- ants, high affinity and an extremely high binding capacity.
  • Immobilized metal affinity chromatography developed by Porath (Nature 258:598, 1975), has been implemented as a standard method for purification of recombinant proteins, and is still one of the favored methods, when high quantities of recombinant proteins have to be purified.
  • histidine groups consist of consecutive histidine residues, as well as histidine, combined with other amino acids, and usually six to ten histidines.
  • His-tags show a much higher affinity to immobilized metal ions than natural proteins, so that a high degree of purification is possi ⁇ ble, especially, when some small concentrations of imidazole are added to the binding and washing buffer, which reduces non-specific interactions of proteins without his-tag.
  • IMAC can be used for the purification of phosphoproteins, where the metal ion directly binds to phosphate groups.
  • phosphoproteins can be separated from proteins without phosphate groups, which show no or almost weak binding to metal ions.
  • IMAC resins can be used for reversibly binding metal- conjugating proteins, such as zinc finger proteins and copper-interacting proteins like cupredoxin or hemocyanin.
  • the ligands, used for IMAC can be classified by the number of coordinating positions, which the lig- and can occupy on the metal ion. So, chelators are called tridentate, when they obtain three metal ion- binding groups, such as amine, carboxy, phosphate, and the like. Tetradentate chelators can coordinate onto four positions on the metal ion, and pentadentate chelators can occupy five positions.
  • IDA iminodiacetic acid
  • NTA nitrilotriacetic acid
  • ED tris-carboxymethyl ethylene diamine
  • metal ions are fixed to the solid phase with only three functionaliza- tions and so it is known, that metal ions are leached into the buffers, so that the protein binding capacity is reduced and the elution buffer can contain small traces of metal ions. And, with almost three free coordination sites for a his-protein at the metal ion some proteins without his-tag are also bound to the resin, so that after purification contaminated eluates are obtained.
  • Tetradentate chelators such as NTA
  • NTA Tetradentate chelators
  • EDTA as a stronger chelator, which removes membrane-stabilizing mag ⁇ nesium ions and is necessary for inhibiting metalloproteases in lysis buffer, removes almost all the metal ions from NTA resin, when its concentration is higher than 2 mM.
  • DTT dithio- threitol
  • metal ions such as nickel
  • Metal affinity chromatography for purifying recombinant his-tagged proteins was introduced by Po- rath et al. (Nature 258, 598-599, 1975).
  • This new technology uses the discovery, that metal ions, such as Cu 2+ and In 2 *, bound to a solid phase via a chelator, interact with donor groups of histidine amino acids of protein side chains. This interaction is effective at a higher pH value, where the histidines remain non-protonated, while at lower pH values, where the histidines are protonated, the proteins can be eluted from the metal ion-modified solid phase.
  • the ligand used for immobilization of metal ions is iminodiacetic acid, but, in case of using nickel as metal, IDA binds nickel with three coordinating groups, so that the stability of the nickel-chelator complex is too low against pH changes, chelating agents and reduction agents in solution, and metal leaching from the surface is observed.
  • EP 0253303 by Hochuli et al. relates to a tetradentate ligand, basing on a substituted nitrilotriacetic acid, which is prepared by carboxymethylation of ⁇ - ⁇ -lysine, deprotection, coupling onto epoxy- activated agarose and charging with nickel.
  • a definite improvement compared to iminodiacetic acid can be achieved, that metal leaching is drastically reduced, while the stability against chelators, such as EDTA, and reducing agents remains limited.
  • Minh in US 6,441,146 described the synthesis of a pentavalent chelator resin by epoxyfunctionaliza- tion, coupling of lysine while blocking the a nitrogen and subsequent carboxymethylation. This substance can be used as material for covalent immobilization by use of e.g. carbodiimide chemistry.
  • a chelator with at least six coordination groups is described in EP 2 183 049 by Goerlich et al.
  • the chelator is immobilized by means of a carboxamide bond through one single carboxyl group, for immobilized metal affinity chromatography (IMAC) in the purification of recombinant proteins with a plurality of histidine residues.
  • IMAC immobilized metal affinity chromatography
  • WO 2009/008802 by Andersson et al. describe the synthesis of a pentadentate chelator via coupling a carboxylic acid anhydride to an amide-functionalized agarose via acid anhydride, so that the product is suitable for detection and purification of recombinant proteins.
  • the present invention provides an easy and convenient way to prepare a chelating resin via binding of two and more polymeric chelators, which are connected by di- or trifunctional linkers.
  • chelator means a polydentate ligand, which can be involved in formation of at least two coordinative bonds.
  • the chelators are polycarboxylic acids, such as EDTA (ethylenediamine tetraacetic acid), DTPA (diethylene triamine pentaacetic acid), TTHA (triethylenetetramine hexaacetic acid), EDDS (Ethylenediamine-/V,W'-disuccinic acid), DOTA (1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-triacetic acid), and the like.
  • One carboxy group of the first chelator is coupled to the solid phase via ester, thioester- or amide bond.
  • Another carboxy group of the first chelator is coupled with a linker via ester, thioester or amide, and the linker is covalently coupled to a second or even to a third chelator, so that the chelator is bound to the solid phase in linear or branched chains, bound to a solid phase via a single carboxy group of only one chelator. So only one modified chelator per chain is bound to the solid phase, so that the sterical demand of the chelator chains is comparable with a single chelator.
  • the first aspect of the present invention relates to a solid-phase bound multimeric chelator, where one aminopolycarboxylic acid is coupled to the solid phase via one carboxylic group and connected to other aminocarboxylic acids via di- or trifunctional linkers.
  • the chelator bound to the solid phase has the following formula:
  • Z is a solid phase
  • S is an optional spacer molecule, able to bind both the solid phase and the chelator
  • APA1 is a tetradentate aminopolycarboxylic acid
  • K is a difunctional linker molecule
  • L is a trifunctional linker molecule
  • APA2 is a pentadentate aminopolycarboxylic acid
  • n is 1 - 50.
  • This invention is relating to materials with a chain of chelators covalently bound to a solid phase.
  • the chelator chains are formed by coupling chelators with linker molecules, where the chain length can be regulated.
  • the number of bound chelators can be determined by different approaches for both ways, evident for a person skilled in the art:
  • the ratio of linker to chelator influences the chain length of the chelator-linker-chain for way b). So, for instance, when a linker is slowly added to a solution of the chelator in the ratio 1:2, a molecule with the formula APA-L-APA is obtained, while, when adding a linker to the chelator in the ratio 2:3, a substance with the formula APA-L-APA-L-APA is generated.
  • long chains (APA-L) n are formed, as described e.g. in . Reactive and Functional Polymers, 29 (1996) 29-39 by Brosse et al.
  • One method to couple the chelator onto the solid phase or linker contains activation of the chelator by carbodiimide chemistry with e.g. EDC, or with other condensing agents, such as carbonyl imidazol, disuccinimidyl carbonate, and other peptide bond forming agents, such as PyBOP and HBTU, and the like, and mixing of the reactants. It is also feasible to add the linker to the activated chelator, as well as the activated chelator to the linker. The linker can also be activated for coupling onto carboxylic groups via transformation to tosyl, triflate- or mesyl functionalities, or the like. A good overview of common methods can be found in Hermanson et al., Bioconjugate Techniques, 3 rd edition, ISBN: 978- 0-12-382239-0.
  • Another way of forming chelator chains is basing upon addition of linker or the solid phase to a solution of chelator dianhydride preferably in an aprotic solvent, such as N, N-dimethylformamide, dimethyl sulfoxide, diethyl ether, or tetrahydrofuran. Coupling of an amino-, hydroxy- or thiol group with an anhydride leads to the formation of an amide, ester, or thioester bond.
  • an aprotic solvent such as N, N-dimethylformamide, dimethyl sulfoxide, diethyl ether, or tetrahydrofuran.
  • the solid phase optimized for covalent coupling of chelator chains is amine-, thiol- or hydroxy- functionalized.
  • the solid phase may be a chromatographic support, such as agarose, cellulose, dex- tran, chitosan, or alginate, preferably agarose.
  • the support can consist of porous or non-porous silica, aluminum oxide, titanium oxide, zirconium oxide, or the like, preferably silica.
  • Other solid phases contain gold layers, glass, polymers such as polystyrene, methacry!ates, acrylates, acrylamide, vinyl acetate or the like.
  • the solid phase can be, but is not limited to a porous or non- porous particle, magnetic silica, agarose, polystyrene, methacrylate or polyvinyl alcohol particle.
  • the solid phase can also be a sensor surface, membrane, a coated plastic surface of an eppendorf or falcon polypropylene tube, a coated microtiter plate, as well as an array plate.
  • amine modified agarose there are many ways to prepare amine modified agarose, such as reacting with epichiorohydrine and chemical reaction with e.g. ammonia, ethylene diamine, 1,6-diamino hexane or other polyamines.
  • carboxymethylated agarose obtained by reaction with sodium chloroacetate is being modified to amines by reaction with di-, tri- or polyamines.
  • the chemical reaction of the amino groups with the carboxy functions of the chelators can be done via activation with EDC or other condensing agents, or by direct contact of the chelator anhydride with the amine.
  • Hydroxy agarose can be prepared by reacting agarose with epichiorohydrine and acid hydrolysis of the epoxy groups, by covalent coupling of agarose with 3-chloropropanol, or by reaction of epoxy agarose with diethanolamine, N-methyl ethanolamine, or ethylene glycol. Another way to obtain hydroxy agarose is covalent modification of agarose with ethylene oxide or propylene oxide. Alternatively, agarose can be used without modification with a spacer, due to existing OH groups of the polymeric carbon hydrate.
  • the covalent coupling of chelators to hydroxyl functions can be performed by reaction of a chelator dianhydride preferably in aprotic media, or with condensing agents, such as EDC.
  • Amine-modified silica, glass and other metal oxides can be prepared by immobilizing e.g. 3-amino- propylsilane, and hydroxy-modified silica is synthesized by coating with 3-glycidyltriethoxysilane and hydrolysis of the epoxides.
  • Gold surfaces can be modified by coating long, thiol-functionalized linear molecules, so-called "self-assembling monolayer", which can be hydroxy- or amine functionalized.
  • the bifunctional linkers used in this invention for connecting chelators have the following general formula:
  • a and B are functional groups, which are able to react with carboxylate groups and are selected from -OH, NH 2 , NHR, CI, Br, I, OMs, OTs, N 3 , m is 1 -12, n is 2-12,
  • L is a straight or branched configuration of 2-100 atoms, which can contain C, H, N, O and S, including cyclic compounds, such as carbon hydrates.
  • Trifunctional linkers for connecting chelators have the following general formulas:
  • A,D and E are functional groups, which are able to react with carboxylate groups and are selected from OH, NH 2 , NHR, CI, Br, I, OMs, OTs, N 3 ,
  • B is a branching atom, preferably C or N, which is connected with at least three stable bonds preferably to carbon atoms, connected with functional groups,
  • N, m, o are 2-30
  • trifunctional linkers examples include trialkoxylated glycerol, triethanolamine, tris(3-aminopropyl)- amine, tris(2-aminoethyl)amine, diethylenetriamine.
  • the linker used for synthesis of multimeric aminocarboxylic acid resins are selected from ethylene glycol, propylene glycol, ethylene diamine, 1,3-diaminopropane, or triethanolamine.
  • the aminopolycarboxylic acid is coupled onto the solid phase, as well as to the linker molecules via ester, thioester, and amide moieties.
  • the reaction product can be charged with metal ions, which can bind the desired target proteins.
  • metal ions which can bind the desired target proteins.
  • Ni, Cu, Co, Zn, Fe, Eu, Sc are suitable for reversible protein binding
  • nickel and cobalt are preferred for purification of his-tagged proteins
  • iron and alumina are preferred for the isolation of phosphoproteins.
  • IMAC of poly- his proteins the affinity of the metal follows the sequence Cu > Ni > Zn > Co, while specificity of the purification follows the sequence Cu ⁇ Ni ⁇ Zn ⁇ Co.
  • Polyhistidine-tags are common in molecular biology and can be used for separating recombinant proteins, expressed in bacteria, such as E coli, yeast, and mammalia. So his-tagged proteins bind with metal ion-loaded chelators at pH values, where the histidine groups are non-protonated, while the great majority of the remaining proteins don't interact with the metal ions. When the pH value is lowered and histidine becomes protonated, the interaction of the protein to the metal ion is cleared, and the protein can be eluted from the column. Alternatively, the elution can also be performed with applying high concentrations of imidazole, which binds onto the metal ions and replaces the his-tags, which leads to elution of the protein. For this application, imidazole concentrations of 100 to 300 mM are commonly used.
  • the solid-phase immobilized aminopolycarboxylic acid compound can also be used for metal affinity purification of his-tagged proteins. Due to the fact, that the chelators are connected with linkers to chain-like molecules with only one connection to the solid phase, the steric demand is comparable to mono-EDTA, while the number of chelators immobilized is much higher. So the metal binding capacity, determined with Cu 2+ , shows very high values, so 75 - 100 ⁇ /ml can be reached. In addition to that, extremely high quantities of his-tagged proteins can be purified with the material according to this invention. So, in the following examples capacities of up to 100 mg protein per ml can be obtained.
  • the chelators because of the short distance between the chelators and adjacent metal ions, they are able to bind his-tagged proteins with a cooperative effect, which gives a much higher affinity than monomeric chelators. And although a mixture of tetradentate and pentadentate chelators is used, due to the strong affinity binding, the material according to this invention shows an extremely good resistance against solution-based chelating agents and reductants. So these extremely high protein binding capacities are also maintained with drastic conditions, e.g. when 20 mM Imidazol, 20 mM EDTA, and 10 mM DTT are added to the binding buffer.
  • the material according to this invention is particularly suitable for the purification of membrane proteins, especially GPCRs.
  • detergents such as dodecyl maltoside, or n-tetradecyl phospho- choline (FOS-14) are used to stabilize the protein in aqueous solution against precipitation and dena- turation.
  • FOS-14 n-tetradecyl phospho- choline
  • the material according to this invention has been tested for the purification of membrane proteins, and it showed high binding capacity and a good tolerance against detergents.
  • the specific phosphorylation of serine, threonine, or tyrosine residues is the most common mechanism for the regulation of cellular protein activity.
  • kinases catalyze the addition of a phosphate moiety to the hydroxyl group of the respective amino acid.
  • the activity of protein kinases is regulated by various intracellular key signals, e.g., the concentration of cyclic AMP or Ca2+.
  • Phosphatases catalyze the specific dephosphorylation of protein, allowing enzymes to switch between phosphorylated and dephosphorylated states.
  • Reversible protein phosphorylation has been known for some years to control a wide range of cellular processes and activities such as transmembrane signaling, intracellular amplification of signals, and cell-cycle control. The analysis of such phosphorylated residues forms the core of signal-transduction studies.
  • Proteins that carry a phosphate group on any amino acid are bound with high specificity to the solid phase, while proteins without phosphate groups do not bind to the resin and can therefore be found in the flow-through.
  • a gentle lysis procedure is carried out in a 25 mM MES buffer, pH 6.0, that contains CHAPS, a zwitterionic detergent, protease inhibitors, and, optionally, benzonase or another DNase or RNase in order to remove protein-nucleic acid complexes.
  • the washing steps are performed with lysis buffer, and the purified phosphoprotein is eluted with potassium phosphate buffer, pH 7.5.
  • the material according to this invention allows an effective purification even in the presence of strong chelators and reductants.
  • metal-binding proteins show affinity to IMAC resins, given that the suitable metal cation is immobilized. So the affinity of different immobilized metal ions against zinc finger domains was examined in protein Expr. Purif. 2011, 79(1): 88-95, and it turned out, that different affinities could be monitored, depending from the element. Copper-binding proteins can be purified using Cu- and Zn IMAC columns, as presented in Proteomics, 2006, May; 6(9):2746-2758. The high ligand density together with a strong binding to metal cations give the material according to this invention a high potential for this application.
  • metal ion-loaded chelator resins are suited for nucleic acid purification.
  • Biotechnol. Prog. 2005, 21, 1472-1477 a method of separating single- and double-stranded nucleic acids is presented. The principle of this procedure is basing upon reversible adsorption of imidazyl moieties onto immobilized metal ions. So partially denatured genomic DNA can be bound to IMAC resin, while double-stranded plasmid DNA, without accessible imidazyl groups of purine bases, cannot be immobilized. With this method, a 1.000.000 fold clearance could be achieved by using Cu IDA agarose. Further applications contain the removal of PCR error products, as described in Plos ONE, 2011, 6, 1, el5412. The extraordinary high stability and affinity of the material according to this invention makes this material suitable for this application.
  • a further application of the inventive material is the removal of metal ions from solutions.
  • EDTA forms metal complexes with stability constants of about 14 to 25 (Martell A.E. & Smith R.M. (1982), Critical Stability Constants, Vol. 5: First Supplement, Plenum Press, New York), so a aminopolycarbox- yli acid chain-modified solid phase can efficiently be used to bind metal ions, such as Ni 2 ⁇ Co 2 ⁇ Mn 2 *, Cr 3 *, Pb 2+ and to drastically reduce their concentration in a solution by simply contacting the non- charged chelator-bound resin with the liquid.
  • chelator-modified agarose particles which can be filled into a cartridge, and the heavy metal concentration of the liquid is reduced during passing through the column.
  • the chelator chains can be coupled to magnetic agarose particles, which can be added to a solution, mixed and removed by a magnetic separator after binding to metal ions.
  • Example 1 Formation of dimeric EDTA chains by sequential coupling of EDTA, ethylene glycol and EDTA
  • 10 ml agarose particles (WorkBeads 40 SEC, BioWorks Sweden AB, Uppsala) are resuspended in 10 ml 1M caustic soda solution and incubated on a thermoshaker for two hours. Then 5 ml epichlorohy- drine are added and the suspension is heated at 30 "C for four hours. The suspension is filtered via suction filtration and washed six times with dd water. Then the agarose is resuspended in 20 ml of a 5% N-methylethanolamine solution, pH 10.5, and incubated for twenty hours at 65 °C. The agarose is suction-dried, washed four times with dd water, four times with phosphate-buffered saline and stored in anhydrous dimethylformamide.
  • 10 ml EDTA agarose particles are resuspended in 10 ml dimethylformamide, and a solution of 2 ml ethylene glycol in 10 ml dimethylformamide is added. After efficient mixing on a thermoshaker the suspension is allowed to react six hours at 65 °C. Then the reaction product is suction-filtered, washed six times with dry dimethylformamide, and resuspended in dimethylformamide.
  • 10 ml agarose particles (WorkBeads 40 SEC, BioWorks Sweden AB, Uppsala) are resuspended in 10 ml 1 caustic soda solution and incubated on a thermoshaker for two hours. Then 5 ml epichlorohy- drine are added and the suspension is heated at 30 °C for four hours. The suspension is filtered via suction filtration and washed six times with dd water. Then the agarose is resuspended in 20 ml of a 5% 1,6-diaminohexane solution, pH 10.5, and incubated for twenty hours at 65 °C. The agarose is suction-dried, washed four times with dd water, four times with phosphate-buffered saline and stored in anhydrous dimethylformamide.
  • Example 3 Formation of dimeric EDTA chains by synthesis of glycol-bridged EDTA, and coupling onto solid phase
  • 10 ml agarose particles (WorkBeads 40 SEC, BioWorks Sweden AB, Uppsala) are resuspended in 10 ml 1M caustic soda solution and incubated on a thermoshaker for two hours. Then 5 ml epichlorohy- drine are added and the suspension is heated at 30 °C for four hours. The suspension is filtered via suction filtration and washed six times with dd water. Then the agarose is resuspended in 20 ml of a 5% diethanolamine solution, pH 10.5, and incubated for twenty hours at 65 °C. The agarose is suction-dried, washed four times with dd water, four times with phosphate-buffered saline and stored in anhydrous dimethylformamide.
  • Example 4 Formation of dimeric EDTA chains by synthesis of Polyethylene Glycol-bridged EDTA, and coupling onto solid phase
  • 10 ml agarose particles (WorkBeads 40 SEC, BioWorks Sweden AB, Uppsala) are resuspended in 10 ml 1M caustic soda solution and incubated on a thermoshaker for two hours. Then 5 ml epichlorohy- drine are added and the suspension is heated at 30 °C for four hours. The suspension is filtered via suction filtration and washed six times with dd water. Then the agarose is resuspended in 20 ml of a 5% N-methyl-ethanolamine solution, pH 10.5, and incubated for twenty hours at 65 °C. The agarose is suction-dried, washed four times with dd water, four times with phosphate-buffered saline and stored in anhydrous dimethylformamide.
  • Example 5 Formation of dimeric EDTA chains by synthesis of ethylene diamine-bridged EDTA, and coupling onto solid phase
  • Example 6 Formation of dimeric EDTA chains by synthesis of Jeffamine-bridged EDTA, and coupling onto solid phase
  • Example 7 Formation of trimeric EDTA chains by synthesis of ethylene diamine-bridged EDTA, and coupling onto solid phase
  • Example 8 Formation of branched trimeric EDTA chains by synthesis of triethanolamine-bridged EDTA, and coupling onto solid phase
  • Example 9 Formation of dimeric DTPA chains by sequential coupling of DTPA, ethylene diamine and DTPA
  • Example 10 Formation of dimeric EDTA chains by sequential coupling of EDTA, ethylene diamine and EDTA with EDC
  • 10 ml amino agarose are resuspended in 10 ml 0.1 M sodium phosphate, pH 7.4, and a solution of 500 mg EDTA disodium salt in 10 ml 0.1 M sodium phosphate, pH 7.4, is added.
  • the reaction mixture is incubated for two hours at ambient temperature and suction dried. After filtration, the product is washed three times with 0.1 M sodium phosphate, pH 7.4, and three times with dd water.
  • 10 ml agarose particles (WorkBeads 40 SEC, BioWorks Sweden AB, Uppsala) are resuspended in 10 ml 1M caustic soda solution and incubated on a thermoshaker for two hours. Then 5 ml epichlorohy- drine are added and the suspension is heated at 30 °C for four hours. The suspension is filtered via suction filtration and washed six times with dd water. Then the agarose is resuspended in 20 ml of a 5% 1,6-diaminohexane solution, pH 10.5, and incubated for twenty hours at 65 °C. The agarose is suction-dried, washed four times with dd water, four times with phosphate-buffered saline and stored in anhydrous dimethylformamide.
  • 10 ml amino agarose are resuspended in 10 ml dimethylformamide, and a solution of 500 mg 4,4'- Ethylenebis(2,6-morpholinedione in 10 ml dimethylformamide is added.
  • the reaction mixture is incubated for six hours at 60 °C and suction dried. After filtration, the product is washed two times with dimethylformamide, two times with dd water, two times with 100 mM acetate buffer, pH 6.0, and two times with dd water.
  • the resin may be loaded with cobalt, iron, aluminum, zinc, copper, and other transition metals or lanthanides by simply exchanging nickel sulfate against cobalt sulfate, iron sulfate, aluminum chloride, zinc chloride, copper sulfate, europium sulfate, and the like.
  • Example 11 Determination of the metal ion binding capacity
  • GFP green fluorescent protein
  • the resin was washed two times with 2.5 ml NPI-20 (50 mM NaH 2 P0 4 , 300 mM NaCI, 20 mM imidazole, pH 7.4) and eluted three times with 1 ml NPI-250 (50 mM NaH 2 P0 4 , 300 mM NaCI, 250 mM imidazole, pH 8.0) .
  • the eluate fractions were combined, measured with 488nm and the yields were calculated and summarized in Table Nr. 1.
  • the chelating additives (20 mM EDTA and lOmM DTT) were added to NPI-10 to demonstrate the resistance of the tested resin to chelating agents and reductants.
  • example 1 Cube 35C
  • resins which probably are EDTA functionalized
  • GFP green fluorescent protein
  • FIG. 1 Graphical visuaiiiation of purifications results
  • the binding capacity of the new Cube Biotech Material is 2,8 time higher than from the Ge materia! (Ni-Sepharose excel) and 9,5 time higher than Roche materia! complete His-Tag Purification Resin, it could be shown that the chelating agent EDTA and the reductant DTT have no influence of the high capacity of the material according to this invention.
  • a solid phase-imroohiiued aminopolycarboxylic acid compound having the general formula: Z-S-(APAM ⁇ resort-APA2

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Abstract

La présente invention concerne un procédé de préparation d'un chélateur multimère lié à une phase solide où le chélateur est couplé par l'intermédiaire d'un groupe carboxylique. Le chélateur est constitué d'acide aminopolycarboxylique, connecté à d'autres acides aminocarboxyliques dpi de lieurs di- ou trifonctionnels. Des exemples de la présente invention ont les formules (I) : dans lesquelles Z est une phase solide, S est une molécule d'espaceur facultative, capable de se lier à la fois à la phase solide et au chélateur, APA1 est un acide aminopolycarboxylique tétradenté, K est une molécule de lieur bifonctionnelle, L est une molécule de lieur trifonctionnelle, APA2 est un acide aminopolycarboxylique pentadenté, n est 1 à 50. En outre, l'invention concerne un procédé permettant de purifier des polypeptides et des protéines recombinants à étiquettehis et des protéines, des phosphoprotéines et des protéines à métal de coordination de métal liées avec des chélateurs di -, tri- et tétra- et polymères. Ces chélateurs sont des polycarboxylates, tels que l'EDTA, le DTPA, et similaire, et ils sont reliés par l'intermédiaire de lieurs, en formant des chaînes. Les groupes de couplage au lieur sont choisis parmi des fonctionnalités ester, thioester ou amide. Les chaînes de chélateur sont liées à une phase solide par l'intermédiaire d'un groupe ester, thioester d'un chélateur terminal. Les chaînes de chélateur liées à une phase solide, chargées avec des ions métalliques, constituent une liaison métallique forte, une stabilité élevée contre les chélateurs et les réducteurs, une affinité élevée et une capacité de liaison extrêmement élevée.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109621912A (zh) * 2018-12-21 2019-04-16 重庆希尔康血液净化器材研发有限公司 一种血液灌流用活性炭吸附剂的包膜方法
WO2020051498A1 (fr) * 2018-09-06 2020-03-12 Cidara Therapeutics, Inc. Compositions et procédés pour le traitement d'infections virales
WO2020109162A1 (fr) 2018-11-28 2020-06-04 Cube Biotech Gmbh Matériau chélateur en phase solide, son procédé de production et son utilisation pour la purification de protéines
CN113351191A (zh) * 2021-05-10 2021-09-07 翌圣生物科技（上海）有限公司 多齿配体的新型imac色谱介质及其制备方法
WO2022167246A1 (fr) * 2021-02-02 2022-08-11 Cube Biotech Gmbh Chélateur soluble pour le ciblage de protéines recombinantes
US11510992B1 (en) 2019-09-06 2022-11-29 Cidara Therapeutics, Inc. Compositions and methods for the treatment of viral infections
CN115888815A (zh) * 2022-10-25 2023-04-04 湖北工程学院 一种n-马来酰化壳聚糖铜催化剂的制备及其在硼加成反应中的应用
CN115894301A (zh) * 2022-10-25 2023-04-04 武汉大学中南医院 一种二聚化钆基t1磁共振对比造影剂及其制备方法和用途
RU2816717C2 (ru) * 2018-09-06 2024-04-03 Сидара Терапьютикс, Инк. Композиции и способы для лечения вирусных инфекций
EP4417697A1 (fr) 2023-02-17 2024-08-21 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Système de conjugaison modulaire

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992017403A1 (fr) * 1991-04-05 1992-10-15 Brigham Young University Acide polycarboxylique de polyalkylene-polyamine fixe a un support et extraction d'ions metalliques au moyen de cet element
EP1199312A2 (fr) * 2000-10-11 2002-04-24 Sumitomo Chemical Company, Limited Procédé de préparation d'un amide
WO2002041989A1 (fr) * 2000-11-27 2002-05-30 Prometic Biosciences Ltd. Adsorbants chelateurs de metaux et utilisations de ceux-ci
WO2009008802A1 (fr) * 2007-07-09 2009-01-15 Ge Healthcare Bio-Sciences Ab Procédé pour la préparation d'un agent d'adsorption biomoléculaire
WO2011152782A1 (fr) * 2010-06-01 2011-12-08 Ge Healthcare Bio-Sciences Ab Nouveau chélateur et son utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992017403A1 (fr) * 1991-04-05 1992-10-15 Brigham Young University Acide polycarboxylique de polyalkylene-polyamine fixe a un support et extraction d'ions metalliques au moyen de cet element
EP1199312A2 (fr) * 2000-10-11 2002-04-24 Sumitomo Chemical Company, Limited Procédé de préparation d'un amide
WO2002041989A1 (fr) * 2000-11-27 2002-05-30 Prometic Biosciences Ltd. Adsorbants chelateurs de metaux et utilisations de ceux-ci
WO2009008802A1 (fr) * 2007-07-09 2009-01-15 Ge Healthcare Bio-Sciences Ab Procédé pour la préparation d'un agent d'adsorption biomoléculaire
WO2011152782A1 (fr) * 2010-06-01 2011-12-08 Ge Healthcare Bio-Sciences Ab Nouveau chélateur et son utilisation

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2816717C2 (ru) * 2018-09-06 2024-04-03 Сидара Терапьютикс, Инк. Композиции и способы для лечения вирусных инфекций
WO2020051498A1 (fr) * 2018-09-06 2020-03-12 Cidara Therapeutics, Inc. Compositions et procédés pour le traitement d'infections virales
US11833213B2 (en) 2018-09-06 2023-12-05 Cidara Therapeutics, Inc. Compositions and methods for the treatment of viral infections
WO2020109162A1 (fr) 2018-11-28 2020-06-04 Cube Biotech Gmbh Matériau chélateur en phase solide, son procédé de production et son utilisation pour la purification de protéines
CN113164915A (zh) * 2018-11-28 2021-07-23 库贝生物科技有限公司 固相螯合剂材料，其制备方法及其用于纯化蛋白质的用途
JP2022509239A (ja) * 2018-11-28 2022-01-20 キューブ バイオテック ゲーエムベーハー 固相キレート材、その製造方法およびタンパク質の精製におけるその使用
JP7544704B2 (ja) 2018-11-28 2024-09-03 キューブ バイオテック ゲーエムベーハー 固相キレート材、その製造方法およびタンパク質の精製におけるその使用
CN109621912A (zh) * 2018-12-21 2019-04-16 重庆希尔康血液净化器材研发有限公司 一种血液灌流用活性炭吸附剂的包膜方法
US11510992B1 (en) 2019-09-06 2022-11-29 Cidara Therapeutics, Inc. Compositions and methods for the treatment of viral infections
WO2022167246A1 (fr) * 2021-02-02 2022-08-11 Cube Biotech Gmbh Chélateur soluble pour le ciblage de protéines recombinantes
CN113351191A (zh) * 2021-05-10 2021-09-07 翌圣生物科技（上海）有限公司 多齿配体的新型imac色谱介质及其制备方法
CN113351191B (zh) * 2021-05-10 2023-12-01 翌圣生物科技（上海）有限公司 多齿配体的新型imac色谱介质及其制备方法
CN115888815A (zh) * 2022-10-25 2023-04-04 湖北工程学院 一种n-马来酰化壳聚糖铜催化剂的制备及其在硼加成反应中的应用
CN115888815B (zh) * 2022-10-25 2024-03-19 湖北工程学院 一种n-马来酰化壳聚糖铜催化剂的制备及其在硼加成反应中的应用
CN115894301A (zh) * 2022-10-25 2023-04-04 武汉大学中南医院 一种二聚化钆基t1磁共振对比造影剂及其制备方法和用途
EP4417697A1 (fr) 2023-02-17 2024-08-21 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Système de conjugaison modulaire
WO2024170792A1 (fr) 2023-02-17 2024-08-22 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Système de conjugaison modulaire

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