Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to compositions, which can be used for purifying and crystallizing molecules of interest.
• Proteins and other macromolecules are increasingly used in research, diagnostics and therapeutics. Proteins are typically produced by recombinant techniques on a large scale with purification constituting the major cost (up to 60 % of the total cost) of the production processes. Thus, large-scale use of recombinant protein products is hindered because ofthe high cost associated with purification.
• Current protein purification methods are dependent on the use of a combination of various chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity or size among other characteristics.
• Smart polymers or stimuli-responsive “intelligent” polymers or Affinity Macro Ligands (AML)] are polymers that respond with large property changes to small physical or chemical stimuli, such as changes in pH, temperature, radiation and the like. These polymers can take many forms; they may be dissolved in an aqueous solution, adsorbed or grafted on aqueous-solid interfaces, or cross-linked to form hydrogels [Hoffman J Controlled Release (1987) 6:297-305; Hoffman Intelligent polymers. In: Park K, ed. Controlled drug delivery. Washington: ACS Publications, (1997) 485-98; Hoffman Intelligent polymers in medicine and biotechnology.
• Smart polymers may be physically mixed with, or chemically conjugated to, biomolecules to yield a large family of polymer-biomolecule systems that can respond to biological as well as to physical and chemical stimuli.
• Biomolecules that may be polymer-conjugated include proteins and oligopeptides, sugars and polysaccharides, single- and double-stranded ohgonucleotides and DNA plasmids, simple lipids and phospholipids, and a wide spectrum of recognition ligands and synthetic drug molecules.
• smart polymers should contain reactive groups for ligand coupling; not interact strongly with the impurities; make the ligand available for interaction with the target protein; give complete phase separation ofthe polymer upon a change of medium property; form compact precipitates; exclude trapping of impurities into the gel structure and be easily solubilized after the precipitate is formed.
• Crystal growth of protein-detergent complexes can be considered equivalent to that of soluble proteins only the solute being crystallized is a complex of protein and detergent, rather than solely protein.
• the actual lattice contacts are formed by protein-protein interactions, although crystal packing brings the detergent moieties into close apposition as well.
• Studies suggested adding an antibody fragment which will increase the chances of producing crystals [Hunte and Michel (2002) Curr. Opin. Struct. Biol. 12:503-508].
• applying this technology to various membrane proteins is difficult as it requires the generation of monoclonal antibodies, which are specific to each membrane protein.
• no detergent micelle can fully and accurately reproduce the lipid bilayer environment of the protein.
• composition of matter comprising at least one ligand capable of binding a target molecule or cell of interest, the at least one ligand being attached to at least one coordinating moiety selected capable of directing the composition of matter to form a non-covalent complex when co-incubated with a coordinator ion or molecule.
• a method of purifying a target molecule or cell of interest comprising: (a) contacting a sample including the target molecule or cell of interest with a composition including: (i) at least one ligand capable of binding the target molecule or cell of interest, the at least one ligand being attached to at least one coordinating moiety; and (ii) a coordinator capable of non-covalently binding the at least one coordinating moiety, the at least one coordinating moiety and the coordinator being capable of forming a complex when co-incubated; and (b) collecting a precipitate including the complex bound to the target molecule or cell of interest, thereby purifying the target molecule or cell of interest.
• the method further comprising recovering the molecule of interest from the precipitate.
• a method of detecting predisposition to, or presence of a disease associated with a molecule of interest in a subject comprising contacting a biological sample obtained from the subject with a composition including: (i) at least one ligand capable of binding the molecule of interest, the at least one ligand being attached to at least one coordinating moiety; and (ii) a coordinator capable of non-covalently binding the at least one coordinating moiety, the at least one coordinating moiety and the coordinator being capable of forming a complex when co-incubated, wherein formation of the complex including the molecule of interest is indicative of predisposition to, or presence ofthe disease associated with the molecule of interest in the subject.
• compositions for crystallizing a molecule of interest comprising: (i) at least one ligand capable of binding the molecule of interest, the at least one ligand being attached to at least one coordinating moiety; and (ii) a coordinator capable of non-covalently binding the at least one coordinating moiety, wherein the at least one coordinating moiety and the coordinator are capable of forming a complex when co- incubated and whereas the composition is selected so as to define the relative spatial positioning and orientation of the molecule of interest when bound thereto, thereby facilitating formation of a crystal therefrom under inducing crystallization conditions.
• a method of crystallizing a molecule of interest comprising contacting a sample including the molecule of interest with a crystallizing composition including: (i) at least one ligand capable of binding the molecule of interest, the at least one ligand being attached to at least one coordinating moiety; and (ii) a coordinator capable of non-covalently binding the at least one coordinating moiety, wherein the at least one coordinating moiety and the coordinator are capable of forming a complex when co-incubated and whereas the crystallizing composition is selected so as to define the relative spatial positioning and orientation of the molecule of interest when bound thereto, thereby facilitating formation of a crystal therefrom under inducing crystallization conditions.
• composition-of-matter comprising a molecule having a first region capable of binding a molecule of interest and a second region capable of binding a coordinator ion or molecule, the second region being designed such that the molecule forms a polymer when exposed to the coordinator ion or molecule.
• a method of depleting a target molecule or cell of interest from a sample comprising: (a) contacting the sample including the target molecule or cell of interest with a composition including: (i) at least one ligand capable of binding the molecule of interest, the at least one ligand being attached to at least one coordinating moiety; and (ii) a coordinator capable of non-covalently binding the at least one coordinating moiety, the at least one coordinating moiety and the coordinator being capable of forming a complex when co-incubated; and (b) removing a precipitate including the complex bound to the target molecule or cell of interest to thereby deplete the target molecule or cell of interest from the sample.
• a method of enhancing immunogenicity of a target molecule of interest comprising contacting the target molecule of interest with a composition including: (i) at least one ligand capable of binding the target molecule of interest, the at least one ligand being attached to at least one coordinating moiety; and (ii) a coordinator capable of non-covalently binding the at least one coordinating moiety, wherein contacting is effected such that the at least one coordinating moiety and the coordinator forms a complex including the target molecule of interest, thereby enhancing immunogenicity ofthe target molecule of interest.
• the molecule of interest is selected from the group consisting of a protein, a nucleic acid sequence, a small molecule chemical and an ion.
• the at least one ligand is selected from the group consisting of a growth factor, a hormone, a nucleic acid sequence, an antibody, an epitope tag, an avidin, a biotin, a enzymatic substrate and an enzyme.
• the at least one ligand is attached to the at least one coordinating moiety via a linker.
• the coordinating moiety is selected from the group consisting of a chelator, a biotin, a nucleic acid sequence, an epitope tag, an electron poor molecule and an electron-rich molecule.
• the coordinator ion or molecule is selected from the group consisting of a metal ion, an avidin, a nucleic acid sequence, an electron poor molecule and an electron-rich molecule.
• FIGs. 2a-b schematically illustrate precipitation of a target molecule using the compositions of the present invention.
• a ligand covalently attached to a bis-chelator is incubated in the presence of a target molecule ( Figure 2a).
• Addition of a metal (M " , M 2+ , M 3+ , M 4 *) binds the chelator and forms a matrix including the target molecule non-covalently bound to the metal ion ( Figure 2b).
• FIG. 3a-e schematically illustrate stepwise recovery of the target molecule from the precipitate.
• Figure 3 a shows the addition of a free chelator, which competes with the binding of the ligand-bound chelator to the metal.
• Figure 3b shows gravity- based separation of the ligand-bound target molecule from the free competing chelator and the complexed metal (Figure 3c).
• Figure 3d shows loading ofthe ligand- bound target molecule on an immobilized metal column to allow binding of the complex. Under proper elution conditions the target molecule is eluted while the ligand-coordinating moiety molecule is not. A desalting stage may be added for further purification of the target molecule.
• FIG. 4 schematically illustrates direct elution of the target molecule from the precipitate, wherein the chelator-metal complex is maintained, while binding between the target molecule and the ligand decreases.
• FIG. 5 schematically illustrates regeneration of the precipitating unit (i.e., ligand-coordinating moiety) following elution of the target molecule. In this case, recovery is achieved by the addition of a competing chelator and application of an appropriate separation procedure, such as, dialysis and ulfrafiltration.
• FIGs. 6a-c schematically illustrate precipitation of a target molecule using nucleic acid sequences as the coordinating moiety.
• a ligand with a covalently bound bis-nucleotide sequence (coordinating moiety ) is incubated in the presence of a target molecule ( Figure 6a).
• Addition of a complementary sequence results in the formation of matrix including ligand-coordinating moiety:target molecule:the complementary sequence (coordinator molecule, Figure 6b).
• Non-symmetrical coordinating sequences are shown as well ( Figure 6c).
• FIGs. 7a-b schematically illustrate precipitation of a target molecule using biotin as the coordinating moiety.
• a ligand with a covalently bound bis-biotin or biotin derivative such as: DSB-X Biotin is incubated in the presence of a target molecule ( Figure 7a).
• Introduction of avidin (or its derivatives) creates a network comprising ligand-coordinating moiety (biotin): target molecule: avidin ( Figure 7b).
• FIGs. 8a-c schematically illustrate precipitation of a target molecule using electron rich molecules as the coordinating moiety.
• a ligand with a covalently bound bis-elecfron rich entity is incubated in the presence of a target molecule ( Figure 8a).
• FIG. 9 schematically illustrates precipitation of a target antibody with protein A (ProA) bound used as a ligand. Addition of an appropriate coordinator results in a network of: Protein A-coordinating moiety : coordinator : target molecule.
• FIGs. lOa-b schematically illustrate the use of the complexes of the present invention for crystallization of membrane proteins.
• FIGs. 1 la-b schematically illustrate the use of metallo complexes (Figure 11a) and nucleo-complexes (Figure l ib) for the formation of crystals of membrane proteins.
• FIG. l ie schematically illustrates a three-dimensional membrane complex using the compositions of the present invention. The hydrophobic domain of the protein is surrounded by detergent micelles.
• FIG. 12 schematically illustrates the formation of a non-covalent composition consisting of three ligands bound to a single metal coordinator, through suitable chelators which are bound to the ligands through covalent linkers.
• FIGs. 13a-b schematically illustrate the modification of three ligands of interest to include the hydroxamate derivatives ( Figure 13 a), such that a tri-non- co valent ligand complex is formed in the presence of Fe 3+ ions ( Figure 13b).
• FIG. 14 schematically illustrates a two-step synthesis procedure for the generation of ligand-chelator molecules.
• FIGs. 16a-b schematically illustrate the formation of di ( Figure 15a) and tri ( Figure 15b) non-covalent ligands, by utilizing the same ligand-linker-chelator molecule, while changing only, the cation present in the medium.
• FIGs. 16a-c schematically illustrate the compositions of the present invention coordinated by electron poor / rich relations. By modifying a ligand with an electron poor moiety (Figure 16a) and synthesizing a tri covalent electron rich moiety (Figure 16b), a complex ofthe structure seen in Figure 16c is formed.
• FIG. 17 schematically illustrates a two step synthesis process for the preparation of ligand-elecfron rich or ligand-elecfron poor derivatives.
• FIG. 17 schematically illustrates a two step synthesis process for the preparation of ligand-elecfron rich or ligand-elecfron poor derivatives.
• FIG. 18 schematically illustrates the use of peptides for the formation of ligand complexes utilizing electron rich and electron poor moieties.
• FIG. 19 schematically illustrates the formation of ligand complexes which utilize a chelator-metal as well as electron rich and poor relationships.
• FIG. 20 schematically illustrates a single step synthesis procedure for the preparation of a chelator-elecfron poor derivative.
• FIGs. 2 la-b schematically illustrate formation of di and tri non-covalent electron poor moieties by utilizing the same chelator-elecfron poor (catechol-TNB) derivative and changing only the cation in the medium.
• FIGs. 22a-b schematically illustrate the addition of a peptide containing an electron rich moiety to form a dimer and a trimer.
• FIGs. 23a-b schematically illustrate the formation of a polymer complex by the addition of a composition including ligand attached to two chelators which are coordinated through electron rich/poor relations.
• FIG. 24 schematically illustrates one possibility of limiting the freedom of motion of non-covalent protein dimers.
• FIG. 25 schematically illustrates chelators and metals, which can be used as the coordinating moiety and coordinator ion, respectively, in the compositions of the present invention.
• FIG. 26 schematically illustrates electron rich and electron poor moieties which can be used as the coordinating moiety in the compositions ofthe present invention.
• the present invention is of compositions, which can be used for purifying and crystallizing molecules of interest.
• the principles and operation ofthe present invention may be better understood with reference to the drawings and accompanying descriptions.
• the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples.
• the invention is capable of other embodiments or of being practiced or carried out in various ways.
• the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
• Affinity Precipitation which is based on the use of "smart" polymers coupled to a recognition unit, which binds the protein of interest. These smart polymers respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties, resulting in phase separation from aqueous solution or order-of- magnitude changes in hydrogel size and precipitation of the molecule of interest.
• compositions of the present invention specifically bind target molecules to form noncovalent complexes which can be precipitated and collected under mild conditions.
• compositions of the present invention are not immobilized (such as to a smart polymer) which reduces affinity of the ligand towards the target molecule, limits the amount of ligand used, necessitates the use of sophisticated laboratory equipment (HPLC) requiring high maintenance, leads to column fouling and limits column usage to a single covalently bound ligand.
• HPLC laboratory equipment
• the target molecule can be a macromolecule such as a protein, a carbohydrate, a glycoprotein or a nucleic acid sequence (e.g., DNA such as plasmids, RNA) or a small molecule such as a chemical. Although most of the examples provided herein describe proteinacious target molecules, it will be appreciated that the present invention is not limited to such targets.
• the target cell can be a eukaryotic cell, a prokaryotic cell or a viral cell.
• the composition-of-matter ofthe present invention includes at least one ligand capable of binding the molecule or cell of interest and at least one coordinating moiety which is selected capable of directing the composition of matter to form a noncovalent complex when co-incubated with a coordinator ion or molecule.
• ligand refers to a synthetic or a naturally occurring molecule preferably exhibiting high affinity (e.g., K D ⁇ 10 "5 ) binding to the target molecule of interest and as such the two are capable of specifically interacting.
• the ligand is selected capable of binding a protein, a carbohydrate or chemical, which is expressed on the surface of the cell (e.g., cellular marker).
• ligand binding to the molecule or cell of interest is a non- covalent binding.
• the ligand according to this aspect of the present invention may be mono, bi (antibody, growth factor) or multi-valent ligand and may exhibit affinity to one or more molecules or cells of interest (e.g., bi-specific antibodies).
• ligands which may be used in accordance with the present invention include, but are not limited to, antibodies, mimetics (e.g., Affibodies® see: U.S. Pat. Nos.
• dyes which often interact with the catalytic site of an enzyme mimicking the structure of a natural substrate or co-factor and consisting of a chromophore (e.g., azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (e.g., a mono- or dichlorotriazine ring, see, Denzili (2001) J Biochem Biophys Methods.
• a chromophore e.g., azo dyes, anthraquinone, or phathalocyanine
• a reactive group e.g., a mono- or dichlorotriazine ring
• small molecule chemicals small molecule chemicals, receptor ligands (e.g., growth factors and hormones), mimetics having the same binding function but distinct chemical structure, or fragments thereof (e.g., EGF domain), ion ligands (e.g., calmodulin), protein A, protein G and protein L or mimetics thereof (e.g., PAM, see Fassina (1996) J. Mol. Recognit.
• receptor ligands e.g., growth factors and hormones
• mimetics having the same binding function but distinct chemical structure, or fragments thereof (e.g., EGF domain), ion ligands (e.g., calmodulin), protein A, protein G and protein L or mimetics thereof (e.g., PAM, see Fassina (1996) J. Mol. Recognit.
• coordinating moiety refers to any molecule having sufficient affinity (e.g., K D ⁇ 10 "5 ) to a coordinator ion or molecule.
• the coordinating moiety can direct the composition of matter of this aspect of the present invention to form a non-covalent complex when co-incubated with a coordinator ion or molecule.
• coordinating moieties which can be used in accordance with the present invention include but are not limited to, epitopes (antigenic determinants antigens to which the paratope of an antibody binds), antibodies, chelators (e.g., His-tag, see other example in Example 1 of the Examples section which follows, Figures 1, 25 and 26), biotin (see Figure 7), nucleic acid sequences
• the coordinating moiety is selected so as to negate the possibility of coordinating moiety-ligand interaction or coordinating moiety-target molecule interaction.
• the ligand is an antigen having an affinity towards an immunoglobulin of interest than the coordinating moiety is preferably not an epitope tag or an antibody capable of binding the antigen.
• coordinator ion or molecule refers to a soluble entity (i.e., molecule or ion), which exhibits sufficient affinity (i.e., KD ⁇ 10 "5 ) to the coordinating moiety and as such is capable of directing the composition of matter of this aspect of the present invention to form a non-covalent complex.
• coordinator molecules which can be used in accordance with the present invention include but are not limited to, avidin and derivatives thereof, antibodies, elecfron rich molecules, electron poor molecules and the like.
• coordinator ions which can be used in accordance with the present invention include but are not limited to, mono, bis or tri valent metals.
• Figure 25 illustrates examples of chelators and metals which can be used as a coordinator ion by the present invention.
• Figure 26 lists examples of electron rich molecules and elecfron poor molecules which can be used by the present invention.
• Methods of generating antibodies and antibody fragments as well as single chain antibodies are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference; Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein; See also Porter, R. R. [Biochem. J. 73: 119-126 (1959);
• the composition of this aspect ofthe present invention includes the coordinator ion or molecule.
• the ligand of this aspect ofthe present invention may be bound directly to the coordinating moiety, depending on the chemistry of the two. Measures are taken, though, to maintain recognition (e.g., affinity) of the ligand to the molecule of interest.
• the ligand may be bound to the coordinating moiety via a linker.
• a general synthetic pathway for modification of representative chelators with a general ligand is shown in Figure 14.
• Margherita et al. (1993) J. Biochem. Biophys. Methods 38:17-28 provides synthetic procedures which may be used to attach the ligand to the coordinating moiety ofthe present invention.
• Complexes of the present invention be of various complexity levels, such as, monomers (see Figures 12 and 13a-b depicting a three ligand complex), dimers, polymers (see Figure 23a-b depicting formation of a polymer via a combined linker as described in Example 3 of the Examples section), sheets (see Figure 24 in which sheets are formed when a single surface exposed Trp residue of a target molecule forms electron rich poor relations with a TNB — TNB entity) and lattices which may form three dimensional (3D) structures (such as when more than one surface exposed Trp residues form electron rich/poor relations).
• monomers see Figures 12 and 13a-b depicting a three ligand complex
• dimers see Figure 23a-b depicting formation of a polymer via a combined linker as described in Example 3 of the Examples section
• sheets see Figure 24 in which sheets are formed when a single surface exposed Trp residue of a target molecule forms electron rich poor relations with a TNB — TNB entity
• compositions of the present invention can be packed in a purification kit which may include additional buffers and additives, as described hereinbelow. It will be appreciated that such kits may include a number of ligands for purifying a number of molecules from a single sample. However, to simplify precipitation (e.g., using the same reaction buffer, temperature conditions, pH and the like) and further purification steps, the coordinating moieties and coordinator ions or molecules are selected the same.
• the compositions of the present invention may be used to purify a molecule or cell of interest from a sample.
• a method of purifying a molecule of interest refers to at least separating the molecule of interest from the sample by changing its solubility upon binding to the composition ofthe present invention and precipitation thereof (i.e., phase separation).
• the method of this aspect of the present invention is effected by contacting a sample including the molecule of interest with a composition of the present invention and collecting a precipitate which includes a complex formed from the composition- of-matter of the present invention and the molecule of interest, thereby purifying the molecule of interest.
• sample refers to a solution including the molecule of interest and possibly one or more contaminants (i.e., substances that are different from the desired molecule of interest).
• the sample can be the conditioned medium, which may include in addition to the recombinant polypeptide, serum proteins as well as metabolites and other polypeptides, which are secreted from the cells.
• purifying refers to concentrating.
• the composition-of-matter of the present invention is first contacted with the sample.
• This is preferably effected by adding the ligand attached to the coordinating moiety to the sample allowing binding of the molecule of interest to the ligand and then adding the coordinator ion or molecule to allow complex formation and precipitation of the molecule of interest.
• the coordinator In order to avoid rapid formation of complexes (which may result in the entrapment of contaminants) slow addition of the coordinator to the sample while stirring is preferred. Controllable rate of precipitation can also be achieved by adding free coordinating entity (i.e., not bound to the ligand), which may also lead to the formation of smaller complexes which may be beneficial in a variety of applications such as for the formation of immunogens, further described hereinbelow.
• precipitation of the complex may be facilitated by centrifugation (e.g., ultra-centrifugation), although in some cases (for example, in the case of large complexes) centrifugation is not necessary.
• the precipitate may be subjected to further purification steps in order to recover the molecule of interest from the complex. This may be effected by using a number of biochemical methods which are well known in the art. Examples include, but are not limited to, fractionation on a hydrophobic interaction chromatography (e.g.
• any of the above-described purification procedures may be repetitively applied on the sample (i.e., precipitate) to increase the yield and or purity ofthe target molecule.
• the composition of matter and coordinator ion or molecule are selected so as to enable rapid and easy isolation of the target molecule from the complex formed.
• the molecule of interest may be eluted directly from the complex, provided that the elution conditions employed do not disturb binding of the coordinating moiety to the coordinator (see Figures 4-5).
• the coordinating moiety used in the complex is a chelator
• high ionic strength may be applied to elute the molecule of interest, since it is well established that it does not effect metal-chelator interactions.
• Altematievly elution with chaotropic salt may be used, since it has been shown that metal-chelator interactions are resistant to high salt conditions enabling elution of the target molecule at such conditions [Porath (1983)
• the complex can be re-solubilized by the addition of free (unmodified) chelator (i.e., coordinating moiety), which competes with the coordinator metal ( Figure 3). Ulfrafiltration or dialysis may be used, thereafter, to remove most of the chelated metal and the competing chelator.
• the solubilized complex i.e., molecule of interest:ligand-coordinating moiety
• an immobilized metal affinity column e.g., iminodiacetic acid (EDA) and nitrilotriacetic acid (NT A)].
• EDA iminodiacetic acid
• NT A nitrilotriacetic acid
• regeneration ofthe ligand-coordinating moiety can be achieved by loading the above-described column with a competing chelator or changing column pH followed by ultrafilfration that may separate between the free chelator and the desired ligand-coordinating moiety.
• the above-described purification methodology can be applied for the isolation of various recombinant and natural substances which are of high research or clinical value such as recombinant growth factors and blood protein products (e.g., von Willebrand Factor and Factor VIII which are therapeutic proteins effective in replacement therapy for von Willebrand's disease and Hemophilia A, respectively).
• the compositions of the present invention may be used to isolate particular populations of cells as well.
• stem cells which are capable of differentiating to any desired cell lineage must be isolated.
• a number of ligands may be employed which bind to surface markers which are unique to this cell population, such as CD34 and CD105 [see Pierelli (2001) Leuk. Lymphoma 42(6): 1195-206].
• Another example is the isolation of erythrocytes using lectin ligands, such as concanavalin A [Sharon (1972) Science 177:949; Goldstein (1965) Biochemistry 4:876].
• Viral cell isolation may be effected using various ligands which are specific for viral cells of interest [see http://www.bdbiosciences.com/clontech/ archive/JAN04UPD/Adeno-X.shtml] .
• retroviruses may be isolated by the compositions of the present invention which are designed to include a heparin ligand [Kohleisen (1996) J Virol
• Methods 60(1):89-101 Cell isolation using the above-described methodology may be effected with preceding steps of sample de-bulking which is effected to isolate cells based on cell density or size (e.g., centrifugation) and further steps of selective cell-enrichment (e.g., FACS).
• the compositions of the present invention may also be used to deplete a sample from undesired molecules or cells. This is effected by contacting the sample including the undesired target molecule or cell of interest with the composition of the present invention such that a complex is formed (described above) and removing the precipitate. The clarified sample is the supernatant.
• This method have various uses such as in depleting tumor cells from bone marrow samples, depleting B cells and monocytes for the isolation and enrichment of
• T cells and CD8 + cells or CD 4 + cells from peripheral blood, spleen, thymus, lymph or bone marrow samples, depleting pathogens and unwanted substances (e.g., prions, toxins) from biological samples, protein purification (e.g., depleting high molecular weight proteins such as BSA) and the like.
• protein purification e.g., depleting high molecular weight proteins such as BSA
• multiple ligands may be employed for the depletion of a number of targets from a given sample such as for the removal of highly abundant proteins from biological fluids (e.g., albumin, IgG, anti-trypsin, IgA, fransferrin and haptoglobin, see http://www.chem.agilent.com/cag prod/ca/51882709small.pdf).
• novel compositions of the present invention provide numerous advantages over prior art precipitation compositions (e.g., smart polymers), some of these advantages are summerized infra.
• Low cost purification the present methodology does not rely upon sophisticated laboratory equipment such as HPLC, thereby circumventing machine maintenance and operating costs.
• Easy up scaling the present methodology is not restricted by limited capacity of affinity columns having diffusion limitations. Essentially, the amount of added precipitating complex is unlimited.
• Mild precipitation process averts limitations resulting from substantial changes in pH, ionic strength or temperature.
• Control over the precipitation process may be governed by, slow addition of an appropriate coordinator ion or molecule to the precipitation mixture; use of mono and/or multi-valent coordinators; use of coordinator ions or molecules with different affinities towards the coordinating moiety; addition ofthe non- immobilized free coordinating moieties to avoid non-specific binding and entrapment of impurities prior to, during or following formation of a non-covalent polymer, sheet or lattice [Mattiasson et al., (1998) J. Mol. Recognit. 11:211-216; Hilbrig and Freitag (2003) J. Chromatogr. B 790:79-90]; as well as by varrying temperature conditions.
• contaminations deriving from the ligand biological background may become modified as well as the ligand itself [provided that the ligand and the contaminants share the same chemistry (e.g., both being proteins)], and might become part of the precipitating complex. Under suitable elution conditions, the target molecule will be recovered, while the modified contaminations will not.
• Binding in homogenous solutions it is well established that binding in homogeneous solution is more rapid and more effective than in heterogeneous phases such as in affinity chromatography [AC, Schneider et al., (1981) Ann. NY Acad. Sci. 369, 257-263; Lowe (2001) J. Biochem. Biophys.
• compositions of the present invention Sanitizing under harsh conditions; the composition is not covalently bound to a matrix and as such can be removed from any device, allowing application of sanitizing conditions to clean the device (column) from non-specifically bound impurities.
• the ability of the compositions of the present invention to arrange molecules of interest in ordered complexes such as in dimers, trimers, polymers, sheets or lattices also enables use thereof in facilitating crystallization of macromolecules such as proteins, in particular membraneous proteins.
• a crystal structure represents ordered arrangement of a molecule in a three dimensional space. Such ordered arrangement can be egenerated by reducing the number of free molecules in a given space (see Figures lOa-b and 1 la-c).
• composition for crystallizing a molecule of interest refers to the solidification of the molecule of interest so as to form a regularly repeating internal arrangement of its atoms and often external plane faces.
• the composition of this aspect of the present invention includes at least one ligand capable of binding the molecule of interest, wherein the ligand is attached to at least one coordinating moiety; and a coordinator capable of non-covalently bmding the at least one coordinating moiety, wherein the at least one coordinating moiety and the coordinator are capable of forming a complex when co-incubated and whereas the composition is selected so as to define the relative spatial positioning and orientation of the molecule of interest when bound thereto, thereby facilitating formation of a crystal therefrom under inducing crystallization conditions.
• compositions of the preset invention are contacted with a sample, which includes the molecule of interest preferably provided at a predetermined purity and concentration.
• the crystallization sample is a liquid sample.
• the crystallization sample is a membrane preparation.
• the hanging drop method is the most commonly used method for growing macromolecular crystals from solution; this approach is especially suitable for generating protein crystals.
• a droplet containing a protein solution is spotted on a cover slip and suspended in a sealed chamber that contains a reservoir with a higher concentration of precipitating agent.
• compositions of the present invention may have evident utility in assaying analytes from complex mixtures such as serum samples, which may have obvious diagnostic advantages.
• the present invention envisages a method of detecting predisposition to, or presence of a disease associated with a molecule of interest in a subject.
• compositions of the present invention are contacted with a biological sample obtained from the subject whereby the level of complex formation including the molecule of interest is indicative of predisposition to, or presence of the disease associated with the molecule of interest in the subject.
• biological sample refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, neuronal tissue, organs, and also samples of in vivo cell culture constituents.
• the biological sample or the composition is preferably labeled (e.g., fluorescent, radioactive labeling).
• compositions of the present invention may also be utilized to qualify and quantify substances present in a liquid or gaseous samples which may be of great importance in clinical, environmental, health and safety, remote sensing, military, food/beverage and chemical processing applications.
• Abnormal protein interaction governs the development of many pathogenic disorders. For example, abnormal interactions and misfolding of synaptic proteins in the nervous system are important pathogenic events resulting in neurodegeneration in various neurological disorders. These include Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy bodies (DLB).
• AD Alzheimer's disease
• PD Parkinson's disease
• DLB dementia with Lewy bodies
• misfolded amyloid beta peptide 1-42 (Abeta), a proteolytic product of amyloid precursor protein metabolism, accumulates in the neuronal endoplasmic reticulum and extracellularly as aggregates (i.e., plaques).
• the compositions of the present invention can be used to disturb such macromolecular complexes to thereby treat such disorders. Methods of administration and generation of pharmaceutical compositions are described by, for example, Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l.
• the compositions of the present invention can be included in a diagnostic or therapeutic kits.
• compositions of a specific disease can be packaged in a one or more containers with appropriate buffers and preservatives and used for diagnosis or for directing therapeutic treatment.
• the ligand and coordinating moiety can be placed in one container and the coordinator molecule or ion can be placed in a second container.
• the containers include a label.
• Suitable containers include, for example, bottles, vials, syringes, and test tubes.
• the containers may be formed from a variety of materials such as glass or plastic.
• other additives such as stabilizers, buffers, blockers and the like may also be added. A number of methods are known in the art for enhancing the immunogenic potential of antigens.
• hapten carrier conjugation which involves cross- linking of the antigenic molecule (e.g., peptides) to larger carriers such as KLH, BSA thyroglobulin and ovalbumin is used to elevate the molecular size of the molecule, a parameter known to govern immunogenicity [see Harlow and Lane (1998) A laboratory manual Infra].
• covalent cross-linking of the antigenic molecule leads to structural alterations therein, thereby limiting antigenic presentation.
• Noncovalent immobilization of the antigenic molecule to various substrates have been attempted to circumvent this problem [Sheibani Frazier (1998) BioTechniques 25:28]. Accordingly, compositions of the present invention may be used to mediate the same.
• the present invention also envisages a method of enhancing immunogenicity of a molecule of interest using the compositions of the present invention.
• immunogenicity refers to the ability of a molecule to evoke an immune response (e.g., antibody response) within an organism.
• the method is effected by contacting the molecule of interest with the composition of the present invention whereby the complex thus formed serves as an immunogen.
• Such a complex can be injected to an animal host to generate an immune response.
• the above-described immunogenic composition is subcutaneously injected into the animal host (e.g., rabbit or mouse).
• compositions of the present invention may have numerous other utilities, which are not distinctly described herein such as those utilities, which are attributed to affinity chromatography [see e.g., Wen-Chien and Kelvin (2004) Analytical Biochemistry 324:1-10]. Additional objects, advantages, and novel features ofthe present invention will become apparent to one ordinarily skilled in the art upon examination ofthe following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
• Figure 12 For example, a hydroxamate (which is a known Fe 3+ chelator) derivative is synthesized (Figure 13 a) such that in the presence of Fe 3+ ions, a non-covalent multi- ligand complex is formed ( Figure 13b).
• Figure 14 A general synthetic pathway for modification of representative chelators with a general ligand is shown in Figure 14. Such a synthesis can be similar to the one presented by Margherita et al., 1999 supra.
• chelators for the preparation of a non-covalent multi-ligand complex, may have an additional advantage which arises from the ability of some chelators to bind different metals with different stochiometries, as in the case of [1,10- phenanthroline] 2 -Cu 2+ , or [l,10-phenanthroline] 3 -Ru 3+ [Onfelt et al., (2000) Proc. Natl. Acad. Sci. USA 97:5708-5713].
• This phenomenon can be utilized for formation of di ( Figure 15a) and tri ( Figure 15b) non-covalent multi-ligand complexes, utilizing the same: ligand — linker — chelator derivative.
• EXAMPLE 2 Synthesis of non-covalent multi ligand complexes utilizing electron rich-poor complexes Electron acceptors form molecular complexes readily with the " excessive" heterocyclic indole ring system. Indole picric acid was the first complex of this type to be described nearly 130 years ago [Baeyer, and Caro, (1877) Ber. 10:1262] and the same electron acceptor was used a few years later to isolate indole from jasmine flower oil. Picric acid had since been used frequently for isolating and identifying indoles as complexes from reaction mixtures.
• Figure 16a illustrates one example of a ligand — linker — elecfron poor (E. poor) derivative
• Figure 16b presents an example of an electron rich covalent trimer that could be used.
• Figure 18 shows an example of a synthetic peptide with four Tip residues (four electron rich moieties) that can be formed, a tetra-non-covalent- ligand in the presence of a ligand derivative modified with an electron poor moiety (trinitrobenzene).
• EXAMPLE 3 Synthesis of non-covalent multi ligand complexes utilizing a combination of electron rich-poor and chelator-metal relationships One can combine the two complexing abilities as described in Examples 1 and 2 above, so as to form non-covalent multi ligand complexes.
• An example of the general structure of such a non-covalent multi ligand complex is shown in Figure 19. To this end, a chelator that is covalently bound to an electron poor moiety is desired.
• a synthetic pathway for generating such a combination is presented in
• a chelator e.g., catechol
• M 2+ M 2+
• M + metals is capable in the presence of M 2+ and M + metals, to form a non-covalent- di-ligand, ( Figure 21a), or a non-covalent-tri-ligand ( Figure 21b).
• a peptide (or polypeptide) with a Trp residue might lead to the formation of the structures shown in Figures 22a-b.
• the combination of the two above binding relationships may introduce additional advantages. For example, the ability to form non-covalent-multi-ligand-polymeric complexes. This may be achieved by synthesizing two chelators and an electron rich moiety between them ( Figure 23a). In the presence of a ligand — E.

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