EP1155327A1 - Purification et affichage differentiel de composants d'echantillons biologiques - Google Patents

Purification et affichage differentiel de composants d'echantillons biologiques

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Publication number
EP1155327A1
EP1155327A1 EP00976796A EP00976796A EP1155327A1 EP 1155327 A1 EP1155327 A1 EP 1155327A1 EP 00976796 A EP00976796 A EP 00976796A EP 00976796 A EP00976796 A EP 00976796A EP 1155327 A1 EP1155327 A1 EP 1155327A1
Authority
EP
European Patent Office
Prior art keywords
biological sample
affinity
hydrophilic
peptoid
groups
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00976796A
Other languages
German (de)
English (en)
Inventor
Ronald N. Zuckermann
Eric Beausoleil
Matthew c/o Chiron Corporation WACHOWICZ
Srinivas c/o Chiron Corporation KOTHAKOTA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chiron Corp filed Critical Chiron Corp
Publication of EP1155327A1 publication Critical patent/EP1155327A1/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • 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
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    • 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
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions 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
    • B01J20/3202—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
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • 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
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • 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
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    • 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
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
    • 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
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
    • 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
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • 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
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • 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
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis

Definitions

  • the present invention relates to techniques and materials for the purification of biological samples and the differential display and analysis of components of such biological samples.
  • the invention is directed to protein purification and proteome differential display.
  • chromatography supports include anionic exchange supports, such as diethylaminoethyl (DEAE) Sepharose, etc., cationic exchange supports, for example having carboxymethyl functional groups, reverse phase and hydrophobic interaction supports.
  • anionic exchange supports such as diethylaminoethyl (DEAE) Sepharose, etc.
  • cationic exchange supports for example having carboxymethyl functional groups
  • reverse phase and hydrophobic interaction supports are able to distinguish between biological sample components, such as proteins, only very generally and are therefore limited in their ability to separate biological sample components, particularly proteins of similar anionic, cationic, etc. character.
  • affinity chromatography supports based on antibodies, proteins, sugars, etc., are available, such as described by Use of antibody fragments in immunoaffinity chromatography: comparison of FV fragments, VH fragments and paralog peptides. Berry, M. J.; Davies, J. J. Chromatogr. (1992), 597(1-2), 239-45.
  • conventional chromatography supports are too general to provide more than limited separation of the complex mixtures of chemicals that typically comprise biological samples, conventional affinity supports are too specific to provide useful separation of the many components of such mixtures beyond a specific corresponding target or antigen.
  • Two-dimensional gels may be run for biological samples, such as cell lysates or tissue samples, separating on the basis of pH in one dimension and on the basis of molecular weight in the other.
  • biological samples such as cell lysates or tissue samples
  • This technique has severe specificity limitations due to the fact that very abundant proteins in the mixture often obscure less abundant proteins.
  • this technique has low throughput and is not amenable to large scale production.
  • a comparison of the components of two biological samples having different phenotypes may allow for the identification of the source(s) of the difference between the samples. For example, by conducting such a differential analysis of a healthy tissue sample and a diseased tissue sample of the same type, it may be possible to identify the cause of the disease and/or characterize the phenotypic symptoms of the disease. This approach has received significant attention at the genetic (nucleic acid) level, and has seen the development of the field of "genomics.”
  • proteomics This approach has received less attention at the gene product (protein) level.
  • proteomics is attractive. While there may be correlation between genetic differences in different biological samples and the different phenotypes observed in their associated cells, tissues, or organisms (e.g., healthy vs. diseased states), genomic differences are not do not necessarily manifest themselves as differences in gene expression.
  • Proteomics directly addresses differences in gene expression by focussing the analysis on the protein constituents of the sample under study. The effectiveness of proteomic approaches, however, are limited by the ability of currently available materials and techniques to provide high resolution differential displays of biological samples under study. For example, Kauvar et al. (e.g., US Patent No.
  • the present invention provides affinity support materials having intermediate binding affinity for biological samples, relative to conventional materials having only very general or very specific binding affinities.
  • materials provided by the present invention are hydrophilic solid supports composed of hydrophilic ligands coupled to hydrophilic matrixes which are compatible with biological samples.
  • Suitable biological samples applicable to the present invention may be of virtually any type or source including for example, cells derived from defined cell lines (e.g., tumor cell lines, bacteria, virus-infected cells, replicons, and the like); biological fluids, such as blood, urine, saliva, or mucus; cells collected from biological surfaces such as from washes (e.g., lavages) or swabes (e.g., pap smears or throat cultures); and - cells derived from tissue samples (e.g., cells obtained from biopsies of diseased tissues and/or undiseased "normal" tissues).
  • defined cell lines e.g., tumor cell lines, bacteria, virus-infected cells, replicons, and the like
  • biological fluids such as blood, urine, saliva, or mucus
  • cells collected from biological surfaces such as from washes (e.g., lavages) or swabes (e.g., pap smears or throat cultures)
  • the biological samples can homogeneous (i.e., from a single source) or heterogeneous (i.e., two or more unrelated sources).
  • the ligands described herein may include affinity property groups and hydrophilic groups pendent from a backbone, and be configured to at least partially resolve components of a biological sample as defined above. Such affinity property groups and hydrophilic groups can be selected using methods described hereinbelow.
  • affinity supports in accordance with the present invention may be used in a variety of techniques and apparatuses to achieve improved separations of complex biological samples and thereby enhance the results of biological sample component resolutions, fractionations, enrichments, purifications, expression product determinations and comparisons, and other biological sample processing and analysis techniques.
  • the affinity supports may be included in kits useful in processing biological samples for applications such as novel protein purification.
  • the hydrophilic ligands of the affinity supports may be peptoids with both affinity property groups and hydrophilic groups pendent from their peptoid backbones.
  • peptoid structures, as well as parameters for designing and fabricating peptoids having suitable properties and arrays of peptoids having suitable ranges of properties are also provided.
  • the present invention provides a method for providing a biological sample component expression pattern for a biological sample involving applying a biological sample to one or more ligands coupled to a biological sample- compatible matrix.
  • the ligand may include a plurality of affinity property groups and hydrophilic groups pendent from a backbone, and configured to at least partially resolve biological sample components of a biological sample.
  • the components of the sample may be fractionated using one or more hydrophilic ligands to provide an enriched fraction, and a biological sample component expression pattern for the biological sample may be determined using the enriched fraction.
  • the invention provides, methods of comparing biological sample phenotypes, reducing complexity of biological samples, massively parallel processing a biological sample on a number of affinity supports both one- and two-dimensionally, and materials and kits incorporating affinity supports useful in accomplishing these methods. Also provided are methods of making affinity supports in accordance with the present invention, in particular supports including peptoids having both affinity property groups and hydrophilic groups pendent from their peptoid backbones and coupled to hydrophilic matrixes.
  • Figure 1 schematically depicts the structure of a hydrophilic peptoid in accordance with one embodiment of the present invention.
  • FIGS. 2A and 2B schematically depict alternative processes for synthesizing a hydrophilic affinity chromatography support in accordance with the present invention.
  • FIGS 3A-C schematically depict the molecular structures of an array of 25 12mer peptoids in accordance with one embodiment of the present invention.
  • Figure 4 schematically a kit and its mode of operation in accordance with one embodiment of the present invention.
  • Figure 5 schematically depicts a process for conducting a differential protein expression (proteomic) analysis in accordance with one embodiment of the present invention.
  • Figure 6 schematically depicts a process for synthesizing hydrophilic peptoids on a hydrophilic substrate in accordance with one embodiment of the present invention.
  • Figure 7 depicts an electrophoretic gel illustrating the reproducibility of chromatography columns prepared using supports in accordance with the present invention.
  • Figure 8 depicts electrophoretic gels illustrating the binding capacity of chromatography columns prepared using supports in accordance with the present invention.
  • Figure 9 depicts an electrophoretic gel illustrating the specificity and enrichment achievable by chromatography columns prepared using supports in accordance with the present invention.
  • Figure 10 depicts an electrophoretic gel illustrating the identification of a peptoid ligand that selectively binds a protein in a cell lysate in accordance with the present invention.
  • Figure 11 depicts electrophoretic gels illustrating the results of a parallel processing of a cell lysate in accordance with the present invention.
  • the present invention provides affinity support materials having intermediate and controllable binding affinity for biological samples, relative to conventional materials.
  • Intermediate and controllable binding affinity support materials refers to materials capable of interacting with the constituents of a biological sample by a combination of ionic, van der Waal's, and hydrogen bond interactions akin to protein-protein, protein-oligonucleoside (oligonucleotide), or oligonucleoside (oligonucleotide)-oligonucleoside (oligonucleotide) interactions.
  • the support materials of the present invention differ significantly from the materials taught by the prior art for separating the components of biological samples, as the materials taught by the prior art use only very general and uncontrollable interactions with biological sample components (e.g., charge-based) or very specific and controlled (e.g., antibody-based) binding affinities, but nothing intermediate these extremes.
  • the supports provided by the present invention combine the attributes of general resins typically based on one functional group (like anion or cation exchange resins) and highly specific affinity resins based on a particular combination of several functional groups chosen for a single, specific target constituent.
  • the supports described herein will have many functional groups, but, on average, they may be less specifically defined than the highly specific arrays of the prior art. Therefore, the supports will still yield high affinities in many cases, but may have a broader specificity than an affinity resin.
  • hydrophilic solid supports composed of hydrophilic ligands coupled to hydrophilic matrixes which are compatible with biological samples, for example, those derived from a cell line, blood or a tissue, or cell lysate.
  • a biological sample-compatible hydrophilic matrix is a matrix that is effective to minimize substantially interactions between components of a biological sample and the matrix.
  • compatible matrixes as described herein will be recognized by those having skill in the chemistry arts as being resistant to proteolytic degradation and providing a relatively low non-specific binding (also sometimes referred to as resistant to protein adsorption) by the components of the biological sample (for example proteins).
  • the ligands may include affinity property groups and hydrophilic groups pendent from a backbone, and be configured to at least partially resolve components of a biological sample.
  • Affinity supports in accordance with the present invention may be used in a variety of techniques and apparatuses to achieve improved separations of complex biological samples and thereby enhance the results of biological sample component resolutions, fractionations, enrichments, purifications, expression product determinations and comparisons, and other biological sample processing techniques.
  • the affinity supports may be included in kits useful in processing biological samples.
  • the present invention provides ligand molecules which can be configured to display a wide variety of chemical functionalities or properties, suitable for incorporation into an affinity separation support having an intermediate binding affinity for biological samples within a hydrophilic context.
  • This hydrophilic context is provided by intercalating hydrophilic property and affinity property groups along a molecular backbone. The function of the hydrophilic groups is to provide substantially aqueous solubility of the affinity ligand in the presence of a diversity of side chains that are often hydrophobic.
  • hydrophilic groups also prevent aggregation of the affinity ligand by preventing large hydrophobic patches.
  • Aqueous solubility allows the efficient derivatization of hydrophilic solid support used in biological sample component, particularly protein, separations. Hydrophilic ligands so formed are then linked to hydrophilic solid phase substrates to form affinity supports.
  • the present invention provides a method of synthesizing a diversity of ligands by solid-phase organic synthesis in organic solvent and on hydrophobic resins such as polystyrene.
  • the ligands are then cleaved and dissolved in aqueous buffer to facilitate their attachment to hydrophilic substrates, such as sepharose, suitable for application of biological samples.
  • the hydrophilic ligands of the affinity supports may be "peptoids" with both affinity property groups and hydrophilic groups pendent from their backbones.
  • a "peptoid” is a poly (N-substituted glycine), sharing the backbone structure of a protein, but having pendant groups that differ in whole or in part from conventional amino acid groups. Peptoids are known in the art and have been described in various publications, including U.S. Patents Nos. 5,877,278, 5,977,301, 5,831,005 and 5,811,387 each of which is incorporated by reference herein for all purposes.
  • ligands in accordance with the present invention may be conferred with a hydrophilic character by intercalating hydrophilic property and affinity property groups along the ligand' s molecular backbone.
  • Hydrophilic peptoids in accordance with the present invention are particularly applicable to separation, purification and other processing of the protein component of biological samples. While the remainder of this disclosure focuses on the structure, synthesis and application of hydrophilic peptoids in relation to various protein processing techniques, it should be understood that the present invention is more generally applicable to the processing of biological sample components with intermediate affinity chromatography supports in accordance with the principles provided herein. Thus, the invention is intended to cover hydrophilic ligand molecules other than peptoids coupled to biological sample-compatible matrixes and their use for processing biological sample components beyond just proteins, and including nucleic acids, lipids, etc.
  • Peptoids are particularly good candidates for chromatography support materials. They are amenable to combinatorial synthesis, and with hundreds of diverse sidechains available it is possible to finely tailor support properties. Peptoids are protease stable, and can thus be used with crude cell lysates, for example, without risk of significant degradation by proteases typically present in such samples. They are chemically and thermally stable, allowing for the regeneration and reuse of supports. In addition, peptoids have relatively simple immobilization chemistry which is easily scaled-up for large scale production.
  • Fig. 1 schematically depicts a hydrophilic peptoid 100 in accordance with one embodiment of the present invention.
  • the methoxyethyl side chain 101 is used as the hydrophilic element 106.
  • This side chain is desirable because it is inexpensive and requires no protection.
  • hydroxyl monomers such as hydroxyethylamine 103, l-hydroxyethyl-2-hydroxyethylamine 104 and the 2,3-dihydroxypropylamine 105, among many others may be used.
  • hydroxyl monomers such as hydroxyethylamine 103, l-hydroxyethyl-2-hydroxyethylamine 104 and the 2,3-dihydroxypropylamine 105, among many others may be used.
  • Fig. 1 schematically depicts a hydrophilic peptoid 100 in accordance with one embodiment of the present invention.
  • the methoxyethyl side chain 101 is used as the hydrophilic element 106.
  • hydroxyl monomers such as
  • the hydrophilic element 106 is intercalated between each property (affinity) element 107, however, this is not necessarily the case and the hydrophilic and affinity groups may be distributed on the backbone 102 in any configuration that achieves the function of ensuring aqueous solubility of the ligand.
  • hydrophilic groups in a particular peptoid will depend upon the hydrophilic/phobic character of the affinity groups included in that peptoid. It should also be noted that the placement of the hydrophilic and hydrophobic affinity groups should be such that highly amphipathic peptoid structures are avoided. Such structures may aggregate to form micelles thus detracting from their ability to achieve separations and/or couple efficiently with the substrate.
  • the peptoid ligand 100 is typically attached to a hydrophobic substrate (not shown) in order to facilitate organic synthetic reactions.
  • the ligand 100 may also have a chemoselective ligation group 108, such as maleimidopropionyl, attached to its free N-terminus to enable subsequent attachment to a hydrophilic support.
  • a chemoselective ligation group 108 such as maleimidopropionyl
  • the peptoid ligand 100 may be cleaved from the hydrophobic resin and dissolved in aqueous buffer, to facilitate its attachment to a hydrophilic support suitable for application of aqueous biological samples, such as sepharose.
  • the peptoid ligand may have a suitable chemoselective terminal group, for example, a maleimide such as maleimidopropionyl, which can form a covalent bond upon reaction with a thiol presented on the surface of a hydrophilic support.
  • a maleimide such as maleimidopropionyl
  • hydrazide which can react to form covalent bonds with aldehyde or ketone moieties on the support
  • aminooxy which can react to form covalent bonds with aldehyde or ketone (or synthetically equivalent) moieties on the support
  • aldehyde which can react to form covalent bonds with hydrazides or aminooxy groups
  • disulfides which can react to form covalent bonds with thiol groups
  • thiols which can react to form covalent bonds with disulfide or maleimide groups, azide, phosphine, or avidin which can react to form a stable non-covalent bonding complex with biotin moieties on the support.
  • the hydrophobic substrate may be converted to a hydrophilic substrate obviating the need to transfer the peptoid from one substrate to another.
  • the substrate can include various removable hydrophobic moieties.
  • a hydrophobic substrate is formed using hydroxyalkylacrylamides protected with alkylsilylethers (e.g., tert- butyldimethylsilane (TBDMS))that can be removed using standard conditions to provide a hydrophilic substrate.
  • TDMS tert- butyldimethylsilane
  • Figs. 2A and 2B depict two processes for producing affinity supports composed of hydrophilic ligands on hydrophilic substrates. In the process of Fig. 2 A
  • a ligand 214 which may be a peptoid as described above or another suitable molecule, is synthesized on a hydrophobic substrate 212, e.g., polystyrene.
  • a chemoselective ligation group (Ri) 216 is attached to the free N-terminus of the hydrophobic substrate-bound ligand 214.
  • the C-terminus or any suitable side-chain can also serve as a point of attachment for the chemoselective ligation group.
  • the ligand 214 is cleaved from the hydrophobic substrate 212 and reattached to a hydrophilic substrate 218, e.g., thiosepharose, via the chemoselective ligation group 216.
  • a hydrophilic substrate 218, e.g., thiosepharose e.g., thiosepharose
  • the hydrophobic and hydrophilic substrates may be composed of a variety of materials as will be apparent to those of skill in the art.
  • a ligand 224 is synthesized on a hydrophobic substrate 222, as in the first process 210 but absent a chemoselective ligation group.
  • the hydrophobic substrate 222 is modified to convert it into a hydrophilic substrate 222m.
  • the substrate may have been made hydrophobic by the presence of protecting groups on available hydroxyl moieties (e.g., -OSiR 3 ).In that case, the substrate can be converted to a hydrophilic state by removal of the protecting groups to provide free hydroxyl (-OH) groups following completion of the organic synthesis.
  • no cleavage and reattachment is required.
  • peptoids are particularly good candidates for chromatography support materials, in part because it is possible to finely tailor support properties by selecting from among the hundreds of diverse sidechains available during the synthesis process.
  • peptoids may be used in order to mimic some of the principal chemical functionalities found in proteins.
  • a biological sample for example a cell lysate, may then be run in parallel on the array of peptoids and the separation results compared. The peptoid or peptoids with the best performance may then be selected for use in the actual separation.
  • One technique for building an array of peptoids possessing a range of affinities is to identify sidechains having physicochemical properties representative of one or more affinity properties commonly found in proteins, and then to combine those sidechains in various permutations in combination with hydrophilic side chains.
  • five different sidechains representing five different physicochemical properties were incorporated into an array of twenty- five 12mer peptoids. The five sidechains selected for this approach are depicted below:
  • Table 1A indicates that each of the affinity properties is represented in the array at low (2 of 12 sidechains in a 12mer), medium (4 of 12), and high (6 of 12) levels (15 12mers).
  • Figure IB shows how the array also includes all pairwise combinations of the five affinity properties (10 12mers).
  • the ligands of the array are schematically depicted in Figs. 3A-C.
  • the ligands of Fig. 3A have a dominant anionic character.
  • the ligands of Fig. 3B have a dominant hydrophobic character (five of the ligands have both strong anionic and hydrophobic character).
  • the ligands of Fig. 3C have a dominant cationic character.
  • the array described above is representative of a range of affinities possible with peptoids synthesized in accordance with the present invention.
  • the affinity groups representing each of the affinity properties are just examples of some of the possible sidechains that may be used.
  • hundreds of diverse sidechains are available for peptoid synthesis, including, for example, those derived from many natural or nor-amino acid, unnatural amino acid, nucleotide base, enzyme cofactor, or sugar.
  • sidechains having isosteric and/or isoelectronic properties of known amino acid sidechains and/or nucleotide bases, or surrogates thereof may be used. Examples of non-natural amino acid sidechains are described in U.S. Patent No. 5,656,660, incorporated by reference herein.
  • sidechains may include, without limitation, those derived from the following: acetichydrazide, N-acetylethylenediamine, 1-adamantamine, 2-
  • adamantamine-HCL 1-adamantanemethylamine, alanine, beta-alanine, alaninamide
  • allylamine O-allylhydroxylamine, 3-amino-l,2-propandiol, 2-amino-l,3-propanediol, 4-amino-l-benzylpiperidine, 2-amino-l-butanol, 4-amino-l-butanol, 6-amino-l- indanone, 5-amino-l-naphthol, 5-amino-l-pentanol, 2-amino-l-phenylethanol, 3- amino-1-propanol, 5-amino-2-methoxypyridine, l-amino-2-propanol, aminoacetonitrile, 4-aminobenzamide, 4-aminobenzylcyanide, 4-aminobiphenyl, 4- aminobutyricacid, 6-aminocaproicacid, l-aminocyclohexan-2,3-diol, 1- aminocyclohexan-3,4,5-triol, trans-4-aminocycl
  • Sidechains which may be used as affinity groups for synthesizing hydrophilic peptoids in accordance with the present invention include: groups selected from the group of alkyl, (cycloalkyl)alkyl, (cycloheteroalkyl)alkyl, aralkyl, and heteroaralkyl, each substituted optionally from oxo, thia, halo, amino, hydroxy, cyano, nitro, thio, aminocarbonyl, carboxy, and imino.
  • (cycloheteroalkyl)alkyl groups may be further selected from methyl, hydroxymethyl, prop-2-yl, 2-methylpropyl, pyrrolidylmethyl, methylthioethyl, 1 -hydroxyethyl, thioethyl, aminocarbonylmethyl, aminocarbonylethyl, carboxymethyl, carboxyethyl,
  • aralkyl and heteroaralkyl groups may be further selected from phenylalkyl, hydroxyphenylalkyl, imidazolylalkyl, purinylalkyl, pyrimidinylalkyl, and indolylalkyl.
  • sugars include furanosylalkyl, pyranosylalkyl, furanosyl, or pyranosyl, attached at any suitable atom.
  • Sidechains which may be used as hydrophilic groups for synthesizing hydrophilic peptoids in accordance with the present invention include those selected from the group of alkyloxyalkyl, hydroxyalkyl, thioalkyl, alkylthioalkyl, alkylsulfinylalkyl, alkyloxycarbonylalkyl, and aminocarbonylalkyl.
  • the sidechain may be further substituted, for example with one of methoxyethyl, hydroxyethyl, l,3-dihydroxyprop-2-yl, 2-(hydroxym ethyl)- 1,3- dihydroxoxyprop-2-yl, and 2,3-dihydroxypropyl, alkylsulfoxidoalkyl, as well as side chains reported, or identified using the techniques described, in Chapman, R.G.; Ostuni, E.; Takayama, S.; Holmlin, R.E.; Yan, L.; Whitesides, G.M.; J. Am. Chem. Soc. 2000, 122, 8303-8304.
  • kits containing a plurality of hydrophilic chromatography supports in accordance with the present invention having a spectrum of affinity properties In one embodiment, a screening kit for affinity chromatography is provided.
  • Fig. 4 illustrates a kit in accordance with such an embodiment of the present invention.
  • the kit 400 includes an array of intermediate affinity hydrophilic chromatography supports 402, such as described above. Supports from the array 402 are dispensed into receptacles 404 suitable for conducting affinity chromatography.
  • the receptacles 404 may be separate compartments of a multi-compartment containment structure.
  • One typical format for such a structure is a 96-well plate adapted for miniature affinity chromatography in that the wells are not closed at the bottom, but instead are instead suited for miniature affinity chromatography, for example fritted well plates.
  • Such miniature chromatography fritted well plates are known to those of skill in the art.
  • a biological sample for example a cell lysate
  • the supports 402 in the receptacles 404 may be applied to the supports 402 in the receptacles 404, and the eluates may be run on an electrophoretic gel in order to screen for the support(s) that provide the best separation for the target biological sample component 408 in the sample. That support or supports may then be selected and used for large scale processing of the biological sample.
  • the present invention when configured in such a kit, advantageously allows for parallel processing of a biological sample to efficiently determine a support or supports having desirable affinity properties for conducting an affinity separation.
  • Suitable chromatography conditions are well known to those of skill in the art.
  • Sample conditions for an array of peptoid ligand-based hydrophilic supports used to separate proteins may be as follows:
  • kits in accordance with the present invention may also have an array of hydrophilic supports deployed on nano-scale chips, for example chips composed of silicon, gold on glass, or aluminum on glass.
  • the present invention may be implemented with technologies such as are described in U.S. Provisional Patent Application Nos. 60/141,469 and 60/209,711, U.S. Patent Nos. 5,744,305 and 5,874,219, all of which are incorporated herein by reference.
  • Affinity supports in accordance with the present invention may be loaded into affinity chromatography columns according to methods well known to those of skill in the art, and may then be used to conduct separations for a variety of purposes. These applications include biological sample component (e.g., protein) purification and determination and comparison of differential protein expression patterns (e.g., proteomics). These techniques provide useful tools in diagnostics and drug development, among other research and production applications.
  • biological sample component e.g., protein
  • differential protein expression patterns e.g., proteomics
  • Fig. 5 illustrates an example of a proteomic application of the present invention.
  • a biological sample 502 is applied to a hydrophilic affinity support 504, such as described above, composed of a hydrophilic ligand coupled to a biological sample-compatible hydrophilic matrix (substrate).
  • the ligand includes affinity property groups and hydrophilic groups pendent from a backbone configured to at least partially resolve components of the biological sample.
  • the biological sample components of the sample are fractionated by the hydrophilic support to provide an enriched fraction 506.
  • the enriched fraction 506 may be that portion of the applied biological sample that does not bind to the affinity support, or, more frequently, a or the part which initially binds but is subsequently eluted from the support.
  • the enriched fraction 506 is then analyzed to determine a biological sample component expression pattern 508, and possibly other features 510 (e.g., protein expression level and protein identity) for the biological sample.
  • the expression pattern may be determined by running the enriched fractions obtained from the chromatography using the hydrophilic affinity supports on an electrophoretic gel.
  • a label for example a radioactive label or dye, may be added to the enriched fractions prior to loading of the gel so that separate bands of biological sample component (e.g., protein) may be seen following processing of the completed gel.
  • Techniques used to determine other features of the biological sample from the enriched fractions obtained from affinity chromatography using the affinity supports of the present invention include mass spectrometry (MS), bio-informatics (comparison of data obtained with relational databases of biological and biochemical information and macromolecular structure analysis techniques, such as are described in U.S. Patent Application No. 09/580,380, filed May 26, 2000, incorporated by reference herein
  • the biological sample applied to the affinity support may be a single sample or may be two or more different samples obtained from different biological sources (e.g., cell lines, tissue samples, biological fluids, such as blood, etc.).
  • the affinity support to which the one or more biological samples are (separately) applied may be a single support, or may be two or more supports having different properties.
  • the chromatography conditions used may vary.
  • an expression pattern may be obtained for a single biological sample run on a single affinity support.
  • a sample may be run on a plurality of supports and/or using a number of different chromatography conditions and their expression patterns compared in order to determine the best support and/or conditions with which to process a given sample.
  • the present invention provides a kit for making such a determination.
  • a plurality of biological samples may be run on one or more affinity supports and their expression patterns compared in order to determine expression differences that correlate with phenotypic differences in the samples.
  • healthy and diseased samples of the same tissue may be processed in this manner in an attempt to elucidate a phenotype of the disease state.
  • tissues of different types but subject to the same disease state may be processed in this manner in order to determine if agents associated with the disease state are manifested in different tissues.
  • Hydrophilic affinity supports in accordance with the present invention may also be used to reduce the complexity of a biological sample by performing affinity chromatography.
  • a biological sample may be applied to a hydrophilic affinity support in accordance with the present invention resulting in the fractionation of the biological sample's components.
  • Such an approach may be used to separate and purify a component, such as a protein, from the biological sample.
  • a particular support or supports suitable for performing such a separation may be selected as described above with reference to the kits provided by the present invention.
  • One particular embodiment of this approach involves a two-stage process.
  • a biological sample may be applied to a multi- compartment containment structure wherein one or more of the compartments contains a plurality of peptoids from an array of peptoids having a range of intermediate affinities for biological sample components, as described above.
  • peptoids having affinity properties suitable for achieving good separation or complexity reduction of a sample may be preliminarily identified in a group.
  • peptoids from a compartment from the multi-compartment containment structure may be selected for further processing.
  • Another sample of the same biological sample may then be applied to a second multi-compartment containment structure having separate compartments containing the peptoids from the selected peptoid-containing compartment from the first multi-compartment containment structure.
  • a second multi-compartment containment structure having separate compartments containing the peptoids from the selected peptoid-containing compartment from the first multi-compartment containment structure.
  • Differences in complexity reduction achieved by the different peptoids contained in the second multi-compartment containment structure may then be determined and a peptoid or peptoids providing the best complexity reduction may be selected for further processing of a particular biological sample component.
  • the optimal affinity substrate for performing a protein separation or purification may be efficiently determined.
  • a further application of the present invention in the reduction of the complexity of biological samples may be in the preparation of samples for mass spectra-based identification and/or quantitation of the components of the biological samples, e.g., using commercially available ICAT TM processing (Applied Biosystems, Foster City, CA).
  • ICAT TM processing Applied Biosystems, Foster City, CA
  • a complex biological sample may be fractionated prior to the ICAT procedure so that ICAT results may be obtained for less populous sample components.
  • the ICAT procedure is further described in the following documents, each of which is incorporated by reference herein in its entirety for all purposes: Measuring gene expression by quantitative proteome analysis Gygi SP, Rist B, Aebersold R, Current Opinion in Biotechnology 11 : (4) 396-401 AUG 2000; and International Patent Application No. WO 00/11208.
  • the present invention also provides a method for serially processing a biological sample using hydrophilic affinity supports in accordance with the present invention in order to provide enhanced separations by using a plurality of supports in the separation process.
  • the supports to be used may be selected by use of a kit as described above.
  • a biological sample may be applied to a first hydrophilic affinity support such as described above, and fractionated to provide an enriched fraction.
  • the enriched fraction may then applied to a second affinity support and fractionated again to provide a further enriched fraction. This process may be repeated as many times as are desired, with the enriched fraction of each fractionation being desalted and placed in a new buffer compatible with the next affinity support.
  • an ELISA or other antibody- based assay e.g., radio-immuno assay, "RIA" could be performed to determine if a chromatography column packed with a peptoid affinity support in accordance with the present invention was able to bind and release the protein of interest.
  • a chromatography column packed with a peptoid affinity support in accordance with the present invention was able to bind and release the protein of interest.
  • Peptoid affinity supports in accordance with the present invention can be packed into a chromatography column (e.g., a medium pressure ( ⁇ 150 psi.) liquid chromatography (MPLC) column) and used in a standard gradient elution mode.
  • a chromatography column e.g., a medium pressure ( ⁇ 150 psi.) liquid chromatography (MPLC) column
  • MPLC liquid chromatography
  • the sample to be separated may be loaded on the column under one set of non- eluting (or low-eluting) conditions and the analytes of interest eluted by running a gradient of an eluting solvent or buffer. This allows complex samples to be fractionated.
  • Peptoids were prepared on Rink amide polystyrene resin as illustrated and described in Fig. 6, part A, Peptoid Synthesis. The synthesis procedure is also reported in Figliozzi, G. M., Goldsmith, R., Ng, S. C, Banville, S. C, Zuckermann,
  • the peptoids on Rink resin were treated with N-FMOC- aminohexanoic acid (0.4M), HOBT (0.4M), DIC (0.44M) at 35 °C for lhr. Double coupling was performed when the ninhydrin test exhibit blue colored beads. After piperidine treatment to remove the FMOC protecting group, the beads were treated with 3-maleimido propionic acid (0.4M), HOBT (0.4M), DIC (0.44M) for lhr. at 35 °C. After cleavage from the resin using 95 % TFA [v/v] in H O the resulting solution was filtered, mixed with H 2 O, frozen and lyophilized. The resulting product was dissolved in 100% AcOH, frozen, lyophilized and dissolved again in 50% [v/v] CH 3 CN in H 2 O, frozen and lyophilized.
  • the peptoid purity was assessed using HPLC and mass spectrometry typically indicating >90%.
  • the resulting product was then dissolved in a NaH PO 4 (0.1 M), buffered at pH 7 and added to thiosepharose resin.
  • the amount of free thiols before and after addition of the maleimido peptoid was determined using the Ellmans reagent, DTNB. When the remaining free thiols were less the 10% of the initial amount of free thiols, the remaining free thiols were capped with maleimide.
  • Each solid phase attached peptoid was packed in a column and loaded with a cell lysate. After a certain period, the column was washed with the loading buffer. The resin bound proteins were eluted using a variety of elution buffers. The fractions collected were analyzed using polyacrylamide gel electrophoresis.
  • Figs. 3A-C shows the structures of the various peptoids used in these evaluation.
  • Fig. 7 shows the elution product reproducibility of peptoid/sepharose column after three loadings, washings and elutions of the HeLa cell lysate.
  • the gel shows a high degree of reproducibility.
  • Fig. 8 shows the binding capacity of peptoid sepharose columns EB8224 and EB8225 toward protein sets of a HeLa cell lysate.
  • EB8224 bound 7% of the cell lysate
  • EB8225 bound 11% of the cell lysate.
  • Fig. 9 shows enrichment and specificity of peptoid sepharose columns EB8217-8221 in a proteomic differential display of breast cancer tissue (low metastatic vs. high metastatic).
  • An array of supports was used to fractionate proteins of high and low metastatic biological samples (in this case cell lysates) and analyze the differences between their phenotypes by looking at differences in the protein expression pattern (the proteome) for the presence or the absence of a protein and the abundance of specific proteins.
  • This gel demonstrates the range of binding affinities achievable with an array of supports in accordance with the present invention, as evidenced, for example, by the specificity differences that may be observed between
  • the present invention provides the ability to cover the space of the proteome in a substantially resolvable manner.
  • the gel also demonstrates the ability of affinity substrates in accordance with the present invention to enrich components of a biological sample and enable analysis of differences in protein expression between, in this example, high and low metastatic tissues, as evidenced by the observable differences in expression between the high and low metastatic tissues samples in the expression patterns provided by support EB8220.
  • the low metastatic tissue sample can be seen to have a much higher expression of a particular protein than the high metastatic tissue. Such a finding may allow an researcher to investigate a possible role for the identified protein in suppressing metastasis, for example.
  • peptoid-based affinity supports prepared in accordance with the present invention were used to purify a protein from a crude cell lysate.
  • the four columns used in this example identified as 01, 02, 03 and 04 in Figure 10, were the columns identified as EB8201, EB8202, EB8203 and EB8204, respectively, in Figures 3A and 3C.
  • a peptoid that binds selectively to the protein FGF (Fibroblast Growth Factor) was located.
  • Each different column was loaded with a FGF-containing cell lysate and run using the cationic conditions described above in Table 1.
  • a cell lysate of a yeast extract with overexpression of bFGF was processed in parallel on several different intermediate affinity chromatography supports in accordance with the present invention.
  • the load, flow through, and eluates were then run on electrophoretic gels as described above. Results and elution conditions are shown in Figure 11.
  • the legend in Figure 11 indicates the columns and conditions used by providing a key to the alphanumeric references appearing below each gel panel.
  • the various conditions referred to are noted in Table 1, and the various column compositions referred to are illustrated in Figures 3 A-3C.

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Abstract

L'invention concerne des matières de support à affinité possédant une affinité de liaison différentielle vis-à-vis des échantillons biologiques. Parmi ces matières on compte des supports solides hydrophiles composés de ligands hydrophiles couplés à des matrices hydrophiles qui sont compatibles avec les échantillons biologiques, par exemple, une lignée cellulaire, un liquide biologique tel que le sang ou un lysat de cellules de tissu. Les ligands peuvent comprendre des groupes de propriétés d'affinité et des groupes hydrophiles attachés à un squelette et être configurés pour résoudre au moins partiellement les composants d'un échantillon biologique. Selon l'invention, les supports à affinité peuvent être utilisés dans diverses techniques et divers appareils pour assurer une meilleure séparation d'échantillons biologiques complexes et améliorer ainsi les résultats d'opérations telles que l'enrichissement, la purification, la détermination et la comparaison des produits d'expression et autres qui portent sur des échantillons biologiques. En outre, les supports d'affinité peuvent être compris dans des kits utilisés dans le traitement d'échantillons biologiques.
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