WO2023056530A1 - Conjugaison de biomolécules - Google Patents

Conjugaison de biomolécules Download PDF

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Publication number
WO2023056530A1
WO2023056530A1 PCT/AU2022/051212 AU2022051212W WO2023056530A1 WO 2023056530 A1 WO2023056530 A1 WO 2023056530A1 AU 2022051212 W AU2022051212 W AU 2022051212W WO 2023056530 A1 WO2023056530 A1 WO 2023056530A1
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Prior art keywords
biomolecule conjugate
metal coordination
biomolecules
coordination complex
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PCT/AU2022/051212
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English (en)
Inventor
Nobuyoshi Joe Maeji
Chang-Yi Huang
Manuel Christoph Wieser
Kai-Anders HANSEN
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Anteo Technologies Pty Ltd
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Priority claimed from AU2021903234A external-priority patent/AU2021903234A0/en
Application filed by Anteo Technologies Pty Ltd filed Critical Anteo Technologies Pty Ltd
Publication of WO2023056530A1 publication Critical patent/WO2023056530A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F11/00—Compounds containing elements of Groups 6 or 16 of the Periodic Table
    • C07F11/005—Compounds containing elements of Groups 6 or 16 of the Periodic Table compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow

Definitions

  • the disclosure relates to a method of bioconjugation where two or more biomolecules are conjugated via metal coordination complexes. More particularly, the disclosure relates to the use of certain metal coordination complexes to directly conjugate two or more biomolecules, including peptides, polypeptides, proteins and the like.
  • Cross-linking agents are used in a wide variety of life sciences applications.
  • cross-linking describes the formation of a covalent bond between two polymer chains and, depending on the type of cross-linker and its cross-linking density, different material properties can be created from the same starting polymers.
  • proteins and other biomolecules are cross-linked the technique may be referred to as bioconjugation.
  • three different types or cross-linkers are described: homobifunctional, heterobifunctional and photoreactive.
  • These cross-linking agents have been used for protein interaction studies, histochemistry, sterilisation, conjugate formation and many other applications. In some applications, it is important to maintain the functionality of the protein after cross-linking.
  • One example is to maintain antibody - antigen recognition in challenging areas such as targeted nanomedicine.
  • technologies such as targeted nanomedicine.
  • more sophisticated strategies that increase the likelihood of maintaining protein functionality are generally more synthetically complex leading to challenges such as high manufacturing costs as well as achieving consistency and reproducibility of product.
  • Homobifunctional cross-linking agents are those that contain two or more identical reactive ends capable of coupling to specific functional groups such as carboxylic acids, primary amines, sulfhydryls, etc. , on proteins or other molecules of interest.
  • One such cross-linker is glutaraldehyde, a linear dialdehyde, but there are many others commercially available. These cross-linkers randomly cross-link like functional groups and, while useful in limited situations, can easily create a broad range of poorly defined conjugates including large, polymerised protein aggregates.
  • Cross-linking agents that may respond to inherent stresses or changes in the system are generally classed under reversible cross-linking chemistries and are often applied to self-healing materials.
  • Metal ions have been included as reversible crosslinking agents with different binding strengths depending on the metal ion and the polymer being cross-linked.
  • IMAC Immobilised Metal ion Affinity Chromatography
  • Metal complex activated particles were understood to provide for rapid kinetics of binding of target molecules such that if two different target molecules were added as a mixture in a certain ratio then the rate of incorporation onto such an activated particle would largely reflect this ratio, thereby providing for simple batch production and improved reproducibility.
  • the approach could not employ metal complexes for cross-linking proteins or other biomolecules in solution as the reaction kinetics were simply too rapid to allow any control over such solution phase cross-linking of proteins or other biomolecules.
  • the present disclosure addresses one or more of these needs or provides a solution or a useful alternative to one or more of the problems or approaches of the prior art.
  • the present disclosure provides simple, one step cross-linking agents which are suitable for cross-linking biomolecules, such as proteins, in the solution phase to form uniform bioconjugates, protein clusters and/or protein gels of advantageously uniform cross-linking density while still maintaining protein function.
  • the approach employs suitable metal complexes with a modified or tailored reactivity to provide for appropriate stable cross-linking while minimising any functional/conformational damage and, optionally, employs suitable buffer conditions to assist on modification of reactivity.
  • biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex.
  • a method of forming a biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex, the method including the steps of:
  • the liquid formulation comprising two or more biomolecules may be a liquid formulation comprising a population of biomolecules.
  • the population of biomolecules may be population of biomolecules which are all of a single type or the population may be a mixed population of two or more populations of different biomolecules.
  • the two or more biomolecules or population of biomolecules are in the solution phase in the liquid formulation at the time of forming the biomolecule conjugate.
  • a functional substrate comprising at least one biomolecule conjugate, the biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex, and the at least one biomolecule conjugate associated with a substrate material.
  • a method of forming a functional substrate comprising at least one biomolecule conjugate including the steps of:
  • the two or more biomolecules may be the same or different.
  • the two or more biomolecules may be selected from the group consisting of proteins, polypeptides, oligopeptides and peptides.
  • the metal coordination complex is an oligomeric metal coordination complex.
  • FIG 1 shows the zeta size of Bovine Serum Albumin (BSA) aggregates formed when unmodified oligomeric metal complexes (Solution 1) is added after (a) 1 minute, (b) 30 minutes.
  • BSA Bovine Serum Albumin
  • FIG 2 shows the zeta size of Bovine Serum Albumin (BSA) clusters after the addition of different concentrations modified oligomeric metal complexes, (a) Solution 3A and (b) Solution 3B over 7 hours.
  • BSA Bovine Serum Albumin
  • FIG 3 shows the zeta size of Bovine Serum Albumin (BSA) clusters formed when modified oligomeric metal complexes (Solution 4) is added at room temperature after (a) 1 minute, (b) 10 minutes, (c) 20 minutes and (d) 60 minutes.
  • BSA Bovine Serum Albumin
  • FIG 4 shows the zeta size of Bovine Serum Albumin (BSA) clusters formed when modified oligomeric metal complexes (Solution 4) is added at 37 °C after (a) 1 minute, (b) 10 minutes, (c) 60 minutes, and (d) 120 minutes.
  • BSA Bovine Serum Albumin
  • FIG 5 shows the zeta size of the Bovine Serum Albumin (BSA) clusters formed using different concentrations of modified oligomeric metal complexes, (a) Solution 4, (b) Solution 5A and (c) Solution 5B over 7 hours.
  • BSA Bovine Serum Albumin
  • FIG 6 shows the absorbance readings of lgM:biotinylated Goat anti-rat antibody cross-linked with Solution 4 compared to an antibody mixture without the cross-linker in a lateral flow half strip model.
  • the Streptavidin-RPE reporter can only detect IgM if the IgM is conjugated to the biotinylated antibody.
  • the wicking of the antibody complex was slower than its individual components but was not too big ( «1 micron) so as to prevent efficient flow through the membrane.
  • FIG 7 shows the absorbance readings of IgM: biotinylated anti-HCG antibody cross-linked with Solution 4 compared to an antibody mixture without the cross-linker in a lateral flow half strip model.
  • the Streptavidin-RPE reporter can only detect IgM if the IgM is conjugated to the biotinylated antibody.
  • the wicking of the antibody complex was slower than its individual components but was not too big ( «1 micron) so as to prevent efficient flow through the membrane.
  • FIG 8 compares the performance differences in a COVID-19 antigen test of antibody clusters formed using three different concentrations of modified oligomeric metal complex (Solution 4) compared to Control.
  • the clusters and control were immobilised onto Eu particles activated with unmodified oligomeric metal coordination complexes.
  • FIG 9 compares the performance of a COVID-19 antigen test for one example of an antibody cluster compared to a Control when using different amounts of conjugate particles; 2pg particles per strip, 0.1 pg particles per strip, 0.05pg particles per strip and 0.025pg particles per strip.
  • FIG 10 compares the performance of a COVID-19 antigen test for one example of an antibody cluster compared to a Control when using 50pg COVID-19 detection mAb per mg of Eu particles.
  • FIG 11 compares the performance of a COVID-19 antigen test for antibody clusters formed with different excesses of capping groups (Solution 5A) compared to a Control when using 50pg COVID-19 detection mAb per mg of Eu particles on a test line formed with 1mg/ml SARS-CoV Ab + 1 mg/ml BSA. Three different Solution 5 concentrations, 0.125mM, 0.0625mM and 0.03125mM were compared.
  • FIG 12 compares the performance of a COVID-19 antigen test for antibody clusters formed with different excesses of capping groups (Solution 5A) compared to a Control when using 150
  • FIG 13 compares the performance differences in a COVID- 19 antigen test of antibody clusters formed on gold nanoparticles using modified oligomeric metal complex under two conditions compared to Control.
  • metal coordination complexes particularly certain oligomeric metal coordination complexes
  • the oligomeric metal coordination complexes employed for this purpose must be modified.
  • the applicant has previously demonstrated the rapid binding of target molecules to unmodified complexes in, for example, Conjugating Molecules to Particles (International publication no. WO 2015/021509).
  • This document demonstrated the extremely rapid kinetics of binding of proteins and the like to the unmodified metal complexes. For this reason, in that approach, it was essential to allow the metal coordination complexes to bind to a surface of a particle and subsequently wash the particle to ensure all unbound complexes were removed.
  • the positive charge of the metal complex helps maintain good dispersion of particles and it was only at this point that the metal coordination complex activated particle could be exposed to a solution comprising the target molecules which had to be in the desired ratio.
  • the rapid kinetics of binding then allowed those target molecules to bind to the particle surface via the metal coordination complexes in generally the same ratio as they were present in the solution, thereby joining the target molecules via the metal complex activated particle which was effectively acting as a linker.
  • the present disclosure presents for the first time the knowledge that metal coordination complexes can have their reactivity modified or tuned down to thereby provide for a much better controlled solution phase bioconjugation approach. This can result in the formation of biomolecule conjugates, without the need for any substrate or support to be present during the conjugation process, which maintain an appropriate level of functionality of the original unlinked biomolecules.
  • biomolecule conjugates described herein will not, in embodiments, likely simply be two biomolecules joined by a single strand of metal coordination complex. Rather, the metal coordination complexes will generally be in oligomeric form and will bond with multiple different biomolecules thereby providing for a biomolecule network which is formed from multiple biomolecules interconnected with metal coordination complexes.
  • biomolecule refers to any compound isolated from a living organism, as well as analogs (including engineered and/or synthetic analogs), derivatives, mutants or variants and/or biologically active fragments of the same.
  • the biomolecule can be a protein (e.g., enzyme), nucleic acid, nucleotide, carbohydrate or lipid.
  • the biomolecule can be an engineered or synthetic analog of a compound isolated from a living cell that is structurally different from the compound but retains a biological activity characteristic of that compound.
  • the “biomolecule” or “biomolecules” refer to such molecules which are made up, at least in part, of amino acids.
  • the terms refer to biological molecules which are peptides or proteins or fragments of either.
  • biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex.
  • the two or more biomolecules may be the same or different.
  • At least one of the biomolecules comprises amino acids.
  • each biomolecule comprises amino acids.
  • the two or more biomolecules may each comprise at least one peptide or a fragment thereof.
  • the two or more biomolecules may each comprise at least one peptide or a fragment thereof
  • the at least one peptide or fragment thereof may be associated with one or more further peptides, ligands, coenzymes, cofactors and/or saccharides.
  • the two or more biomolecules may be independently selected from the group consisting of proteins, polypeptides, oligopeptides, peptides, glycoconjugates, globulins, steroid-binding proteins, antibodies, antigens, haptens, enzymes, or fragments thereof.
  • the two or more biomolecules comprise a peptide or polypeptide or protein secondary structure.
  • the two or more biomolecules comprise a polypeptide or protein tertiary structure.
  • the two or more biomolecules comprise a polypeptide or protein quarternary structure.
  • the two or more biomolecules are proteins, or fragments thereof, they may be the same or different. If the biomolecule conjugates are formed in the presence of a range of biomolecules then a bioconjugate population may be formed in which biomolecules may be joined in all possible combinations and/or in which a bioconjugate network is formed interconnecting a number of different biomolecules.
  • the two or more biomolecules may be independently selected from the group consisting of an antibody, an antigen, a monoclonal antibody, a polyclonal antibody, an antibody fragment, an antibody peptide, an antibody mimetic, an antibody fusion protein, a phage display, a nucleic acid aptamer, a fibronectin display, a peptide-nucleic acid aptamer, and a non-antibody protein scaffold.
  • the two or more biomolecules may be independently selected from the group consisting of an antigen, an epitope of an antigen, an antibody, and an antigenically reactive fragment of an antibody.
  • the two or more biomolecules may be independently selected from antigen binding proteins, such as polyclonal antibodies, monoclonal antibodies and antigen binding fragments thereof, that bind specifically to one or more of: SARS-CoV-2, human immunodeficiency virus (HIV), hepatitis, malaria, respiratory syncytial virus (RSV), Ebola virus (EBOV), human cytomegalovirus (HCMV) and influenza.
  • antigen binding proteins such as antibodies and antigen binding fragments thereof, that specifically bind to CoV spike or nucleocapsid protein, influenza hemagglutinin or nucleocapsid, or an antigen fragment thereof.
  • biomolecules of the disclosure are directly conjugated, one to the other.
  • biomolecules are conjugated directly by the metal coordination complex to form an interconnected network of biomolecule conjugates.
  • the metal coordination complex is bonded only to the biomolecules and additional metal coordination complex at the point of the bioconjugates being formed. That is, the metal coordination complexes are not bonded to a substrate or support at the point of exposure to the two or more biomolecules.
  • the biomolecule conjugate is a solution-phase bioconjugate. That is, the biomolecule conjugate was formed with each biomolecule and the metal coordination complex purely in the solution phase.
  • the two or more biomolecules may be bonded to any region of the metal coordination complex. This is so because the metal coordination complex was not bonded to a surface, substrate or particle and so all ‘surfaces’ or regions not bound to other metal coordination complexes are available for bonding to the biomolecules.
  • the biomolecule conjugate is not bound to a physical support or substrate or particle at formation.
  • the biomolecule conjugate is not bound to a polymer that is not a metal coordination complex bonding the two or more biomolecules.
  • the biomolecule conjugate substantially comprises only biomolecules and metal coordination complex.
  • the biomolecule conjugate consists or consists essentially of the two or more biomolecules and metal coordination complex.
  • the two or more biomolecules are conjugated by the metal coordination complex through non-covalent bonds. Bioconjugation is often described as involving covalent bonding between cross-linking agent and biomolecule but, as discussed further herein, the present disclosure advantageously relies on multiple dative or coordinate bonds between the metal coordinate complex and the biomolecules.
  • the biomolecule conjugate may be viewed as a biomolecule conjugate network or cluster or polymer, which terms may be used interchangeably herein.
  • the biomolecule conjugate network or cluster or polymer is a discrete biomolecule conjugate network or cluster or polymer.
  • the biomolecule conjugate network or cluster or polymer only comprises bonds between biomolecules and metal coordination complex and between metal coordination complex and metal coordination complex.
  • the biomolecule conjugate network or cluster or polymer does not comprise a solid support, substrate or particle.
  • the biomolecule conjugate network or cluster or polymer has an average diameter of between 10 nm to less than 1000 nm, 10 nm to less than 900 nm, 10 nm to less than 800 nm, 10 nm to less than 700 nm, 10 nm to less than 600 nm, 10 nm to less than 500 nm, 10 nm to less than 400 nm, 10 nm to less than 300 nm, 10 nm to less than 200 nm, 10 nm to less than 175 nm, between 20 nm to less than 1000 nm, 20 nm to less than 900 nm, 20 nm to less than 800 nm, 20 nm to less than 700 nm, 20 nm to less than 600 nm, 20 nm to less than 500 nm, 20 nm to less than 400 nm, 20 nm to less than 300 nm, 20 nm to less than 200 nm, 20 nm to
  • the biomolecule conjugate network or cluster or polymer has an average diameter of between 20 nm to 500 nm.
  • nanosized clusters or networks can be consistently formed with peptides and proteins.
  • the applicant has demonstrated the ability to form such nanosized clusters with synthetic polymers, such as PAA and CMC, but doing so with peptides and, particularly, large proteins is much more challenging.
  • the approach disclosed herein employing modified oligomeric metal coordination complexes, for example modified with carboxylate ligands, provides for consistent outcomes which are further enhanced in terms of PDI with the use of elevated temperatures and appropriate buffer conditions as described herein.
  • the biomolecule conjugate network or cluster or polymer may comprise a water-soluble polymer.
  • the water-soluble polymer may be included in the formation of the biomolecule conjugate network or cluster or polymer to add an additional functional or structural property to the biomolecule conjugate network or cluster or polymer.
  • the water-soluble polymer may, in one embodiment, be polyacrylic acid (PAA) but a range of water-soluble polymers are known in the art which may be incorporated to provide a variety of functionalities.
  • biomolecule conjugate network or cluster or polymer is, as described, a solution phase ligation approach and so only water-soluble polymers may be included when the biomolecule conjugate network or cluster or polymer is forming.
  • the metal ion of the metal coordination complex is selected from the group consisting of chromium, ruthenium, iron, cobalt, titanium, aluminium, zirconium, and combinations thereof.
  • the metal ion of the oligomeric metal coordination complex is selected from the group consisting of chromium, ruthenium, titanium, iron, cobalt, aluminium, zirconium, rhodium and combinations thereof.
  • the metal ion of the metal coordination complex is chromium.
  • the metal ion of the metal coordination complex may be present in any applicable oxidation state.
  • the metal ion may have an oxidation state selected from the group consisting of I, II, III, IV, V, or VI, as appropriate and obtainable under standard conditions for each individual metal.
  • the person of skill in the art would be aware of which oxidation states are appropriate for each available metal.
  • the metal ion is a chromium ion
  • it is preferred that the chromium has an oxidation state of III.
  • the metal ion may be associated with any suitable counter-ions such as are well-known in metal-ligand coordination chemistry.
  • mixtures of different metal ions may be used, for example, to form a plurality of different oligomeric metal coordination complexes. In such cases, it is preferred that at least one metal ion is chromium.
  • Metals are known to form a range of oligomeric metal coordination complexes.
  • Preferred ligands for forming the oligomeric metal coordination complex are those that include nitrogen, oxygen, or sulfur as dative bond forming groups. More preferably, the dative bond forming groups are oxygen or nitrogen. Even more preferably, the dative bond forming group is an oxygen-containing group which assist in olation to form the oligomeric complexes. In embodiments, the oxygen-containing group is selected from the group consisting of oxides, hydroxides, water, sulphates, phosphates, or carboxylates.
  • the metal coordination complex is a chromium oligomeric metal coordination complex such as a chromium (III) oligomeric metal coordination complex.
  • the metal coordination complex is an oxo-bridged chromium (III) oligomeric coordination complex. This complex may optionally be further oligomerised with one or more bridging couplings such as carboxylic acids, sulphates, phosphates and other multi-dentate ligands.
  • Exemplary oxo-bridged chromium structures are provided below, albeit without indication of any appropriate modification of reactivity for biomolecule conjugate formation: [0085] On contact with some substrate particle, such as described in Conjugating to Particles (PCT/AU2014/050181), at least one of the water or hydroxyl groups (or whatever ligands may be present) on the oligomeric metal coordination complexes is replaced by a dative bond with the particle surface. This is illustrated below wherein “X” represents the dative bond to the particle surface.
  • the oligomeric metal coordination complexes to which the two or more biomolecules are exposed are modified in terms of a ‘tuning down’ of their reactivity.
  • the capping agents and its excess set some threshold by which only a limited number of more strongly coordinating ligands on the biomolecule can coordinate to the metal complexes.
  • uncontrolled coordination of metal complexes to any ligand in the biomolecule is minimised to encourage intermolecular cross-linking of biomolecules by the oligomeric metal complexes.
  • stability is maintained by a multiplicity of a small set of coordinate bonds, each individual coordinate bond compared to covalent coupling is relatively weak and reversible allowing for better maintenance of biomolecule functional structure.
  • the capping agents may be part of the oligomeric metal complex or may be present in the solvent/buffer that contains the oligomeric metal complexes and/or the biomolecules before they are combined.
  • a metal coordination complex preferably an oligomeric metal coordination complex, directly and non-covalently bonded to the at least two biomolecules
  • the oligomeric metal coordination complexes may no longer exist as discrete oligomeric complexes or may not be able to be truly identified as such once the biomolecule conjugate has formed. This is because the bonding with the biomolecules will result in multiple previously separate oligomeric metal coordination complexes being bound to each biomolecule.
  • biomolecule conjugate network or cluster or polymer is formed with the formerly oligomeric metal coordination complexes essentially forming the strands or connections of the network between separate biomolecules as hubs.
  • the biomolecule conjugate network or cluster or polymer may be viewed as polymeric, or at least ‘extended’ in nature even though it has been formed, in part, from metal coordination complexes, such as oligomeric metal coordination complexes.
  • the interconnected biomolecule conjugate network or cluster or polymer formed by crosslinking of the biomolecules and the metal coordination complexes is a dynamic system due to the nature of the association between the components.
  • the oligomeric metal coordination complexes are directly bonded with the biomolecules through avidity or multi-component bonding and so each biomolecule is directly bonded to what becomes a metal coordination complex network through multiple coordinate bond interactions the accumulated strength of which results in anchoring of the biomolecules to the oligomeric metal coordination complex as if they were bonded via standard covalent bonding. As discussed, this may be viewed as forming an interconnected biomolecule conjugate network or cluster or polymer.
  • any individual coordination bond between the metal ion in the oligomeric metal coordination complexes and the two or more biomolecules is relatively weak (in comparison to covalent) and can break as a result of a local stressor such as the biomolecules preference to maintain its preferred conformational structure. This also allows for desirable freedom of movement or orientation allowing the biomolecules to be advantageously functionally available.
  • a biomolecule conjugate network comprising two or more peptide, polypeptide or protein biomolecules directly conjugated, one to the other, and interconnected through non- covalent bonds by an oligomeric chromium metal coordination complex.
  • the biomolecule conjugate network may have been formed in the absence of a solid support, substrate or particle. That is, all components may have been in the solution phase when the biomolecule conjugate network formed.
  • the biomolecule conjugate network is not bonded through a metal coordination complex to a solid support, substrate or particle.
  • the biomolecule conjugate of the network, the biomolecules, and the metal coordination complexes may be of a nature and interact in manners as described in any other embodiment of the first aspect described.
  • a method of forming a biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex, the method including the steps of:
  • the two or more biomolecules and oligomeric metal coordination complex and biomolecule conjugate may be as described for any embodiment, or combination of embodiments, of the first aspect.
  • the advantages of the present disclosure relate, at least in part, to the ability to have the metal coordination complexes linking the two or more biomolecules to form those bonds in solution in a controlled fashion by reducing the reaction kinetics and adjusting the competition rates between the ligands on the biomolecule with those not on the biomolecule.
  • This approach is demonstrated within the examples to provide for improvements in functional availability and/or mobility of the biomolecules versus bonding with unmodified metal coordination complexes.
  • modified oligomeric metal coordination complexes being relatively unreactive, or reduced reactivity, metal complexes which will form bonds to the two or more biomolecules at an appropriate rate to allow sufficient time for uniform mixing, leading to controlled size and conjugate uniformity of the interconnected biomolecule conjugate network.
  • modified oligomeric metal coordination complexes being relatively unreactive, or reduced reactivity, metal complexes which will form bonds to the two or more biomolecules at an appropriate rate to allow sufficient time for uniform mixing, leading to controlled size and conjugate uniformity of the interconnected biomolecule conjugate network.
  • the degree of modification of the oligomeric metal coordination complexes for example the extent or excess of capping or coordinating agents resulting in the modified oligomeric metal coordination complex, the presence of other competing ligands in solution such as those provided by buffer salts, the concentration of all of these components and the pH of the reaction can be controlled in tandem to modify the speed of formation of the biomolecule conjugate network. As shown in the experimental section, adjustment of these parameters, alone or in concert, can have a direct effect on the biomolecule functionality in the final biomolecule conjugate network.
  • the modified oligomeric metal coordination complex may be defined as a reduced reactivity oligomeric metal coordination complex, especially relative to the same metal ion which is fully hydrated (for example a hexahydrate).
  • the modified oligomeric metal coordination complex is modified such that its reactivity is reduced as compared with the same oligomeric metal coordination complex which has not been so modified, for example the same metal coordination complex but in a fully hydrated state (for example in the form of a hexahydrate).
  • the unmodified metal coordination complex has non- or weakly coordination anions as ligands.
  • the reduced reactivity of the modified oligomeric metal coordination complex may be defined as a reduced level of reactivity as compared with an unmodified metal complex, for example an unmodified oxo-bridged chromium (III) complex.
  • the unmodified metal complex may be a fully hydrated metal complex.
  • the oxo-bridged chromium (III) complex may be a fully hydrated oxo-bridged chromium (III) complex.
  • the reduced reactivity of the modified oligomeric metal coordination complex may be defined as a reduced level of reactivity due to the presence of stronger coordinating capping agents at appropriate concentration and strength compared with an unmodified metal complex, for example an unmodified oxo- bridged chromium (III) complex having only weakly or non-coordinating anions or ligands.
  • an unmodified metal complex for example an unmodified oxo- bridged chromium (III) complex having only weakly or non-coordinating anions or ligands.
  • the unmodified oxo-bridged chromium (III) complex used for comparison purposes may be that as formed in Solution 1 of Example 1 in the examples section.
  • the modified oligomeric metal coordination complex is modified such that its reactivity to, or speed to bond with, one of at least one of the biomolecules is reduced as compared with the same oligomeric metal coordination complex which has not been so modified.
  • the biomolecule used to assess the reduced reactivity by comparison to that with an unmodified oligomeric metal coordination complex is an antibody such as monoclonal antibodies to virus antigens such as SARS-CoV2, Flu A/B viruses or polyclonal antibodies such as goat anti-mouse antibodies.
  • the reduced reactivity of the modified oligomeric metal coordination complex may be defined as a reduced level of reactivity with monoclonal antibodies to virus antigens such as SARS-CoV2, Flu A/B viruses or polyclonal antibodies such as goat anti-mouse antibodies as compared with that of a corresponding unmodified metal complex, especially a corresponding fully hydrated metal complex (such a complex has non- or weakly coordination anions as ligands).
  • virus antigens such as SARS-CoV2
  • Flu A/B viruses or polyclonal antibodies such as goat anti-mouse antibodies
  • the reduced reactivity of the modified oligomeric metal coordination complex may be defined as a reduced level of reactivity with monoclonal antibodies to virus antigens such as SARS-CoV2, Flu A/B viruses or polyclonal antibodies such as goat anti-mouse antibodies as compared with that of an oxo-bridged chromium (III) complex.
  • virus antigens such as SARS-CoV2, Flu A/B viruses or polyclonal antibodies such as goat anti-mouse antibodies
  • the oxo-bridged chromium (III) complex used for comparison purposes may be that as formed in Solution 1 of Example 1 in the examples section.
  • the at least one modified metal coordination complex is a capped metal coordination complex, which may otherwise be referred to as one having stronger coordinating ligands as capping agents and/or having stronger competing coordinating ligands provided in solution.
  • the modified oligomeric metal coordination complex has been modified to display capping agent groups coordinately bound to the metal of the oligomeric metal coordination complex.
  • the capping agents will alter the reaction kinetics of the now modified oligomeric metal coordination complex with ligands on two or more biomolecules as they will be more resistant to being displaced (due to their greater relative coordinating potential) than, for example, simple counterions.
  • the moieties of the metal coordination complexes will therefore react more slowly and a more limited selection of ligands on the biomolecules depending on the type and concentration of capping agent to form an appropriate biomolecule conjugate network.
  • the method may further include the step of selecting or controlling the relative extent of the total coordination capacity of the oligomeric metal coordination complex which is taken up by the capping agent groups, such as carboxylate or phosphate capping or coordinating groups. That is, there may be benefits in choosing or modifying the % of the total coordination capacity of the metal ions of the oligomeric metal coordination taken up by capping agents (as measured by that remaining following formation of the oligomeric metal coordination complex itself - as a coordination interaction is reversible, this percentage is the starting percentage taken up by the capping agents).
  • the % of the total coordination capacity taken up by capping or coordinating agents may be greater than 10%, or 20% or 30% or 40% or 50% any of which values may be combined to form a range with a maximum value of less than 100%, 200%, 400% or 600%.
  • the capping or coordination agents is in excess of the available coordination potential of the oligomeric metal complex, this excess leads to greater competition for coordination to the available oligomeric metal complex.
  • the degree of excess also changes the reaction kinetics of the now modified oligomeric metal coordination complex with the biomolecules as there are more capping agents in competition.
  • competing coordinating ligands may be supplied as part of the buffer solution in which the biomolecule conjugation is performed.
  • Components comprising different buffer salts or other additives can also function as capping or coordinating agents when they exchange with the original ligand on the metal coordination complex and become bound and can replace or augment capping agents on the oligomeric metal complex to further tune the reaction kinetics of the now modified oligomeric metal coordination complex.
  • Appropriate capping agents will therefore be those which slow down coordination of the modified oligomeric metal coordination complexes with the biomolecules but do not prevent it.
  • affinity agent clusters of any desired size can be formed with some appropriate level of intermolecular coordination to maintain a stable cluster. Without this control, such as in the approach of binding biomolecules using standard unmodified oligomeric metal coordination complexes, the metal complexes will simply form tightly bound aggregates with the biomolecules and will not provide for appropriate functionality of said biomolecules.
  • the displacement of the capping agents should occur over an appropriate commercial timeframe which can be easily tested by running parallel reactions of oligomeric metal coordination complexes modified with different capping agents and exposed to the same biomolecules. Similarly, the level of functionality of the biomolecules can be tested by running parallel reactions with different ratios of metal complex and capping groups relative to the amount of biomolecule that was used.
  • useful capping or coordinating agents may be those that include nitrogen, oxygen, or sulphur as dative bond forming groups. More preferably, the dative bond forming groups of the capping agent are oxygen or nitrogen. Even more preferably, the capping or coordinating group is one comprising a dative bond forming group which is an oxygen containing group.
  • the oxygen containing group of the capping or coordinating agent is selected from the group consisting of sulphates, phosphates, carboxylates, sulphonic acids and phosphonic acids.
  • the capping or coordinating agent may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, maleate, sulphate, phosphate, and hydroxyacetate.
  • the capping group may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, maleate, citrate, sulphate, phosphate, an amino acid, naphthalene acetate, and hydroxyacetate.
  • the capping or coordinating agent may be selected from the group consisting of formate, propionate, oxalate, malonate, succinate, glutarate, maleate, citrate, aconitate, sulphate, phosphate, and hydroxyacetate.
  • the capping group may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, maleate, citrate, sulphate, phosphate, an amino acid, and hydroxyacetate.
  • the capping group may be selected from the group consisting of oxalate, malonate, succinate, glutarate, adipate, maleate, citrate, and aconitate.
  • the capping group may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, glutarate, maleate, citrate, and aconitate.
  • the capping group may be selected from the group consisting of acetate, oxalate, malonate, succinate, and citrate.
  • the capping group may be selected from the group consisting of acetate, oxalate, phosphate and succinate.
  • the capping group may be selected from the group consisting of acetate, oxalate, and succinate.
  • the capping group may be oxalate.
  • the capping group may be succinate.
  • the capping or coordinating agent may be carboxylate or phosphate, preferably carboxylate.
  • the carboxylate may be a dicarboxylate or a tricarboxylate, preferably a dicarboxylate.
  • the capping or coordinating agent is a monodentate, bidentate or multidentate capping agent. In embodiments, the capping agent is a monodentate or bidentate capping agent.
  • the capping or coordinating agent has a molecular mass of less than 1000 Daltons, or less than 500 Daltons, or less than 400 Daltons, or less than 300 Daltons. Any of these values may be combined with a lower value of 10, 30 or 50 Daltons to form a range of molecular mass values for the capping agent such as 10 to 1000, 10 to 500, 10 to 400 or 10 to 300 Daltons.
  • the capping group or coordinating agent has a molecular mass of less than 1000 Daltons, or less than 500 Daltons, or less than 400 Daltons, or less than 300 Daltons. Any of these values may be combined with a lower value of 10, 30 or 50 Daltons to form a range of molecular mass values for the capping agent such as 10 to 1000, 10 to 500, 10 to 400 or 10 to 300 Daltons.
  • the capping or coordinating agent is not simply a counterion of the oligomeric metal coordination complex or a group donated by a base.
  • oligomeric metal coordination complexes it is common to expose the metal complex to a base, such as ethylene diamine, which simply encourage formation of the desired complexes. While the amine nitrogen may be, to a small degree, incorporated into the formed oligomeric metal coordination complex it does not have a significant enough effect on the subsequent reactivity of the oligomeric metal coordination complex to be considered a capping agent. Therefore, in one embodiment, the capping agent is not one donated by a base, including ethylene diamine.
  • the step of forming the modified oligomeric metal coordination complex may include contacting the oligomeric metal coordination complex with a solution comprising a capping agent, such as a carboxylate or phosphate ligand-containing solution.
  • a capping agent such as a carboxylate or phosphate ligand-containing solution.
  • the method may further include the step of adjusting the pH of a liquid solution comprising the modified metal coordination complex and/or controlling the temperature of the liquid solution to be between 15 to 45 °C or 15 to 40 °C or 15 to 38 °C or 15 to 30 °C.
  • the step of contacting the liquid formulation comprising the population of two or more biomolecules with a metal coordination complex may also include contacting the liquid formulation comprising the population of two or more biomolecules with a water-soluble polymer.
  • the water-soluble polymer may be selected from any available in the prior art and which would have coordinating ligands to bond to the metal coordination complex and so become a component of the forming biomolecule conjugate network or cluster or polymer.
  • the step of adjusting the pH may include adjusting the pH of the solution in which the oligomeric metal coordination complexes themselves are forming (prior to exposure to the biomolecules) to ensure the desired degree of modification.
  • This may comprise allowing the pH to become more acidic due to the release of hydrogen ions by the metal salts employed or it may comprise the addition of a base, such as ethylene diamine or a metal hydroxide, to mop up some of the released hydrogen ions to prevent the solution becoming too acidic. If a base is added then the amount will be such that the solution is still acidic, as defined above.
  • the modified metal coordination complex can be formed via the direct reduction of chromium (VI) oxide in the presence of suitable capping groups, such as carboxylate groups including acetate and oxalate groups from the corresponding acids.
  • suitable capping groups such as carboxylate groups including acetate and oxalate groups from the corresponding acids.
  • the modified oligomeric metal coordination complex may include various capping groups having on/off rates which are appreciably slower than pre-existing water and other ligand groups, and hence will affect coordination with an additional component of the formulation.
  • the oligomeric metal coordination complexes can be formed by providing conditions for forming electron donating groups for bridging or otherwise linking or bonding two or more metal ions. When not already commercially available, this can be done by providing a pH above pH 1 , and preferably between about 1 to 5, or about 2 to 5 to the solution when forming the complexes.
  • a pH above pH 1 and preferably between about 1 to 5, or about 2 to 5 to the solution when forming the complexes.
  • the chosen pH will depend on the approach by which modification of the oligomeric metal coordination complex is to be achieved. That is, while a pH above 3.8 may be appropriate for forming the oligomeric metal coordination complex when they are to be modified by use of capping groups, a pH below 3.8 is highly desirable for the oligomeric metal coordination complexes formed in aqueous solutions.
  • chromium salts such as chromium chloride, chromium nitrate, chromium sulphate, chromium acetate, chromium perchlorates, may be used to form a chromium-based oligomeric metal coordination complex. Unless pre-existing in some oligomeric form and used ‘as is’, these salts are mixed with an alkaline solution, such as sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium bicarbonate, sodium sulphite and ammonium hydroxide to form different metal coordination complexes.
  • an alkaline solution such as sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium bicarbonate, sodium sulphite and ammonium hydroxide to form different metal coordination complexes.
  • Organic reagents that can act as bases such as ethylene diamine, bis(3- aminopropyl)diethylamine, pyridine, imidazoles, can also be used.
  • bases such as ethylene diamine, bis(3- aminopropyl)diethylamine, pyridine, imidazoles.
  • the size and structure of the oligomeric metal coordination complex can vary with pH, temperature, choice of solvent and other conditions.
  • a liquid carrier or solvent forming the liquid formulation comprising the two or more biomolecules may be an aqueous or organic solvent, or mixture thereof.
  • the liquid carrier has at least some aqueous component.
  • the liquid carrier may preferably be an aqueous solution.
  • the liquid carrier may be water or an alcohol or a mixture thereof.
  • the alcohol may be methanol, ethanol, propanol, isopropanol or butanol.
  • the liquid carrier is water or isopropanol.
  • the liquid carrier is water.
  • the liquid carrier is an aqueous carrier.
  • the liquid carrier is or comprises a buffer salt.
  • the liquid carrier is or comprises a buffer solution.
  • Buffer salts and solutions comprising same are well-known in the art and may comprise, for example, phosphate salts.
  • the method further includes the step of warming the liquid formulation during or following bioconjugate formation.
  • the method may include the step of contacting the liquid formulation comprising the population of two or more biomolecules with the metal coordination complex at a temperature above 20 °C, above 25 °C, or above 26 °C, or above 27 °C, or above 28 °C, or above 29 °C or above 30 °C, or above 31 °C, or above 32 °C, or above 33 °C or above 34 °C, or above 35 °C all of which are considered to form the lower end of a temperature range with an upper limit being less than 45°C, less than 42 °C or less than 40 °C.
  • the conjugation between metal coordination complex and the two or more biomolecules may occur at a temperature between 20 to 45 °C or between 30 to 40 °C.
  • the experiments described herein demonstrate that an advantageous level of polydispersity or uniformity can be achieved, in combination with the use of modified metal complexes, if the conjugation is carried out at these temperatures which are elevated above room temperature (23 °C) but below temperatures which are likely to damage the biomolecule functionality or structure.
  • a method of forming a biomolecule conjugate comprising two or more peptide, polypeptide or protein biomolecules directly conjugated, one to the other, and interconnected through non-covalent bonds by an oligomeric chromium metal coordination complex, the method including the steps of:
  • the two or more peptide, polypeptide or protein biomolecules may be populations of such species which may be the same or different.
  • the biomolecule conjugate is a biomolecule conjugate network.
  • the modified oligomeric chromium metal coordination complex is an oligomeric chromium metal coordination complex comprising a degree of carboxylate or phosphate capping agents at the time of contacting the biomolecules.
  • the liquid formulation comprising the population of two or more peptide, polypeptide or protein biomolecules is contacted with the modified oligomeric chromium metal coordination complex at a temperature of greater than 15 °C and less than 45 °C, or greater than 20 °C and less than 45 °C or greater than 25 °C and less than 42 °C or greater than 30 °C and less than 40 °C, or greater than 33 °C and less than 45 °C or greater than 33 °C and less than 42 °C or greater than 33 °C and less than 40 °C, or greater than 35 °C and less than 45 °C or greater than 35 °C and less than 42 °C or greater than 35 °C and less than 40 °C.
  • a functional substrate comprising at least one biomolecule conjugate, the at least one biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non- covalent bonds by a metal coordination complex, and the at least one biomolecule conjugate associated with a substrate material.
  • biomolecule conjugate, two or more biomolecules, and metal coordination complex may be as described in any embodiment, or combination of embodiments, of the first aspect.
  • a method of forming a functional substrate comprising at least one biomolecule conjugate including the steps of:
  • biomolecule conjugate, two or more biomolecules, and metal coordination complex may be as described for any embodiment, or combination of embodiments, of the first aspect.
  • the substrate may be a polymeric substrate.
  • the substrate is a gel or resin.
  • Example 1 Preparation of metal coordination complex solutions.
  • oligomeric metal coordination complexes are described. Depending on the metal ion, salt, the base, the final pH and other ligands used, the metal coordination complex solutions exhibit different binding properties.
  • chromium perchlorate hexahydrate 45.9 g was dissolved into 480 mL of purified water and mixed thoroughly until all solid dissolved.
  • 8 mL of ethylene diamine (EDA) solution was added to 490 mL of purified water. The solutions were combined by the dropwise addition of the EDA solution into the chromium salt solution and stirred overnight at room temperature, and then left to equilibrate to a pH of approximately 4.5.
  • chromium perchlorate and ethylenediamine solution can be used to generate solutions having a different pH such as pH 3.0, 4.0, pH 5.0 or some other pH.
  • chromium perchlorate hexahydrate (103.5 g) was dissolved into 1000 mL of purified water and mixed thoroughly until all solid dissolved. 8 mL of ethylene diamine solution was added to 1000 mL of purified water. The solutions were combined by the dropwise addition of the EDA solution into the chromium salt solution, and stirred overnight at room temperature, and then left to equilibrate to the desired pH.
  • BSA Bovine Serum Albumin
  • Table 1 shows the trends with metal complex to oxalic acid ratio of 1 :4 (Solution 5B) and Table 2 shows the trends with metal complex to succinic acid ratio of 1 :4 (Solution 6C).
  • the rate of BSA cluster formation is different and changes with not only the capping agent but also the ratio between the protein and the modified oligomeric metal complex.
  • Table 1- the zeta size in nanometres of the Bovine Serum Albumin (BSA) clusters formed using oxalic acid capped (Solution 5B) at different ratios to BSA.
  • BSA Bovine Serum Albumin
  • Table 2- the zeta size in nanometres of the Bovine Serum Albumin (BSA) clusters formed using succinic acid capped (Solution 6C) at different ratios to BSA.
  • BSA Bovine Serum Albumin
  • the capping groups allow fine control over the rate of protein cluster formation and its final size. At some set concentration of oligomeric metal complex, a lower excess of suitable capping groups increases the availability of ligands on the protein that can potentially coordinate with the metal complex. The trend would be towards more intramolecular interactions within a protein which can lead to functional damage. At the extreme, it results in insoluble protein aggregates. Alternatively, larger excess of capping groups will restrict coordination only to the most reactive of ligands on the protein. In this case, the trend is towards more intermolecular cross-linking between proteins.
  • a conventional lateral flow half strip test was used to exemplify the present invention.
  • An anti-IgM test line and a biotinylated antibody control lines were stripped onto nitrocellulose membranes. If an IgM antibody was conjugated to a biotinylated antibody, such a protein cluster would be captured on the test line and can be reported using Streptavidin-RPE (Code:016-110-084, Jackson Immunoresearch) with a fluorescence and absorbance reader (Axxin). If the lgM:biotinylated Ab cluster was not formed, the IgM captured on the test line would not have any biotinylated Ab to report with Streptavidin-RPE. Two different conjugates, a., an IgM: biotinylated anti-HCG Ab and b., lgM:biotinylated goat anti-rat Ab complexes were formed using Solution 4.
  • Two IgM-Biotinylated IgG complexes were formed using modified oligomeric metal complex, Solution 4.
  • one complex was 0.2 mg/ml of Human IgM (No. 18260, Sigma Aldrich) mixed with 0.2mg/ml of Biotinylated Goat anti-rat IgG (No. 112-065-003, Jackson ImmunoReseach), and cross-linked with 2mM Solution 4.
  • the other complex was a 0.2 mg/ml of Human IgM mixed with 0.2mg/ml of Biotinylated anti- HCG (Cat#2H8B, HyTest), cross-linked with 2mM Solution 4.
  • the antibody cross-linked with Solution 4 was compared to the antibody mixture which was not cross-linked with Solution 4 using a half strip model.
  • IgM cross-linked with biotinylated IgG was reported on the test line when streptavidin-RPE was added.
  • the mixture of IgM and biotinylated IgG without Solution 4 would have captured IgM but would not have been observable without the cross-linked biotinylated IgG.
  • These cross-linked proteins wicked easily through the porous membrane confirming that insoluble aggregates were not forming but the flow rate was slower when compared to non-cross-linked mixtures.
  • a suspension of Europium-chelate latex particles (Merck ref# F1-Eu-030) was sonicated and then the particles separated from the supernatant by centrifugation at 12000 g for 10 minutes. After removing the supernatant, the particles were redispersed in an equal volume of Solution 1. After constant mixing on a rotary mixer for 3 hours, the particles were then separated from Solution 1 by centrifugation and washed twice with DI water. Particles were then checked for monodispersity and size using laser diffraction using a zetasizer (Malvern: Model nanoseries Z). Activation was demonstrated by a change in charge from negative to positive. Their concentration was evaluated against a known standard using the fluorescent readout of a spectrophotometer (Tecan model Infinite M200 Pro). The final concentration was adjusted to 10mg/mL.
  • Stock COVID-19 mAb (Cat#40143-MM08, Sino Biological) was diluted to 600pg/mL in MES buffer.
  • the activated europium particle suspension was mixed with an equal volume of 600pg/mL COVID-19 mAb was sonicated and vortexed to fully disperse the particles.
  • the conjugate particle was left on a tube rotator for 1 hour and then 25pL of blocking buffer (10% BSA in MES buffer, Bovine Serum Albumin, A7030, Sigma) was added. After fully dispersing, the blocked conjugate particle was left for another hour on a tube rotator.
  • the suspension was again centrifuged to remove supernatant and resuspended in an equal volume of 50mM TRIS buffer, pH8.0. After repeating this wash step, the conjugated particle was left in TRIS buffer at a concentration of 10mg/mL.
  • Example 5 Binding Antibody Clusters to Eu Particles (Cluster Conjugates).
  • Stock Solution 4 (oxalic modified oligomeric metal coordination complexes) was diluted to 2mM with DI water and stock COVID-19 mAb was diluted to 1.2mg/mL in 25mM MES buffer, pH6.0.
  • Antibody Cluster binding to activated Europium Particle [00174] The Antibody Cluster formed above conjugated to unmodified metal coordination complex-activated Europium particles in the manner as described in Example 3.
  • a lateral flow half test strip was used to compare any differences between the Control (Example 4) and antibody cluster (Example 5) conjugates.
  • Goat anti Mouse IgG was diluted to 0.2 mg/mL in carbonate buffer (pH8,5).
  • COVID-19 mAb capture was diluted to 1 mg/mL in carbonate buffer and then mixed with 1 L of 100mg/mL BSA to give a BSA concentration of 1 mg/mL BSA.
  • the COVID-19 mAb and GAM solutions were striped onto nitrocellulose membrane on a plastic support (HF090 card HF090MC100, Millipore) using the Linomat V (CAMAG). After striping, the ligand membrane was dried for 2 hours at 37°C in a fan forced incubator. The membrane was then stored in a sealed foil pouch with desiccant until use.
  • the COVID-19 antigen was used at 5 different concentrations; 25, 50, 100, 200 and 400 pg/ml, and Blank was also included.
  • the stock 10mg/mL of conjugate particles were diluted to 2pg/mL in TRIS buffer, pH 8.0 with 1% TWEEN + 0.25% BSA. 20pL was used for each strip.
  • Antigen capture was detected using a fluorescent reader (Axxin). As shown in Table 4 and Figure 8, the antibody cluster conjugates, formed with Solution 4, had different outcomes indicating that the functional activity of antibodies in the clusters were different.
  • Example 9 Comparison of Antibody Cluster vs Control (IV): The Effect of different Ligand Concentrations to Metal Coordination Complex.
  • a lateral flow half test strip was used to compare any differences in outcome with Solution 5A (in comparison to Solution 4) in the formation of antibody clusters.
  • conjugates Control and Solution 5A mediated antibody clusters
  • 50pg Ab per mg of Eu particles on a test line formed with 1 mg/ml SARS-CoV Ab + 1mg/ml BSA and as well, 150
  • three different Solution 5 concentrations, 0.125mM, 0.0625mM and 0.03125mM were compared.
  • Example 10 Binding Antibody Clusters to Gold Nanoparticles.
  • antibody clusters formed from, A., 10pL of 1mM Solution 4 with 10pL of 1200pg/ml COVID-19 mAb, and B., 10pL of 0.5mM Solution 5A with 10pL of 1200pg/ml COVID-19 mAb, mixed for 30 mins and then diluted to a final concentration of 3.2pg/ml. These two clusters were compared to the Control (mAb at 3.2pg/ml without any metal complex).
  • Gold colloids (1mL at OD1) were added to 3 tubes and centrifuged to form pellets. After removing 500 pL of supernatant, the pellet was vortexed to resuspend and disperse the colloids. After adding a 500 pL of mAb solution (clusters and Control), the tubes were vortexed to fully disperse the colloids and left on a tube rotator at 25rpm for 1 hr at room temperature. 10%BSA (50pL) blocking buffer was then added and the tubes vortexed and left on a rotator for another 1 hr. The tubes were placed in the centrifuge and the samples were spun down to remove supernatant. The pellet was resuspended in 1000 pL 2mM Boric acid, 0.05% sodium azide buffer, pH 9.0. As before, the size and zeta potential were measured using a Zeta Sizer and shown in Table 5.

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Abstract

L'invention concerne de manière générale un procédé de bioconjugaison dans lequel deux biomolécules ou plus sont conjuguées par l'intermédiaire de complexes de coordination métalliques. Plus particulièrement, l'invention concerne de manière générale l'utilisation de certains complexes de coordination métalliques pour conjuguer directement deux biomolécules ou plus, y compris des peptides, des polypeptides, des protéines et analogues.
PCT/AU2022/051212 2021-10-08 2022-10-07 Conjugaison de biomolécules WO2023056530A1 (fr)

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