Method for determining the presence of an analyte in a sample

Abstract

A method for measuring the degree of aggregation of a protein or polypeptide in a liquid sample, the method comprising the steps of mixing an aggregated protein or polypeptide in the liquid sample with a predetermined amount of a fluorophore molecule; and measuring the fluorescence polarization value of the mixture to determine the degree of aggregation; wherein the fluorophore molecule is a diazaoxatriangulenium of formula (I) or a derivative thereof.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to determining the presence of an analyte in a sample using fluorescence polarization (FP). In particular, the present invention relates to detecting non-monomer formation in a sample using fluorescent dyes and FP. [Background technology]

[0002] 2. Background of the Invention Aggregation of therapeutic proteins during production poses a significant risk to the success of the product, due to the formation of high molecular weight species (HMWS), i.e. non-monomeric IgG molecules.

[0003] Aggregates are known to potentially affect the safety of therapeutic drugs and are therefore considered a "critical quality attribute (CQA)" during manufacturing. Aggregation can occur at many steps during the manufacturing process, from cell culture to purification to drug product. The ability to measure aggregates quickly and economically is therefore important.

[0004] The classical gold standard method for measuring aggregates is by high performance liquid chromatography (HPLC) size exclusion chromatography (SEC), but despite its accuracy, this has low throughput and requires highly trained personnel.

[0005] There are several dye-based methods to measure levels of aggregation, such as Sypro Orange, Thioflavin T, and the commercially available Proteostat® (Enzo life sciences, Exeter, UK), which allow high-throughput quantification of aggregates by measuring fluorescence, but these are less accurate when measuring low percentages of naturally occurring aggregates.

[0006] A study published by Sheun Oshinbolu et al. (Journal of Chemical Technology and Biotechnology. Vol. 93(3), pp. 909-917 (2018)) describes the measurement of protein aggregation using aggregation binding fluorophores. However, this work was performed with non-native aggregates generated by thermal denaturation. These dyes perform poorly with naturally occurring aggregates. Furthermore, methods based on commercially available fluorescent dyes are less accurate at low levels of aggregation with both natural and non-native aggregates. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sheun Oshinbolu, et al., Journal of Chemical Technology and Biotechnology, vol. 93(3), 909-917 (2018) [Non-Patent Document 2] Haug-land, MOLECULAR PROBES HANDBOOK, supra, (2002) [Non-Patent Document 3] JM Stewart and JD Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984) [Non-Patent Document 4] M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984) [Non-Patent Document 5] Dinon et al., J. Mol.Recognit.2011 Nov-Dec; 24(6) Summary of the Invention [Problem to be solved by the invention]

[0008] SUMMARY OF THE PRESENT EMBODIMENT An object of the present invention is to overcome at least one of the above mentioned problems. [Means for solving the problem]

[0009] The invention will be more clearly understood from the following description of embodiments thereof, given by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows that high molecular weight species (HMWS) of IgG (non-monomeric) were spiked into monomeric IgG at various ratios in a total volume of 120 ul of medium (CD-CHO, ThermoFisher, UK). 0.1 ug of N'-propyl, N-propyl derivative of diazaoxatriangulenium (N'PNP-DAOTA) was added and incubated for 5 minutes in the dark before measuring the polarization signal. [Diagram 2] Figure 2 shows that high molecular weight species (HMWS) of IgG (non-monomeric) were spiked into the culture medium at various ratios to monomeric IgG, highlighting the relationship between aggregation and the assay signal of the competitive Proteostat Enzo dye. [Diagram 3] FIG. 3 compares various derivatives of diazaoxatriangulenium (DAOTA) and their binding properties as indicated by their polarized signals. [Figure 4] FIG. 4 shows a comparison of the aggregation of different derivatives of azadioxatriangulenium (ADOTA) with monomeric IgG by polarization signals. [Diagram 5]Figure 5 shows performance testing with commercially available aggregation standards: (A) predicted % aggregation for DAOTA vs. known % aggregation, and (B) predicted % aggregation for the Enzo Proteostat® assay vs. known % aggregation. As clearly shown in Figure 5, when the Enzo Proteostat® assay measures non-native aggregates in solution, (A) DAOTA used in the claimed invention outperformed (B), showing an R2 of 0.99 vs. 0.93, respectively. [Figure 6] FIG. 6 is a pair of graphs showing (A) the predicted % of natural non-monomer DAOTA vs. known % of non-monomer (using the method of the claimed invention) and (B) the predicted % of non-monomer vs. known % of non-monomer by the Enzo Proteostat® assay. Quite clearly, DAOTA can measure native aggregates in solution (see (A)). FIG. 6(A) shows the performance of DAOTA to quantify the % of dimer from monomeric samples spiked with various % of dimer (characterized and separated by HPLC-SEC). The correlation between DAOTA and the known % of dimer is excellent with R2=0.99. In contrast, the Enzo Proteostat® assay (FIG. 6(B)) is unable to distinguish dimer from monomer or native aggregates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Applicants have discovered that derivatives of the fluorophore molecule diazaoxatriangulenium (DAOTA - Formulas (I) and (III)), such as the N'-propyl, N-propyl derivative of diazaoxatriangulenium (N'PNP-DAOTA; Formula (II)), bind to non-monomeric IgG in cell culture medium, and that this binding can be measured with high sensitivity using fluorescence polarization (FP). Thus, this method can be used to quantitate levels of non-monomeric (or other analytes) in cell culture supernatants.

[0012] [ka] Formula (I)

[0013] where R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, C4-C8 cycloalkyl, C1-C6 aminoalkyl, C1-C6 alkylenesulfonate, C1-C 18 Alkyl, C1-C 22 Acryl, methylenesulfonate, ethylenesulfonate, C1-C6 alkylsulfonyl, trifluoromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, sulfonyl, sulfonyloxy, thioacyl, thiol, thiocarbonyl, C1-C6 alkylthio, heteroaryl, cycloalkyl, phenyl, hydroxyphenyl The substituents are independently selected from: aryl, aminophenyl, amino-C1-C6-alkyl, heterocyclyl, polyethylene glycol, carboxylic acid, alkyl halide, acrylamide, activated ester of carboxylic acid, hydroxy, aldehyde, sulfonate, amine, antigen, anhydride, aniline, aryl halide, azide, aziridine, boronate, carbodiimide, diazoalkane, epoxide, glycol, haloacetamide, halotriazine, hydrazine, hydroxylamine, imidoester, isocyanate, isothiocyanate, ketone, maleimide, phosphoramidite, sulfonyl halide, thiol group, butyric acid, butanoic acid.

[0014] [ka] Formula (II)

[0015] According to the appended claims, there is provided a method of measuring the degree of agglutination binding using fluorescence polarization by adding the above-mentioned fluorophores to an antibody sample of interest, and the quantitative degree of agglutination of the sample can be determined by interpolation from a standard curve of known degrees of agglutination.

[0016] There is provided a method for measuring the degree of aggregation of a protein or polypeptide in a liquid sample, the method comprising: adding a predetermined amount of a fluorophore molecule to a liquid sample containing the aggregated protein or polypeptide; measuring the fluorescence polarization value of the liquid sample; and comparing the measured fluorescence polarization value of the liquid sample with a reference fluorescence polarization value to determine the degree of aggregation of proteins or polypeptides in the liquid sample; and the fluorophore molecule is a diazaoxatriangulenium of formula (I) or a derivative thereof.

[0017] [ka] Formula (I)

[0018] where R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, C4-C8 cycloalkyl, C1-C6 aminoalkyl, C1-C6 alkylenesulfonate, C1-C 18 Alkyl, C1-C 22Acryl, methylenesulfonate, ethylenesulfonate, C1-C6 alkylsulfonyl, trifluoromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, sulfonyl, sulfonyloxy, thioacyl, thiol, thiocarbonyl, C1-C6 alkylthio, heteroaryl, cycloalkyl, phenyl, hydroxyphenyl The substituents are independently selected from: aryl, aminophenyl, amino-C1-C6-alkyl, heterocyclyl, polyethylene glycol, carboxylic acid, alkyl halide, acrylamide, activated ester of carboxylic acid, hydroxy, aldehyde, sulfonate, amine, antigen, anhydride, aniline, aryl halide, azide, aziridine, boronate, carbodiimide, diazoalkane, epoxide, glycol, haloacetamide, halotriazine, hydrazine, hydroxylamine, imidoester, isocyanate, isothiocyanate, ketone, maleimide, phosphoramidite, sulfonyl halide, thiol group, butyric acid, butanoic acid.

[0019] In some embodiments, the derivative is the following diazaoxatriangulenium:

[0020] [ka] Formula (III).

[0021] In one embodiment, the fluorophore molecule is selected from N-propyl, N'-propyl derivatives of diazaoxatriangulenium. Preferably, the fluorophore molecule is an N-propyl, N'-propyl derivative of diazaoxatriangulenium of formula (II).

[0022] [ka] Formula (II).

[0023] In one embodiment, the protein or polypeptide is selected from an antibody, an antibody fragment, an enzyme, an amyloid. Preferably, the antibody is selected from IgG, IgM, IgE, IgD and IgA.

[0024] In one aspect, a method for determining the presence of an aggregated analyte in a sample is provided, the method comprising: contacting the sample with a compound of formula (I) or a derivative thereof

[0025] [ka] Formula (I)

[0026] (Wherein, R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, C4-C8 cycloalkyl, C1-C6 aminoalkyl, C1-C6 alkylenesulfonate, C1-C 18 Alkyl, C1-C 22Acryl, methylenesulfonate, ethylenesulfonate, C1-C6 alkylsulfonyl, trifluoromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, sulfonyl, sulfonyloxy, thioacyl, thiol, thiocarbonyl, C1-C6 alkylthio, heteroaryl, cycloalkyl, phenyl, hydroxyphenyl , aminophenyl, amino-C1-C6-alkyl, heterocyclyl, polyethylene glycol, carboxylic acid, alkyl halide, acrylamide, activated ester of carboxylic acid, hydroxy, aldehyde, sulfonate, amine, antigen, anhydride, aniline, aryl halide, azide, aziridine, boronate, carbodiimide, diazoalkane, epoxide, glycol, haloacetamide, halotriazine, hydrazine, hydroxylamine, imide ester, isocyanate, isothiocyanate, ketone, maleimide, phosphoramidite, sulfonyl halide, thiol group, butyric acid, butanoic acid); irradiating the sample; measuring the fluorescence polarization of the illuminated sample; comparing the fluorescence polarization value of the illuminated sample with a reference fluorescence polarization value to determine the degree of aggregation of the analyte in the sample. Includes.

[0027] In one embodiment, the derivative is a diazaoxatriangulenium of formula (III).

[0028] [ka] Formula (III).

[0029] In one embodiment, the compound is selected from N-propyl, N'-propyl derivatives of diazaoxatriangulenium. Preferably, the fluorophore molecule is an N-propyl, N'-propyl derivative of diazaoxatriangulenium of formula (II).

[0030] [ka] Formula (II).

[0031] In one embodiment, the analyte is selected from an antibody, an antibody fragment, an enzyme, and an amyloid.

[0032] In one embodiment, the derivative of formula (I) can be conjugated to a carrier molecule, which is preferably selected from the group consisting of amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acid polymers, drugs, hormones, lipids, lipid assemblies, synthetic polymers, polymeric microparticles, biological cells, or viruses. Other carrier molecules are also suitable.

[0033] In one embodiment, the derivative of formula (I) can be conjugated to solid support by well-established methods known to those skilled in the art.Useful solid supports include solid and semi-solid matrices such as sol-gel, aerogel and hydrogel, resin, beads, biochip (including thin-film coated biochip), microfluidic chip, silicon chip, multi-well plate (also called microtiter plate or microplate), membrane, conductive and non-conductive metal, glass (including microscope slide) and magnetic support. More specific examples of useful solid supports include silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides such as sepharose, poly(acrylates), polystyrene, poly(acrylamide), polyols, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinyl chloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, starch, and the like.

[0034] Conjugates of carrier molecules, such as drugs, peptides, toxins, nucleotides, phospholipids, proteins and other organic molecules, comprising the derivatives of formula (I) of the present invention are generally prepared by means well known in the art (Haug-land, MOLECULAR PROBES HANDBOOK, supra, (2002)). Preferably, conjugation to form a covalent bond consists of simply mixing the reactive derivatives of the present invention in a suitable solvent in which both the reactive derivative and the substance to be conjugated are soluble. The reaction preferably proceeds spontaneously at room temperature or below without the addition of reagents. For photoactivated reactive derivatives, the conjugation is facilitated by irradiating the reaction mixture with light to activate the reactive derivative. Chemical modification of water-insoluble substances to prepare the desired derivative-conjugates is preferably carried out in aprotic solvents such as dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform. Similar modification of water-soluble substances is easily achieved by using readily available reactive derivatives to render the water-soluble substances more soluble in organic solvents.

[0035] definition In this specification, the terms "protein" and "polypeptide" should be understood to mean large biomolecules and macromolecules consisting of a long chain of one or more amino acid residues. Proteins differ from each other mainly in the sequence of amino acids. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides containing less than 20-30 residues are rarely considered proteins and are generally called peptides, or often oligopeptides. Individual amino acid residues are linked to adjacent amino acid residues by peptide bonds. Examples of proteins and / or polypeptides include antibodies, antibody fragments, enzymes, Fc fusion proteins, anticoagulants (coumarins (vitamin K antagonists such as warfarin), heparin and derivative substances (unfractionated heparin (UFH), low molecular weight heparin (LMWH), ultra-low molecular weight heparin (ULMWH)), synthetic pentasaccharide inhibitors of factor Xa (fondaparinux, idraparinux, idrabiotaparinux), direct acting oral anticoagulants (DOACs; dabigatran, rivaroxaban, Antithrombin), Direct Acting Factor Xa Inhibitors (such as rivaroxaban, apixaban and edoxaban), Blood Factors (I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII), Bone Morphogenetic Proteins, Artificial Protein Scaffolds, Growth Factors, Hormones, Interferons, Interleukins, Thrombolytics, Amyloid (such as amyloid beta), Tau, α-synuclein, TAR DNA-binding protein 43, C9orf72-related protein, Islet Amyloid Polypeptide and Transthyretin.

[0036] As used herein, the term "Fc fusion protein" (also known as Fc chimeric fusion protein, Fc-Ig, Ig-based chimeric fusion protein and Fc-tag protein) should be understood to mean the Fc domain of IgG genetically linked to a peptide or protein of interest.

[0037] Protein and polypeptide for use in the present invention (including its variant and fragment) can be produced in whole or in part by chemical synthesis or by expression from nucleic acid.Protein and peptide for use in the present invention and protein and peptide for use in the present invention can be easily prepared according to the well-established standard liquid or preferably solid phase peptide synthesis method known in the art (see, for example, JM Stewart and JD Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984) (Non-Patent Document 3); M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984) (Non-Patent Document 4)).

[0038] As used herein, the term "variant" should be understood to mean a protein or peptide obtained by introducing one or more substitutions, additions, and / or deletions into the sequence of a wild-type protein or peptide. The term "variant" is also intended to include mimetics (i.e., peptidomimetics) and chemical derivatives of generic antibody binding proteins, i.e., one or more residues of the generic antibody binding protein are chemically derivatized by reaction of a functional side group. Also included in the term variant are generic antibody binding proteins in which naturally occurring amino acid residues are replaced by amino acid analogues. Examples of variants of generic antibody binding proteins are described in Dinon et al. (J. Mol.Recognit.2011 Nov-Dec; 24(6)).

[0039] As used herein, the term "fluorescent compound" or "fluorophore molecule" should be understood to mean a compound whose presence can be detected by fluorescence when illuminated with light of the appropriate wavelength. A detectable label can be a fluorescent dye molecule or a fluorophore molecule.

[0040] As used herein, the term "antibody" should be understood to mean an immunoglobulin, such as an IgG, IgA, IgE or IgM immunoglobulin, in humanized or non-humanized, monoclonal or polyclonal form, or a fragment thereof. In one embodiment, the antibody is an IgG molecule, preferably a monoclonal IgG molecule. In one embodiment, the antibody is a human antibody.

[0041] As used herein, the term "amyloid" should be understood to mean protein aggregates characterized by extracellular proteinaceous fibrillar deposits exhibiting a β-sheet secondary structure and the ability to stain with certain dyes such as Congo Red, distinguishable by apple green birefringence under polarized light.) Amyloid is essentially any polypeptide that polymerizes to form cross-β structures, either in vivo or in vitro, inside or outside a cell.

[0042] As used herein, the term "enzyme" should be understood to mean a protein that acts as a biological catalyst.

[0043] The term "fluorescence polarization" should be understood to mean exciting a sample with plane polarized light at a wavelength corresponding to the excitation wavelength of a fluorochrome and detecting the light intensity emitted by the fluorochrome at the appropriate emission wavelength in two planes: one parallel to the excitation plane and one perpendicular to the excitation light plane. The excitation plane can be vertical or horizontal, and the emission light is detected in the vertical and horizontal planes. The degree to which the emission intensity is transferred from the excitation plane (i.e., the vertical plane) to the vertical plane (i.e., the horizontal plane), i.e., the change in polarization between the excitation and emission light, is a function of the degree of rotation of the fluorochrome.

[0044] In this specification, the term "triangulenium" should be understood to mean dyes that constitute a family of versatile chromophores with impressive light absorption and emission properties. Representative members of the trianguleniums are the aza / oxa-triangulenium dyes azadioxatriangulenium (ADOTA + ) and diazaoxatriangulenium (DAOTA + ) (see formula (III) below), which have extremely low non-radiative deactivation rates and low susceptibility to quenching by oxygen. These organic dyes have a fluorescence peak in the range of 550 nm to 600 nm, can exhibit an unusually long fluorescence lifetime of nearly 20 ns, and have high quantum yields. Aza / oxa-triangulenium dyes are highly stabilized carbenium ions and have a rigid, planar heterocyclic skeleton.

[0045] As used herein, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group. 16 -alkyl" and "C1-C 12 The term "-alkyl" denotes an alkyl group having 1 to 6 or 1 to 12 carbon atoms, respectively, unless otherwise specified. Suitable alkyl groups include linear or branched C1-C6-alkyl, which denotes an alkyl group having 1 to 6 carbon atoms, unless otherwise specified. Such suitable C1-C6-alkyl groups include, for example, methyl, ethyl, propyl, such as n-propyl and isopropyl, butyl, such as n-butyl, iso-butyl, sec-butyl and tert-butyl, pentyl, such as n-pentyl and hexyl (e.g. n-hexyl). Suitable alkyl groups include linear C1-C6-alkyl, such as n-butyl, iso-butyl, sec-butyl and tert-butyl, pentyl, such as n-pentyl and hexyl (e.g. n-hexyl). 12 -alkyl, which refers to a straight alkyl chain having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, unless otherwise specified.

[0046] As used herein, the term "alkenyl" refers to an aliphatic hydrocarbyl group having at least one double bond. The term "C2-C6-alkenyl" can be interpreted similarly to the term "alkyl" unless otherwise specified. Suitable alkenyl groups include, for example, ethenyl, propenyl, 1-butenyl, and 2-butenyl.

[0047] In this specification, the term "alkynyl" refers to an aliphatic hydrocarbyl group having at least one double bond. The term "C2-C6-alkynyl" can be interpreted similarly to the term "alkyl" unless otherwise specified. An alkenyl group has at least one triple bond. The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I), preferably F, Cl or Br, unless otherwise specified. The compounds of formula (I) and compounds of formula (III) may be substituted with one, two, three, four, five, six or more halogens, preferably Cl or Br, more preferably Cl.

[0048] As used herein, the term "C1-C6-alkoxy" refers to the group -O-C1-C6-alkyl, where C1-C6-alkyl is defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, f-butoxy, sec-butoxy, and n-pentoxy.

[0049] As used herein, "C1-C 12 The term "alkanoic acid" refers to C1-C 12 Refers to the alkylCOOH group.

[0050] As used herein, the term "acyl" refers to the groups HC(O)-, alkyl-C(O)-, alkenyl-C(O)-, alkynyl-C(O)-, cycloalkyl-C(O)-, cycloalkenyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)-, and heterocycle-C(O)-, where alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are as otherwise defined herein. Acyl includes the "acetyl" group CHC(O)-.

[0051] As used herein, the term "acylamino" refers to the groups -NRC(O)alkyl, -NRC(O)cycloalkyl, -NRC(O)cycloalkenyl, -NRC(O)alkenyl, -NRC(O)alkynyl, -NRC(O)aryl, NRC(O)heteroaryl, and -NRC(O)heterocycle, where R is hydrogen or alkyl, and alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are as otherwise defined herein.

[0052] As used herein, the term “acyloxy” refers to the groups alkyl-C(O)O—, alkenyl-C(O)O—, alkynyl-C(O)O—, aryl-C(O)O-cycloalkyl-C(O)O—, cycloalkenyl-C(O)O—, heteroaryl-C(O)O—, and heterocycle-C(O)O—, where alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are as otherwise defined herein.

[0053] As used herein, the term "aryl" refers to a monovalent aromatic carbocyclic group of 5 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, etc.), provided that the point of attachment is at an aromatic carbon atom. Preferred aryl groups include phenyl and naphthyl.

[0054] As used herein, the terms "carboxyl" or "carboxy" refer to --COOH.

[0055] As used herein, the term "carboxyl ester" or "carboxy ester" refers to the groups -C(O)O-alkyl, -C(O)O-alkenyl, -C(O)O-alkynyl, -C(O)O-aryl, -C(O)O-cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-heteroaryl, and -C(O)O-heterocycle, where alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are as defined herein.

[0056] As used herein, the term "(carboxyl ester)amino" refers to the groups -NR-C(O)O-alkyl, -NR-C(O)O-alkenyl, -NR-C(O)O-alkynyl, -NR-C(O)O-aryl, -NR-C(O)O-cycloalkyl, -NR-C(O)O-cycloalkenyl, -NR-C(O)O-heteroaryl, and -NR-C(O)O-heterocycle, where alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are as defined herein.

[0057] As used herein, the term "(carboxylester)oxy" refers to the groups -OC(O)O-alkyl, sOC(O)O-alkenyl, -OC(O)O-alkynyl, -OC(O)O-aryl, -OC(O)O-cycloalkyl, -OC(O)O-cycloalkenyl, -OC(O)O-heteroaryl, and -OC(O)O-heterocycle, where alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are as defined herein. "Cycloalkyl" refers to cyclic alkyl groups having from 3 to 10 carbon atoms having single or multiple cyclic rings, including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.

[0058] As used herein, the term "cycloalkenyl" refers to a non-aromatic cyclic alkyl group having from 3 to 10 carbon atoms having a single or multiple cyclic rings and having at least one >C=C< cyclic unsaturation, preferably having 1 to 2 sites of >C=C< cyclic unsaturation.

[0059] As used herein, the term "heteroaryl" refers to an aromatic group having 5-10 carbon atoms and 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl), which condensed rings may or may not be aromatic and / or may or may not contain heteroatoms, provided that the point of attachment is through an atom of the aromatic heteroaryl group. Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.

[0060] As used herein, the term "heterocycle" or "heterocyclic" or "heterocycloalkyl" or "heterocyclyl" refers to saturated or unsaturated groups having a single ring or multiple condensed rings, including fused bridge systems, having 1-10 carbon atoms and 1-4 heteroatoms selected from the group consisting of nitrogen, sulfur, or oxygen within the ring, where in a fused ring system, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In one embodiment, the nitrogen and / or sulfur atoms of the heterocycle group are optionally oxidized to provide N-oxide, sulfinyl, or sulfonyl moieties.

[0061] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, phenylpyridine ... These include aryl, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, and tetrahydrofuranyl.

[0062] As used herein, the term "5- or 6-membered heterocyclyl containing at least one nitrogen or sulfur" includes, but is not limited to, benzofuran, indole, pyrrolidine, pyrrole, thiolane, thiophene, imidazolidine, pyrazolidine, imidazole, pyrazole, oxazolidine, isoxazolidine, oxazole, isoxazole, thiazolidine, isothiazolidine, thiazole, isothiazole, dithiolane, triazole, furazan, oxadiazole, thiadiazole, dithiazole, tetrazole, piperidine, pyridine, thiane, thiopyran, piperazine, diazine, morpholine, oxazine, thiomorpholine, thiazine, dithiane, dithiin, triazine, or tetrazine.

[0063] As used herein, the term "sulfonate" refers to the group -S(O)3-, while the term "sulfonyl" refers to the divalent group -S(O)2-.

[0064] As used herein, the term "alkylsulfonyl" refers to the group -S(O)2-alkyl, where alkyl is as defined herein. Preferably, the alkyl group is a small group having less than 6 carbon atoms, and preferably the alkyl group is methyl or ethyl.

[0065] As used herein, the term "sulfonyloxy" refers to the groups -OSO2-alkyl, -OSO2-alkenyl, -OSO2-cycloalkyl, -OSO2-cycloalkenyl, -OSO2-aryl, -OSO2-heteroaryl, and -OSO2-heterocycle, where alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are as defined herein.

[0066] As used herein, the term "thioacyl" refers to the groups HC(S)-, alkyl-C(S)-, alkenyl-C(S)-, alkynyl-C(S)-, cycloalkyl-C(S)-, cycloalkenyl-C(S)-, aryl-C(S)-, heteroaryl-C(S)-, and heterocycle-C(S)-, where alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are as defined herein.

[0067] As used herein, the term "thiol" refers to the group --SH.

[0068] As used herein, the term "thiocarbonyl" refers to the divalent group -C(S)-, which is equivalent to -C(=S)-.

[0069] As used herein, the term "alkylthio" refers to the group --S-alkyl, where alkyl is as defined herein.

[0070] As used herein, the term "conjugated substance" refers to a biological or non-biological moiety that is attached or becomes attached to a compound of formula (I) of the present invention.

[0071] As used herein, the term "reference fluorescence polarization values" refers to a list, standard curve, or database of known fluorescence polarization values ​​measured when a fluorophore molecule or compound used in the present invention is mixed and bound to known concentrations of aggregated proteins, aggregated polypeptides, and aggregated analytes in a liquid sample. This standard curve or database of known fluorescence polarization values ​​is used as a reference point in the methods of the claimed invention to determine the concentration of aggregated proteins, polypeptides, or analytes in the liquid sample being tested.

[0072] In this specification, the term "database" should be understood to mean a standard curve of known fluorescence polarization values ​​for known % aggregation in a sample, insofar as calibration for measuring the degree of aggregation in a sample is concerned. The use of a standard curve (calibration curve or reference curve) is a common and standard operation when using an assay to determine the concentration or % aggregation of a protein of interest in an unknown sample. Thus, the term "database" should also be referred to as a standard curve or reference curve or calibration curve of known fluorescence polarization values ​​for known % aggregation in a sample. The database used in this application is determined as follows: To create a standard curve or calibration curve, a range of proteins of interest with known degrees of aggregation (e.g., the degree of aggregation has been previously quantified using size-exclusion high performance liquid chromatography (HPLC-SEC)) is prepared, the assay is performed, and the results are plotted as in FIG. 1. As shown in FIG. 1, the mP value for each degree of aggregation is measured. This is the operation of creating a "standard curve" or "calibration curve", a method well known to those skilled in the art of performing typology. Then, when a sample of unknown aggregation is measured, the mP value can be interpolated to the percentage of aggregation, for example, using a standard curve or calibration curve as provided in FIG. 1. The specific values ​​of the standard curve (e.g., percent aggregation and corresponding mP values ​​for the protein of interest) can be saved (computerically or manually) and reused as a reference, or the standard curve can be generated again with unknown samples in the same experiment.

[0073] Detailed Description of the Drawings Materials and Methods N'PNP-DAOTA (see formula (II)) was purchased from Ku-Dyes (Copenhagen, Denmark).

[0074] [ka] Formula (II)

[0075] Derivatives of DAOTA were generated by adding different R groups to the structure of formula (I).

[0076] [ka] Formula (I)

[0077] Derivatives of ADOTA were generated by adding different R groups to the structure of formula (IV).

[0078] [ka] Formula (IV)

[0079] Four different R groups were used in this study: polyethylene glycol (PEG), butyric acid, N'PNP, and C18 (a chain of 18 carbon atoms).

[0080] Non-monomeric IgG was sourced in-house and was obtained from Chinese hamster ovary (CHO) cell supernatant by size exclusion chromatography (this is a naturally occurring non-monomeric IgG).

[0081] FP readings were performed in black half-area NBS plates in a total volume of 120 ul (Corning, New York, USA) using a BMG Pherastar plate reader (BMG, Berlin, Germany).

[0082] Fluorescence was measured according to the Enzo Proteostat® assay instructions. Briefly, 60 ul of IgG sample, 38 ul of medium, and 2 ul of probe solution were added to each well. This was incubated at room temperature for 15 minutes before reading the fluorescence.

[0083] For the DAOTA FP assay, high molecular weight species (HMWS) of IgG (non-monomeric) were spiked at various ratios to monomeric IgG at a constant concentration of 200mg / L in a total volume of 120ul of culture medium (CD-CHO, ThermoFisher, UK). 0.1ug of N'-propyl, N-propyl derivative of diazaoxatriangulenium (N'PNP-DAOTA) was added and incubated for 5 minutes in the dark before measuring the polarization signal.

[0084] result As is evident from Figure 1, N'PNP-DAOTA can be used to accurately measure the level of non-monomeric IgG in cell culture media. Figure 2 shows a comparison with the Enzo Proteostat® assay, which is methodologically similar but relies on a fluorescent signal other than a fluorescent polarization signal.

[0085] Interestingly, when a very similar triangulenium molecule, N-aza-di-oxa-triangulenium butanoate (N-phenyl-ADOTA), was used instead of N'PNP-DAOTA, N-phenyl-ADOTA did not bind to IgG aggregates despite its similar structure.

[0086] To determine what effect the side groups attached to the fluorophore have, various side chains were added to the fluorophores of formula (I) and formula (IV). We considered whether the effect was limited to compounds with N'PNP side groups. The aggregate binding effect of the side groups on the DAOTA molecule was compared to the aggregate binding effect of the side groups on the ADOTA molecule (see formula (IV)). Figures 3 and 4 show that the aggregate binding is highly specific to the DAOTA molecule. All side chains tolerate IgG aggregate binding with DAOTA, albeit with changes in shift. Conversely, no IgG aggregate binding was observed with ADOTA derivatives, regardless of side chain modification. This is due to the lack of a clear relationship between the proportion of non-monomers and the FP(mP) signal.

[0087] Performance tests performed with commercially available aggregation standards demonstrated that the DAOTA (Figure 5(A)) outperforms the Enzo Proteostat® assay when following the protocol considered for the measurement of non-native aggregates in solution (Figure 5(B)), with an R of 0.99 vs. 0.93, respectively. 2 As shown by.

[0088] As shown in Figure 6, the DAOTA molecule can also measure native aggregates in solution. Figure 6(A) shows the performance of the DAOTA molecule to quantify the % dimer from monomer samples spiked with varying % dimer (characterized and separated by HPLC-SEC). The correlation between the DAOTA molecule and the known % dimer is much better, with R 2 = 0.99. In contrast, the Enzo Proteostat® assay (Figure 6(B)) had an R 2 The value of β-amino acid is low at 0.57, which does not allow us to distinguish between dimers and monomers or any native aggregates.

[0089] Discussion We have shown that N'PNP-DAOTA and various mutants of DAOTA bind to non-monomeric IgG in cell culture medium and that this binding can be measured with high sensitivity using FP. Therefore, this method can be used to quantify the levels of non-monomeric aggregates of proteins or polypeptides in cell culture supernatants.

[0090] R between the results shown in Figs. 2 Comparing the values, R achieved using the method of the claimed invention 2 The R value of 0.99 is the R obtained using the Enzo Proteostat® assay, the current commercially available gold standard assay. 2This is far superior to the value of 0.72. This increase in precision at the low end is also evident from Figure 5. These results clearly show an increase in precision at the low range of aggregation, and this effect is highly specific to DAOTA and DAOTA derivatives. This is a surprising and unexpected result, since no aggregation binding is observed, regardless of the R group used, even with ADOTA, a very similar molecule.

[0091] The increased accuracy of the claimed invention compared to the current gold standard fluorescent assay, Enzo Proteostat®, is even more pronounced when using naturally occurring IgG aggregates (see Figure 6). 2 The values ​​are 0.996 and 0.571, respectively. It is clear that quantification of naturally occurring aggregates is a more accurate representation of the usefulness of the assay, since the aggregates measured in this assay are naturally occurring, e.g., from cell culture supernatants. Of note, Enzo Proteostat® is claimed to be much superior to Thioflavin T (discussed in Oshinbolu et al. (Non-Patent Document 1)) from the company's website (https: / / www.enzolifesciences.com / ENZ-51023 / proteostat-protein-aggregation-assay / ).

[0092] The particular fluorophores used in the claimed invention are better than expected at discriminating between monomers and dimers (aggregates), especially when compared to known fluorophore assay products.

[0093] As used herein, the terms "comprise", "comprises", "consisting", "comprising" or any variation thereof and the terms "include", "includes", "included", "including" or any variation thereof are fully interchangeable and all are to be given the broadest possible interpretation and vice versa.

[0094] The invention is not limited to the embodiments described herein, which may vary both in construction and detail.

Claims

1. 1. A method for measuring the degree of aggregation of a protein or polypeptide in a liquid sample, comprising: The method comprises: adding a predetermined amount of fluorophore molecules to a liquid sample containing said aggregated proteins or polypeptides; measuring the degree of fluorescence polarization of the liquid sample; and comparing the measured fluorescence polarization value of the liquid sample with a reference fluorescence polarization value to determine the degree of aggregation of the protein or polypeptide in the liquid sample; Including, The method wherein said fluorophore molecule is a diazaoxatriangulenium of formula (I) or a dibasic salt thereof: 【Chemical 1】 Formula (I) (Wherein R is hydrogen, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, phenyl, C 4 -C 8 Cycloalkyl, C 1 -C 6 Aminoalkyl, C 1 -C 6 Alkylene sulfonates, C 1 -C 18 Alkyl, C 1 -C 22 Acrylic, methylene sulfonate, ethylene sulfonate, C 1 -C 6 Alkylsulfonyl, trifluoromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, sulfonyl, sulfonyloxy, thioacyl, thiol, thiocarbonyl, C 1 -C 6 -Alkylthio, heteroaryl, cycloalkyl, phenyl, hydroxyphenyl, aminophenyl, amino-C 1 -C 6 -independently selected from alkyl, heterocyclyl, polyethylene glycol, carboxylic acid, alkyl halide, acrylamide, activated ester of carboxylic acid, hydroxy, aldehyde, sulfonate, amine, antigen, anhydride, aniline, aryl halide, azide, aziridine, boronate, carbodiimide, diazoalkane, epoxide, glycol, haloacetamide, halotriazine, hydrazine, hydroxylamine, imidoester, isocyanate, isothiocyanate, ketone, maleimide, phosphoramidite, sulfonyl halide, thiol group, butyric acid, butanoic acid.

2. 2. The method of claim 1, wherein the derivative is a diazaoxatriangulenium: 【Chemistry 2】 Formula (III)

3. 2. The method of claim 1, wherein the fluorophore molecule is selected from N-propyl, N'-propyl derivatives of diazaoxatriangulenium.

4. 4. The method of claim 3, wherein the fluorophore molecule is an N-propyl, N'-propyl derivative of the diazaoxatriangulenium of formula (II). 【Chemistry 3】 Formula (II).

5. 2. The method of claim 1, wherein the protein or polypeptide is selected from an antibody, an antibody fragment, an enzyme, an amyloid, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an artificial protein scaffold, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic agent, tau, alpha-synuclein, TAR DNA binding protein 43, C9orf72-related protein, islet amyloid polypeptide, and transthyretin.

6. 6. The method of claim 5, wherein the antibody is selected from IgG, IgM, IgE, IgD, and IgA.

7. 1. A method for determining the presence of an aggregated analyte in a sample, the method comprising: contacting said sample with a compound of formula (I) or a derivative thereof 【Chemistry 4】 Formula (I) (Wherein R is hydrogen, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, phenyl, C 4 -C 8 Cycloalkyl, C 1 -C 6 Aminoalkyl, C 1 -C 6 Alkylene sulfonates, C 1 -C 18 Alkyl, C 1 -C 22 Acrylic, methylene sulfonate, ethylene sulfonate, C 1 -C 6 Alkylsulfonyl, trifluoromethyl, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, sulfonyl, sulfonyloxy, thioacyl, thiol, thiocarbonyl, C 1 -C 6 -Alkylthio, heteroaryl, cycloalkyl, phenyl, hydroxyphenyl, aminophenyl, amino-C 1 -C 6 -independently selected from alkyl, heterocyclyl, polyethylene glycol, carboxylic acid, alkyl halide, acrylamide, activated ester of carboxylic acid, hydroxy, aldehyde, sulfonate, amine, antigen, anhydride, aniline, aryl halide, azide, aziridine, boronate, carbodiimide, diazoalkane, epoxide, glycol, haloacetamide, halotriazine, hydrazine, hydroxylamine, imidoester, isocyanate, isothiocyanate, ketone, maleimide, phosphoramidite, sulfonyl halide, thiol group, butyric acid, butanoic acid. irradiating the sample; measuring the fluorescence polarization of the illuminated sample; and comparing the fluorescence polarization value of the illuminated sample with a reference fluorescence polarization value to determine the degree of aggregation of the analyte in the sample. A method comprising:

8. 8. The method of claim 7, wherein the derivative is a diazaoxatriangulenium of formula (III). 【Chemistry 5】 Formula (III).

9. 8. The method of claim 7, wherein the compound is an N-propyl, N'-propyl derivative of a diazaoxatriangulenium.

10. 10. The method of claim 9, wherein the compound is an N-propyl, N'-propyl derivative of the diazaoxatriangulenium of formula (II). 【Chemistry 6】 Formula (II)

11. 8. The method of claim 7, wherein the analyte is selected from an antibody, an antibody fragment, an enzyme, an amyloid, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an artificial protein scaffold, a growth factor, a hormone, an interferon, an interleukin, a thrombolytic agent, tau, alpha-synuclein, TAR DNA binding protein 43, C9orf72-related protein, islet amyloid polypeptide, and transthyretin.

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