METHOD FOR QUANTITATIVE DETERMINATION OF A THERAPEUTIC TNF-ALPHA INHIBITOR

Description

[0001] The invention lies in the field of in vitro diagnostics and relates to a method for the quantitative determination of therapeutic TNF-alpha inhibitors.

[0002] Tumor necrosis factor-alpha protein inhibitors (TNF-alpha, TNF-α, cachectin) are used as medications to treat inflammatory diseases such as Crohn's disease, ankylosing spondylitis, psoriasis, and especially rheumatoid arthritis. TNF-alpha inhibitors are TNF-alpha binding proteins, such as anti-TNF-alpha antibodies or fragments thereof, or genetically engineered fusion proteins that encompass the extracellular TNF-alpha binding domain of the human TNF receptor. All therapeutically effective TNF-alpha inhibitors bind to TNF-alpha protein and inactivate the TNF-alpha protein by preventing the binding of TNF-alpha protein to the TNF receptors.

[0003] Various TNF-alpha inhibitors are approved for the treatment of chronic inflammatory diseases, such as infliximab, etanercept, adalimumab, certolizumab pegol or golimumab and various biosimilars (biopharmaceutical copycat products).

[0004] The problem is that not all patients respond equally to TNF-alpha inhibitors. It is known that serum or plasma concentrations of TNF-alpha inhibitors correlate significantly with the clinical symptoms of the treated patients. The effectiveness of TNF-alpha inhibitor therapy generally correlates with the amount of therapeutic antibody detectable in the patient's serum or plasma shortly before the next drug administration, the so-called trough level.

[0005] Individual differences in pharmacokinetics and the presence of antibodies against the therapeutic TNF-alpha inhibitor (so-called anti-drug antibodies, ADAs) can influence the trough concentration and thus the efficacy of TNF-alpha inhibitor therapy. The lack of therapeutic success can have various causes, such as underdosing or the presence of anti-drug antibodies.

[0006] In order to optimize TNF-alpha inhibitor therapy, e.g. by changing the dosage or switching to another preparation, it is necessary to determine the serum or plasma concentration of the therapeutic TNF-alpha inhibitor in a treated patient.

[0007] Various methods are known for the quantitative determination of therapeutic TNF-alpha inhibitors in patient samples. Firstly, there are commercial ELISA tests for the determination of specific TNF-alpha inhibitors. These tests use monoclonal antibodies specific for the respective TNF-alpha inhibitor fixed on a microtiter plate (IDKmonitor®< tests for adalimumab, infliximab, and etanercept, Immundiagnostik AG, Bensheim, Germany, and a test for adalimumab according to WO-A1-2009 / 032128 A1) or in which TNF-alpha protein is fixed directly or indirectly on a microtiter plate (IDKmonitor®< test for golimumab, Immundiagnostik AG, Bensheim, Germany, and Level Adalimumab and Level Infliximab tests from Sanquin, Amsterdam, Netherlands). Finckh, A, et al.(Joint Bone Spine 77 (2010) 313-318) disclose an ELISA test for the determination of infliximab in which TNF-alpha protein is fixed on the microtiter plate using an anti-TNF-alpha antibody.

[0008] WO-A1-2016 / 110595 and WO-A1-2018 / 007327 disclose particle-enhanced turbidimetric methods for the quantitative determination of therapeutic TNF-alpha inhibitors in patient samples. In these methods, polystyrene particles coated with TNF-alpha protein are mixed with the sample, and the agglutination in the reaction mixture is measured. The advantage is that this is a universal test setup with which any therapeutic TNF-alpha inhibitor can be detected. For precise quantification of a specific TNF-alpha inhibitor, only a calibration curve generated with the same specific TNF-alpha inhibitor is required. However, the disadvantage is that it requires the additional use of a nonspecific, TNF-alpha inhibitor-crosslinking ligand from the group of anti-Ig antibodies (e.g.Fc- or Fab-specific antibodies), protein G, protein A, protein H, protein L and protein A / G fusion protein are required to achieve sufficient sensitivity of the test so that all therapeutic TNF-alpha inhibitors, in particular etanercept (brand name Enbrel, Pfizer), can be detected.

[0009] The object underlying the present invention was therefore to provide a universal method for the quantitative determination of therapeutic TNF-alpha inhibitors in patient samples that is sufficiently sensitive to detect all therapeutic TNF-alpha inhibitors.

[0010] With regard to the known particle-enhanced methods, the object underlying the present invention was in particular to provide a universal, particle-enhanced method for the quantitative determination of therapeutic TNF-alpha inhibitors in patient samples, which is sufficiently sensitive to be able to detect all therapeutic TNF-alpha inhibitors without the need for the use of a non-specific, TNF-alpha inhibitor-crosslinking ligand.

[0011] It has been found that by contacting the sample with isolated, free TNF-alpha protein and subsequently adding a TNF-alpha binding partner, a sufficiently sensitive method for the quantitative determination of therapeutic TNF-alpha inhibitors is achieved, with which all therapeutic TNF-alpha inhibitors can be detected. The therapeutic TNF-alpha inhibitor contained in the sample binds to the added TNF-alpha protein and thus prevents the binding of the subsequently added TNF-alpha binding partner. The more of a therapeutic TNF-alpha inhibitor present in a sample, the more strongly it inhibits the formation of a complex between TNF-alpha protein and the added TNF-alpha binding partner. This is essentially a competitive test method.

[0012] With regard to particle-enhanced methods, it has been found in particular that by contacting the sample with isolated, free TNF-alpha protein and subsequently adding a particulate solid phase coated with a TNF-alpha binding partner, a sufficiently sensitive method for the quantitative determination of therapeutic TNF-alpha inhibitors is achieved, with which all therapeutic TNF-alpha inhibitors can be detected without the need for a nonspecific, TNF-alpha inhibitor-crosslinking ligand. The therapeutic TNF-alpha inhibitor contained in the sample binds to the added TNF-alpha protein and thus prevents the binding of the subsequently added solid-phase-coupled TNF-alpha binding partner. The more of a therapeutic TNF-alpha inhibitor present in a sample, the more strongly it inhibits the agglutination reaction.

[0013] The present invention therefore relates to a method for the quantitative determination of a therapeutic TNF-alpha inhibitor in a sample as described in claims 1 to 6. The method comprises, among other steps: a) Providing a reaction mixture by contacting the sample i. with isolated, free TNF-alpha protein and then ii. with a first TNF-alpha binding partner; b) Determining the amount of a complex of TNF-alpha protein and the first TNF-alpha binding partner formed in the reaction mixture; and then c) Determining the amount of the therapeutic TNF-alpha inhibitor in the sample by comparing the thus determined amount of the complex of TNF-alpha protein and the first TNF-alpha binding partner in the reaction mixture with amounts of a complex of TNF-alpha protein and the first TNF-alpha binding partner in reaction mixtures containing samples with known concentrations of a TNF-alpha binding partner.

[0014] The sample may be a body fluid sample, preferably a human body fluid sample, such as whole blood, plasma, serum, or urine. Typically, the body fluid sample comes from a patient receiving a therapeutic TNF-alpha inhibitor.

[0015] The method is suitable for the quantitative determination of therapeutic TNF-alpha inhibitors that bind to TNF-alpha protein, such as anti-TNF-alpha antibodies or fragments thereof, or genetically engineered fusion proteins that encompass the extracellular TNF-alpha binding domain of the human TNF receptor. All therapeutically effective TNF-alpha inhibitors bind to TNF-alpha protein and inactivate the TNF-alpha protein by preventing the binding of TNF-alpha protein to the TNF receptors. The method is particularly suitable for the quantitative determination of therapeutic TNF-alpha inhibitors from the group of infliximab, etanercept, adalimumab, certolizumab pegol, and golimumab, as well as their biosimilars.

[0016] The isolated, free TNF-alpha protein can be recombinantly or synthetically produced TNF-alpha protein, or a native TNF-alpha protein, meaning it can originate from natural sources, e.g., TNF-alpha protein purified from human blood. Known prokaryotic or eukaryotic expression systems are suitable for the production of recombinant TNF-alpha protein, such as expression in bacteria (e.g., E. coli), in yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris), in plant, animal, or human cell cultures (e.g., HEK cells). Known techniques for in vitro protein synthesis, such as solid-phase synthesis (e.g., Merrifield synthesis), are suitable for the production of synthetic TNF-alpha protein.

[0017] The TNF-alpha protein is preferably human TNF-alpha protein. The term "TNF-alpha protein" encompasses not only the complete TNF-alpha protein, but also fragments of the complete TNF-alpha protein to which therapeutic TNF-alpha inhibitors can bind. The term "TNF-alpha protein" further encompasses not only the wild-type TNF-alpha protein or fragments thereof, but also TNF-alpha proteins with one or more amino acid substitutions to which therapeutic TNF-alpha inhibitors can bind. The TNF-alpha protein or a TNF-alpha protein fragment can be fused at the N-terminus to a heterologous signal sequence, i.e., to a polypeptide that is not normally present in the human TNF-alpha protein, but which positively influences the expression and / or secretion of the recombinantly expressed TNF-alpha protein in the chosen expression system. Furthermore, the TNF-alpha protein ora TNF-alpha protein fragment can be fused at the C-terminus to one or more affinity tags that enable the binding of, for example, the recombinantly expressed protein to an affinity carrier, thereby enabling, for example, the purification of recombinantly expressed TNF-alpha protein. Small affinity tags with a length of no more than 12 amino acids are preferred. Affinity tags from the group consisting of His-tag, Flag-tag, Arg-tag, c-Myc-tag, and Strep-tag are particularly preferred. Suitable affinity carriers that bind to an affinity tag with high affinity include, for example, specific antibodies, immobilized cations (e.g., Ni 2+< with affinity for His-tags) or other types of binding partners (e.g., streptavidin with affinity for Strep-tags).The term "isolated, free TNF-alpha protein" refers to an unbound, unconjugated TNF-alpha protein that is, for example, neither associated with a binding partner nor with a solid phase, nor with a detectable label, nor with a component of a signal-generating system.

[0018] After the sample has been mixed or contacted with isolated, free TNF-alpha protein, the reaction mixture is preferably first incubated for a period of 1 second to 10 minutes, particularly preferably for a period of 5 seconds to 5 minutes, before the first TNF-alpha binding partner is added. The reaction mixture is preferably incubated at a temperature of approximately 37°C. This ensures that all molecules of a therapeutic TNF-alpha inhibitor present in the sample bind to the added free TNF-alpha protein, thus increasing the precision of the quantitative determination.

[0019] The first TNF-alpha binding partner is associated with a particulate solid phase, and the agglutination of the particulate solid phase in the reaction mixture is measured to determine the amount of the complex formed in the reaction mixture between TNF-alpha protein and the first TNF-alpha binding partner.

[0020] For the purposes of this invention, the term "particulate solid phase" refers to non-cellular particles having an approximate diameter of at least 20 nm and not more than 20 µm, typically between 200 nm and 350 nm, preferably between 250 and 320 nm, particularly preferably between 270 and 290 nm, and most preferably 280 nm. The microparticles can be regularly or irregularly shaped. They can be spheres, spheroids, or spheres with more or less large cavities or pores. The microparticles can consist of organic or inorganic material, or a mixture or combination of both. They can consist of a porous or non-porous, swellable or non-swellable material. In principle, the microparticles can have any density, but particles with a density close to that of water, such as about 0.7 to about 1.5 g / ml, are preferred.The preferred microparticles are suspendable in aqueous solutions and have the longest possible suspension stability. They may be transparent, partially transparent, or opaque. The microparticles can consist of multiple layers, such as so-called "core-and-shell" particles with a core and one or more enveloping layers. The term microparticles encompasses, for example, dye crystals, metal sols, silica particles, glass particles, and magnetic particles. Preferred microparticles are particles that are suspendable in aqueous solutions and consist of water-insoluble polymer material, in particular substituted polyethylenes. Very particular preference is given to latex particles, e.g., made of polystyrene, acrylic acid polymers, methacrylic acid polymers, acrylonitrile-butadiene-styrene polymers, polyvinyl acetate-acrylate, polyvinylpyridine, and vinyl chloride-acrylate.Of particular interest are latex particles with reactive groups on their surface, such as carboxyl, amino, or aldehyde groups, which allow covalent binding of an anti-TNF-alpha binding partner to the latex particles. Human, animal, plant, or fungal cells, or bacteria, are explicitly not included in the term "particulate solid phase" for the purposes of this invention.

[0021] The term "associated" is to be understood broadly and includes, for example, covalent and non-covalent binding, direct and indirect binding, adsorption to a surface and inclusion in a recess or cavity, etc. In covalent binding, the antibodies are bound to the solid phase or to a component of a signaling system via a chemical bond. Examples of non-covalent binding include surface adsorption, inclusion in cavities, or the binding of two specific binding partners. In addition to direct binding to the solid phase or a component of a signaling system, the antibodies can also be bound to the solid phase or the label indirectly via specific interactions with other specific binding partners.This will be further illustrated by examples: The biotinylated antibody can be bound to the label via label-bound avidin, or a fluorescein antibody conjugate can be bound to the solid phase via solid phase-bound anti-fluorescein antibodies, or the antibody can be bound to the solid phase or the label via immunoglobulin-binding proteins.

[0022] The TNF-alpha binding partner coated on the particulate solid phase can be an anti-TNF-alpha antibody, a peptide or nucleic acid aptamer, or a peptide affimer.

[0023] The anti-TNF-alpha antibody coated on the particulate solid phase is an immunoglobulin, for example, an immunoglobulin of the class or subclass IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3, IgG4, or IgM. The antibody has at least one binding site (often called a paratope) for an epitope (often also called an antigenic determinant) in the TNF-alpha protein. Such an epitope is characterized, for example, by its spatial structure and / or by the presence of polar and / or apolar groups. The binding site of the antibody is complementary to the epitope. The antigen-antibody reaction works according to the so-called "lock-and-key principle" and is usually highly specific, i.e., the antibodies are able to distinguish small deviations in the primary structure, charge, spatial configuration, and steric arrangement of the TNF-alpha protein.

[0024] For the purposes of this invention, the term "antibody" refers not only to complete antibodies, but also explicitly to antibody fragments, such as Fab, Fv, F(ab') 2 , Fab'; as well as chimeric, humanized, bi- or oligospecific, or "single-chain" antibodies; and also to aggregates, polymers, and conjugates of immunoglobulins and / or their fragments, provided the binding properties to the TNF-alpha protein are retained. Antibody fragments can be produced, for example, by enzymatic cleavage of antibodies with enzymes such as pepsin or papain. Antibody aggregates, polymers, and conjugates can be generated by a variety of methods, e.g., by heat treatment, reaction with substances such as glutaraldehyde, reaction with immunoglobulin-binding molecules, biotinylation of antibodies and subsequent reaction with streptavidin or avidin, etc.

[0025] An anti-TNF-alpha antibody within the meaning of this invention can be a monoclonal or a polyclonal antibody. The antibody can be produced by conventional methods, e.g., by immunizing an animal such as a mouse, rat, guinea pig, rabbit, horse, donkey, sheep, goat, chicken, etc., with TNF-alpha protein and subsequently obtaining the antiserum; or by establishing hybridoma cells and subsequently purifying the secreted antibodies; or by cloning and expressing the nucleotide sequences, or modified versions thereof, that encode the amino acid sequences responsible for the binding of the natural antibody to the TNF-alpha protein.

[0026] If the sample contains a therapeutic TNF-alpha inhibitor, it binds to the added free TNF-alpha protein in the reaction mixture, thus preventing the binding of the subsequently added solid-phase-coupled TNF-alpha binding partner, thus preventing the formation of a complex of TNF-alpha protein and solid-phase-coupled TNF-alpha binding partner. Only free, unbound TNF-alpha protein is now bound by the solid-phase-coupled TNF-alpha binding partner and causes agglutination of the particulate solid phase in the reaction mixture. The more of a therapeutic TNF-alpha inhibitor a sample contains, the more strongly it inhibits the formation of a complex of TNF-alpha protein and solid-phase-coupled TNF-alpha binding partner, i.e., the agglutination reaction.

[0027] The measurement of agglutination of the particulate solid phase in the reaction mixture can be carried out photometrically, for example turbidimetrically or nephelometrically. Binding tests based on the principle of particle-enhanced light scattering have been known since around 1920 (for an overview see Newman, DJ et al., Particle enhanced light scattering immunoassay. Ann Clin Biochem 1992; 29: 22-42). In this context, polystyrene particles with a diameter of 0.1 to 0.5 µm are preferably used, particularly preferably with a diameter of 0.15 to 0.35 µm. Polystyrene particles with amine, carboxyl, or aldehyde functions are preferably used. Shell / core particles are also preferred. The synthesis of the particles and the covalent coupling of ligands is described, for example, in Peula, JM et al., Covalent coupling of antibodies to aldehyde groups on polymer carriers. Journal of Materials Science: Materials in Medicine 1995; 6: 779-785.

[0028] Alternatively, the agglutination of the particulate solid phase in the reaction mixture can be measured by measuring a signal generated by a signal-generating system when a first and a second component of the signal-generating system are brought into spatial proximity to one another. In this context, a first fraction of the particulate solid phase is associated with a first component of a signal-generating system, and a second fraction of the particulate solid phase is associated with a second component of the signal-generating system, wherein the first and second components of the signal-generating system interact to produce a detectable signal when the first and second components of the signal-generating system are brought into spatial proximity to one another, and the agglutination of the particulate solid phase in the reaction mixture is measured based on the resulting signal.

[0029] In this embodiment of the method according to the invention, the signal-generating system comprises at least a first and a second component which interact in such a way that a detectable signal is generated when they are brought into spatial proximity to one another and can therefore interact with one another. An interaction between the components is understood to mean, in particular, an energy transfer - i.e. the direct transmission of energy between the components, e.g. through light or electron radiation or via reactive chemical molecules such as short-lived singlet oxygen. The energy transfer can occur from one component to another, but a cascade of different substances through which the energy transfer takes place is also possible.For example, the components can be a pair of an energy donor and an energy receiver, such as photosensitizer and chemiluminescent agent (EP-A2-0515194, LOCI ®< technology) or photosensitizer and fluorophore (WO 95 / 06877) or radioactive iodine <125> and fluorophore (Udenfriend et al. (1985) Proc. Natl. Acad. Sci. 82: 8672-8676) or fluorophore and fluorescence quencher (US Pat. No. 3,996,345). Particularly preferably, the first component of the signal-generating system is a chemiluminescent agent and the second component of the signal-generating system is a photosensitizer, or vice versa, and the chemiluminescence in the reaction mixture is measured.

[0030] After the agglutination in the reaction mixture has been measured (e.g., by determining the maximum absorption change of the reaction mixture), the measured agglutination is compared with the agglutination in reaction mixtures containing samples with known concentrations of a TNF-alpha binding partner. For this purpose, samples with various known concentrations of a therapeutic TNF-alpha inhibitor, an anti-TNF-alpha antibody, a TNF-alpha-specific peptide or nucleic acid aptamer, or a TNF-alpha-specific peptide affiner (so-called calibrators) are usually measured in reaction mixtures using the same method, and a calibration curve is generated from which the concentration of the sample under investigation can then be read or determined after a conversion step.

[0031] The particle-based agglutination tests described above are so-called homogeneous methods, which are characterized by the fact that no separation of unbound TNF-alpha protein or unbound solid-phase-coupled TNF-alpha binding partner is required for the quantitative detection of the complex formed in the reaction mixture between TNF-alpha protein and solid-phase-coupled TNF-alpha binding partner. The amount of the complex formed in the reaction mixture between TNF-alpha protein and solid-phase-coupled TNF-alpha binding partner is measured after or even during the binding reaction without a further separation or washing step to separate unbound binding partners in the reaction mixture.

[0032] The component of a signal-generating system can be a label that itself generates a detectable signal, so no additional components are necessary. Many organic molecules absorb ultraviolet and visible light. These molecules can enter an excited energy state through light absorption and release the absorbed energy in the form of light of a different wavelength than the incident light. Still other labels, such as radioactive isotopes or dyes, can directly generate a detectable signal.

[0033] However, the component of a signal-generating system can also be a label that requires further components for signal generation, such as substrates, coenzymes, quenchers, accelerators, additional enzymes, substances that react with enzyme products, catalysts, activators, cofactors, inhibitors, ions, etc. Suitable labels are, for example, enzymes including horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, glucose oxidase, β-galactosidase, luciferase, urease and acetylcholinesterase; dyes; fluorescent substances including fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, ethidium bromide, 5-dimethylaminonaphthalene-1-sulfonyl chloride and fluorescent chelates of rare earth elements; chemiluminescent substances including luminol, isoluminol, acridinium compounds, olefin, enol ethers, enamine, aryl vinyl ethers, dioxene, arylimidazole, lucigenin, luciferin and aequorin;Sensitizers including eosin, 9,10-dibromoanthracene, methylene blue, porphyrin, phthalocyanine, chlorophyll, rose bengal; coenzymes; enzyme substrates; radioactive isotopes including 125I, 131I, 14C, 3H, 32P, 35S, 14C, 51C, 59Fe, 57Co and 75Se; particles including magnetic particles or particles, preferably latex particles, which may themselves be labeled with, for example, dyes, sensitizers, fluorescent substances, chemiluminescent substances, isotopes or other detectable labels; sol particles including gold or silver sols, etc.

[0034] The second TNF-alpha binding partner can be, as already described above for the first TNF-alpha binding partner, an anti-TNF-alpha antibody, a peptide or nucleic acid aptamer, or a peptide Affimer. The first and second TNF-alpha binding partners can be the same binding partner, for example, the same monoclonal anti-TNF-alpha antibody, or they can be different binding partners, for example, a monoclonal anti-TNF-alpha antibody and a peptide aptamer, or two monoclonal anti-TNF-alpha antibodies that bind to different epitopes of the TNF-alpha protein.

[0035] It has been found that the method according to the invention allows the use of a single universal calibrator for the determination of any therapeutic TNF-alpha inhibitor. The universal calibrator can contain either a single therapeutic TNF-alpha inhibitor, preferably from the group consisting of infliximab, etanercept, adalimumab, certolizumab pegol, and golimumab and their biosimilars, or a mixture of at least two different therapeutic TNF-alpha inhibitors or their biosimilars, or an anti-TNF-alpha antibody, or a TNF-alpha-specific peptide or nucleic acid aptamer, or a TNF-alpha-specific peptide affimer. This has the advantage that a single test kit and a single calibrator can be used for the determination of any therapeutic TNF-alpha inhibitor, eliminating the need for inhibitor-specific test kits or calibrators.A "universal calibrator" also refers to a set of calibrator materials that differ only in the concentration of the therapeutic TNF-alpha inhibitor(s), anti-TNF-alpha antibody, TNF-alpha-specific peptide or nucleic acid aptamer, or TNF-alpha-specific peptide Affimer contained therein. While not every combination of therapeutic TNF-alpha inhibitor and calibrator to be determined exhibits the same reactivity, the different reactivities of the TNF-alpha inhibitors can be compensated for by using linear conversion functions or polynomial conversion functions (second or third order, respectively), thus calculating correct concentrations.

[0036] Another object of the present invention is the use of a test kit in a method for the quantitative determination of a therapeutic TNF-alpha inhibitor in a sample according to one of claims 1 to 6. The test kit contains at least the following components: a) a first vessel containing a reagent, the reagent containing 50 µg - 2 mg / L of isolated free TNF-alpha protein; and b) a second vessel containing a first TNF-alpha binding partner associated with a particulate solid phase.

[0037] The reagent in the first vessel contains an isolated, free TNF-alpha protein of the type already described above, particularly preferably a recombinant TNF-alpha protein. The reagent contains the TNF-alpha protein in a final concentration of 50 µg - 2 mg / L, preferably 50 µg - 1 mg / L, and most preferably 100 µg - 0.5 mg / L. The reagent can be provided in liquid form in the vessel. Alternatively, for the purpose of long-term stabilization, the reagent can also be provided as a lyophilisate, which is resuspended with a suitable volume of solvent shortly before use to achieve the desired final concentration.

[0038] In case some or all of the reagents of the test kit are present as lyophilisates, the test kit may additionally contain the solvents required to dissolve the lyophilisates, such as distilled water or suitable buffers.

[0039] The second tube of the test kit, which contains a first TNF-alpha binding partner, contains a particulate solid phase, preferably latex particles, to which the first TNF-alpha binding partner is associated. The reagent can be a liquid suspension or a resuspendable lyophilizate thereof. Such a test kit is suitable for measuring agglutination using photometric methods.

[0040] Another embodiment of the test kit contains, in addition to the first vessel and the second vessel, a third vessel containing a second TNF-alpha binding partner. This third vessel of the test kit also contains a particulate solid phase coated with the second TNF-alpha binding partner, preferably with an anti-TNF-alpha antibody. In this context, the particulate solid phase of the reagent in the second vessel is associated with a first component of a signal-generating system, and the particulate solid phase of the reagent in the third vessel is associated with a second component of the signal-generating system, wherein the first and second components of the signal-generating system interact such that a detectable signal is generated when the first and second components of the signal-generating system are brought into spatial proximity to one another.Preferably, the first component of the signal-generating system is a chemiluminescent agent, and the second component of the signal-generating system is a photosensitizer, or vice versa. Such a test kit is suitable for measuring agglutination using chemiluminescence.

[0041] A first or second anti-TNF-alpha antibody coated on the solid phase in the reagent in the second and / or third vessel is preferably a monoclonal antibody.

[0042] The following examples serve to illustrate the present invention and are not to be construed as limiting. EXAMPLES Example 1: Homogeneous agglutination tests for the quantitative determination of various therapeutic TNF-alpha inhibitors Reagent 1:

[0043] 500 µg of human, recombinant, lyophilized TNF-alpha protein (Active Bioscience GmbH, Hamburg, Germany) was dissolved in 500 µL of water and stored at 2–8 °C (TNF-alpha stock solution 1 mg / mL). To prepare Reagent 1, the TNF-alpha stock solution was diluted to a final concentration of 250 µg / L TNF-alpha protein in lipoprotein-free human citrated plasma.

[0044] Reagent 2: To prepare Reagent 2, approximately 1.5 mg of the monoclonal anti-TNF-alpha antibody MAK 1D4 was mixed with 1 mL of polystyrene latex particles (40 mg / mL, particle diameter 0.2-0.3 µm) in a buffer solution and incubated. After washing the particles several times, they were then resuspended in 60 mL of a buffer solution.

[0045] The monoclonal anti-TNF-alpha antibody MAK 1D4 was produced by immunizing a mouse with human TNF-alpha protein and subsequent hybridoma cell establishment.

[0046] To prepare samples containing a therapeutic TNF-alpha inhibitor, human serum samples from normal donors were added with different amounts of Adalimumab (Humira ®< , Abbvie Inc., USA), infliximab (Remicade ®< , MSD SHARP & DOHME GmbH, Germany), etanercept (Enbrel ®< , Pfizer Inc., USA), certolizumab pegol (Cimzia ®< , UCB Pharma GmbH, Germany) or golimumab (Simponi ®< , Janssen Biologics BV, Netherlands) added (final concentration in the sample 0.8-25 mg / L).

[0047] To prepare calibrators, a pool of human normal serum 100 mg / L adalimumab (Humira ®< ), or 100 mg / L infliximab (Remicade ®< )

[0048] added. Calibrators with lower inhibitor concentrations were generated by dilution with normal serum or a buffer solution.

[0049] The serum samples and calibrator samples were diluted 1:5 and 1:20, respectively, with a buffer solution. 50 µL of serum sample and calibrator sample were mixed with 10 µL of REAGENT 1 and incubated for one minute at 37 °C. Then, 35 µL of REAGENT 2 were added to the mixture, and the light scattered by the antigen-antibody complexes was measured over a period of 6 minutes at a wavelength of 840 nm in a nephelometric analyzer (BN II System, Siemens Healthcare Diagnostics Products GmbH, Germany). The change in the measurement signal (in bits) after 6 minutes is determined as the measurement result. Example la: Determination of adalimumab using an adalimumab calibration curve

[0050] Six serum calibrators with different adalimumab concentrations were measured using the method of the invention, and a calibration curve was generated.

[0051] Furthermore, 14 serum samples with different adalimumab concentrations were measured using the method according to the invention. The adalimumab concentrations corresponding to the measurement results were read from the previously created calibration curve.

[0052] Table 1 shows, for each sample, the expected adalimumab concentration based on the adalimumab dosage (theoretical concentration) and the concentration determined using the method according to the invention in duplicate measurements (measured concentration). It can be seen that the theoretical adalimumab concentrations are recovered in the concentration range of 0.8–3 mg / L with a maximum deviation of 0.44 mg / L and in the concentration range of 3–25 mg / L with a maximum deviation of 27.5%. This allows for precise quantification of adalimumab for therapy monitoring. Table 1 Adalimumab [mg / L] theoretical Adalimumab [mg / L] measured (n=2) 25 18,1 22,5 16,5 20 15,3 17,5 13,5 15 12,3 12,5 9,8 10 8,0 7,5 6,1 5 3,7 3 2,6 2,5 2,1 2 1,9 1 1,1 0,8 0,9 Example 1b: Determination of infliximab using an infliximab calibration curve

[0053] Six serum calibrators with different infliximab concentrations were measured using the method of the invention, and a calibration curve was constructed.

[0054] Furthermore, 14 serum samples with different infliximab concentrations were measured using the method according to the invention. The infliximab concentrations corresponding to the measurement results were read from the previously generated calibration curve.

[0055] Table 2 shows, for each sample, the infliximab concentration expected based on the infliximab dosage (theoretical concentration) and the concentration determined using the method according to the invention in a duplicate measurement (measured concentration). It can be seen that the theoretical infliximab concentrations are recovered in the concentration range of 0.8–5 mg / L with a maximum deviation of 0.47 mg / L and in the concentration range of 5–25 mg / L with a maximum deviation of 24.5%. This allows for precise quantification of infliximab for therapy monitoring. Table 2 Infliximab [mg / L] theoretical Infliximab [mg / L] measured (n=2) 25 18,9 22,5 17,5 20 16,8 17,5 15,4 15 13,9 12,5 11,7 10 9,2 7,5 6,3 5 4,5 3 2,8 2,5 2,6 2 1,9 1 1,3 0,8 1,1 Example 1c: Determination of various therapeutic TNF-alpha inhibitors using an adalimumab calibration curve

[0056] Six serum calibrators with different adalimumab concentrations were measured using the method of the invention, and a calibration curve was generated.

[0057] Furthermore, 12 to 14 serum samples with different adalimumab, infliximab, etanercept, certolizumab pegol, and golimumab concentrations were measured using the method according to the invention. The adalimumab, infliximab, etanercept, certolizumab pegol, and golimumab concentrations corresponding to the measurement results were read from the previously generated calibration curve.

[0058] Tables 3 to 7 show, for each sample, the concentration expected based on the dosage of the respective inhibitor (theoretical concentration) and the concentration determined using the method according to the invention in a duplicate measurement (measured concentration). It can be seen that the theoretical concentrations of adalumimab and infliximab are recovered even without corrective calculations in the concentration range of 0.8–3 mg / L with a maximum deviation of 0.44 mg / L and in the concentration range of 3–25 mg / L with a maximum deviation of 42.3% (infliximab).

[0059] It was found that by plotting the measured concentration against the theoretical concentration, functions (linear or polynomial) can be calculated that allow the measured values ​​to be corrected, particularly for etanercept, certolizumab pegol and golimumab.

[0060] Using a linear function or a polynomial function (second or third degree), the theoretical concentrations of adalimumab, infliximab, etanercept, certolizumab pegol, and golimumab are recovered with acceptable deviations. It is shown that the theoretical concentrations in the concentration range of 10–25 mg / L are recovered with a maximum deviation of 11.2% (golimumab). In the concentration range of 2–10 mg / L, the absolute deviations are a maximum of 0.61 mg / L (etanercept and infliximab).

[0061] For etanercept, conversion using a polynomial is only possible in the concentration range of 2 - 25 mg / L. Table 3 sample Concentration theoretical Concentration measured (n=2) Concentration calculated linearly [mg / L] [mg / L] [mg / L] Adalimumab 1 25 18,1 24,1 2 22,5 16,5 21,9 3 20 15,3 20,3 4 17,5 13,5 17,8 5 15 12,3 16,1 6 12,5 9,8 12,8 7 10 8,0 10,3 8 7,5 6,1 7,8 9 5 3,7 4,5 10 3 2,6 2,9 11 2,5 2,1 2,3 12 2 1,9 2,0 13 1 1,1 0,9 14 0,8 0,9 0,7 Table 4 sample Concentration theoretical Concentration measured (n=2) Concentration calculated polynomial [mg / L] [mg / L] [mg / L] Infliximab 1 25 14,4 24,2 2 22,5 13,8 21,8 3 20 13,5 20,5 4 17,5 12,7 18,0 5 15 11,9 15,5 6 12,5 10,5 12,4 7 10 8,6 9,5 8 7,5 6,2 6,9 9 5 4,5 5,3 10 3 2,9 3,4 11 2,5 2,6 3,0 12 2 1,9 1,9 13 1 1,4 0,9 14 0,8 1,1 0,4 Table 5 sample Concentration theoretical Concentration measured (n=2) Concentration calculated polynomial [mg / L] [mg / L] [mg / L] Etanercept 1 25 10,0 23,6 2 22,5 10,0 24,0 3 20 9,4 19,8 4 17,5 9,0 17,2 5 15 8,7 15,2 6 12,5 8,1 12,4 7 10 7,6 9,9 8 7,5 7,1 7,6 9 5 6,5 5,4 10 3 5,9 3,4 11 2,5 5,6 2,7 12 2 5,1 1,4 Table 6 sample Concentration theoretical Concentration measured (n=2) Concentration calculated polynomial [mg / L] [mg / L] [mg / L] Certolizumab Pegol 1 25 66,4* 24,4* 2 22,5 56,5* 22,8* 3 20 44,4* 19,8* 4 17,5 37,6* 17,6* 5 15 29,7* 14,8* 6 12,5 23,7* 12,3* 7 10 18,0* 9,7* 8 7,5 15,6* 7,9* 9 5 10,0 5,5 10 3 5,2 2,8 11 2,5 4,5 2,5 12 2 3,5 1,9 13 1 2,1 1,0 14 0,8 1,8 0,9 *=1:20 sample dilution Table 7 sample Concentration theoretical Concentration measured (n=2) Concentration calculated polynomial [mg / L] [mg / L] [mg / L] Golimumab 1 25 18,0 23,7 2 22,5 17,5 21,7 3 20 17,3 21,1 4 17,5 16,2 17,1 5 15 16,1 16,7 6 12,5 14,8 13,0 7 10 13,3 10,0 8 7,5 11,3 7,2 9 5 7,7 4,7 10 3 4,8 3,3 11 2,5 3,8 2,7 12 2 3,2 2,2 13 1 1,9 1,0 14 0,8 1,6 0,6

[0062] The method described here allows the automatic determination of the concentrations of different TNF-alpha inhibitors in a single homogeneous assay.

[0063] Using a linear function or a polynomial function (2nd or 3rd degree) to convert the obtained results, the accuracy of the recovery of each individual TNF-alpha inhibitor is sufficient for therapy monitoring and therapy control.

Claims

1. Competitive method for quantitatively determining a therapeutic TNF-alpha inhibitor in a sample, the method comprising the steps of: a) providing a reaction mixture by contacting the sample i. with isolated, free TNF-alpha protein and then ii. with a particulate solid phase associated with a first TNF-alpha binding partner; b) measuring the agglutination of the particulate solid phase in the reaction mixture to determine the amount of a complex which forms in the reaction mixture and is composed of TNF-alpha protein and the first TNF-alpha binding partner; and c) determining the amount of the therapeutic TNF-alpha inhibitor in the sample by comparing the thusly determined amount of the complex composed of TNF-alpha protein and the first TNF-alpha binding partner in the reaction mixture with amounts of a complex composed of TNF-alpha protein and the first TNF-alpha binding partner in reaction mixtures containing samples having known concentrations of a TNF-alpha binding partner.

2. Method according to Claim 1, wherein the agglutination of the particulate solid phase in the reaction mixture is measured photometrically.

3. Method according to Claim 2, wherein a first fraction of the particulate solid phase is associated with a first component of a signal-forming system and a second fraction of the particulate solid phase is associated with a second component of the signal-forming system, and wherein the first and the second component of the signal-forming system cooperate such that a detectable signal is produced when the first and the second component of the signal-forming system are brought into close proximity to one another and the agglutination of the particulate solid phase in the reaction mixture is measured on the basis of the signal produced.

4. Method according to Claim 3, wherein the first component of the signal-forming system is a chemiluminescent agent and the second component of the signal-forming system is a photosensitizer or vice versa and wherein the chemiluminescence in the reaction mixture is measured.

5. Method according to any of the preceding claims, wherein the reaction mixture is incubated for a period of from 1 second to 10 minutes after contacting the sample with isolated, free TNF-alpha protein and before adding the first TNF-alpha binding partner.

6. Method according to any of the preceding claims, wherein the first and / or the second TNF-alpha binding partner is an anti-TNF-alpha antibody.

7. Use of an assay kit in a competitive method for quantitatively determining a therapeutic TNF-alpha inhibitor in a sample according to any of Claims 1-6, the assay kit containing the following components: a) a first vessel containing a reagent, the reagent containing 50 µg - 2 mg / L isolated, free TNF-alpha protein; and b) a second vessel containing a first TNF-alpha binding partner associated with a particulate solid phase.

8. Use according to Claim 7, wherein the particulate solid phase is latex particles.

9. Use according to either of Claims 7 and 8, wherein the assay kit further contains the following component: c) a third vessel containing a particulate solid phase coated with the first TNF-alpha binding partner or with a second TNF-alpha binding partner, wherein the particulate solid phase in the second vessel is associated with a first component of a signal-forming system and the particulate solid phase in the third vessel is associated with a second component of the signal-forming system, and wherein the first and the second component of the signal-forming system cooperate such that a detectable signal is produced when the first and the second component of the signal-forming system are brought into close proximity to one another.

10. Use according to Claim 9, wherein the first component of the signal-forming system is a chemiluminescent agent and the second component of the signal-forming system is a photosensitizer or vice versa.

11. Use according to any of Claims 7 to 10, wherein the TNF-alpha protein present in the first reagent is a recombinant TNF-alpha protein.

12. Use according to any of Claims 7 to 11, wherein the first TNF-alpha binding partner in the second vessel is an anti-TNF-alpha antibody, preferably a monoclonal anti-TNF-alpha antibody.