METHOD FOR QUANTITATIVELY DETERMINING A THERAPEUTIC TNF-ALPHA INHIBITOR

Abstract

The invention is in the field of in vitro diagnostics and relates to a method for quantitatively determining therapeutic TNF-alpha inhibitors.

Claims

1. A 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 first TNF-alpha binding partner; b) determining 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 amount of the complex composed of TNF-alpha protein and the first TNF-alpha binding partner determined 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. The method as claimed in claim 1, wherein the first TNF-alpha binding partner is associated with a particulate solid phase and wherein the amount of the complex which forms in the reaction mixture and is composed of TNF-alpha protein and the first TNF-alpha binding partner in the reaction mixture is determined by measuring agglutination of the particulate solid phase in the reaction mixture.

3. The method as claimed in claim 2, wherein the agglutination of the particulate solid phase in the reaction mixture is measured photometrically.

4. The method as claimed in 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 component and the second component of the signal-forming system cooperate such that a detectable signal forms when the first component 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 formed.

5. The method as claimed in claim 4, 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.

6. The method as claimed in claim 1, wherein the first TNF-alpha binding partner is associated with a solid phase and wherein the amount of the complex which forms in the reaction mixture and is composed of TNF-alpha protein and the first TNF-alpha binding partner is determined in step b) by removing the reaction mixture from the solid phase and subsequently contacting the solid phase with a second TNF-alpha binding partner and measuring the amount of the second TNF-alpha binding partner which is bound to the complex which was formed in the reaction mixture and is associated with the solid phase and is composed of TNF-alpha protein and the first TNF-alpha binding partner.

7. The method as claimed in claim 6, wherein the second TNF-alpha binding partner is associated with a component of a signal-forming system.

8. The method as claimed in claim 6, 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.

9. The method as claimed in claim 6, wherein the first and/or the second TNF-alpha binding partner is an anti-TNF-alpha antibody.

10. An assay kit for use in a method for quantitatively determining a therapeutic TNF-alpha inhibitor in a sample, which contains the following components: a) a first vessel containing a reagent, the reagent containing 50 g-2 mg/L isolated, free TNF-alpha protein; and b) a second vessel containing a first TNF-alpha binding partner.

11. The assay kit as claimed in claim 10, wherein the first TNF-alpha binding partner in the second vessel is associated with a particulate solid phase.

12. The assay kit as claimed in claim 11, wherein the particulate solid phase are latex particles.

13. The assay kit as claimed in claim 11, further comprising: c) a third vessel containing a particulate solid phase which is coated with the first 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 component and the second component of the signal-forming system cooperate such that a detectable signal forms when the first component and the second component of the signal-forming system are brought into close proximity to one another.

14. The assay kit as claimed in claim 13, 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.

15. The assay kit as claimed in claim 10, wherein the first TNF-alpha binding partner in the second vessel is associated with a surface of the second vessel.

16. The assay kit as claimed in claim 15, further comprising: c) a third vessel containing a second TNF-alpha binding partner.

17. The assay kit as claimed in claim 16, wherein the second TNF-alpha binding partner is associated with a component of a signal-forming system.

18. The assay kit as claimed in claim 10, wherein the TNF-alpha protein present in the first reagent is a recombinant TNF-alpha protein.

19. The assay kit as claimed in claim 10, wherein the first TNF-alpha binding partner in the second vessel is an anti-TNF-alpha antibody.

20. The assay kit as claimed in claim 19, wherein the anti-TNF-alpha antibody is a monoclonal anti-TNF-alpha antibody.

Description

EXAMPLES

Example 1: Homogeneous Agglutination Assays for Quantitatively Determining Various Therapeutic TNF-Alpha Inhibitors

Reagent 1:

[0056] 500 g of human, recombinant, lyophilized TNF-alpha protein (Active Bioscience GmbH, Hamburg, Germany) were dissolved in 500 L 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 g/L TNF-alpha protein in a lipoprotein-free human citrate plasma.

Reagent 2:

[0057] To prepare Reagent 2, about 1.5 mg of the monoclonal anti-TNF-alpha antibody MAK 1D4 were mixed and incubated with 1 mL of polystyrene latex particles (40 mg/mL, particle diameter 0.2-0.3 m) in a buffer solution. After repeated washing of the particles, the particles were subsequently resuspended in 60 mL of a buffer solution.

[0058] The monoclonal anti-TNF-alpha antibody MAK 1D4 was produced by immunization of a mouse with human TNF-alpha protein and subsequent establishment of hybridoma cells.

[0059] To prepare samples containing a therapeutic TNF-alpha inhibitor, different amounts of [0060] adalimumab (Humira, Abbvie Inc., USA), [0061] infliximab (Remicade, MSD SHARP & DOHME GmbH, Germany), [0062] etanercept (Enbrel, Pfizer Inc., USA), [0063] certolizumab pegol (Cimzia, UCB Pharma GmbH, Germany) or [0064] golimumab (Simponi, Janssen Biologics B.V., the Netherlands)
were added to human serum samples from normal donors (final concentration in the sample 0.8-25 mg/L).

[0065] To prepare calibrators, [0066] 100 mg/L adalimumab)(Humira or [0067] 100 mg/L infliximab)(Remicade
were added to a pool of human normal serum. Calibrators having lower inhibitor concentrations were generated by dilution with normal serum or a buffer solution.

[0068] The serum samples and the calibrator samples were diluted 1:5 and 1:20, respectively, with a buffer solution. 50 L of serum sample or calibrator sample were mixed with 10 L of REAGENT 1 and incubated at 37 C. for one minute. Then, 35 L of REAGENT 2 were added to the mixture, and the light which is scattered at the antigen-antibody complex was measured within 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 measurement result ascertained is the change in the measurement signal (in bit) after 6 minutes.

Example 1a: Determination of Adalimumab with the Aid of an Adalimumab Calibration Curve

[0069] Six serum calibrators having different adalimumab concentrations were measured using the method according to the invention and a calibration curve was prepared.

[0070] Furthermore, 14 serum samples having different adalimumab concentrations were measured using the method according to the invention. The adalimumab concentrations corresponding to the measurement results were read off on the previously prepared calibration curve.

[0071] Table 1 shows, for each sample, the adalimumab concentration to be expected according to adalimumab dosage (theoretical concentration) and the concentration ascertained in duplicate using the method according to the invention (measured concentration). It becomes apparent that the theoretical adalimumab concentrations are recovered with a maximum deviation of 0.44 mg/L in the concentration range of 0.8-3 mg/L and with a maximal deviation of 27.5% in the concentration range of 3-25 mg/L. This allows a precise quantification of adalimumab for therapy monitoring.

TABLE-US-00001 TABLE 1 Adalimumab [mg/L], Adalimumab [mg/L], theoretical 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 with the Aid of an Infliximab Calibration Curve

[0072] Six serum calibrators having different infliximab concentrations were measured using the method according to the invention and a calibration curve was prepared.

[0073] Furthermore, 14 serum samples having different infliximab concentrations were measured using the method according to the invention. The infliximab concentrations corresponding to the measurement results were read off on the previously prepared calibration curve.

[0074] Table 2 shows, for each sample, the infliximab concentration to be expected according to infliximab dosage (theoretical concentration) and the concentration ascertained in duplicate using the method according to the invention (measured concentration). It becomes apparent that the theoretical infliximab concentrations are recovered with a maximum deviation of 0.47 mg/L in the concentration range of 0.8-5 mg/L and with a maximum deviation of 24.5% in the concentration range of 5-25 mg/L. This allows a precise quantification of infliximab for therapy monitoring.

TABLE-US-00002 TABLE 2 Infliximab [mg/L], Infliximab [mg/L], theoretical 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 with the Aid of an Adalimumab Calibration Curve

[0075] Six serum calibrators having different adalimumab concentrations were measured using the method according to the invention and a calibration curve was prepared.

[0076] Furthermore, 12 to 14 serum samples having different concentrations of adalimumab, infliximab, etanercept, certolizumab pegol or golimumab were in each case measured using the method according to the invention. The concentrations of adalimumab, infliximab, etanercept, certolizumab pegol or golimumab belonging to the measurement results were read off on the previously prepared calibration curve.

[0077] Tables 3 to 7 show, for each sample, the concentration to be expected according to dosage of the particular inhibitor (theoretical concentration) and the concentration ascertained in duplicate using the method according to the invention (measured concentration). It becomes apparent that, even without a corrective calculation, the theoretical concentrations of adalimumab and infliximab are recovered with a maximum deviation of 0.44 mg/L in the concentration range of 0.8-3 mg/L and with a maximum deviation of 42.3% in the concentration range of 3-25 mg/L (infliximab).

[0078] It was found that it is possiblewhen the measured concentration is plotted against the theoretical concentrationto calculate functions (linear or polynomial) which allow correction of the measured values, particularly for etanercept, certolizumab pegol and golimumab.

[0079] Using a linear function or a polynomial function (2nd or 3rd degree), the theoretical concentrations of adalimumab, infliximab, etanercept, certolizumab pegol and golimumab are recovered with acceptable deviations. It becomes apparent that the theoretical concentrations are recovered with a maximum deviation of 11.2% in the concentration range of 10-25 mg/L (golimumab). In the concentration range of 2-10 mg/L, the absolute deviations are maximally 0.61 mg/L (etanercept and infliximab).

[0080] For etanercept, a polynomial conversion is only possible in the concentration range of 2-25 mg/L.

TABLE-US-00003 TABLE 3 Concentration, Concentration, Concentration, calculated, theoretical measured (n = 2) linear Sample [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-US-00004 TABLE 4 Concentration, Concentration, Concentration, calculated, theoretical measured (n = 2) polynomial Sample [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-US-00005 TABLE 5 Concentration, Concentration, Concentration, calculated, theoretical measured (n = 2) polynomial Sample [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-US-00006 TABLE 6 Concentration, Concentration, Concentration, calculated, theoretical measured (n = 2) polynomial Sample [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-US-00007 TABLE 7 Concentration, Concentration, Concentration, calculated, theoretical measured (n = 2) polynomial Sample [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

[0081] The method described here allows the automatic determination of the concentrations of various TNF-alpha inhibitors in a single homogeneous assay.

[0082] Using a linear function or a polynomial function (2nd or 3rd degree) for converting the results obtained, the accuracy of the recovery of each individual TNF-alpha inhibitor is sufficient for therapy monitoring and therapy control.