Validation of a biolayer interferometry-based method for the determination of binding (total) antibodies to romiplostim in human serum

INTRODUCTION. Romiplostim treatment of patients with idiopathic thrombocytopenic purpura is associated with formation of anti-drug antibodies (ADA), which often leads to the serious adverse events. A method based on biolayer interferometry is proposed for determination of binding (total) ADA (bADA) to romiplostim in human serum. From a technological point of view, instruments using this principle have some advantages (higher throughput, prolonged service intervals, low level of equipment contamination during analysis of biosamples) over those based on surface plasmon resonance, which are used to monitor bADA levels during clinical trials of the original drug. The method should be validated to ensure reproducibility of measurements and stan dardise clinical laboratory trials of the immunogenicity of a biosimilar drug.

AIM. To establish the key validation characteristics of a biolayer interferometry-based method for the determination of total anti-drug antibodies to romiplostim in human serum.

MATERIALS AND METHODS. The method is used to detect the specific interaction of binding antibodies with biotinylated romiplostim immobilised on the streptavidin-coated biosensors. The method includes a screening test to determine bADA presence or absence in the samples; a confirmatory test to check the specificity of bADA binding to romiplostim; determination of antibody titer and maximal dilution, that allows to detect bADA in the samples. Positive control samples containing different concentrations of polyclonal rabbit antibodies to romiplostim were used in the work. Detection of protein complex formation was performed in real time mode using an Octet® QKe interferometer.

RESULTS. The biological variability factor and the limit of not significant inhibition were 1.481 and 34.2%, respectively. The precision and specificity of the method were confirmed.The lower limit of bADA detection in the absence of romiplostim was 622 ng/mL. The analytical signal of 8 out of 10 blood serum samples after the addition of bADA increased by more than 1.481 times when assessing the matrix effect in the screening test; the percentage inhibition value exceeded 34.2% when analysing 8 out of 10 samples with the addition of bADA in the confirmatory test. There was no high-dose effect at bADA concentrations from 0.8 to 50 μg/mL. The results of bADA determination in the screening test were robust to the presence of 1 ng/mL romiplostim in the samples. Streptavidin-coated biosensors with immobilised romiplostim maintained stability after 14 days of storage at a temperature of (5±3) °С and at least 20 regeneration cycles.

CONCLUSIONS. Analysis of the validation results obtained confirms the suitability of the method for reliable and reproducible determination of binding (total) antibodies to romiplostim in human serum.

1. Melikyan AL, Egorova EK, Pustovaya EI, Kolosheynova TI, Volodicheva EM, Kaporskaya TS, et al. Interim results of epidemiological study of idiopathic thrombocytopenic purpura in adults in the Russian Federation: intermediate results. Hematology and Transfusiology. 2019;64(4):436–46 (In Russ.). https://doi.org/10.35754/0234-5730-2019-64-4-436-446

2. Neunert C, Terrell DR, Arnold DM, Buchanan G, Cines DB, Cooper N, et al. American Society of Hematology 2019 guidelines for immune thrombocytopenia. Blood Adv. 2019;3(23):3829–66. https://doi.org/10.1182/bloodadvances.2019000966

3. Barger TE, Boshier A, Jawa V, Kim J, Mytych DT, Park J, et al. Assessment of romiplostim immunogenicity in adult patients in clinical trials and in a global registry. Blood. 2018;132 (Supplement 1):2427. https://doi.org/10.1182/blood-2018-99-113484

4. Swanson SJ. What are clinically significant anti-drug antibodies and why is it important to identify them. Front Immunol. 2024;15:1401178. https://doi.org/10.3389/fimmu.2024.1401178

5. Shankar G, Devanarayan V, Amaravadi L, Barrett YC, Bowsher R, Finco-Kent D, et al. Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal. 2008;48(5):1267–81. https://doi.org/10.1016/j.jpba.2008.09.020

6. Hagman C, Chasseigne G, Nelson R, Anlauff F, Kagan M, Goldfine AB, et al. Immunogenicity assessment strategy for a chemically modified therapeutic protein in clinical development. Front Immunol. 2024;15:1438251. https://doi.org/10.3389/fimmu.2024.1438251

7. Shibata H, Nishimura K, Miyama C, Tada M, Suzuki T, Saito Y, et al. Comparison of different immunoassay methods to detect human anti-drug antibody using the WHO erythropoietin antibody reference panel for analytes. J Immunol Methods. 2018;452:73–7. https://doi.org/10.1016/j.jim.2017.09.009

8. Li J, Schantz A, Schwegler M, Shankar G. Detection of low-affinity anti-drug antibodies and improved drug tolerance in immunogenicity testing by Octet® biolayer interferometry. J Pharm Biomed Anal. 2011;54(2):286–94. https://doi.org/10.1016/j.jpba.2010.08.022

9. Jug A, Bratkovič T, Ilaš J. Biolayer interferometry and its applications in drug discovery and development. Trends Anal Chem. 2024;176:117741. https://doi.org/10.1016/j.trac.2024.117741

10. Wadhwa M, Knezevic I, Kang H-N, Thorpe R. Immunogenicity assessment of biotherapeutic products: An overview of assays and their utility. Biologicals. 2015;43(5):298–306. https://doi.org/10.1016/j.biologicals.2015.06.004

11. Jani D, Marsden R, Mikulskis A, Gleason C, Klem T, Fiorotti CK, et al. Recommendations for the development and validation of confirmatory anti-drug antibody assays. Bioanalysis. 2015;7(13):1619–31. https://doi.org/10.4155/bio.15.96

12. Smith HW, Moxness M, Marsden R. Summary of confirmation cut point discussions. AAPS J. 2011;13(2):227–9. https://doi.org/10.1208/s12248-011-9263-z

13. Gunn GR, Sealey DC, Jamali F, Meibohm B, Ghosh S, Shankar G. From the bench to clinical practice: understanding the challenges and uncertainties in immuno-genicity testing for biopharmaceuticals. Clin Exp Immunol. 2016;184(2):137–46. https://doi.org/10.1111/cei.12742

14. Tada M, Suzuki T, Ishii-Watabe A. Development and characterization of an anti-rituximab monoclonal antibody panel. MAbs. 2018;10(3):370–9. https://doi.org/10.1080/19420862.2018.1424610

15. Namburi RP, Kancherla V, Ponnala AR. High-dose hook effect. J Dr NTR Univ Health Sci. 2014;3(1):5–7. https://doi.org/10.4103/2277-8632.128412

16. Varghese LN, Defour J-P, Pecquet C, Constantinescu SN. The Thrombopoietin receptor: structural basis of traffic and activation by ligand, mutations, agonists, and mutated calreticulin. Front Endocrinol (Lausanne). 2017;8:59. https://doi.org/10.3389/fendo.2017.00059

17. Qi J, Zheng L, Hu B, Zhou H, He Q, Liu H, et al. Pharmacokinetics, safety, and pharmacodynamics of romiplostim in chinese subjects with immune thrombocytopenia: a phase I/II trial. Clin Pharmacol Drug Dev. 2022;11(3):379–87. https://doi.org/10.1002/cpdd.1059