Detection of anti-drug antibodies to aflibercept by enzyme-linked immunosorbent assay: Development and validation of the method

INTRODUCTION. Aflibercept is used to treat neovascular age-related macular degeneration, a leading cause of vision loss. Immunogenicity is one of the significant adverse effects that results in the production of anti-drug antibodies (ADAs). The development of a method for detecting ADAs to aflibercept is essential for immunogenicity assessment.

AIM. This study aimed to develop and validate an analytical method for detection and stepwise characterisation of total anti-drug antibodies to aflibercept in human serum utilising the enzyme-linked immunosorbent assay.

MATERIALS AND METHODS. The method was developed using a solid-phase ELISA. Aflibercept (GNR-098, JSC GENERIUM, Russia) and a conjugate of GNR-098 with horseradish peroxidase were employed. Polyclonal antibodies against GNR-098 were obtained by immunising rats, followed by serum collection and antibody purification via affinity chromatography. ADA detection was performed using labelled GNR-098 in a bridging ELISA. Model samples were prepared by spiking pooled sera from healthy donors with rat polyclonal antibodies. Method validation included the following parameters: cut-off points; precision; limit of detection (LOD); drug tolerance; specificity; matrix effect; minimum required dilution; robustness; reagent stability; hook effect.

RESULTS. An analytical method includes a screening assay for the presumptive detection of ADAs; a confirmatory assay to establish ADA specificity and assay for ADA titer determination and IgE isotyping. The cut-off points for the screening assay and IgE-specific assay were 1.3 and 1.2, respectively; for the confirmatory assay, 15.2%. The LOD of the screening assay was 185 ng/mL. The method demonstrated drug tolerance (interference from soluble target, hrVEGF165) at concentrations up to 2000 pg/mL. No matrix effects were observed in the screening or confirmatory assays across 13 test samples containing ADAs at 280 ng/mL. The absence of a hook effect was confirmed in both assays using samples with ADA concentrations five-fold higher than the high-positive control.

CONCLUSIONS. An analytical method was developed for the ELISA-based detection and stepwise characterisation of total anti-drug antibodies to aflibercept (GNR-098) in human serum. The method’s suitability was confirmed through validation. This analytical procedure will facilitate the immunogenicity assessment of aflibercept.

1. Veritti D, Sarao V, Gorni G, Lanzetta P. Anti-VEGF drugs dynamics: Relevance for clinical practice. Pharmaceutics. 2022;14(2):265. https://doi.org/10.3390/pharmaceutics14020265

2. Anguita R, Tasiopoulou A, Shahid S, Roth J, Sim SY, Patel PJ. A review of aflibercept treatment for macular disease. Ophthalmol Ther. 2021;10(3):413–28. https://doi.org/10.1007/s40123-021-00354-1

3. Sharma A, Kumar N, Parachuri N, Bandello F, Kuppermann BD. Immunogenicity: Clouding the future of intravitreal therapy. Ocul Immunol Inflamm. 2022;31(9):1746–9. https://doi.org10.1080/09273948.2022.2139273

4. Gyenes A, Istvan L, Benyo F, Papp A, Resch M, Sandor N, et al. Intraocular neutralizing antibodies against aflibercept in patients with age related macular degeneration. BMC Ophthalmol. 2023;23(1):14. https://doi.org/10.1186/s12886-022-02761-9

5. Fleckenstein M, Schmitz-Valckenberg S, Chakravarthy U. Age-related macular degeneration: A review. JAMA. 2024;331(2):147–47. https://doi.org/10.1001/jama.2023.26074

6. Harris C.T, Cohen, S. Reducing immunogenicity by design: Approaches to minimize immunogenicity of monoclonal antibodies. BioDrugs. 2024;38(3):205–26. https://doi.org/10.1007/s40259-023-00641-2

7. Koren E, Smith HW, Shores E, Shankar G, Finco-Kent D, Rup B, et al. Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products. J Immunol Methods. 2008;333(1–2):1–9. https://doi.org/10.1016/j.jim.2008.01.001

8. Ivanov R, Sekareva G, Kravtsova O, Kudlay D, Lukyanov S, Tikhonova I, et al. Guidelines of research biosimilar drugs. Pharmacokinetics and Pharmacodynamics. 2014;(1):21–36 (In Russ). EDN: SYTXJB

9. Pineda C, Castaneda Hernandez G, Jacobs IA, Alvarez DF, Carini C. Assessing the immunogenicity of biopharmaceuticals. BioDrugs. 2016;30(3):195–206. https://doi.org/10.1007/s40259-016-0174-5

10. 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

11. Carter PJ, Rajpal A. Designing antibodies as therapeutics. Cell. 2022;185(15):2789–805. https://doi.org/10.1016/j.cell.2022.05.029