Development and Validation of Approach for the Detection of Neutralizing Antibodies Against Insulin (Glargine) in Human Blood Plasma

Introduction. IImmunogenicity identification of therapeutic proteins, such as human insulin analogues, is one of the most relevant and significant area in medicine and pharmaceuticals. Determination the possibility of producing neutralizing antibodies to insulin reducing the therapeutic effect of the drug, is an important step to understand the pharmacological profile of the drug. Applying of cell-based methods one allows to determinate neutralizing antibodies to insulin.

Aim. Development and validation methods for detection of neutralizing antibodies against insulin in human plasma.

Materials and methods. The method is based on the use of the iLiteTM Insulin Assay Ready Cells [1], in the genome of which the firefly luciferase reporter gene is introduced under the control of an insulin-dependent promoter. As the insulin concentration increases, the firefly luciferase expression (Firefly) increases, allowing one to use this cell line to estimate the number of neutralizing antibodies against insulin. For normalization by the number of cells and considering the matrix effect of studied samples, the second reporter gene luciferase Renilla is used, which is expressed under the control of a constitutive promoter. The activity of both luciferases was measured using the DualGlo Luciferase Assay System (Promega) assay [2].

Results and discussion. Optimal insulin concentration and plasma/serum dilution were determined to identify neutralizing antibodies to insulin. The long-term stability of neutralizing antibodies to insulin were shown in human plasma for more than 3 months. The developed method was applied in a comparative research of the safety and immunogenicity of insulin analogues (Glargine). Method for the determination of antibodies to insulin was.

Conclusion. A method for determination of neutralizing antibodies to insulin in human K₂EDTA plasma was developed and validated using iLiteTM Insulin Assay Ready Cells system; based on the binding of the insulin alpha chain to the high-affinity heterodimeric CD220 receptor.

1. Dual-GloR Luciferase Assay System Technical Manual (Promega) Available at: https://www.promega.com/-/media/files/resources/protocols/technical-manuals/0/dual-luciferase-reporter-assaysystem-protocol.pdf?la=en.

2. iLiteTM Insulin Assay Ready Cells (REF: BM3060), Product specification (Euro Diagnostica AB, Doc No: E-165-GB01). Available at: https://www.eurodiagnostica.com/downloadFile.php?fileName=upload/files/fileLibrary/BM3060%20Product%20Specification%20iLite%20Insulin%20Assay%20Ready%20Cells%20LABEL-DOC-0362%20v1%200.pdf.

3. Ametov A. S., Karpova E. V. A modern view on insulin therapy in patients with type 2 diabetes in the daily clinical practice of an endocrinologist. Endokrinologiya. 2008; 495: 71–5 (in Russ.).

4. Avakova K. A. Optimization of methods of modern insulin therapy in the treatment of diabetes. PhD thesis. 2009 (in Russ.).

5. McKeage K., Goa K. L. Insulin glargine. Drugs. 2001; 61(11): 1599–1624. https://doi.org/10.2165/00003495-200161110-00007.

6. Davies M., Dahl D., Heise T., Kiljanski J., Mathieu C. Introduction of biosimilar insulins in Europe. Diabetic Medicine. 2017; 34(10): 1340– 1353. https://doi.org/10.1111/dme.13400.

7. European Medicines Agency. Abasaglar Insulin Glargine. EPAR Summary for the Public, 2014. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_Summary_for_thepublic/human/002835/WC500175384.pdf.

8. Determination of anti-Insulin neutralizing antibodies using iLiteTM Insulin Assay Ready Cells, Application note (Euro Diagnostica AB, Doc No: E-183-GB00). Available at: https://www.eurodiagnostica.com/downloadFile.php?fileName=upload/files/fileLibrary/Application%20note%20-%20Determination%20of%20antiinsulin%20neutralizing%20antibodies%20LABEL-DOC-0390%20v1%200.pdf.

9. Quantification of Insulin using iLiteTM Insulin Assay Ready Cells, Application note (Euro Diagnostica AB, Doc No: E-182-GB00). Available at: https://www.eurodiagnostica.com/downloadFile.php?fileName=upload/files/fileLibrary/Application%20note%20-%20Quantification%20of%20Insulin%20LABEL-DOC-0389%20v1%200.pdf.

10. FDA/CDER/CBER. Immunogenicity Testing of Therapeutic Protein Products – Developing and Validating Assays for Anti-Drug Antibody Detection Guidance for Industry. FDA. 2019.

11. Home P., Derwahl K.-M., Ziemen M., Wernicke-Panten K., Pierre S., Kirchhein Y., Garg S. K. Anti-Insulin Antibodies and Adverse Events with Biosimilar Insulin Lispro Compared with Humalog Insulin Lispro in People with Diabetes. Diabetes Technol. Ther. 2018; 20: 160–170. https://doi.org/10.1089/dia.2017.0373.

12. Chatterjee S., Vashishta L., Waichale V. S., Nayak V. G., Melarkode R., Donnelly C. M., Sengupta N. Development and validation of a cellbased assay for the detection of neutralizing antibodies against recombinant insulins. J. Immunol. Methods. 2017; 452: 53–62. doi:10.1016/j.jim.2017.09.004.