Physicochemical and biological characterization of the medicinal product RinLis®

Introduction. Access to insulin is limited by global barriers, the most crucial of which is the pricing monopoly of multinational originator companies. The expiry of patent on the originator product opens the way for the production of biosimilars being affordable products with comparable quality, effectiveness and safety. The imperative for the development of biosimilars is to conduct comprehensive comparability studies with the originator product, including physicochemical and biological characterization. The development of a comparability study program, specifically the selection of attributes and orthogonal methods for analyzing the structure and the functional integrity of the insulin molecule, should be based on a deep understanding of the structure and properties of the molecule, as well as on the latest scientific knowledge.

Aim. To conduct physicochemical and biological characterization of the medicinal product RinLis® (GP40011) in comparison with the originator medicinal product Humalog® (insulin lispro) to establish their comparability within the framework of the biosimilarity assessment.

Materials and methods. The primary protein structure was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide mapping. Higher-order protein structures were investigated using capillary isoelectric focusing, Ellman's method, circular dichroism, fluorescence spectrophotometry and dynamic light scattering. The comparability of impurity profiles of the tested batches was studied using size-exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography (RP HPLC). Insulin receptor (IR-A and IR-B) binding kinetics assay, insulin receptor phosphorylation assay and metabolic cell-based assays (glucose uptake, insulin-induced lipogenesis, inhibition of stimulated lipolysis) were performed to characterize the functional properties and biological activity of the insulin products.

Results and discussion. GP40011 was found to have high similarity to the reference product Humalog® in terms of the primary protein structure, higher-order structures and impurity profile based on the results of the evaluation of the physicochemical properties. Functional characterization studies showed that GP40011 and Humalog® had identical insulin receptor binding kinetics to IR-A, IR-B and comparable biological activity.

Conclusion. The study results demonstrated full analytical similarity of RinLis® to Humalog®. The study made a significant contribution to the scientific substantiation of the equivalence of the biosimilar RinLis® and the reference product Humalog®, thereby providing a reliable basis for successful approval and subsequent clinical use.

1. GBD 2021 Diabetes Collaborators. Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis for the Global Burden of Disease Study 2021. The Lancet. 2023;402(10397):203–234. DOI: 10.1016/S0140-6736(23)01301-6.

2. Flood D., Seiglie J. A., Dunn M., Tschida S., Theilmann M., Marcus M. E., Brian G., Norov B., Mayige M. T., Singh Gurung M., Aryal K. K., Labadarios D., Dorobantu M., Silver B. K., Bovet P., Jorgensen J. M. A., Guwatudde D., Houehanou C., Andall-Brereton G., Quesnel-Crooks S., Sturua L., Farzadfar F., Saeedi Moghaddam S., Atun R., Vollmer S., Bärnighausen T. W., Davies J. I., Wexler D. J., Geldsetzer P., Rohloff P., Ramírez-Zea M., Heisler M., Manne-Goehler J. The state of diabetes treatment coverage in 55 low-income and middle-income countries: a cross-sectional study of nationally representative, individual-level data in 680 102 adults. The Lancet Healthy Longevity. 2021;2(6): E340–E351. DOI: 10.1016/S2666-7568(21)00089-1.

3. Beran D., Ewen M., Laing R. Constraints and challenges in access to insulin: a global perspective. The Lancet Diabetes & Endocrinology. 2016;4(3):275–285. DOI: 10.1016/S2213-8587(15)00521-5.

4. Agbogbo F. K., Ecker D. M., Farrand A., Han K., Khoury A., Martin A., McCool J., Rasche U., Rau T. D., Schmidt D., Sha M., Treuheit N. Current perspectives on biosimilars. Journal of Industrial Microbiology & Biotechnology. 2019;46(9–10):1297–1311. DOI: 10.1007/s10295-019-02216-z.

5. Morolla D., Beran D., Ewen M., Raviglione M., von Schoen-Angerer T. Role of biosimilar introduction on insulin glargine prices: a retrospective analysis in 28 European countries. BMJ Open. 2025;15(1):e090484. DOI: 10.1136/bmjopen-2024-090484.

6. Koivisto V. A. The human insulin analogue insulin lispro. Annals of Medicine. 1998;30(3):260–266. DOI: 10.3109/07853899809005853.

7. Posner B. I. Insulin Signalling: The Inside Story. Canadian Journal of Diabetes. 2017;41(1):108–113. DOI: 10.1016/j.jcjd.2016.07.002.

8. Hsu J.-C., Lai B.-Y., Hsieh W.-L., Wang D.-Y., Shih D. Y.-C., Lo C.-F. Identification of recombinant insulin analogues by peptide mapping method. Journal of Food and Drug Analysis. 2012;20(4):7. DOI: 10.6227/jfda.2012200427.

9. Sklepari M., Rodger A., Reason A., Jamshidi S., Prokes I., Blindauer C. A. Biophysical characterization of a protein for structure comparison: methods for identifying insulin structural changes. Analytical Methods. 2016;8(41):7460–7471. DOI: 10.1039/C6AY01573E.

10. Patil S. M., Keire D. A., Chen K. Comparison of NMR and Dynamic Light Scattering for Measuring Diffusion Coefficients of Formulated Insulin: Implications for Particle Size Distribution Measurements in Drug Products. The AAPS Journal. 2017;19(6):1760–1766. DOI: 10.1208/s12248-017-0127-z.

11. Babina A. V., Drai R. V., Saparova V. B., Afanasyeva A. N., Zaikin P. G., Shmurak V. I., Eltysheva T. E., Makarenko I. E. Physicochemical and functional characterization of medicinal product Rinsulin® R. Drug development & registration. 2023;12(2):124–134. (In Russ.) DOI: 10.33380/2305-2066-2023-12-2-124-134.]

12. Korn M., Wohlfart P., Gossas T., Kullman-Magnusson M., Niederhaus B., Dedio J., Tennagels N. Comparison of metabolic and mitogenic response in vitro of the rapid-acting insulin lispro product SAR342434, and US- and EU-approved Humalog®. Regulatory Toxicology and Pharmacology. 2019;109:104497. DOI: 10.1016/j.yrtph.2019.104497.

13. Somwar R., Sweeney G., Ramlal T., Klip A. Stimulation of glucose and amino acid transport and activation of the insulin signaling pathways by insulin lispro in L6 skeletal muscle cells. Clinical Therapeutics. 1998;20(1):125–140. DOI: 10.1016/s0149-2918(98)80040-4.

14. Owens R. A., Hansen R. J., Kahl S. D., Zhang C., Ruan X., Koester A., Li S., Qian H.-R., Farmen M. W., Michael M. D., Moyers J. S., Cutler G. B. Jr., Vick A., Beals J. M. In Vivo and In Vitro Characterization of Basal Insulin Peglispro: A Novel Insulin Analog. The Journal of Pharmacology and Experimental Therapeutics. 2016;357(3):459-465. DOI: 10.1124/jpet.115.231035.

15. Byrd R. A., Owens R. A., Blackbourne J. L., Coutant D. E., Farmen M. W., Michael M. D., Moyers J. S., Schultze A. E., Sievert M. K., Tripathi N. K., Vahle J. L. Nonclinical pharmacology and toxicology of the first biosimilar insulin glargine drug product (BASAGLAR®/ABASAGLAR®) approved in the European Union. Regulatory Toxicology and Pharmacology. 2017;88:56–65. DOI: 10.1016/j.yrtph.2017.05.013.

16. Ghade N. S., Thappa D. K., Lona J., Krishnan A. R., Sonar S. M. Comparative physicochemical and structural characterisation studies establish high biosimilarity between BGL-ASP and reference insulin aspart. Scientific Reports. 2024;14(1):4224. DOI: 10.1038/s41598-024-54819-x.

17. Goyal P., Pai H. V., Kodali P., Vats B., Vajpai N., Annegowda S., Mane K., Mohan S., Saxena S., Veerabhadraia A. B., Palande M., Nair P. S., More D. C., Rao Karudumpa U., Jyothirmai K., Bhattacharya A., Almeida F., Khyade S. G., Gouda S., Ranayhossaini D. J., Reddy Moole P., Smith J. P., Barve A., Melarkode R., Ullanat R. Physicochemical and functional characterization of MYL-1501D, a proposed biosimilar to insulin glargine. PLoS ONE. 2021;16(6):e0253168. DOI: 10.1371/journal.pone.0253168.

18. Afanasyeva A. N., Saparova V. B., Makarenko I. E., Drai R. V., Selmenskikh T. A. Experimental validation of an in vitro method for assessing insulin-dependent glucose uptake. Bulletin of the Scientific Centre for Expert Evaluation of Medicinal Products. Regulatory Research and Medicine Evaluation. 2023;13(1):77–88. DOI: 10.30895/1991-2919-2022-398.]

19. Zhou C., Qi W., Lewis E. N., Carpenter J. F. Characterization of sizes of aggregates of insulin analogs and the conformations of the constituent protein molecules: a concomitant dynamic light scattering and Raman spectroscopy study. Journal of Pharmaceutical Sciences. 2016;105(2):551–558. DOI: 10.1016/j.xphs.2015.10.023.