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In Vitro Biological Characterization of Recombinant Insulin Aspart from Biogenomics and Originator Insulin Aspart

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Abstract

Background

Bioassays are used to identify the pharmacological activity of new or chemically unknown compounds, as well as their undesirable effect, including toxicity. Biological assays are also required to ensure the quality, safety, and efficacy of recombinant biologics to confirm its biosimilarity to its originator. In the present study, analytical similarity between the biosimilar and its innovator is established by in vitro bioassays.

Objective

The objective of this study was to show the comparative in vitro characterization of the recombinant insulin aspart from BioGenomics with its originator insulin aspart, using relevant biological assays.

Methods

In vitro assays such as receptor binding, receptor autophosphorylation, glucose uptake, and mitogenic potential were analyzed for biological characterization of BioGenomics recombinant insulin aspart (BGL-ASP) manufactured by BioGenomics Limited and NovoRapid® as the reference medicinal product (RMP) manufactured by Novo Nordisk. Insulin receptor binding was studied by a state-of-the-art method, surface plasmon resonance (SPR) for biomolecular interactions. The receptor autophosphorylation assay measures the phosphorylated insulin receptor in cell lysates. The glucose uptake assay measures the uptake of glucose by 3T3-L1 cells in the presence of insulin. Lipogenesis was studied in treated 3T3-L1 cells by detecting the accumulation of lipid droplets in the cells. Mitogenic effect was studied by cell proliferation assay using MCF-7 cells. A rabbit bioidentity test was performed by measuring the sudden decrease in blood glucose in the presence of insulin.

Results

The binding studies showed that the affinity of BGL-ASP was highly comparable to NovoRapid®. Insulin receptor autophosphorylation, glucose uptake, and lipogenesis demonstrated high similarity to the RMP. The mitogenic assay for BGL-ASP did not show any proliferative effect and was comparable to the RMP. The in vivo bioidentity test showed that the BGL-ASP is highly similar to the innovator, NovoRapid®.

Conclusion

The biological characterization studies of BGL-ASP demonstrated high binding and functional similarity to NovoRapid®.

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References

  1. Ghosh S, Bose S, Gowda S, Mukhopadhyay P. Biosimilar insulins—what a clinician needs to know? Indian J Endocrinol Metab. 2019;23(4):400–6. https://doi.org/10.4103/ijem.IJEM_180_19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dolinar RO, Edelman S, Heinemann L, Home P, Goyal S, Polonsky WH, Schellekens H. Impact of biosimilar insulins on clinical practice: meeting report. J Diabetes Sci Technol. 2014;8(1):179–85. https://doi.org/10.1177/1932296813518267.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Rotenstein LS, Ran N, Shivers JP, Yarchoan M, Close KL. Opportunities and challenges for biosimilars: what’s on the horizon in the global insulin market? Clin Diabetes. 2012;30(4):138–50. https://doi.org/10.2337/diaclin.30.4.138.

    Article  Google Scholar 

  4. Kirchhoff CF, Wang XM, Conlon HD, Anderson S, Ryan AM, Bose A. Biosimilars: key regulatory considerations and similarity assessment tools. Biotechnol Bioeng. 2017;114(12):2696–705. https://doi.org/10.1002/bit.26438.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Guideline on non-clinical and clinical development of similar biological medicinal products containing recombinant human insulin and insulin analogues, EMEA/CHMP/BMWP/32775/2005_Rev. 1. https://www.ema.europa.eu/documents/scientific-guideline/guideline-non-clinical-clinical-development-similar-biological-medicinal-products-containing_en-0.pdf. Accessed 26 Feb 2015.

  6. Reflection paper on statistical methodology for the comparative assessment of quality attributes in drug development, EMA/CHMP/138502/2017. https://www.ema.europa.eu/documents/scientific-guideline/reflection-paper-statistical-methodology-comparative-assessment-quality-attributes-drug-development_en.pdf. Accessed 26 July 2021.

  7. Nupur N, Joshi S, Gulliarme D, Rathore AS. Analytical similarity assessment of biosimilars: global regulatory landscape, recent studies and major advancements in orthogonal platforms. Front Bioeng Biotechnol. 2022;10:832059. https://doi.org/10.3389/fbioe.2022.832059.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Tsong Yi, Dong X, Shen M. Development of statistical methods for analytical similarity assessment. J Biopharm Stat. 2015. https://doi.org/10.1080/10543406.2015.1092038.

    Article  PubMed  Google Scholar 

  9. Westermeier F, Sáez T, Arroyo P, Toledo F, Gutiérrez J, Sanhueza C, Pardo F, Leiva A, Sobrevia L. Insulin receptor isoforms: an integrated view focused on gestational diabetes mellitus. Diabetes Metab Res Rev. 2016;32(4):350–65. https://doi.org/10.1002/dmrr.2729.

    Article  CAS  PubMed  Google Scholar 

  10. Moruzzi N, Lazzeri-Barcelo F, Valladolid-Acebes I, Moede T, Paschen M, Leibiger B, Berggren PO, Leibiger IB. Tissue-specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models. J Cell Mol Med. 2021;25(10):4800–13. https://doi.org/10.1111/jcmm.16452.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Belfiore A, Frasca F, Pandini G, Sciacca L, Vigneri R. Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease. Endocr Rev. 2009;30(6):586–623. https://doi.org/10.1210/er.2008-0047.

    Article  CAS  PubMed  Google Scholar 

  12. Ward CW, Lawrence MC. Landmarks in insulin research. Front Endocrinol. 2011. https://doi.org/10.3389/fendo.2011.00076.

    Article  Google Scholar 

  13. Siddle K. Signalling by insulin and IGF receptors: supporting acts and new players. J Mol Endocrinol. 2011. https://doi.org/10.1530/jme-11-0022.

    Article  PubMed  Google Scholar 

  14. Di Camillo B, Carlon A, Eduati F, Toffolo GM. A rule-based model of insulin signalling pathway. BMC Syst Biol. 2016. https://doi.org/10.1186/s12918-016-0281-4.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Hall C, Yu H, Choi E. Insulin receptor endocytosis in the pathophysiology of insulin resistance. Exp Mol Med. 2020. https://doi.org/10.1038/s12276-020-0456-3.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Belfiore A, Malaguarnera R, Vella V, Lawrence CM, Sciacca L, Frasca F, Morrione A, Vigneri R. Insulin receptor isoforms in physiology and disease: an updated view. Endocr Rev. 2017;38:379–431.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Sakoda H, Ogihara T, Anai M, Funaki M, Inukai K, Katagiri H, Fukushima Y, Onishi Y, Ono H, Fujishiro M, Kikuchi M, Oka Y, Asano T. Dexamethasone-induced insulin resistance in 3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction. Diabetes. 2000;49:1700–8.

    Article  CAS  PubMed  Google Scholar 

  18. Subramanian K, Fee CJ, Fredericks R, Stubbs RS, Hayes MT. Insulin receptor-insulin interaction kinetics using multiplex surface plasmon resonance. J Mol Recognit. 2013;26:643–52.

    Article  CAS  PubMed  Google Scholar 

  19. Subramanian K. Kinetics of insulin–insulin receptor interaction using a surface plasmon resonance. 2014. http://hdl.handle.net/10092/9327. Accessed 05 Dec 2013.

  20. Kleiman LB, Maiwald T, Conzelmann H, Lauffenburger DA, Sorger KP. Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding. Mol Cell. 2011;43:723–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Goodyear LJ, Giorgino F, Sherman LA, Carey J, Smith RJ, Dohm GL. Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects. J Clin Investig. 1995;95:2195–204.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Frasca F, Pandini G, Scalia P, Sciacca L, Mineo R, Costantino A, Goldfine ID, Belfiore A, Vigneri R. Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells. Mol Cell Biol. 1999;19:3278–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Murakami SM, Rosen MO. The role of insulin receptor autophosphorylation in signal transduction. J Biol Chem. 1991;266(33):22653–60.

    Article  CAS  PubMed  Google Scholar 

  24. Vishwanath D, Srinivasan H, Patil MS, Seetarama S, Agrawal SK, Dixit MN, Dhar K. Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog. J Cell Commun Signal. 2013;7:129–40.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Zhang LM, Hu JJ, Du G. Establishment of a cell-based assay to screen insulin-like hypoglycemic drugs. Drug Discov Ther. 2008;24:229–33.

    Google Scholar 

  26. Madsen L, Petersen RK, Kristiansen K. Regulation of adipocyte differentiation and function by polyunsaturated fatty acids. Biochim Biophys Acta. 2005;1740:266–86.

    Article  CAS  PubMed  Google Scholar 

  27. Krycer JR, Quek LE, Francis D, Zadoorian A, Weiss FC, Cooke KC, Nelson ME, Diaz-Vegas A, Humphrey SJ, Scalzo R, Hirayama A, Ikeda S, Shoji F, Suzuki K, Huynh K, Giles C, Varney B, Nagarajan SR, Hoy AJ, Soga T, Meikle PJ, Cooney GJ, Fazakerley DJ, James DE. Insulin signaling requires glucose to promote lipid anabolism in adipocytes. J Biol Chem. 2020;295(38):13250–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hui C, Zhang ZW, Guo Y, Wang Y, Liu Y, Luo N, Zhu Y. The proliferative role of insulin and the mechanism underlying this action in human breast cancer cell line MCF-7. J BUON. 2012;17:658–62.

    Google Scholar 

  29. Tennagels N, Werner U. The metabolic and mitogenic properties of basal insulin analogues. Arch Physiol Biochem. 2013;119(1):1–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wei ML, Duan P, Wang ZM, Ding M, Tu P. High glucose and high insulin conditions promote MCF-7 cell proliferation and invasion by upregulating IRS1 and activating the Ras/Raf/ERK pathway. Mol Med Rep. 2017;16(5):6690–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Milazzo G, Giorgino F, Damante G, Sung C, Stampfer MR, Vigneri R, Goldfine ID, Belfiore A. Insulin receptor expression and function in human breast cancer cell lines. Cancer Res. 1992;52:3924–30.

    CAS  PubMed  Google Scholar 

  32. USP-NF 〈121〉 INSULIN ASSAYS. https://www.uspnf.com/sites/default/files/usp_pdf/EN/USPNF/gc_121_insulin_assays_03012015.pdf. Accessed 01 May 2019.

  33. Yie J, Dey M, Su J, Sergi J, Zhang Y, Le TH, Kashi S, Gurney K. Development of a robust functional cell-based assay for replacing the rabbit blood sugar bioidentity test of insulin glargine drug substance. J Pharm Biomed Anal. 2020;186: 113328. https://doi.org/10.1016/j.jpba.2020.113328.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr. K. R. Srinivasan for the support and guidance throughout the process of submission.

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Authors and Affiliations

  1. BioGenomics Ltd., Thane, Maharashtra, 400610, India

    Akshay G. Mishra, Rutuja B. Deshmane, Damodar K. Thappa, Jeseena Lona, Nikhil S. Ghade, Sanjay M. Sonar & Archana R. Krishnan

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  1. Akshay G. Mishra
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  2. Rutuja B. Deshmane
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Corresponding author

Correspondence to Akshay G. Mishra.

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Funding

This research was partially funded by USV Pvt. Ltd., India.

Conflicts of interest

All authors are employed by BioGenomics Ltd.

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All data generated or analyzed during this study are included in this published article.

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Author contributions

AGM and RBD contributed towards the experimental setup, analysis, and interpretation of the data. DKT, NSG, and JL contributed in conception, design and development of the molecule. SMS and ARK contributed in development of the molecule, reviewed the article critically for important intellectual content, and gave their final approval of the version to be published.

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Mishra, A.G., Deshmane, R.B., Thappa, D.K. et al. In Vitro Biological Characterization of Recombinant Insulin Aspart from Biogenomics and Originator Insulin Aspart. BioDrugs 37, 709–719 (2023). https://doi.org/10.1007/s40259-023-00607-4

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