(T1530-06-40) Analyzing Insulin Glargine Biosimilars: Stability, Degradation, and Structural Characterization

Purpose: Insulin glargine became the first insulin analogue with a biologic product definition, paving the way for the emergence of biosimilar alternatives such as Semglee (insulin glargine-yfgn) and Rezvoglar (insulin glargine-aglr) to Lantus. However, due to differences in manufacturing processes and pharmaceutical formulations, innovator insulins may undergo distinct chemical modifications compared to their biosimilars. Moreover, insulin glargine is highly sensitive to environmental stresses and is prone to structural changes, unfolding, and aggregation. Even minor alterations that affect the structural integrity of insulin could have profound implications on its biological efficacy, particularly in terms of physiological and pharmacological activities. Therefore, it is crucial to thoroughly understand the potential differences in aggregation propensity among biosimilar versions of insulin glargine. We have chosen Basaglar as a model drug to investigate these implications and established analytical methods for evaluation critical quality attributes. Analytical characterization of Basaglar provides insights into its comparability to innovator insulin glargine, aiding in the assessment of biosimilar insulin products.

Methods: Basaglar was incubated in various temperatures (37°C and 50°C) and shaking conditions to induce degradation. Samples were collected from the insulin pens at experimental time points (0, 1, 3, 7, 14, 21, 28 and 35 days) to monitor the degradation. To evaluate the concentration of insulin glargine, we developed an ultra-performance liquid chromatography (UPLC-UV) method with a 17.6 min run time. Compared to traditional high-performance liquid chromatography (HPLC-UV) methods, UPLC-UV allowed us to monitor the changes in insulin glargine in approximately 30% less time. After detecting some impurities using UPLC-UV, we employed liquid chromatography-mass spectrometry (LC/MS) analysis to reveal the structural differences between stressed Basaglar and fresh Basaglar, providing insights into any potential modifications or degradation. In addition, dynamic light scattering (DLS) was employed to monitor changes in particle size and assessed the potential aggregation of insulin glargine during the incubation process, providing valuable information on the formation of larger particles. Furthermore, gel permeation chromatography (GPC) was used to examine the formation of high molecular weight products (HMWP). We previously optimized the application of thioflavin T (ThT), a widely-used fluorescent dye, to assess the formation of amyloid fibrils in stressed insulin lispro. We applied that method to the stressed Basaglar in the study. By employing a combination of UPLC-UV, LC/MS, DLS, GPC and ThT techniques, we aimed to comprehensively assess the degradation, particle formation, aggregation, and structural changes in Basaglar under different stressed conditions.

Results: The effective concentration range of insulin is typically considered to be 95-105 units/ml. Upon subjecting insulin glargine to incubation at 37°C and 80 rpm for 35 days, a significant decrease in concentration was observed compared to the fresh sample, although the concentration remained above 95 units/mL throughout the incubation period (Figure 1). However, after being incubated at 50°C for 14 days, the insulin glargine in Basaglar showed a significant decrease in concentration, below 95 units/mL, indicating the loss of potency. Notably, the particle size exhibited significant changes during the incubation both at 37°C and 50°C. Following incubation at 80 rpm and 37°C for 7 days or 50°C for 3 days, the GPC chromatogram displayed the presence of HMWP at approximately 8.5 minutes. However, no obvious amyloid fibrils were observed in Basaglar after incubation under these conditions. In the LC/MS data, we have already seen 4 peaks of degradation in the stressed samples and we are in the process of identifying these.

Conclusion: Using Basaglar as a model drug, we investigated the possible chemical and physical changes that may occur in insulin glargine under different stressed conditions. These established methodologies will be applied to evaluation and comparison of Lantus and it’s FDA approved biosimilars. This research enhances our understanding of biosimilar insulin glargine products and their aggregation propensity, ultimately benefiting individuals with diabetes worldwide.

The changes of Basaglar under different stressed conditions. A. The concentration of insulin glargine after incubating at 80 rpm at 37°C for different periods. n = 3, *P < 0.05. B. The size of Basaglar after incubating at 80 rpm at 37°C for different periods. C. GPC chromatograms of Basaglar after incubating at 80 rpm at 37°C for different periods. D. The fluorescence intensity of stressed Basaglar at 80 rpm at 37°C detected by thioflavin T. n = 3. E. The concentration of insulin glargine after incubating at 50°C for different periods. n = 3, ****P < 0.0001. F. The size of Basaglar after incubating at 50°C for different periods. C. GPC chromatograms of Basaglar after incubating at 50°C for different periods. D. The fluorescence intensity of stressed Basaglar at 50°C. n = 3.