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  1. Regulatory Science in Action

Establishment and validation of an in vitro cell-based assay to assess the biological activity of insulin products

CDER researchers develop, validate, and publish an easily performed, in vitro, cell-based assay for the biological activity of insulin analogs that will support the transition away from animal testing of these important medical products.

Why move away from animal testing methods for drugs?

In vivo testing during foundational investigations has historically supported many advancements in medicine; however, alternative in vitro testing methods are becoming more accessible thanks to increased understanding of molecular mechanisms underlying biological pathways, and modern developments in both bench laboratory science as well as analytical and computational approaches. Currently, there is global support to significantly reduce the reliance on in vivo testing, which is fueled by both ethical and scientific rationale.1 To support the transition from in vivo to in vitro testing of the biological activity of insulin products and expand the availability of publicly available in vitro cell-based assay protocols, CDER researchers have expanded the utility of an in vitro assay derived from USP <121> to cover additional types of insulin. In doing so, they also added the use of alternate fluorescent reagents that can be detected by commonly used plate readers.

The scientific challenges and evolution of insulin products

Insulin was discovered in 1921 by Dr. Frederick Banting, a surgeon, and Charles Best, a medical student.  Insulin is a 51 amino acid peptide hormone, which plays a critical role in regulating glucose metabolism. Insulin binds to insulin receptors, triggering a cascade of cellular responses, including glucose uptake, lipid metabolism regulation, and energy management. According to the CDC, 1 in 10 Americans has diabetes,2 a chronic condition characterized by insulin deficiency or resistance. Patients with type 1 diabetes are unable to synthesize insulin and are thus dependent on daily insulin injections to manage their blood glucose levels. Patients with type 2 diabetes either do not produce enough insulin or have a resistance to insulin; they often rely on insulin treatment along with lifestyle changes to manage their blood glucose levels. Diabetes is among the top 10 causes of death in the U.S., resulting in hundreds of billions of dollars in medical costs and lost wages. Insulin has been a key tool in managing diabetes and increasing the length and quality of life of those who live with the disease.

Since insulin discovery, pork and beef glands became the primary commercial sources of insulin. However, the purification process presented variable yields, and the strengths and concentrations of insulin present in each batch were inconsistent. To standardize the biological activity of each insulin batch, the "rabbit blood glucose assay" was developed. This in vivo assay allowed the quantification of a standard amount of insulin (an international unit of insulin3) required to have a defined biological effect. The international unit of insulin is still used today by patients to manage their blood glucose.

Insulin products have significantly evolved since the 1920s, including, for example, the development of longer acting insulins in the 1940s and the approval of a recombinant insulin in 1982. Insulin manufacturing has also improved with the use of modern biotechnology-based processes and physicochemical methods; for example, reverse-phase HPLC (RP-HPLC) can be used to control insulin product purity. Human insulin is a relatively simple molecule with limited heterogeneity and few isoforms.4 The RP-HPLC method effectively separates human insulin and traditional insulin analogs from major degradation products and eliminates interference from phenolic preservatives. Therefore, insulin potency is often inferred from content after purification using physicochemical methods, such as RP- HPLC. However, this approach may not fully capture the biological activity (or bioidentity) of insulin products, particularly for new and emerging insulins, and the rabbit assay is still largely used to evaluate the biological activity of insulin products.

Because of the worldwide aim to reduce animal use in testing, multiple industry members and academic researchers have developed in vitro cell-based assays to assess the biological activity of insulin analogs. In December 2020, USP chapter <121>, which until then only described the rabbit assay as a method to assess the biological activity or bioidentity of insulin products, was updated to include an in vitro cell-based assay for two common analogs: insulin glargine and insulin lispro.

A method to assess the biological activity or bioidentity of insulin analogs in vitro

The method established by CDER researchers is based on the measurement of the phosphorylation of the insulin receptor following treatment with insulin products. Binding of human insulin or insulin analogs to the human insulin receptor on cells induces the auto-phosphorylation of the receptor’s kinase domain, a modification which is necessary for kinase activity and receptor activation. Hence, quantification of insulin-induced auto-phosphorylation of the human insulin receptor is a mechanistically sound and objective read-out for the biological activity of insulin and insulin analogs (Figure 1). CDER researchers have validated this assay for three common types of insulin: insulin glargine, insulin aspart, and insulin lispro.

CHOK1 overexpressing human insulin receptor

Using this approach, the biological activity of insulin samples of interest could be easily compared to the biological activity of a USP reference standard or other standardized reference product in 4 to 5 days. As shown in Figure 2 (reproduced from Garige et al. Methods Protoc. 2023 Mar 24;6(2):33), cells overexpressing the human insulin receptor are treated with insulin samples of interest as well as a reference standard, then fixed and permeabilized. Receptor activation is detected with a primary antibody to phosphorylated tyrosine and a secondary antibody linked to a fluorescent dye. Fluorescent signal can be detected with a classic plate reader, and a fluorescent stain that binds to DNA allows one to correct for different cell numbers.   

Infographic illustrating a step-by-step protocol for an in vitro cell-based assay to test insulin activity. Steps include plating ATCC #CRL-3307 cells, preparing and treating cells with insulin standards and samples, incubation, washing, fixation, permeabilization, blocking, antibody application, secondary antibody and Hoechst 33342 staining, and final analysis including washing, reading, and data analysis. Duration for each step is specified, from 30 minutes up to overnight processes.

Conclusion

In conclusion, by establishing, expanding, and publishing a step-by-step protocol of this method, CDER researchers are providing public resources to manufacturers and researchers to support the transition from in vivo to in vitro testing of the biological activity of insulin analogs.

This Impact Story is based on: “Protocol to Assess the Biological Activity of Insulin Glargine, Insulin Lispro, and Insulin Aspart In Vitro”, by Garige M, Ghosh S, Roelofs B, Rao VA & Sourbier C. Methods Protoc. 2023 Mar 24;6(2):33. doi: 10.3390/mps6020033. PMID: 37104015.

How can this work help advance public health?

Development of in vitro bioassays that reliably monitor the biological activity of biotechnology products in lieu of animal testing is a critical step towards the implementation of the 3R’s initiative, that aims to replace, reduce, and refine animal use in testing. CDER researchers have established and validated an in vitro insulin cell-based assay, derived from the USP chapter <121> “insulin assays”, to expand the ability to assess the biological activity of insulin glargine, insulin aspart, and insulin lispro in vitro. This method is easily performed with commonly used equipment and amenable to adaptation for assessing the biological activity of other insulin analogs. It was published in an open access journal to provide a methodological option to drug developers and regulators with interests in assessing the biological activity of insulin analogs.

 

References

[1] US agency seeks to phase out animal testing, Identifying Key Factors for Accelerating the Transition to Animal-Testing-Free Medical Science through Co-Creative, Interdisciplinary Learning between Students and Teachers

[2] https://www.cdc.gov/diabetes/basics/quick-facts.html

[3] An international unit (IU) of insulin corresponds to the amount of insulin required to lower the blood sugar by a standard amount. One IU corresponds to the activity contained in 0.0347mg of human insulin or 0.0363 mg of insulin glargine.

[4] Isoforms are functionally similar proteins with amino acid sequences that are not identical.

 
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