According to the WHO, diabetes is the 9th leading cause of death, with an increasing number of deaths associated with the rising cost and inaccessibility of proper insulin therapy [1,2]. These rising costs are a result of a monopoly formed by leading insulin manufacturers [3,4]. Additionally, with the number of diabetic patients expected to grow in the coming decade, current manufacturing will likely not be sufficient to accommodate the projected demand, making accessibility even more challenging [5]. We propose an alternative to the established insulin manufacturing protocols by leveraging the utility of cell-free systems (CFS), which allow for reduced upstream processing times and enhanced feasibility of biomanufacturing at the point-of-care (POC). Additionally, by performing post translational modification (PTM) and purification of proinsulin in one coupled step, our bioprocess could significantly reduce the downstream processing time.
Currently, insulin manufacturing is achieved through two well established fermentation processes. These fermentation processes are typically performed at large scale (5,000-25,000 Liters) and typically take days to weeks to yield a final product which is deemed safe and effective for use [6]. Additionally, the costly facilities which these products are manufactured in, are required to comply with good manufacturing practices (GMP) established by the regulatory agencies (e.g., FDA). To date there are no bioprocesses available for production of insulin at the POC. We seek to build on the currently established methods and develop a low-cost POC manufacturing platform, which can produce biosimilar insulin, safely and effectively, on the time scale of hours to days. This will not only motivate a reduction in insulin prices but also improve accessibility in areas of the world where insulin is hard to access.
To this end, we will start by designing a genetically engineered proinsulin analogue which can be expressed in both E. coli and N. tabacum CFS. The engineered proinsulin construct will then be cloned into an expression vector conducive for rapid transcription and translation in each of the respective host CFS on a time scale of hours. To enhance protein yield, a series of DOE methods assessing critical cell-free process parameters will be applied to both CFS to ensure maximum yield of soluble proinsulin. The proinsulin harvest will then be subjected to affinity purification to remove host cell impurities which may perturb subsequent enzymatic conversion steps. Upon achieving near homogeneity of proinsulin, a series of endoproteases and carboxypeptidase will be introduced into the bioprocess to convert the proinsulin into mature human insulin. The mature insulin product can then be purified to greater homogeneity via a series of polishing steps, yielding human insulin ready for formulation.
The proposed bioprocess will then be subject to a technoeconomic analysis which will take into consideration all the materials and reagents needed to manufacture insulin at POC. With the goal of displaying the practical nature of manufacturing insulin using CFS, a comparative analysis of the cost of goods sold (COGS, $/g) for the proposed bioprocess will be made against the current state of the art. We hypothesize that this technoeconomic analysis, in conjunction with the proposed expression, purification, and conversion methods outlined, will allow for the manufacturing of insulin exhibiting similar safety and efficacy profiles, at a cost equivalent or less than that observed by current manufacturing approaches.
References:
1. WHO: The top 10 causes of death. Available from: https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death
2. Lin, X., Xu, Y., Pan, X., Xu, J., Ding, Y., Sun, X., … Shan, P. F. (2020). Global, regional, and national burden and trend of diabetes in 195 countries and territories: an analysis from 1990 to 2025. Scientific Reports. https://doi.org/10.1038/s41598-020-71908-9
3. Rajkumar, S. V. (2020). The High Cost of Insulin in the United States: An Urgent Call to Action. Mayo Clinic Proceedings. https://doi.org/10.1016/j.mayocp.2019.11.013
4. Rathore, A. S., & Shaheef, F. (2019). Shadow pricing and the art of profiteering from outdated therapies. Nature Biotechnology. https://doi.org/10.1038/s41587-019-0049-7
5. Basu, S., Yudkin, J. S., Kehlenbrink, S., Davies, J. I., Wild, S. H., Lipska, K. J., … Beran, D. (2019). Estimation of global insulin use for type 2 diabetes, 2018–30: a microsimulation analysis. The Lancet Diabetes and Endocrinology. https://doi.org/10.1016/S2213-8587(18)30303-6
6. Conner, J., Wuchterl, D., Lopez, M., Minshall, B., Prusti, R., Boclair, D., … Allen, C. (2014). The Biomanufacturing of Biotechnology Products. In Biotechnology Entrepreneurship: Starting, Managing, and Leading Biotech Companies. https://doi.org/10.1016/B978-0-12-404730-3.00026-9