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On-demand insulin manufacturing using cell-free systems with an "on-column" conversion approach
Shayan G Borhani1, Max Z Levine2, Chandrasekhar Gurramkonda1
1Center for Advanced Sensor Technology, Baltimore, MD 21250, USA; Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
Cell-free protein synthesis (CFPS) rapidly produces insulin for diabetes treatment. This advancement decentralizes manufacturing, improving patient access to this vital medication.
Area of Science:
- Biotechnology
- Biochemistry
- Endocrinology
Background:
- Diabetes prevalence is rising, increasing demand for insulin.
- Current insulin production faces supply chain challenges, limiting global accessibility.
- Insulin is a critical recombinant therapeutic, yet access remains a significant issue.
Purpose of the Study:
- To develop a rapid and efficient method for producing mature insulin using cell-free protein synthesis (CFPS).
- To advance decentralized insulin manufacturing closer to the point-of-care.
- To improve accessibility of insulin for diabetic patients globally.
Main Methods:
- Utilized PUREfrex® 2.1 cell-free extract to synthesize a tagged proinsulin construct.
- Employed on-column affinity chromatography for capturing and converting proinsulin to mature insulin.
- Investigated the role of chaperones (FkpA, Skp) in cell-free proinsulin expression.
Main Results:
- Successfully synthesized and purified desB30-insulin in less than a day using CFPS.
- Demonstrated sequential purification and conversion of proinsulin on a Ni-NTA column.
- Identified critical roles for FkpA and Skp chaperones in the process.
Conclusions:
- CFPS offers a time and resource-efficient alternative to current insulin manufacturing methods.
- Simultaneous cell-free expression and on-column conversion enable rapid, on-demand insulin production.
- This approach can significantly improve insulin accessibility and prevent future shortages.
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