ALTERNATIVE APPROACHES TO INSULIN MANUFACTURING USING CELL-FREE SYSTEMS

Abstract

The modular nature of cell-free protein synthesis (CFPS) has resulted in a paradigm shift in the way scientists can design, discover, and manufacture therapeutic proteins. This dissertation reports on three advances made in point-of-care (POC) manufacturing of therapeutics by using CFPS systems to address current challenges around pandemic preparedness and future therapeutic shortages. First, the utility of both prokaryotic and eukaryotic cell-free systems (CFS) was exploited to synthesize human proinsulin to assess the extent of post-translational modification and product quality achieved by each CFS. Second, an Òon-columnÓ purification approach was developed for post-translational conversion of proinsulin into mature human insulin using a specialized set of proteases. Third, a technoeconomic model was utilized to assess the cost effectiveness of cell-free manufacturing of insulin in comparison to the current state of the art. A less complex protein target, griffithsin (GRFT), was also tested using similar approaches. According to a 2022 study, approximately 1.3 million diabetic Americans are currently rationing insulin due to cost. Additionally, microsimulations have shown that by the year 2030, half of Type II diabetic patients are expected to face challenges accessing insulin. Currently, insulin manufacturing takes place in good manufacturing practice (GMP) facilities which utilize in vivo fermentations expressing proinsulin using E. coli and P. pastoris cell lines. While efficient, these fermentations take days to weeks to complete and are not achievable at the POC. For this reason, the in vitro approach enabled by CFPS systems offers a significant advantage, as it is more rapid and can be utilized for production of therapeutics at the POC and on-demand. This dissertation reports the reproducible and soluble expression of difficult-to-express proinsulin, as well as antiviral GRFT, in under 24h using both E. coli and ALiCE? (N. tabacum) CFS. Specifically, a series of cell-free reaction parameters were adjusted including, plasmid concentration, temperature, reaction time, chaperone concentration, and redox potential to achieve optimal protein titer. This dissertation also highlights a cost reduction in purification and post-translational conversion of proinsulin into recombinant human insulin by using an Òon-columnÓ immobilized metal affinity chromatography (IMAC) approach. The results suggest that by reducing the number of unit operations using this approach, a lower cost of goods sold (COGS) for insulin could be achievable with further optimization. To evaluate and optimize the manufacturing cost of the bioprocess outlined in this study, SuperPro Designer software was utilized to identify which components within the process were cost drivers. Additionally, a sensitivity analysis was performed around unit operations and parameters which were a source of increased cost and compared to the COGS of insulin using current manufacturing approaches. Keywords: human insulin; proinsulin; cell-free protein synthesis, CFPS; cell-free; transcription, translation, in vitro; protein engineering; biomanufacturing; bioprocess; point-of-care, POC; post-translational modification, PTM; biologics; pandemic preparedness; antivirals; next-generation biomanufacturing; proteases; enzymes; purification; immobilized metal affinity chromatography, IMAC