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16 Pharmaceutical Technology APIs, EXCIPIENTS, AND MANUFACTURING eBOOK 2021 P h a r mTe c h . c o m APIs Immobilizing a biocatalyst also makes it compatible with a flow chemistry process, either using a cartridge of packed beads or lining the inside surface of a flow reactor with the biocatalyst, so the starting materials simply have to flow through it at an appropriate rate for reactions to occur. Immobilization in action The challenges of developing a synthetic route for an API in development are illustrated in a case study. The current process uses one of two commercial routes; however, both have issues, particularly relating to in- tellectual property (IP). A biocatalyzed reaction would offer advantages not only in process efficiency and cost benefits but would also avoid the IP issues. The original, classical route to the API works well. The two-step process involves a coupling followed by a hydrolysis; however, one step is protected by a patent. The second route uses a slightly different synthetic path to avoid this IP-protected step; however, the product yield of approximately 25% is not ideal as the two principal starting materials are expensive. Research was conducted to determine if the first route was viable by replacing the hydrolysis step with a biocatalyzed reaction; however, there were no natu- ral enzymes in the literature that could perform the step. In a collaboration with Northumbria University, in-silico screening of other potential enzymes was conducted to create a shortlist of alternative enzymes known to work on molecules with similar substitu- tion patterns. However, none showed activity when trialed on the actual molecule. Further in-silico modeling showed that changing the order of the reactions gave potentially more op- tions for biocatalysis and circumvented the IP pro- tection. Using standard chemical reagents, perform- ing the steps in reverse was feasible; however, in this revised approach, issues with product stability and yield on the hydrolysis step were identified. The stability issues were overcome when using a lipase-catalyzed hydrolysis as the first of the two steps. Research on the second step using both chemical and biocatalytic processes is ongoing, as are efforts to determine how best to immobilize en- zymes for scale-up to a commercially viable synthe- sis. Early indications and calculations suggest that to manufacture a ton of the API material would need an enzyme created via directed evolution that would be cost prohibitive unless immobilization and reuse was possible. While the project is still embryonic, it does demonstrate the challenges of biocatalysis and the potential of immobilized enzymes to open up new areas of chemistry and manufacturing. More work to be done The potential of biocatalysis in pharmaceutical man- ufacturing has been well-established, and much re- search and academic discovery has assisted in its prog- ress. While there are examples of processes proven to be wholly possible using only enzymes, such as the human immunodeficiency virus drug islatravir (9), the commercial reality is that the technology will only be embraced where it is economically viable to do so, and this will involve specific synthetic steps. References 1. Research and Markets, World Biocatalysis & Biocatalyst Market Analysis to 2024–Market Forecast to Grow at a CAGR of 15% from 2019 to 2024, Report (2020). 2. E. Abdelraheem, et. al., React Chem Eng, 4, 1878-1894 (2019). 3. I. Schomburg, et.al., Journal of Biotechnology 261, 194–206 (2017). 4. BRENDA, "The Comprehensive Enzyme Information System," brenda-enzymes.org, accessed Sept. 19. 221. 5. UniProt Knowledgebase, "UniProtKB 2020_06 Results", uniprot. org/uniprot/. 6. A. Kan and N. Joshi, MRS Commun. 9 (2) 441–445 (2019). 7. S. More, Chem. Eng. Journal 15 (334) 1977–1987 (2018). 8. A.A. Homaei et. al., J Chem Biol 6,185–205 (2013). 9. M. Satyanarayana, M. "Scientists Made an HIV Drug Using Noth- ing but Enzymes," Chemical & Engineering News, Dec. 20, 2019. PT