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Pharmaceutical Technology APIs, EXCIPIENTS, AND MANUFACTURING eBOOK 2021 13 The pace of discovery of new enzymes has risen with their use in the pharmaceutical industry. Ap- proximately 1000 new enzymes have been intro- duced since 2017 across a number of classes, and with research continuing, many more can be ex- pected in coming years (3–4). Biocatalysis is now a mainstay of the manufacture of products ranging from foodstuffs to cosmetics to medicines. Multiple scientific advances underpin the growth in biocatalysis. First, the advent of ultra-high through- put next-generation sequencing has led to vast data stores of protein and gene sequences. The open-source protein database TrEMBL, for example, now contains substantially more than 200 million entries (5). Advances in protein engineering have also had a dramatic effect. Natural enzymes have evolved with very specific functions over billions of years and, for the most part, work most efficiently under very specific and mild conditions, such as ambient temperature and pressure, and a relatively neutral pH. Directed evolu- tion has allowed DNA to be manipulated in such a way that tailored enzymes can be created (6), with prop- erties optimized for specific reactions and, potentially, more forcing conditions. It is for this evolution of the technology that the American scientist Frances Arnold was awarded the Nobel Prize in Chemistry in 2018. The advantages of immobilization Despite the advantages that biocatalysis offers, en- zymes are expensive and are difficult to recover and reuse. One solution to these downsides is to immobi- lize the enzyme in some way, so that it can be filtered off from the reaction solvent and washed for reuse, rather than used in solution. Immobilization can also improve an enzyme's sta- bility. If an enzyme is immobilized onto and within cavities of a porous surface—such as a polymer or in- organic material—a micro-environment can be cre- ated around it, effectively encapsulating the enzyme in a small pocket of solvent (usually water). This helps to stabilize the enzyme if the macro environment has a pH that is too high or low for the enzyme to oper- ate efficiently, or if the presence of organic solvent is inhibiting its activity. The reaction can then occur in a heterogeneous environment, with the substrate diffusing into the pores where the reaction happens, followed by the product diffusing out. Immobilizing the enzyme has the potential to be advantageous in terms of cost. For cheap, commodity enzymes, this is not such an issue as for novel catalysts generated by directed evolution, where the ability to re- cover and reuse the enzyme has a huge bearing on the economics of a reaction. If an enzyme is reused once, the cost will be halved; this calculation must be offset with the cost of immobilization and the medium onto which it is immobilized. If the enzyme can be reused several times, process costs can fall dramatically. The technology for immobilizing biocatalysts exists and is proven, with examples cited in the literature of multiple cycles of enzyme use, followed by filtration and reuse (7). In a confidential project, a nutraceutical ingredient has been manufactured using an enzyme that has been immobilized onto beads and enclosed within a cartridge that resembles a water filter. Both starting materials for the process and the product are liquids, allowing the catalyst cartridge to be removed once the reaction is complete and put into storage until it is needed again. The reuse of the enzyme has been demonstrated on more than 20 process cycles and has the potential to be used for many more. Immobilization can also reduce concerns about cross-contamination, particularly in multipurpose pharma facilities. An immobilized enzyme is far less likely to cause contamination, although a key part