Pharmaceutical manufacturers are showing increased interest in incorporating biocatalysis into manufacturing processes. Using enzymatic approaches in place of traditional chemical synthesis can offer a number of advantages. Enzymes are naturally occurring catalysts that can often provide higher activity and selectivity compared with chemical catalysts, producing a higher yield of chemical product. Enzyme-based processes can be faster and less onerous than traditional syntheses, achieving results with fewer processing steps. Reducing the number of steps can also save considerably on manufacturing costs as a result of the reduced labour and materials requirements, and mean faster delivery of product.
Enzymes are particularly useful for generating isomerically pure pharmaceuticals and fine chemicals. Their chiral active sites can discriminate between different stereoisomers and regioisomers for enantio- and regioselective chiral chemistry.1,2 These advantages can help further to reduce costs and offer considerable benefits for drug development and manufacturing.
Despite the many advantages of biocatalysis, manufacturers have been slow to adopt industrial-scale enzymatic approaches. A significant hurdle has been the requirement for most naturally existing enzymes to undergo artificial optimisation in vitro before they can be used in industrial process conditions. These conditions may involve high temperatures, extreme pH levels and the use of organic solvents and detergents that disrupt natural functioning of enzymes.
Researchers in the 1980s developed protein engineering techniques to produce enhanced enzymes that could function under unfavourable conditions.3,4 As these techniques gained credibility, scientists developed these methods to alter the actual functionality and capabilities of enzymes, initially through targeting specific regions of enzymes’ active sites. This site-directed mutagenesis approach3 enabled development of enzymes with new substrate ranges that were suitable for more unusual synthetic processes, including those required by the pharma industry.
In the 1990s, alternative approaches of directed evolution through random mutagenesis were developed, where enzymes are put through sequential rounds of random mutagenesis combined with high-throughput screening and selection for specific traits.5 The process creates very large libraries of mutant enzymes with great diversity that can be searched for properties of interest. Small changes in enzyme properties accumulate over time, resulting in larger changes in function.
Now, full R&D programmes can be completed in a matter of months through using the latest enzyme optimisation technology platforms
In the past, enzyme development and optimisation programmes took several years to complete, requiring significant investment. Now, however, full R&D programmes can be completed in a matter of months through using the latest enzyme optimisation technology platforms, such as CodeEvolver Directed Evolution Technologies. These platforms exploit recent significant advances in sequence data collection and understanding, including from next generation sequencing technologies, improvements in bioinformatics and novel computational algorithms, all of which have increased data access and analysis.
These developments provide valuable new information for enzyme optimisation, enabling researchers to focus on and identify optimal mutations more efficiently, and greatly accelerate the speed and success rates of enzyme optimisation programmes. The CodeEvolver platform now enables rapid development of custom-designed enzymes for specific chemical reactions and manufacturing processes.
Directed evolution technologies have been critical in reducing the timescales and investment required for enzyme optimisation programmes. Other enzyme technologies are also being investigated for commercial manufacturing, as organisations look for further ways to improve their cost-effectiveness and reduce use of materials and resources. One approach that is gaining traction is enzyme immobilisation, which is particularly useful for enhancing enzyme stability under various process conditions and allows for easier separation of enzymes from a reaction mixture. Immobilisation can also allow enzymes to be re-used at a commercial scale, which could bring major cost and resource savings.
Enzyme immobilisation is not a new concept: it has been used for industrial applications since 1967, when immobilised aminoacylase was first applied for resolution of amino acids.6 Immobilisation generally works by introducing additional binding forces (covalent and non-covalent) to the enzyme’s external matrix. Various methods for immobilisation have been developed over the years; common methods include covalently binding to a solid support, or using carriers through crosslinking, adsorption or entrapment.
Different immobilisation processes can result in differing effects on the same enzyme’s stability
Immobilisation serves two broad purposes: first, by tightly fixing the enzyme, the protein cannot unfold or become denatured during manufacturing processes; and second, it actually improves the stability of enzymes beyond that of free form enzymes, enhancing the activity and performance under different pH, temperature and storage conditions, such that the enzyme can maintain high turnover rate and catalytic activity over time.6,7
The immobilised enzyme’s stability can be affected by a number of factors (interaction with support, binding position, number of bonds, etc). Ultimately, however, the degree to which an enzyme’s stability is improved has been shown to depend on the actual immobilisation process that is employed.8 Accordingly, different immobilisation processes can result in differing effects on the same enzyme’s stability. It has been shown that immobilisation can improve enzyme stability and activity in organic solvents up to 100-fold.9 A number of approaches have been developed over the past decade for a variety of enzyme types in diverse processes.10,11,6
Another key performance feature of enzymes is their selectivity. When developing enzymes for novel substrates, achieving the necessary selectivity can be a considerable issue. As mentioned previously, the enzyme’s stereo- and regioselectivity are particularly important for the pharma industry. Immobilisation has been shown to alter enzyme selectivity, or even to change a non-selective enzyme into a stereoselective enzyme.11
Many studies have also demonstrated higher activity of immobilised enzymes compared with unfixed forms. It has additionally been shown that immobilisation can decrease substrate inhibition, which also has the effect of increasing activity.6 Activity can be further improved by using oriented immobilisation, as opposed to random immobilisation, to ensure that modifications to the enzyme don’t inadvertently affect its active site. In some cases, targeted immobilisation can enhance activity through improved access to the enzyme’s active site. Immobilisation of lipases, for example, has been shown to enhance their activity in a number of applications.12,13,6
Immobilisation can also allow for the enzyme to be re-used, which considerably improves the efficiency and costs of a process
Immobilisation can also allow for the enzyme to be re-used, which considerably improves the efficiency and costs of a process.10 Recently, Merck scientists developed an immobilised form of CAL-B (Candida antarctica lipase B) with 15 times greater stability and enhanced activity compared with previously available preparations. The immobilised CAL-B was used for dynamic kinetic resolution of an azlactone, yielding a chiral starting material for manufacturing odanacatib (a cathepsin K inhibitor that is in clinical trials for treatment of osteoporosis). The enzyme’s improved stability enabled the development of a continuous process: running the reaction in continuous mode provides significant production cost savings.14
Transaminases are commonly used for the direct synthesis of chiral intermediates and APIs.2 The optimisation of transaminases for new chemical processes has contributed to significantly improved synthetic routes for manufacturing pharmaceuticals. For example, a transaminase enzyme was recently developed to replace a rhodium-catalysed asymmetric enamine hydrogenation, in large-scale manufacture of sitagliptin, an anti-diabetic treatment.15
Using computer-aided molecular modelling, Codexis researchers designed an engineered transaminase with marginal activity for the desired substrate and then used CodeEvolver directed evolution technology to fully optimise the enzyme with high activity and selectivity for the target chiral amine product. The resulting biocatalytic process has been incorporated into sitagliptin manufacturing for a more efficient, economical and environmentally cleaner process. In a further development, the sitagliptin transaminase was immobilised on a polymer resin, for enzyme reuse. This resulted in a highly active and stable immobilised enzyme that can be used to synthesise sitagliptin in organic solvents for 10 cycles, with minimal performance deterioration.14
Applying immobilised enzymes to different screening processes will potentially accelerate new-molecule and API development
Codexis and Purolite are now collaborating to further develop enzyme immobilisation technologies, starting with a new kit of immobilised transaminases. These screening kits will allow scientists to apply immobilised enzymes to different screening processes, and will potentially accelerate new-molecule and API development.
Incorporating enzyme-based processes into pharmaceutical manufacturing now offers many cost advantages. By developing improved new synthetic routes, with smarter efficiency and higher yield, enzymatic approaches have been shown to reduce steps and offer cleaner, cheaper alternatives to many traditional industrial processes.
References
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