Use of new biocatalysts is making many industrial processes more effcient. Dr Jim Lalonde, Senior Vice President, R&D, Codexis, looks at the logistics and drivers behind pharma’s increasing interest in biocatalysis for drug development and manufacturing
Dr Jonathan Vroom (scientist at Codexis) preparating a library of diverse biocatalysts
In July 2014 Codexis, a biotechnology company based in Redwood City, California, US, granted GlaxoSmithKline (GSK) a licence to use its CodeEvolver platform technology to develop novel enzymes for use in the manufacture of GSK’s human pharmaceutical and healthcare products. As part of the agreement, which is worth US$25m plus milestones and potential royalties, the CodeEvolver protein engineering platform will be installed at GSK’s research and development site at Upper Merion, Pennsylvania.
This is the first time that Codexis has licensed its protein engineering platform technology to any party in the healthcare field, but it is likely to be the first of a series of similar licences as the pharma industry increasingly recognises the advantages of incorporating enzymatic processes into the manufacture of drugs.
Over the past few decades, the pharma industry has been under pressure to reduce its use and generation of hazardous substances, and limit the environmental impact of manufacturing
The manufacture of active pharmaceutical ingredients (APIs) is renowned for being complex and materials-intensive, requiring many processing steps and generating higher levels of waste/kg output than other chemical manufacturing industries.1 Over the past few decades, the pharma industry has been under pressure to reduce its use and generation of hazardous substances, and limit the environmental impact of manufacturing. Interest has grown in the development of Green Chemistry technologies, particularly since the US Pollution Protection Act (1990).
The principles of Green Chemistry aim to ‘design and produce cost-competitive chemical products and processes that attain the highest level of the pollution-prevention hierarchy by reducing pollution at its source’.2 Adopting Green Chemistry could not only provide more sustainable manufacturing options, but could also bring significant cost-savings to the pharma industry through more efficient manufacturing with minimal hazardous waste products.
Companies are likely to adopt greener chemistries only if the cost-savings exceed the cost of implementation and maintenance
Previous reports have indicated potential cost-savings for the combined chemical industries in the region of $65bn by 2020, but adoption of Green Chemistry principles by the pharma industry has been limited so far.3 The reality is that companies are likely to choose to adopt greener chemistries only if the cost-savings exceed the cost of implementation and maintenance4.
However, biocatalysis is one green technology that is seeing greater adoption across the pharma industry. Since the 1980s, biocatalytic methods have developed from bench-scale experimental research tools into powerful methods that are now being applied on an industrial scale for alternative chemical synthesis. The use of enzymes can provide higher activity and selectivity, as well as operate under easier and safer conditions that are less environmentally harmful or energy-intensive than some currently used chemical processes.
For pharmaceutical companies such as GSK, biocatalysts can be deployed for individual transformations to offer more efficient and sustainable manufacturing processes with lower costs than traditional chemical reactions. Enzymes are useful for generating isomerically pure compounds because their chiral active sites can discriminate between different stereoisomers and regioisomers for enantio- and regioselective chiral chemistry.5,6 Enzymes can also be used to design shorter and simpler synthetic routes that are not accessible by classical chemical approaches. By reducing the number of steps needed to make an active pharmaceutical, the cost-savings and sustainability benefits provided by enzymatic routes are significant.
Developing biocatalytic approaches requires the identification and optimisation of enzymes specifically for the required reaction and industrial processing conditions. Codexis has a significant track record of delivering evolved biocatalysts for pharmaceutical and fine chemical applications over an extended period. Indeed, Codexis has won the US EPA Presidential Green Chemistry Award three times: in 2006, 2010 and 2012.
Developing biocatalytic approaches requires the identification and optimisation of enzymes specifically for the required reaction and industrial processing conditions
Some well-known examples of green processes developed by the company include development of a bacterial halohydrin dehalogenase (HHDH) for use as a biocatalyst in the manufacture of ethyl (R)-4-cyano-3-hydroxybutyrate (HN), which is the starting material for production of atorvastatin (the cholesterol-lowering drug marketed as Lipitor).7 The resulting biocatalyst had 35 amino acid changes from the starting enzyme, and was suitable for commercial use, including improving the volumetric productivity of the process over the wild-type enzyme by 4,000-fold.
Elsewhere, Codexis was involved in the development of a new biocatalytic process in the production of montelukast sodium (Singulair, an antiallergy and antiasthmatic treatment).8 Directed evolution was used to engineer a novel ketoreductase (KRED) to catalyse the asymmetric reduction of (E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-oxopropyl) benzoate to the corresponding (S)-alcohol, a key intermediate in the synthesis of montelukast sodium. The catalytic reduction replaced the use of DIP-chloride, an expensive, stoichiometric asymmetric reductant. The engineered KRED’s biocatalytic activity was improved by 3,000-fold over the enzyme from nature.
In another example, a biocatalyst was developed to replace a rhodium-catalysed asymmetric enamine hydrogenation for the large-scale manufacture of sitagliptin (Januvia, an antidiabetic treatment)9. The resulting biocatalytic process was incorporated into sitagliptin manufacturing for a more efficient, economical and environmentally cleaner process. In this case, no enzyme from nature was found to be capable of the desired transamination and so one was designed using proprietary molecular modelling methods followed by CodeEvolver directed evolution technology to improve the catalytic efficiency of the starting enzyme by more than 25,000-fold.
Over the past 12 years, Codexis has developed industry-leading, proprietary technologies for custom development of ‘ideal’ biocatalysts by combining cutting-edge advances in computational molecular modelling, molecular biology, high throughput chemistry and bioinformatics. The CodeEvolver directed evolution technology platform licensed to GSK merges capabilities in all of these fields to enable the rapid development of custom-designed biocatalysts that are highly optimised for efficient chemical transformations and manufacturing processes. This technology platform is comprised of a) proprietary methods for the design and generation of diverse genetic libraries, b) automated high throughput screening techniques suitable for testing thousands of the best candidate variants, and c) computational algorithms for the interpretation of screening data, thereby eliminating the need to run tens of thousands of additional costly experiments.
The CodeEvolver technology is enzyme agnostic, which allows it to be applied to a wide range of chemical transformations
There are several protein engineering technologies available for enzyme optimisation, but the CodeEvolver technology offers the ability to design performing proteins more effectively than other engineering tools on the market, both in terms of its speed and the specificity of the results it delivers.
‘We chose the Codexis platform after a thorough evaluation of the enzyme evolution landscape. The CodeEvolver technology is enzyme agnostic, which allows it to be applied to a wide range of chemical transformations. Codexis is also constantly improving the platform, which has allowed the company to stay at the cutting edge of enzyme evolution technology,’ said Doug Fuerst, Technology Development Lead, Synthetic Biology at GSK.
The proteins designed by this technology can perform multiple potential functions, ranging from biocatalysts that will catalyse chemical reactions more cost-effectively than alternative chemistries, to enzymes useful in the production of therapeutics, diagnostic or prophylactic products.
Our goal is to manufacture small molecules more efficiently and sustainably and this platform will assist us to do that
‘Our goal is to manufacture small molecules more efficiently and sustainably and this platform will assist us to do that,’ said John Baldoni, Senior Vice President, Platform Technology and Science of GSK. ‘We are evaluating opportunities and planning to use Codexis technology across the entire GSK development portfolio of small molecule assets. This includes all therapeutic classes,’ added Fuerst.
Mark Buswell, Head of Advanced Manufacturing Technologies at GSK, pointed out that one can evolve an enzyme randomly, but it takes a tremendous amount of time to determine which amino acid to change or whether to change one or more. ‘What Codexis has done is crack the massive bioinformatics challenge,’ he said.
As part of the agreement, Codexis is providing comprehensive technology transfer services to GSK over a two-year period. This includes comprehensive support services, from helping with laboratory design, equipment and bioinformatics software selection, to working directly with GSK’s scientists at Codexis’ Redwood City facilities. This training programme will enable GSK’s scientists to learn the complete process for designing and developing novel biocatalysts. Once GSK’s new lab in Pennsylvania is complete, Codexis’ scientists will continue to provide advisory support, including visiting GSK’s site while the newly-trained GSK scientists take over the leading roles in running the CodeEvolver directed evolution methodologies on additional API projects at GSK.
The collaborative nature of this licensing approach will benefit the pharmaceutical industry as a whole in the long term
The new licensing approach benefits companies by accelerating their ability to apply biocatalysis to their own processes. In addition, licensees will be able to use the CodeEvolver directed evolution technology at their own facilities, providing internal control of their projects and significant cost savings and sustainability benefits over alternative methods. It will also minimise the need to disclose confidential information to third parties. In addition, the company will provide technical support and training to each licensee, enabling them to use the technology independently.
The collaborative nature of this licensing approach will benefit the pharmaceutical industry as a whole in the long term, as other development partners will be working in parallel with Codexis to convert the production of additional drugs so as to incorporate biocatalytic processes.
The company is already in discussion with other companies who may be interested in licensing its CodeEvolver directed evolution technology platform and also foresees licensing agreements with companies in industries beyond pharmaceuticals. The CodeEvolver directed evolution technology platform is suitable for use with practically any enzyme class and can be applied across a wide range of chemical transformations representing numerous manufacturing opportunities. Therefore, any company that can visualise deriving value from using or marketing novel, high performance enzymes or other proteins is a candidate for a CodeEvolver directed evolution technology platform licence from Codexis.
1. Sheldon RA (2007). Green Chemistry 9: 1273–1283.
2. US EPA, Basics of Green Chemistry, pub online: http://www2.epa.gov/green-chemistry/basics-green-chemistry#twelve
3. MacDonald G (2014). In-Pharma Technologist, 1 October, pub online: http://www.in-pharmatechnologist.com/Ingredients/How-green-was-my-Valium-Not-very-despite-industry-claims
4. Ciriminna R, Pagliaro M (2013). Organic Process Research & Development 17: 1479–1484.
5. Barrozo A, Borstnar R, Marloie G, Caroline S, Kamerlin L (2012). Int J Mol Sci 13: 12428–12460
6. Huisman GW, Collier SJ (2013). Curr Op Chem Biol 17: 284–292.
7. Fox RJ, Davis C, Mundorff EC, et al (2007). Nature Biotech 25(3): 338–344.
8. Liang J, Lalonde J, Borup B, et al (2010). Org Process Res Dev 14: 193–198.
9. Savile CK, Janey JM, Mundorff EC, et al (2010). Science 329: 305–309.
CodeEvolver is a registered trademark of Codexis, Inc