A key step in the manufacture of a number of active pharmaceutical ingredients (APIs) and intermediates is the use of an enzyme to provide chirality to the final molecule, typically the biocatalytic reaction may be one part of a multi-reaction process, and so the production of the target enzyme needs to be at extremely lost cost to make the overall process economically viable.
This poster describes the steps taken at Dr. Reddy's Chirotech Technology Centre in Cambridge UK to go from idea (enzyme type) to biocatalyst manufacture (1000's litres fermentation).
Each of the following steps forms part of this process:
1. Screening – commercial versus in-house (or literature) enzymes.
2. Enzyme identification – sequence information.
3. Gene isolation or synthesis – based on sequence and desired recombinant host strain.
4. Cloning – into industrial strain (eg. Escherichia coli BL21(DE3)-T1R).
5. Enzyme engineering if required.
6. Expression studies – shake flask (soluble vs insoluble enzyme).
7. Fermentative growth, optimisation of conditions and scale-up – high cell density, enzyme titre and quality of enzyme.
8. Enzyme isolation – Generic scaleable downstream process.
9. Large scale process transfer – process scale up considerations – final product form etc...
The route we have developed has made possible a 5-day 20 L pilot scale fermentation and downstream process from the initial glycerol stock seed stage to final enzyme product.
The extremely low cost contribution of our enzymes is achieved through the combination of molecular biology, fermentation and an efficient downstream process as described here.