1. Development of an Enzymatic Process for Lipitor
Dave Bauer, Mike Burns*, Simon Davidson, Alastair Denholm, Aoife Fahy,
Cathal Healy, John O’Shaughnessy, Éanna Ó Maitiú, Floriana Stomeo,
Gillian Whittaker, and John Wong
The 12th Annual
Green Chemistry & Engineering Conference
Washington, DC
June 24-26, 2008

2. Biocatalysis Center of Emphasis
Centered in Groton, Connecticut; colleagues in Sandwich, UK
Groton Site
Largest R&D site

3. Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Introduce BCE
18 colleagues, including 2 in Sandwich

4. Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Introduce BCE
18 colleagues, including 2 in Sandwich

5. Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Introduce BCE
18 colleagues, including 2 in Sandwich

6. Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Introduce BCE
18 colleagues, including 2 in Sandwich

7. Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Access to Pfizer resources that include:
Chemical Engineers
Introduce BCE
18 colleagues, including 2 in Sandwich

8. Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Access to Pfizer resources that include:
Chemical Engineers
Statisticians
Introduce BCE
18 colleagues, including 2 in Sandwich

9. Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Access to Pfizer resources that include:
Chemical Engineers
Statisticians
Protein Chemists and Crystallographers
Introduce BCE
18 colleagues, including 2 in Sandwich

10. Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Access to Pfizer resources that include:
Chemical Engineers
Statisticians
Protein Chemists and Crystallographers
Primary function is to develop chemoenzymatic routes to small molecule compounds.
This includes all stages of development and approved products.
Introduce BCE
18 colleagues, including 2 in Sandwich

11. New Process for Atorvastatin
Same route, different process
Implemented 2008
Hydroxynitrile is purchased.
Produced by Codexis, 2006 Green Chemistry Award winner
The reduction of hydroxyketone to cis diol is now a biocatalytic step

12. Comparison of Chemical and Biocatalytic Reactions
Chemical process is slow: 80 hours for 6 x methanol distillations to remove boronic wastes. Enzymatic reaction is faster and has a relatively simple work-up.
Quality: Enzymes are highly selective, giving improved cis: trans ratio.
TEB: pyrophoric and toxic
NaBH4: Safety hazard. H2 source. Toxic
Multiple solvents and low temperature requirement eliminated

13. Biocatalytic Routes to Chiral Alcohols
traditionally = resolution (50% yield)
direct hydroxylation looks great on paper - challenging
reduction – first choice, excellent stereoselectivity, high throughput
Accounts of Chemical Research
Biocatalytic Reductions: From Lab Curiosity to “First Choice”
Oliver May, et. al., Acc. Chem. Res. (2007), 40(12) - DSM
“…ketoreductases are the preferred catalyst for ketone reduction…”
Jeffrey C. Moore, et. al., Acc. Chem. Res. (2007), 40(2) - Merck

14. Reaction Mechanism for Ketone Reduction
Biocatalytic routes to chiral alcohols
traditionally = resolution (50% yield)
direct hydroxylation looks great on paper - challenging
reduction – first choice, excellent stereoselectivity, high throughput
Accounts of Chemical Research

15. Reaction Mechanism for Ketone Reduction
KRED: Ketone reductase
KRED2: Enzyme used for cofactor regeneration
NAD(P)H: Nicotinamide Adenine Dinucleotide
Cofactor in the reduced form
NAD(P)H is “bio borohydride”
- Carlos
The NAD(P)H provides the hydride for the reduction

16. Co-factor Recycling Systems
NAD = $1500/kg
NADP = $8000/kg
Talk about need for co-factor recycle
$$$$
years ago, this was not as efficient and precluded the industrial use of ADHs
other systems: FDH, Phosphite dehydrogenase
Substrate Coupled vs. enzyme coupled

17. Intellectual Property Landscape
Reeve (Zeneca), US Patent 6,001,615
Microbial reductases obtainable from Beauveria, Candida, Kluyveromyces, Debaromyces, and Pichia for reduction of
cyanoketoester to diol
Kizaki (Kaneka), US Patents 6,472,544 and 6,645,746
Recombinant E. coli expressing reductase from Candida magnoliae,
and glucose dehydrogenase from Bacillus megaterium for reduction of chloroketoester
Microorganisms and enzymes from in-house screen – lots of hits.
We needed to develop our own or license a biocatalyst
Patents before BCE
Reeve – cyanoketoester
Kizaki - chloroketoester

18. Development of Biocatalytic Route
Decided not to pursue the development of proprietary biocatalyst
Evaluated biocatalysts from vendors
Licensed Ox28 alcohol dehydrogenase from IEP (Wiesbaden, Germany).
Developed reaction. Main issues were:
Work-up / Biocatalyst formulation
Reaction time – unstable hydroxyketone
Liked substrate coupled approach
No gluconic acid waste

19. Biocatalyst Production
Wild type enzymes cloned into E.coli
pOX28
4909 bp
lacI
Kan
IEP Ox28
par
Promoter
oriV
Ox 28
Explain process
Gel electrophoresis showing Ox28 over-expressed protein

20. Development of the biocatalyst formulation
Whole frozen E. coli cells
Cell debris caused problems with phase splits during product work-up
Also note challenges with working with a GMO – lysate shown to be free of GMO
Lysate stabilized
with isopropanol

21. Biocatalyst Production
E. coli fermentation
Cell separation
Cell slurry
microfiltration or
centrifugation
growth of cells In stirred tank
dilute with water
Centrifugation
Heat Treatment
Homogenisation
Cell Disruption
to break cell wall
Denature unwanted proteins
Separation of
Unwanted biomass
to give clear supernate
30,000L fermentor
Enzyme
Stabilisation
Ultrafiltration
Concentration of Solution
Removal of water
Add Isopropyl
Alcohol
Refined Enzyme

22. Development of the Reaction
Work-up  New Enzyme Formulation
Reaction Time  Reaction Kinetics
Next challenge was to improve the reaction kinetics. We wanted to speed up the reaction because the starting hydroxyester slowly decomposes in aqueous conditions. Goal was to decrease reaction time and/or temperature

23. Development of the Reaction

24. Green Chemistry
Prevent waste
Design safer chemicals and products
Design less hazardous chemical syntheses
Use renewable feedstocks
Use catalysts, not stoichiometric reagents
Avoid chemical derivatives
Maximize atom economy
Use safer solvents and reaction conditions
Increase energy efficiency
Design chemicals and products to degrade after use
Analyze in real time to prevent pollution
Minimize the potential for accidents
Paul Anastas and John Warner in Green Chemistry: Theory and Practice (Oxford University Press: New York, 1998)

25. Twelve Principles of Green Chemistry
Prevent waste
Design safer chemicals and products
Design less hazardous chemical syntheses
Use renewable feedstocks
Use catalysts, not stoichiometric reagents
Avoid chemical derivatives
Maximize atom economy
Use safer solvents and reaction conditions
Increase energy efficiency
Design chemicals and products to degrade after use
Analyze in real time to prevent pollution
Minimize the potential for accidents

26. Solvent Savings

27. Solvent Savings
Organic solvent usage and waste reduced by 65% per annum

28. Elimination of Toxic Chemicals
Triethyborane – pyrophoric and toxic

29. Elimination of Toxic Chemicals
Triethyborane – pyrophoric and toxic

30. Elimination of Toxic Chemicals
Sodium borohydride
Safety hazard. H2 source

31. Elimination of Toxic Chemicals
Sodium borohydride
Safety hazard. H2 source.

32. Elimination of Cryogenic Conditions
-75°C, high energy use

33. Elimination of Cryogenic Conditions
-75°C, high energy use

34. Green Advantages of Biocatalytic Processes
Fix fonts, maybe get logos, maybe put in map of world with arrows

35. Green Advantages of Biocatalytic Processes
Reduce solvent usage
Fix fonts, maybe get logos, maybe put in map of world with arrows

36. Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable resources.
Fix fonts, maybe get logos, maybe put in map of world with arrows

37. Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable resources.
Gentle reaction conditions (pH & temp)
Fix fonts, maybe get logos, maybe put in map of world with arrows

38. Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable resources.
Gentle reaction conditions (pH & temp)
Catalytic
Fix fonts, maybe get logos, maybe put in map of world with arrows

39. Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable resources.
Gentle reaction conditions (pH & temp)
Catalytic
Very regio-selective and functionally selective; may alleviate need for protection and deprotection steps.
Fix fonts, maybe get logos, maybe put in map of world with arrows

40. Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable resources.
Gentle reaction conditions (pH & temp)
Catalytic
Very regio-selective and functionally selective; may alleviate need for protection and deprotection steps.
Biodegradable
Fix fonts, maybe get logos, maybe put in map of world with arrows

41. Summary and Conclusions
Enzymatic Lipitor Process has Advantages

42. Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process

43. Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
Better Selectivity

44. Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
Better Selectivity
Biocatalytic Processes are Commercially Feasible

45. Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
Better Selectivity
Biocatalytic Processes are Commercially Feasible
Pregabalin and Atorvastatin (Pfizer)

46. Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
Better Selectivity
Biocatalytic Processes are Commercially Feasible
Pregabalin and Atorvastatin (Pfizer)
The Future of Biocatalysis is now!
(Ibuprofen, phenylethylamine, acrylamide)

47. Development of an Enzymatic Process for Lipitor
Dave Bauer, Mike Burns*, Simon Davidson, Alastair Denholm, Aoife Fahy,
Cathal Healy, John O’Shaughnessy, Éanna Ó Maitiú, Floriana Stomeo,
Gillian Whittaker, and John Wong
The 12th Annual
Green Chemistry & Engineering Conference
Washington, DC
June 24-26, 2008

48. Questions?

49. Questions?