Process Optimization for R -PAC Production

1. Process Optimization for R-PAC Production N. Leksawasdi1, M. Breuer2, B. Hauer2, P.L. Rogers1, B. Rosche1 1BABS, UNSW, Sydney, NSW, 2052, Australia 2BASF-AG, 67056 Ludwigshafen, Germany

2. What is R-PAC • R-PAC is for R-Phenyl-Acetyl-Carbinol • Precursor for production of ephedrine & pseudoephedrine; used to treat asthma and flu symptoms

3. PDC catalysed reactions PDC

4. Process of model development • Theoretical for general model structure • Experimental for model structure modification & evaluation of constants • Combined theoretical & experimental • Confirmation of model by independent batch biotranformation profile • Optimization by designing feeding profile for fed batch system

5. Theoretical model development According to King and Altman (1956) Full form Simplified form

6. Theoretical model Product Reactants By-products

7. Experimental model development Quantification of kinetics • Enzyme activity • Substrate concentrations • Enzyme deactivation effect • Batch biotransformations for Overall rate of R-PAC formation Rate constants of by-products formation

8. Enzyme activity effect

9. Kb = 1 x 10-4 mM-1.34 h = 2.34 [Benzaldehyde] effect Monod-Wyman-Changeux (MWC) Model R2 = 0.9963

10. [Pyruvate] effect Michaelis – Menten kinetics Model R2 = 0.9973 Km = 10.6 mM

11. Enzyme deactivation effect 0 mM 60 mM Bz

12. 120 100 0 mM 80 Relative enzyme activity (%) 60 40 20 200 mM 0 0 20 40 60 80 100 120 140 Time (h) Enzyme deactivation by benzaldehyde R2 = 0.9827

13. Enzyme deactivation effect Kd1 = 2.64 x 10-3 h-1 kd2 = 1.98 x 10-4 mM -1 h-1 tlag = 5.23 h

14. Overall rate constant & by-product rate constants determination Independent prediction and confirmation k2, Vq, Vr

15. Batch biotransformation R2 = 0.9857

16. Batch biotransformation R2 = 0.9981

17. Overall & by-products rate constants Overall rate constant (k2 ) = 24.8 mmol h-1 U-1 Acetaldehyde rate constant (Vq ) = 0.0156 h-1 (U/ml) -1 Acetoin rate constant (Vr ) = 0.00251 h-1 (U/ml) -1 mM-1

18. Theoretical & experimental model

19. Simulation of biotransformation

20. Confirmation of simulation R2 = 0.9953

21. Model application in fed-batch system • Suggestion of substrates level to be maintained for optimum R-PAC production • Pulse feeding can be designed to achieve optimum R-PAC production • Prediction of fed-batch biotransformation profile

22. Simulation for prediction of optimum substrate level Hourly feed 1.2 Pyr/Bz Initial Volume 1.00 L 4.0 U/ml PDC

23. Feeding profile for 90 mM Bz, 108 mM Pyr Hourly feed 10.3 M Bz Hourly feed 1.4 M Pyr Initial Volume 1.00 L

24. Predictive fed-batch profile 89 mg/U Initial Volume 1.00 L Final Volume 3.39 L

25. Conclusions • Model provides good prediction for batch biotransformation system • Model suggests substrate levels in the range of 90 mM Bz & 108 mM Pyr to be maintained in fed-batch system • Potential for 8-fold higher R-PAC per U than in batch system but verification by experiment is necessary Note : Possible additional effects of inhibition (high R-PAC, acetaldehyde conc.) and inactivation (benzaldehyde droplets) may need to be considered

26. Royal Thai Government, BASF-AG • Professor Peter L. Rogers, Dr. Bettina Rosche • Dr. Vanessa Sandford • Dr. Russell Cail & Malcolm Noble • Dr. Christopher Marquis • Wolfgang Nittel, Sue Jackson • Martin Zarka & Tony Gellert • Mallika Boonmee, Alan Rushby • Lia, Allen, Cindy, Onn, Ronachai, Apple Acknowledgements

27. Questions