From LABS to WORKSHOPS

Transcription

1 Semi Synthetic ARTEMISININ Project From LABS to WRKSPS The 2010 W/MMV Artemisinin Conference ctober 2010 Antananarivo, Madagascar Presented By enri FARRET, Project Director

2 The Challenge Bring a highly sophisticated technology from Academic to Industrial level, in a very short timeframe (3 years) Improve the fermentation performances, in cooperation with Amyris, our biotech partner Increase the recovery yield from 60% to more than 80% from fermentation broth Develop an industrial process for the Chemistry of a very unstable molecule And finally, reach economical performances to be close to the fair price of Artemisinin ( $/Kg)

3 Simple Sugar Acetyl-CoA Acetoacetyl-CoA MG-CoA Mevalonate ERG10 ERG13 ERG12 Mevalonate-P ERG8 Mevalonate-PP tmgr X2 Synthetic Biology ADS Amorphadiene AM/CPR AM/CPR Chemical Conversions xidation and Ring-Closure Peroxidation Microbially Derived Artemisinin Initial route not productive enough to reach economical target Acid Ester ydroperoxide ERG19 IDI1 IPP DMAPP GPP FPP Met erg9::p MET3 -ERG9 Non-Enzymatic AM/CPR Esterification Acid Ester Acid Squalene ERG1,7,11,24,25,6,2,3,5,4 Ergosterol Purification Reduction

4 Simple Sugar Acetyl-CoA ERG10 Acetoacetyl-CoA ERG13 MG-CoA Strain Improvement (Amyris) Adding some genes discovered by Pat Covello tmgr X2 Mevalonate ERG12 Mevalonate-P CYP71AV 1 AaCYB5 CYP71AV 1 AaAD1 AaCYB5 CYP71AV 1 AaALD1 AaCYB5 ERG8 Mevalonate-PP AaCPR 1 AaCPR 1 AaCPR 1 ERG19 IDI1 IPP DMAPP Art. Acid Met GPP FPP erg9::p MET3 -ERG9 Squalene Fermentation improvement: Feeding optimization Cheaper Raw Material Energy adjustment ERG1,7,11,24,25,6,2,3,5,4 Ergosterol

5 Simple Sugar Acetyl-CoA Acetoacetyl-CoA MG-CoA Mevalonate ERG10 ERG13 ERG12 Mevalonate-P ERG8 Mevalonate-PP tmgr X2 Synthetic Biology ADS Amorphadiene AM/CPR AM/CPR Chemical Conversions xidation and Ring-Closure Purification process to be simplified and with a better yield Peroxidation Microbially Derived Artemisinin Acid Ester ydroperoxide ERG19 IDI1 IPP DMAPP GPP FPP Met erg9::p MET3 -ERG9 Squalene ERG1,7,11,24,25,6,2,3,5,4 Non-Enzymatic AM/CPR Purification Esterification Acid Ester Acid Reduction Ergosterol

6 Simple Sugar Acetyl-CoA Acetoacetyl-CoA MG-CoA Mevalonate Mevalonate-P Mevalonate-PP Met IPP ERG10 ERG13 tmgr X2 ERG12 ERG8 ERG19 IDI1 GPP FPP Squalene DMAPP erg9::p MET3 -ERG9 ERG1,7,11,24,25,6,2,3,5,4 Synthetic Biology ADS Amorphadiene AM/CPR AM/CPR Non-Enzymatic AM/CPR Redesign the Recovery Process Direct Extraction with a solvent Removing Impurities Crystallisation in water Performances Yield > 80% Recovery costs divided by 3 Down Stream Process in standard equipments Very safe powder crystallisation igh Quality (Assay > 95%) Purification Reduction Ergosterol

7 Quality of Artemisinic Acid Artemisinic Acid in the fermentation broth Crystallized Artemisinic Acid

8 Simple Sugar Acetyl-CoA Acetoacetyl-CoA MG-CoA Mevalonate ERG10 ERG13 tmgr X2 Synthetic Biology ADS Amorphadiene Initial Route cannot ERG12 reach technical and Mevalonate-P AM/CPR ERG8 economical targets Mevalonate-PP AM/CPR Chemical Conversions xidation and Ring-Closure Peroxidation Microbially Derived Artemisinin Acid Ester ydroperoxide ERG19 IDI1 IPP DMAPP GPP FPP Met erg9::p MET3 -ERG9 Squalene ERG1,7,11,24,25,6,2,3,5,4 Non-Enzymatic AM/CPR Purification Esterification Acid Ester Acid Reduction Ergosterol

9 Simple Sugar Acetyl-CoA ERG10 Acetoacetyl-CoA ERG13 Synthetic Biology ADS Amorphadiene MG-CoA tmgr X2 Mevalonate ERG12 Mevalonate-P ERG8 Mevalonate-PP ERG19 IDI1 IPP DMAPP GPP FPP Met erg9::p MET3 -ERG9 Squalene ERG1,7,11,24,25,6,2,3,5,4 AM/CPR AM/CPR Non-Enzymatic AM/CPR We developed a new route (patent pending) ptimized ydrogenation to Produce DAA Activation of the DAA Photo-oxydation Crystallization of Pure Artemisinin Purification Reduction Ergosterol

10 New Synthesis f Artemisinin by Photochemistry Step 1 Step 2 C Starting Material C Et Step 3 Photooxygenation Photosensitizer Et Step 4 Step 5 Crystallization Artemisinin

11 What are the benefit of this new synthesis? 2009 Partnership Update It is a one pot chemistry: we start from Artemisinic Acid and obtain directly Pure Artemisinin Cycle Time is very short: less than 1 week to produce about 500KG of Pure Artemisinin Chemical Yield is doubled compared to the initial chemical route No very expensive catalyst like Molybdenum salts Improved safety by a better reaction control BUT SCALE UP WAS CMPLEX AND LNGER TAN STANDARD CEMISTRY

12 The scale up of Photo-xydation 2009 Partnership Update

13 And the Photo-xydation tomorrow Partnership Update

14 Quality of semi-synthetic Artemisinin Project utcomes We obtain a high quality product, with assay > 95% verall quality meets W specifications for a Starting Material Many successful tests of transformation into Artesunate (API produced within sanofi-aventis) Current representative quality Artemisinin sample (NRA PFI) AU Artemisiten Artemisinin diastereoisomer Artemisinin Minutes

15 And now, what we plan to do? Project utcomes We are transferring the technology into the plants, in order start building capacity. We can propose samples of Industrial Quality mid First half of 2012, we plan to start integration of semi-synthetic Artemisinin in an ACT Second half of 2012, we will propose commercial quantities.

16 Development Semi-Synthetic Artemisinin Phases & Timing Development Technical Transfer Q2, 2008 Co- Development Pilot Scale Up Production Capacity building 1 st integration in ACT s Dev. Phase L 10,000L 100,000L 2nd source of artemisinin for ACT Dev. Phase 2 Industrialization Manufacturing

17 Thank You