JOURNÉE TECHNIQUE BIOPROCÉDÉS. Biologie de Synthèse, industrialisation du procédé Artémisinine

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1 JURNÉE TECNIQUE BIPRCÉDÉS 2 juillet 2015 Biologie de Synthèse, industrialisation du procédé Artémisinine Paul Baduel 1

The Challenge: Supplying Artemisinin Malaria: 700,000 1,000,000

2007-2008 Source: World Malaria Report 2009 Treating malaria would

needed: 150 to 200 tons of artemisinin per year 2-3X increase in

2 The Challenge: Supplying Artemisinin Malaria: 700,000 1,000,000 deaths in 2008 <15% of children <5y old had access to ACTs in Source: World Malaria Report 2009 Treating malaria would require: 189 to 327 million ACT treatments per year Artemisinin needed: 150 to 200 tons of artemisinin per year 2-3X increase in production Stabilization of supply Stabilization of price From Chris Paddon Amyris 2

3 Semi-synthetic Artemisinin, the project: Cultivation Extraction Artemisinin ACT Therapy Months DERIVATIVES Fermentation Chemistry Yeast + Sugar Artemisinic Acid Artemisinin Weeks 3

4 The Alliance 4

5 The tender from BMGF / iw in 2006 Tilman Achstetter, Roberto Spagnoli, Denis Pompon, Bruno Dumas et al. Nat Biotechnol. (2003), 21,

6 Key intermediate compounds Amorpha-4,11-diene (Amorphadiene) -15 carbon ring compound generated by Amorphadiene Synthase (ADS) from FPP Artemisinic Acid (Art Acid) -15 carbon ring compound generated in 3 oxidative/reduction reactions using Amorphadeine as a substrate -Compound of interest for the generation of Artemisinin Artemisinin -Derived from Artemisinic Acid via defined chemistry steps -Can be further modified for more effective forms of treatment From Chris Paddon 6

7 Engineering Yeast to produce artemisinic acid tmgr C 3 SCoA Acetyl-CoA ERG10 thiolase ERG13 synthase reductase tmgr 2 C 3 C Mevalonate FPP PP ERG20 IDI1 ERG19 ERG8 ERG12 UPC2-1 FPP synthase isomerase decarboxylase P-mev kinase mev kinase ADS CYP71AV1 AaCPR1 CYP71AV1 AaCPR1 CYP71AV1 AaCPR1 Amorphadiene Art. Acid Chris Paddon 7

8 It elps to ave All of the Genes Pat Covello - CNRC-PBI AaAD1 AaALD1 From Chris Paddon 8

9 eterologous Expression of the Entire S. cerevisiae Mevalonate Pathway C 3 SCoA Acetyl-CoA ERG10 thiolase ERG13 synthase tmgr 2 C 3 C Mevalonate FPP PP ERG20 IDI1 ERG19 ERG8 ERG12 FPP synthase isomerase decarboxylase P-mev kinase mev kinase CYP71AV1 ADS Amorphadiene AaCYB5 AaCPR1 AaAD1 AaALD1 Art. Acid 9 From Chris Paddon

10 ptimizing fermentation yield and cost Fermentation Cell Bank Different carbon sources studied central metabolism is key for artemisinic acid production Fed batch process ptimized carbon source feed igh Cell Density cultivation Process ptimization Yield Economic aspects 10 Artemisinic acid production and specific yield 10 Process Robustness Scale-up Artemisinic acid * Yps * Titer Yps 2 2 Artemisinic acid 0 P1 P2 P3 P4 Final lab process industrial scale 0 *arbitrary units From Claus Lattemann 10

11 Recovery and purification of Artemisinic acid from fermentation broth Isolation Extraction Recovery Fermentation broth direct solvent extraction crystallization Purification concentration of organic phase solid-liquid-separation Crystallization Drying Purification buffered aqueous washing P drying Process ptimization alkaline extraction dry artemisinic acid powder Scale-up solvent stripping (distillation) Isolation Yield > 80% Artemisinic acid From Claus Lattemann 11

Purification Crystallization Fermentation broth Drying Process

12 Crystallization Isolation Extraction ptimized Crystallization procedure yields to highly pure artemisinic acid Assay > 95 % Purification Crystallization Fermentation broth Drying Process ptimization Scale-up Crystallized Artemisinic Acid Artemisinic acid Claus Lattemann 12

xidation and Ring closure Yes Process ptimization Scale-up

13 CEMICAL CNVERSIN Evolution of Industrial Photooxygenation Semi-synthesis Reduction Esterification Photooxygenation xidation and Ring closure Yes Process ptimization Scale-up Laboratory scale Artemisinin Pilot scale Claus Lattemann 13

14 CEMICAL CNVERSIN - PTXYGENATIN Industrial skid under reaction in Garessio Semi-synthesis Reduction Esterification Photooxygenation xidation and Ring closure Process ptimization Scale-up Artemisinin Claus Lattemann 14

Commercial Production 80 Produktionsstandorte 30 F&E-Standorte synmikro

15 A Global Project A Global challenge for Technology and Process Transfer Strain Fermentation Biotech/ CM Chemistry Fermentation Scale-up Development Commercial Production 80 Produktionsstandorte 30 F&E-Standorte synmikro "Design von Antibiotika" 31-MAY-2012 Dr. Claus Lattemann SEMI-SYNTETIC AERTEMISININ 15

16 Summary and conclusion Semi-synthetisic artemisinin is available at industrial scale through a global consortium of academia, industry and non profit organizations Annual production volume of up to 60 t will stabilize artemisinin supply and price at sustainable level Production of first commercial batches in 2012 MANUBI 2014, Boston 16

17 Acknowledgements UC Berkeley Eric Paradise Dae-Kyun Ro Mario ulett Jim Kirby Sanofi Corinne Brocard Denis Thibaut Ronan Guevel Marc Ferron Bruno Dumas Paul Baduel enri Farret Philippe Mackiewicz Philippe Charreau einrich Decker Michael Köhler Michelle Chang Syd Withers Jay Keasling Peter Krämer Maxime Bellut elmut Meier Robert Feling Laurence Goldenberg Wolfgang Laux Rudolf Göller Andreas Burgard Frederic Griolet Joel Turconi Roberto Villa Andrea Geatti Amyris Biotechnologies Patrick Westfall Kirsten Benjamin Diana Eng Rika Regentin iroko Tsuruta Doug Pitera Jake Lenihan Jack Newman Neil Renninger Kinkead Reiling Institute for neworld ealth Nina Grove Tue Nguyen Kay Monroe Autumn Ehnow anxiao Jiang Tom Treynor Anna Tai Andrew Main Dan Dengrove Michele Fleck Gordon Dang Stephanie Secrest Tizita orning Karl Fischer Derek McPhee Consultants Jasper Rine (UCB) ans van Dijken (Bird Engineering) Paul rtiz de Montellano (UCSF) Pat Covello (CNRC) 17

From LABS to WORKSHOPS

From LABS to WORKSHOPS Semi Synthetic ARTEMISININ Project From LABS to WRKSPS The 2010 W/MMV Artemisinin Conference 11-13 ctober 2010 Antananarivo, Madagascar Presented By enri FARRET, Project Director The Challenge Bring a