BIOCATALYSIS- AN EFFECTIVE TOOL FOR A COST EFFICIENT, SUSTAINABLE & GREEN MANUFACTURING. December 5, 2015

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1 BICATALYSIS- A EFFECTIVE TL FR A CST EFFICIET, SUSTAIABLE & GREE MAUFACTURIG December 5, 2015

2 Pharmaceuticals & fine chemicals are one of the most polluting industries Environmental impact of some of the chemical industries Industries Production Scale (Tons per Year) E-factor il Refining < 0.1 Bulk Chemicals < 1-5 Fine Chemicals Industry > 50 Pharmaceutical Industry >100 A sustainable and greener footprint in chemical production is increasingly becoming a global need There are existing and emerging solutions to counter pollution Source: R.A.Sheldon, Chem & Ind, 1992, 903 ; 1997, 12, IGCW

3 Improving Manufacturing Sustainability: Enzymatic Solutions can improve the overall sustainability profile of pharmaceutical manufacturing Pharmaceutical manufacture typically has > 100 kg raw material consumption / kg product Disposal costs Infrastructure costs Energy costs I (kg) Raw Material 1 Raw Material 2 Raw Material 3 Raw Material 4 Raw Material 5 PRCESS ut (kg) Product By-products LSSES Reject Waste Re-cycle Down-cycle Re-use Re-sale Source: GSK presentation, 2010

4 4 Life Cycle Assessment (LCA) in ovozymes

5 5 Life cycle assessment (LCA) Raw material production Enzyme production Enzyme use Waste water treatment

6 6 Documenting the benefits of our biological solutions Life cycle assessment enables us to compare environmental impacts of conventional processes (often chemically based) and biological processes Conventional Process 1 process Biological Process 2 process User benefit... same benefit to the user - but different environmental impact

7 7 Changes when enzymes replace tin catalyst in fatty acid ester production.. Coconut oil Temperature Tin catalyst Steam Reaction Deodor. Volatile comp. ovozyme 435 recycling Aqueous waste > 180 C 140 C 100 C 60 C 20 C Bleaching agent Bleaching Conventional Chemical catalysis method Enzymatic catalysis method Drying Aqueous waste Filter aid Filtration Solid waste Fatty acid ester production Packing Fatty acid ester

8 8 Saved and used materials and energy per five ton of fatty acid esters Saved Used eat: 5000 MJ ~ 12 m 3 boiling water 220 kg chemicals Tin oxalate Tin Sodium formiate Calcium hydroxide Sulphuric acid Liquid nitrogen Filter aid Water 700 Water litres 700 litres ovozyme g 100 W 200 days Electricity 500 kwh

9 9 Perspectives of enzyme application in fatty acid ester production World use of fatty acid esters for cosmetics is about 120,000 ton per year If all fatty acid esters used for cosmetics were produced by enzyme technology this would reduce C 2 emissions by 20,000 ton per year This corresponds to 2,300 average persons annual load or the annual load of 5,000 person cars

10 ovozymes & Biocatalysis 10

11 11 There are several benefits of using enzymes over chemical reaction to produce APIs General Advantages using Enzymes in API Manufacturing Improved Productivity igher Yields Shortened synthesis route Greater Savings Potential Replaces costly chiral resolving agents Significant reduction in waste streams Greater Selectivity igh level of Stereo-, regio-, and chemo-selectivity Milder reaction conditions Mostly ambient reaction condition Simplified work-streams Simplified processing and purification Fewer byproducts, reducing impurities

12 Based on the reaction types, enzymes are grouped into six main classes Enzyme Class EC o. Selected Reactions ovozymes Enzymes xidoreductase 1 Transferase 2 C= and C=C reduction Reductive amination of C= xidation of C-,C=C, C- and C- Cofactor reduction/oxidation Transfer of functional groups such as amino,acyl, phosphoryl, methyl, glycosyl, nitro & sulphur groups Peroxidases (Catalases) Glucose oxidases Laccases Fructosyltransferases Glucosyltransferases ydrolase 3 Lyase (synthase) 4 Isomerase 5 Ligases 6 Source: ydrolysis of esters, amides, lactones, lactams, epoxides, nitriles & reverse reactions Addition of small molecules to double bonds such as C=C, C= and C= Isomerisations such as racemizations, epimerizations & rearrangement reactions Amylases Lipases Proteases Pectate lyases Pectinases Cellulases Phytases Pullulanases Acetolactate decarboxylases Glucose isomerases Formation of complex compounds but o products at the moment enzymatically active only in presence of ATP as cofactor 12

13 Lipase Catalysed Reactions ydrolysis Epoxidation R 1 R R 1 + R 2 R 1 R 3 R 1 R 3 Carboxylic acid R 4 22 R 2 R 4 R 2 Esterification R 1 + R 2 R 1 R Polycondensation + (C 2 ) n (C 2 ) n (C 2 ) n (C 2 ) n n Interesterification + R 1 R 2 R 3 R 4 R 1 R 4 + R 3 R 2 Polycondensation + 2 (C 2 ) n (C 2 2 ) n (C 2 ) n (C 2 ) n n 2 Transesterification (e.g. methanolysis) + + R 2 R 1 R 2 R 1 Ring opening lipase n 2 C C 2 n m

14 14 We have the most comprehensive lipase portfolio CalA type of enzymes candida antarctica lipase A Allows branching on C- alpha and bulky side chains on the alcohol part Key Applications Kinetic Resolution of 2- phenylbut-3-yn-2-ol Resolution of 1-methyl- 1,2,3,4-tetrahydronapthalen-1-ol CalB type of enzymes candida antarctica lipase B Allows larger groups on acid part and high specificity on alcohol part Key Applications Kinetic resolution of racemic 1,2,3,4- tetrahydroquoniline-2- acetate Selective hydrolysis of hydroxydiethylcarboxylate s Resolution of Rivastigmineintermediate Alcoholysis of glutaric anhydride TLL type of enzymes Thermomyces lanuginosus lipase Allows larger groups on both alcohol and acid parts Key Applications Kinetic Resolution by desymmetrization of CDE-key intermediate for Pregabalin

15 15 ovozymes Immobilized Lipases ovozym 435 Lipozyme TL IM ovozym CALB enzyme adsorbed on Macroporous Acrylic Resin TL enzyme adsorbed on Silica Gel RM Enzyme adsorbed on Macroporous Anionic Resin

16 16 Some of the other important enzymes in ovozymes portfolio are the proteases- Alcalase & Savinase Alcalase subtilisin based alkaline proteases Available as liquid formulations Alcalase 2.4LFG & Alcalase 2.5L Key applications Dynamic kinetic resolution of protected Amino acids eg. DKR of Benzylated Tyrosine Savinase subtilisin based alkaline proteases Availaible as Savinase 12T solid granulate & liquid formulation Savinase 16L Key applications Kinetic resolution of vince lactam-key intermediate for Carbovir & Abacavir

17 17 Biocatalysis is a proven technology for some prominent API synthesis Case Studies of Application developed by Innovator using ovozymes Enzymes Pregabalin- Lipase from TL Source Darunavir- ovozyme 435/Lipozyme CALBL Moxifloxacin- Lipozyme CALBL Prostaglandin- ovozym 435 (immobilised lipase) Cosmetic Emollient Ester- ovozym 435 (immobilised lipase)

18 18 Pregabalin Biocatalytic Route Improvements due to replacing chemical process with chemoenzymatic reaction Pregabalin Chemical synthesis Pregabalin Biocatalytic Route ovozymes Lipase Lipozyme TL 100 L Inefficient Resolution at rac.pregabalin stage

19 Flow Chart of Pregabalin Process.. Racemisation Pregabalin Recycling (R)-Diester (S)-Diester Water Enzyme (R)-Diester (S)-Monoester Water Enzyme (S)-Monoester Water Enzyme Water Enzyme Biotreatment Key Advantages of Biocatalytic Route Low protein Loading(0.8%) Resolution at first stage-wrong isomer can be recycled igh throughput All reactions conducted in water E-factor improved from 86 to 17 Starting material reduction by 800 Mt (cumulative) Drastic reduction in solvent usage Mandelic acid usage-500mt eliminated (Cumulative) Energy usage reduced by 83% 19

20 20 Pregabalin Chemoenzymatic route used >5x less inputs than Chemical Route Raw Material Inputs for 1000kg API utput Inputs Chemical Route Kg Chemoenzymatic Route Kg Reduction CDE % Enzyme S-Mandelic acid Raney ickel Solvents % 88% Total

21 Substrate concentration can be as high as 100g/L Reaction can be carried out using lipozyme CALBL or ovozym 435 Reaction can be carried out using water as a solvent Reaction is carried out under mild & ambient conditions 21 Darunavir Key Intermediate Biocatalytic Route Lipozyme CALBL is commercially used for stereoselective synthesis of key bicylcic intermediate for Darunavir Darunavir Synthesis Darunavir Intermediate Biocatalytic Route + 2 S 2 Acylation a 2 P 4 buffer, C Lipozyme CALBL or ovozym 435 Key Bicyclic Intermediate + Key Advantages of Biocatalytic Route: S 2 hydrolysis Darunavir Key Intermediate

22 22 Moxifloxacin Key Intermediate Biocatalytic Route Key step for moxifloxacin key intermediate manufacturing is enzymatic Moxifloxacin Side Chain Synthesis Moxifloxacin Synthesis Pd/C, 2 60 C Ac 2, TEA DMAP Toluene,25 C Enzyme F F F + F F Benzylamine Cl Conc.Cl F Moxifloxacin Moxifloxacin Side Chain

23 Moxifloxacin Key Intermediate Biocatalytic Route Lipozyme CALBL or ovozym 435 can be used for desymetrisation of racemic substrate to the required 2S,3R isomer dimethyl 1-acetylpiperidine-2,3-dicarboxylate Lipozyme CALBL/ovozym 435 Phosphate buffer p C Required Isomer + dimethyl (2S,3R)-1-acetylpiperidine- 2,3-dicarboxylate (2R,3S)-1-acetyl-2-(methoxycarbonyl)piperi dine-3-carboxylic acid Key Advantages of Biocatalytic Route: Biocatalytic reaction is carried out at high substrate concentrations ~100g/L Reaction yields are >90% & highly regioselective. Reaction can be carried out using appropriate solvent or without any solvent in water with appropriate buffer Reaction is carried out under mild & ambient conditions 23

24 Biocatalytic reaction is carried out at high substrate concentrations. Reaction yields are >90% & highly regioselective. Reaction can be carried out using appropriate solvent or without any solvent Reaction is carried out under mild & ambient conditions 24 Latanoprost & Travoprost Biocatalytic Route ovozym 435 is used for the key regioselective esterification step Latanoprost & Travoprost Synthesis Multiple Steps Ac Corey Lactone Latonoprost & Travoprost Biocatalytic Route Latanoprost C Ph 3 C 3 C ovozym 435 C Ph C C Travoprost C 3 C 3 C Latanoprost C PhCF 3 Ph PhCF 3 ovozym 435 PhCF 3 Travoprost Latanoprost Travoprost Key Advantages of Biocatalytic Route:

25 25 Case story: Biocatalysis for cosmetic Making chemicals more efficiently Emollient ester Esterification for production of cosmetic fatty acid esters. Chem. Soc. Rev., 2013, 42, lipases and their hydrolytic, esterifying and acylating activities show enormous potential for implementation in the production of cosmetic ingredients

26 26 Future outlook of biocatalysis is very promising Biocatalysis evolution so far and future outlook So Far the first and second wave The third wave: future outlook First wave living cells applied to useful chemical transformations Second Wave Initial protein engineering techniques extended range of enzymes to allow synthesis of unusual synthetic intermediates Directed Evolution Variant Selection or screening Advanced and Emerging Technologies Smarter Libraries of Enzymes DA technology & Protein engineering Bioinformatics Molecular Modelling studies-docking of substrate with proteins Reaction engineering & process intensification using biocatalysts igh throughput enzyme screening Enzyme screening kits

27 ovozymes Introduction 27

28 Everywhere you need us

29 ovozymes in numbers

30 ovozymes in 2014

31 Thank you for attention CTACT AME : DIES AIR TITLE : REGIAL BUSIESS MAAGER DI@VZYMES.CM