December 5, 2015

BIOCATALYSIS- AN EFFECTIVE TOOL FOR A

COST EFFICIENT, SUSTAINABLE & GREEN

MANUFACTURING

Pharmaceuticals & fine chemicals are one of the most

polluting industries

IndustriesProduction Scale

(Tons per Year)E-factor

Oil Refining 106 - 108 < 0.1

Bulk Chemicals 104 - 106 < 1 - 5

Fine Chemicals Industry 102 - 104 5 - > 50

Pharmaceutical Industry 10 - 103 25 - >100

Source: R.A.Sheldon, Chem & Ind, 1992, 903 ; 1997, 12, IGCW 2011

Environmental impact of some of the chemical industries

2

A sustainable and greener footprint in chemical production is increasingly becoming a

global need…

There are existing and emerging solutions to counter pollution

Improving Manufacturing Sustainability: Enzymatic

Solutions can improve the overall sustainability profile

of pharmaceutical manufacturing

IN (kg)

ProductPROCESS

Raw Material 1

Raw Material 5

Raw Material 2

Raw Material 4

Raw Material 3

Out (kg)

LOSSES WasteReject

By-products

Re-cycle

Re-use

Down-cycle

Re-sale

£

££

Pharmaceutical manufacture typically has > 100 kg raw

material consumption / kg product• Disposal costs

• Infrastructure costs

• Energy costs

Source: GSK presentation, 2010

Life Cycle Assessment (LCA) in Novozymes

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5

Raw materialproduction

Enzymeproduction

Enzyme use Waste watertreatment

Life cycle assessment (LCA)

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Process 2Biologicalprocess

Process 1Conventional process

User benefit

Life cycle assessment enables us to compare environmental impacts of conventional processes (often chemically based) and biological processes

Documenting the benefits of our biological solutions

... same benefit to the user - but different environmental impact

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Changes when enzymes replace tin catalyst in fatty acid ester production……..

Reaction

Packing

BleachingBleaching

agent

Tin catalyst

Volatile comp.

Deodor.Steam Aqueous waste

Aqueous wasteDrying

FiltrationFilter aid Solid waste

Fatty acid ester

Coconut oil

Novozyme 435 recycling

> 180°C

140°C

100°C

60°C

20°C

Temperature

Enzymaticmethod

Conventional method

Chemical catalysis

Enzymatic catalysis

Fatty acid ester production

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Used Saved

Saved and used materials and energy per five ton of fatty acid esters

Novozyme 435270 g

Electricity 500 kWh

100 W200 days

Water700 litres

220 kg chemicals

Tin oxalateTinSodium formiateCalcium hydroxideSulphuric acidLiquid nitrogenFilter aid

Heat: 5000 MJ

~ 12 m3

boiling water

Water700 litres

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This corresponds to 2,300 average persons’ annual load or the annual load of 5,000 person cars

If all fatty acid esters used for cosmetics were produced by enzyme technology this would reduce CO2 emissions by 20,000 ton per year

Perspectives of enzyme application in fatty acid ester production

World use of fatty acid esters for cosmetics is about 120,000 ton per year

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Novozymes & Biocatalysis

There are several benefits of using enzymes over

chemical reaction to produce APIs

General Advantages using Enzymes in API Manufacturing

Improved Productivity

− Higher Yields

− Shortened synthesis route

Milder reaction conditions

− Mostly ambient reaction condition

Greater Savings Potential

− Replaces costly chiral resolving agents

− Significant reduction in waste streams

Greater Selectivity

− High level of Stereo-, regio-, and

chemo-selectivity

Simplified work-streams

− Simplified processing and purification

− Fewer byproducts, reducing impurities

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Based on the reaction types, enzymes are grouped into

six main classesEnzyme Class EC No. Selected Reactions Novozymes Enzymes

Oxidoreductase 1

C=O and C=C reduction Reductive amination of C=O Oxidation of C-H,C=C, C-N

and C-O Cofactor reduction/oxidation

Transferase 2

Transfer of functional groups such as amino,Acyl,

phosphoryl, methyl, glycosyl, nitro & sulphur groups

Hydrolase 3 Hydrolysis of esters, amides,

lactones, lactams, epoxides, nitriles & reverse reactions

Lyase(synthase)

4 Addition of small molecules

to double bonds such as C=C, C=N and C=O

Isomerase 5

Isomerisations such as racemizations, epimerizations & rearrangement reactions

Ligases 6

Formation of complex compounds but enzymatically active only in presence of ATP as cofactor

12Source:

Peroxidases (Catalases)

Glucose oxidases

Laccases

Fructosyltransferases

Glucosyltransferases

Amylases Cellulases

Lipases

Pectinases

Proteases Pullulanases

Phytases

Pectate lyases

Acetolactate decarboxylases

Glucose isomerases

No products at the moment

Lipase Catalysed Reactions

Hydrolysis

Esterification

Interesterification

Transesterification(e.g. methanolysis)

R2

O

R1 O

+ OH2

H

O

R1 O

+ OH R2

H

O

R1 O

+ OH R2

R2

O

R1 O

+ OH2

O

R1 O R2

+

R4

O

R1 O

O

R3 O R4

O

R3 O R2

+

O

R1 O R2

+ OH CH3

O

R1 O CH3

+ OH R2

O

O

n

lipaseO

H2C OCH2

n

m

R4

R3

R1

R2 O R

4

R3

R1

R2

+ OHOH (CH2)n

OH

O

OH

O

(CH2)nOH (CH2)n

O

O

O

OH(CH2)nn

+ NH2NH2 (CH2)n

OH

O

OH

O

(CH2)n

OH (CH2)n

O

NH

O

NH2(CH2)nn

Carboxylic acid

H2O2

Epoxidation

Polycondensation

Polycondensation

Ring opening

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-tetrahydronapth-

alen-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 hydroxydiethylcarboxylates

Resolution of Rivastigmineintermediate

Alcoholysis of glutaricanhydride

TLL type of enzymes

Thermomyces lanuginosuslipase

Allows larger groups on both alcohol and acid parts

Key Applications

Kinetic Resolution by

desymmetrization of

CNDE-key intermediate for

Pregabalin

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Novozymes Immobilized Lipases……

Novozym®435 Lipozyme®TL IM

TL enzyme adsorbed on

Silica Gel

Novozym®40086

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Macroporous Acrylic Resin

RM Enzyme adsorbed on

Macroporous Anionic ResinCALB enzyme adsorbed on

Alcalase Savinase

Some of the other important enzymes in Novozymes portfolio

are the proteases- Alcalase & Savinase

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

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

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Biocatalysis is a proven technology for some prominent API

synthesis

Pregabalin- Lipase from TL Source

Darunavir- Novozyme 435/Lipozyme CALBL

Moxifloxacin- Lipozyme CALBL

Prostaglandin- Novozym 435 (immobilised lipase)

Cosmetic Emollient Ester- Novozym 435 (immobilised lipase)

Case Studies of Application developed by Innovator using Novozymes Enzymes

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Pregabalin Biocatalytic Route

Pregabalin Chemical synthesis Pregabalin Biocatalytic Route

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Improvements due to replacing chemical process with chemoenzymatic reaction

InefficientResolution at rac.pregabalin

stage

Novozymes Lipase

Lipozyme TL 100 L

Flow Chart of Pregabalin Process……..

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(R)-Diester

(S)-Diester

Water

Enzyme

(R)-Diester

(S)-Monoester

Water

Enzyme

(S)-Monoester

Water

Enzyme

Water

Enzyme

Racemisation

Recycling

Key Advantages of Biocatalytic Route

Low protein Loading(0.8%)

Resolution at first stage-wrong isomer can be recycled

High 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%

Pregabalin

Biotreatment

23%

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Inputs Chemical Route

Kg

ChemoenzymaticRoute

Kg

CNDE 6212 4798

Enzyme 0 574

S-Mandelic acid 1135 0

Raney Nickel 531 79.5

Solvents 50042 6230

Total 57920 11681.5

85%

88%

Reduction

Pregabalin Chemoenzymatic route used >5x less inputs than

Chemical Route

Raw Material Inputs for 1000kg API Output

Darunavir Key Intermediate Biocatalytic Route

Darunavir Synthesis

Key Advantages of Biocatalytic Route:

Substrate concentration can be as high as 100g/L

Reaction can be carried out using lipozyme CALBL or Novozym 435

Reaction can be carried out using water as a solvent

Reaction is carried out under mild & ambient conditions 21

Lipozyme CALBL is commercially used for stereoselective synthesis of key bicylcic intermediate

for Darunavir

O

O

O

O

ON

O

O +

N

CH3

CH3

NH2

OH

S

O

O

NH2

O

O

O

ON

CH3

CH3NH

OH

S

O

O

NH2

Key Bicyclic Intermediate

Darunavir Intermediate Biocatalytic Route

O O

OH

Acylation

O O

O

O

CH3

Lipozyme CALBL

or Novozym 435

O O

O

O

CH3H

H

+O O

OHH

H

hydrolysis

O O

OHH

H

NaH2PO

4 buffer,

40-45 C

Darunavir Key Intermediate

Moxifloxacin Key Intermediate Biocatalytic Route

Moxifloxacin Side Chain Synthesis Moxifloxacin Synthesis

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F

F

F

N

O O

OH NH

NH

H

H

+

F

N

F

N

O O

OH

NH

H

H

F

N

OCH3

N

O O

OH

NH

H

H

N

CH3OO

OCH3

O

OCH3

N

O

OCH3

O

OCH3

NH

O

OCH3

O

OCH3

N

CH3OO

OCH3

O

OCH3

NH

O

OH

O

OH

HCl

N

CH3O

O

N

O

NH

NH

H

H

Pd/C,H2

60 C

Ac2O, TEA

DMAP

Toluene,25 C

Enzyme

Conc.HClBenzylamine

Moxifloxacin Side Chain

Moxifloxacin

Key step for moxifloxacin key intermediate manufacturing is enzymatic

Moxifloxacin Key Intermediate Biocatalytic Route

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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

N

CH3OO

OCH3

O

OCH3

N

CH3OO

OCH3

O

OCH3

+N

CH3OO

OCH3

O

OH

Lipozyme CALBL/Novozym 435

Phosphate buffer pH 6-9

25-35 C

dimethyl 1-acetylpiperidine-2,3-dicarboxylate

dimethyl (2S,3R)-1-acetylpiperidine-2,3-dicarboxylate

(2R,3S)-1-acetyl-2-(methoxycarbonyl)piperidine-3-carboxylic acid

RequiredIsomer

Lipozyme CALBL or Novozym 435 can be used for desymetrisation of racemic substrate to the

required 2S,3R isomer

Latanoprost & Travoprost Biocatalytic Route

Latanoprost & Travoprost Synthesis

Key Advantages of Biocatalytic Route:

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

Novozym 435 is used for the key regioselective esterification step

Latanoprost

Latonoprost & Travoprost Biocatalytic Route

OH

OH

COOH

Ph

OH

CH3

CH3

OH OH

OH

COO

Ph

OH

CH3

CH3

Latanoprost

Novozym 435

OH

OH

COOH

OPhCF 3

OH

CH3

CH3

OH OH

OH

COO

PhCF 3

OH

CH3

CH3

Travoprost

Novozym 435

Travoprost

OAc

OH

O

O

OH

OH

COO

Ph

OH

CH3

CH3

OH

OH

COO

PhCF 3

OH

CH3

CH3

Travoprost Latanoprost

Corey Lactone

Multiple

Steps

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Case story: Biocatalysis for cosmeticMaking chemicals more efficiently

Esterification for production of cosmetic fatty acid esters.Chem. Soc. Rev., 2013, 42, 6475--6490

lipases and their hydrolytic, esterifying and acylating activities show enormous potential for implementation in the production of cosmetic ingredients

Emollient ester

Future outlook of biocatalysis is very promising

Directed Evolution

Variant Selection or screening

Smarter Libraries of Enzymes

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Advanced and Emerging Technologies

DNA technology & Protein engineering

Bioinformatics

Molecular Modelling studies-Docking of

substrate with proteins

Reaction engineering & process intensification

using biocatalysts

High throughput enzyme screening

Enzyme screening kits

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

Biocatalysis evolution so far and future outlook

So Far… the first and second wave The third wave: future outlook

Novozymes Introduction

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Everywhere you need us

Novozymes in numbers

Novozymes in 2014

CONTACT

NAME : DINESH NAIR

TITLE : REGIONAL BUSINESS MANAGER

EMAIL : DINN@NOVOZYMES.COM

WWW.BIOPHARMA.NOVOZYMES.COM

Thank you for attention