FINE ENZYMES FOR FINE CHEMICALS · Selected chiral intermediates R (CH 2)n R O H O H enantiopure...

FINE ENZYMES FOR FINE CHEMICALS

DR. SASCHA HAUSMANN, CIC 2013, APRIL, 18TH, 2013

THE COMING OF AGE OF INDUSTRIAL BIOCATALYSIS

evocatal facts Introduction

Development and Production of Biocatalysts for the Pharma and Fine Chemicals Industry

Our enzymes and production strains open alternatives to conventional synthetic routes.

• Founded in 2006 as a spin-off from Heinrich-Heine-Universität Dusseldorf

• 23 employees

• 550 m2 of labs and offices + 200 m² production

• Labs are in S1- and S2-standard

• Sustainably financed: High-Tech Grunderfonds

Sirius Seed Fonds

Business Angels

April 18th, 2013 2 CLIB International Conference 2013

evocatal facts Business Model

Customized solutions

Standardized solutions

evocatal has a strong technology background in enzyme production and engineering

evoservices evoproducts


evocatal facts Products

evoproducts

Standardized solutions

• > 60 commercial enzymes available from stock

Enzyme classics 19 Lipases (incl. CalB)

12 ADHs (3 in pipeline)

9 Cofactor regenerating enzymes

6 Transaminases

Enzyme specialities 3 HNLs

2 Aldolases (2 in pipeline)

7 TPP-dependent Lyases

4 Nucleoside Phosphorylases

4 Adenosine Desaminases

• > 50 fine chemicals biocatalytic processes established for chiral chemicals


evocatal facts

> Production of chiral cyanohydrins

Oxynitrilases CHOHCN H

CNHO

*+

evozyme

Lipases

> Esterifications, transesterifications R O

O

R'+ H2O

R OH

O+ R' OH

evozyme

evozyme

> Production of chiral amines

Transaminases evozyme

R

O

R

NH2

*+ +OH

O

NH2

OH

O

O

R

O

R

NH2

*+ +OH

O

NH2

OH

O

O

R

O

R

NH2

*+ +OH

O

NH2

OH

O

O

> Production of chiral alcohols evozyme

Alcohol Dehydrogenases

R R'

O

R R'

OH

*

Products


evocatal facts Products at any scale

from research scale… • …to kg- and industrial (t-) scale


Enzyme production From lab to pilot scale and further

2 x 7.5 L Infors units

150 L Pilot Fermenter


evocatal facts Selected chiral intermediates

R (CH2)n R

OH OH

enantiopure Diols

chiral ligands

O

O

OH

CH3

(S)-Ethyl 2-hydroxy-4-phenylbutyrate

Cilazapril

N

OH

(R)-Quinuclidinol

Solifenacin

CH3 OCH3

OH O

(R)-Methyl-3-hydroxybutyrate

Dorzolamide

CH3

CH3

OH

(S)-2-Octanol

Liquid Crystals

CH3

F3C

CF3

OH

(R)-1-(3,5-Bistrifluoromethylphenyl) ethanol

Aprepitant

2.5 mt

120 kg

in scale-up

30 kg

10 kg (in scale-up)

O OH

COOH

OHNH

OH

OHOH

CH3

O

N-Acetyl-Neuraminic Acid

Zanamivir


evocatal facts Business Model

Bio-Development internal

Chemical Development internal

Bulk- Production (Partners)

Bulk- Production (Partners)

Pilot- Production (inhouse)

Pilot- Production (inhouse)

Bioprocess- Development

Process- Development

Strain development

Enzyme optimization

Enzyme Discovery

external

external


Keysteps Workflow for establishment of bio-based processes

Enzyme discovery

* PoP

* feasibility

Enzyme production

* optimization

* enzyme yield

* robustness

* batch-to-batch

* upscalability

Process development

* optimization

* robustness

* substrate load

* catalyst amount

* space-time yield


Case study I Starting point

target reaction

Dehydrogenase

NADPNADPH + H+

GlucoseGluconolactone

O OH

API IntermediateSterane with keto-function


non optimized process

initial fed-batch fermentations

multi-enzyme

production of biocatalysts

biotransformation

process

Case study I Enzyme production, review of fermentation

● Optimization of parameters: - robustness / batch to batch variation

- upscalability - cost reduction

- increase of yield


Case study I Enzyme production, review of fermentation

0

50

100

150

200

250

volu

met

ric

acti

vity

[U/m

L]

before optimization

after optimization

150 L pilot scale

1.5 m³ / 15 m³ scale

● Upscale successful: - new process defined

- yields increased and robust

- independent of scale


Case study I Biotransformation

● Optimization of parameters: - process efficiency

- reduction of enzyme

- space/time yield

- product purity



● Optimization successful - new process defined

- increased space/time yield

- reduced enzyme and cofactor load - product purity ≥ 99.9 %

0

20

40

60

80

100

120

140

160

before optimization

after optimization



● Conformance batches - production of enzyme for process

- 4 conformance batches on 1 m3 scale successful

- customer ordered enzyme for ton-scale production


Case study II Starting point

Synthesis of (R)-3-quinuclidinol (R-Qol)

● Target Drug:

- Solifencin - Application: Overactive bladder

● Current synthesis of R-Qol

- Racemic resolution applying chiral auxiliary - Process no longer competitive

Alcohol Dehydrogenase

NADPNADPH + H+

Co-substrateCo-product

N

O

N

OH

N O

O

N

Solifenacin


case study II workflow

Enzymatic Synthesis of (R)-3-quinuclidinol (R-Qol)

● Goals to be reached:

- Identification of a suitable enzyme (catalyst) - Optimization of the enzyme - Optimization of enzyme production - Optimization of production process


case study II screening for a suitable biocatalyst

● Identification of suitable enzyme:

- Wild-type enzymes and promising enzyme mutants from evocatal stock libraries were produced on small scale

- Produced enzymes were used in small scale reaction (1 mL)

- Performance of enzymes concerning conversion and selectivity was tested and analyzed

- Best enzyme showed:

- Conversion: 20 % - Enantiomeric excess: 87 %

Fermentation (production) of potential wild-type ADHs on small scale

Screening of enzymes for activity and enantio-selectivity

Analysis of performance

Enzyme X Enzyme Y Enzyme Z


Group 2

Group 3

Group 1

case study II biocatalyst optimization

● Optimization of enzyme: - Enzyme evolution by CASTing

Group 1: 1st sphere red; 2nd sphere orange Group 2: 1st sphere yellow; 2nd sphere cyan Group 3: 1st sphere pink; 2nd sphere magenta

Small and smart library


Case study II

ADH Variant; active site

87 % ee, 20 % conversion 99 % ee, >99 % conversion

ADH wild-type; active site

Biocatalyst optimization


Case study II Optimization of enzyme production

● Optimization of enzyme production: - screening for optimal production parameters


0,00

0,50

1,00

1,50

2,00

2,50

3,00

3,50

4,00

4,50

en

zym

atic

act

ivit

y [U

/ml]

Benchmark

Case study II Optimization of enzyme production

● Optimization of enzyme production:

SDS-PAGE

enzymatic activity

target

0

100

200

300

400

500

600

700

800

rela

tive

act

ivit

y (%

)


Case study II Optimization of production process of R-Qol

● Optimization of production process: - developed up to scale of 1 L at evocatal

- currently in scale-up at project partner


Conclusions Biocatalysis at Industrial Scale

April 18th, 2013 25

● The number of identified enzymes able to conduct industrial relevant reactions is constantly increasing.

» Biocatalysis works at industrial scale and will gain further importance.

● The tools for tailoring the enzymes to the process needs (e.g. activity, stability, selectivity) are well established and allow efficient improvement of the catalyst´s performance.

● The cost-efficient production of biocatalysts at industrial relevant scales is possible.

● The performance of optimized biotransformations (e.g. space-time yields, costs) is competitive to classical chemical reactions.

CLIB International Conference 2013

FINE ENZYMES FOR FINE CHEMICALS

evocatal GmbH Merowinger Platz 1a 40225 Düsseldorf

FINE ENZYMES FOR FINE CHEMICALS · Selected chiral intermediates R (CH 2)n R O H O H enantiopure...

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