fine enzymes for fine chemicals · selected chiral intermediates r (ch 2)n r o h o h enantiopure...
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FINE ENZYMES FOR FINE CHEMICALS
DR. SASCHA HAUSMANN, CIC 2013, APRIL, 18TH, 2013
THE COMING OF AGE OF INDUSTRIAL BIOCATALYSIS
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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
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evocatal facts Business Model
Customized solutions
Standardized solutions
evocatal has a strong technology background in enzyme production and engineering
evoservices evoproducts
April 18th, 2013 3 CLIB International Conference 2013
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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
April 18th, 2013 4 CLIB International Conference 2013
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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
April 18th, 2013 5 CLIB International Conference 2013
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evocatal facts Products at any scale
from research scale… • …to kg- and industrial (t-) scale
April 18th, 2013 6 CLIB International Conference 2013
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Enzyme production From lab to pilot scale and further
2 x 7.5 L Infors units
150 L Pilot Fermenter
April 18th, 2013 7 CLIB International Conference 2013
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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
April 18th, 2013 8 CLIB International Conference 2013
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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
April 18th, 2013 9 CLIB International Conference 2013
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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
April 18th, 2013 10 CLIB International Conference 2013
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Case study I Starting point
target reaction
Dehydrogenase
NADPNADPH + H+
GlucoseGluconolactone
O OH
API IntermediateSterane with keto-function
April 18th, 2013 11 CLIB International Conference 2013
non optimized process
initial fed-batch fermentations
multi-enzyme
production of biocatalysts
biotransformation
process
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Case study I Enzyme production, review of fermentation
● Optimization of parameters: - robustness / batch to batch variation
- upscalability - cost reduction
- increase of yield
April 18th, 2013 12 CLIB International Conference 2013
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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
April 18th, 2013 13 CLIB International Conference 2013
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Case study I Biotransformation
● Optimization of parameters: - process efficiency
- reduction of enzyme
- space/time yield
- product purity
April 18th, 2013 14 CLIB International Conference 2013
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Case study I Biotransformation
● 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
April 18th, 2013 15 CLIB International Conference 2013
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Case study I Biotransformation
● Conformance batches - production of enzyme for process
- 4 conformance batches on 1 m3 scale successful
- customer ordered enzyme for ton-scale production
April 18th, 2013 16 CLIB International Conference 2013
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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
April 18th, 2013 17 CLIB International Conference 2013
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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
April 18th, 2013 18 CLIB International Conference 2013
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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
April 18th, 2013 19 CLIB International Conference 2013
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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
April 18th, 2013 20 CLIB International Conference 2013
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Case study II
ADH Variant; active site
87 % ee, 20 % conversion 99 % ee, >99 % conversion
ADH wild-type; active site
Biocatalyst optimization
April 18th, 2013 21 CLIB International Conference 2013
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Case study II Optimization of enzyme production
● Optimization of enzyme production: - screening for optimal production parameters
April 18th, 2013 22 CLIB International Conference 2013
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
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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 (%
)
April 18th, 2013 23 CLIB International Conference 2013
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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
April 18th, 2013 24 CLIB International Conference 2013
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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
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FINE ENZYMES FOR FINE CHEMICALS
evocatal GmbH Merowinger Platz 1a 40225 Düsseldorf