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
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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
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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
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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
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evocatal facts Products at any scale
from research scale… • …to kg- and industrial (t-) scale
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Enzyme production From lab to pilot scale and further
2 x 7.5 L Infors units
150 L Pilot Fermenter
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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
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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
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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
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Case study I Starting point
target reaction
Dehydrogenase
NADPNADPH + H+
GlucoseGluconolactone
O OH
API IntermediateSterane with keto-function
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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
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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
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Case study I Biotransformation
● Optimization of parameters: - process efficiency
- reduction of enzyme
- space/time yield
- product purity
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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
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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
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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
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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
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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
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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
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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
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Case study II Optimization of enzyme production
● Optimization of enzyme production: - screening for optimal production parameters
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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 (%
)
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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
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Conclusions Biocatalysis at Industrial Scale
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● 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