sustainable production of advanced intermediates and api’s ......sustainable production of...
TRANSCRIPT
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Sustainable Production of Advanced Intermediates and API’s Using Bio- and Homogeneous Catalysis
André H.M. de Vries
DSM Innovative Synthesis B.V.
Geleen, the Netherlands
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Page 1
Outline
1. DSM - Company profile and history
2. API Synthesis in drug development – Technology leadership
3. API synthesis examples@DSM
a. Almorexant (Catalytic Asymmetric Hydrogenation)
b. Statins (Aldolase)
4. Acknowledgements
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Company Profile and History
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Page 3
Driving focused growth!
Successful transformation from Coal mining to a global Life Sciences & Material Sciences company
Coal Mining Commodity Chemicals Specialty
Chemicals Life Sciences &
Materials Sciences
1902 40’s - 50’s 90’s 2011
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Page 4
DSM transformation of last 10 years
2000 2005 H1 2010
Polymer Intermediates
Performance Materials
Pharma
Nutrition
Bre
akdo
wn
DSM
Sal
es (%
)
Elastomersothers
Petrochemicals
Engineering Plastic Products
Base Chemicals & Materials
others
Base Chemicals & Materials
others
Elastomersothers
Petrochemicals
Engineering Plastic Products
Base Chemicals & Materials
others
Base Chemicals & Materials
others
Annual sales in 2011 was ~9 billion Euro. End 2011, ~22.000 Employees
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Page 5 Page 5
Current organization
DSM Managing Board DSM Innovation Center Corporate Staff
Shared Competences & Business Support
INDUSTRIAL CHEMICALS
DSM Innovation Center
Corporate Staff
Shared Competences & Business Support
PERFORMANCE MATERIALS
INDUSTRIAL CHEMICALS
LIFE SCIENCES MATERIAL SCIENCES
EBA
•DSM Nutritional
Products
•DSM Food
Specialties
•DSM Pharma-
ceutical Products
•DSM Sinochem
Pharmaceuticals
•DSM Resins
•DSM Engineering
Plastics
•DSM Dyneema
•DSM Fibre
Intermediates
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Page 6
DSM Pharmaceutical Products (DPP) Global provider of custom manufacturing and development services
Pharma Chemicals
Primary manufacturing of APIs and Intermediates
Dosage Forms
Secondary manufacturing of sterile injectables, non-sterile liquids, and oral dosage forms
Biologics
Custom manufacturing of biopharmaceutical
ingredients
BioSolutions
Microbial fermentation and associated product recovery
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Page 7
Global Presence of DPP
Pharma Chemicals: ::Linz, Austria ::Venlo, Netherlands ::Regensburg,Germany ::Geleen, Netherlands
Dosage Forms: ::Greenville NC, ::Parsippany, NJ (DPP Head Quarters)
Biologics: ::Groningen, Netherlands ::Brisbane, Australia
Biosolutions: ::Capua, Italy ::Delft, The Netherlands
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Page 9
Sustainability “Walking on two legs”
Renewable Raw Materials
Process Technology
Integrated Process - Low Energy and Energy Integration
DSM: Leader in Sustainable Manufacturing
Catalysis
Competence
Chemo-Catalysis
&
Bio-Catalysis
Biotechnology
Competence
Fermentation
&
Enzymology
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Page 10
Fermentation &
metabolic engineering
Bioc
atalyt
icco
uplin
g
Chem
istry
& Bi
ocata
lysis
DSM Sinochem Pharmaceuticals Long history in sustainable production of antibiotics
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Page 11
Sustainability Comparison of pure chemical route vs “walking on two legs” route
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Page 12
API synthesis in drug development
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Page 13
API synthesis in drug development
Drug Development
Lead
Finding
Lead
Optimization
Preclinical
&
Clinical
Phase 1
Clinical
development/
validation
(phase 2+3)
Registration
& Launch
Speed
Diversity
Efficacy
Speed
Diversity
Efficacy
Speed
Route
scouting
Purity
cGMP
Speed
Select route
Changing synthesis requirements
cGMP
Costs
Gx
Costs cGMP
Technology Leadership
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Page 14 Page 14
Technology Leadership
General organic chemistry
and process R&D
Homogeneous catalysis
Biocatalysis
Flow chemistry
Chirality, amino acids &
peptides
Fermentation & downstream processing
Microreactor technology
Route scouting &
retrosynthesis
Catalytic oxidation
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Page 15
• Broadest collection of off the shelf enzymes (>3000)
• Excellent expression and enzyme design capabilities
• Secure supply of enzyme at any scale (with FTO!)
• Unmet track record (also regulatory) in large scale implementation (>30
processes industrialized)
• Critical mass for all important expertises
• Open innovation approach provides access to huge network
Industrial Biocatalysis
D192
V193
V194
A199
G200
T226
Used in tons scale productionUsed in lab and pilot scale
Epoxides, DiolsHaloalcohol dehalogenases
Epoxides, DiolsEpoxide hydrolases
Alcohols, SulfoxidesMonooxygenases (P450, Baeyer-Villiger)
Amides, NitrilesNitrile hydratases
Carboxylic acids, NitrilesNitrilases
Alcohols and Amino acidsDehydrogenases (alcohol & amino acid)
Alcohols, Aldehydes, Carboxylic acidsOxidases
AminesOmega-Transaminases
CyanohydrinsHydroxynitrile lyases
Amino acids, N-Acetyl-Amino acidsAcylases
Amino acids, AmidesAmidases
Amino acidsHydantoinases, Carbamoylases, Racemases
Amino acidsAmmonia lyases
Alcohols, Esters, Carboxylic acidsLipases and Esterases
Peptides, Amines, CarboxyestersProteases
Alcohols, Diols, Amino acoholsAldolases
Product classesEnzyme Platforms
Epoxides, DiolsHaloalcohol dehalogenases
Epoxides, DiolsEpoxide hydrolases
Alcohols, SulfoxidesMonooxygenases (P450, Baeyer-Villiger)
Amides, NitrilesNitrile hydratases
Carboxylic acids, NitrilesNitrilases
Alcohols and Amino acidsDehydrogenases (alcohol & amino acid)
Alcohols, Aldehydes, Carboxylic acidsOxidases
AminesOmega-Transaminases
CyanohydrinsHydroxynitrile lyases
Amino acids, N-Acetyl-Amino acidsAcylases
Amino acids, AmidesAmidases
Amino acidsHydantoinases, Carbamoylases, Racemases
Amino acidsAmmonia lyases
Alcohols, Esters, Carboxylic acidsLipases and Esterases
Peptides, Amines, CarboxyestersProteases
Alcohols, Diols, Amino acoholsAldolases
Product classesEnzyme Platforms
>30 enzym
atic proc
esses im
plemente
d on indu
strial sca
le
0
100
200
300
400
500
600
700
1 2 3 4
Num
ber o
f enz
ymes
DSM plus partnersBiocatalyticsSigma-AldrichCodexis
oxidored
uctases
transfera
ses
hydrolas
eslyas
es
supplier 1supplier 2supplier 3
0
100
200
300
400
500
600
700
1 2 3 4
Num
ber o
f enz
ymes
DSM plus partnersBiocatalyticsSigma-AldrichCodexis
oxidored
uctases
transfera
ses
hydrolas
eslyas
es
supplier 1supplier 2supplier 3
oxidored
uctases
transfera
ses
hydrolas
eslyas
es
supplier 1supplier 2supplier 3
Number of accessible enzymes for rapid screening at DSM
Conti
nuou
s exp
ansio
n
> 1500 enzymes in DSM enzyme collection > 3000
Mutation atcontact pointof subunits
Mutation atcontact pointof subunits
PharmaPLE®
Candida sp.2Corynebacterium
Rhodococcus
Moraxella
Thermoanaerobium
Pseudomonas Candida sp. 1
Sporidiolobus
>500 Enzymes
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Page 16
DSM Enzyme collection > 3000 enzymes ready for screening
0
10
20
30
40
50
60
70
80
90
100
Alcoh
ol de
hydro
gena
ses
P450
Enoa
te red
uctas
es
Oxida
ses
BVMO
s
S-om
ega-T
ransa
mina
ses
R-om
ega-T
ransa
mina
ses
L-Ami
no ac
id tra
nsam
inase
s
D-Am
ino ac
id tra
nsam
inase
s
Trans
amina
ses (
total)
Lipas
es / E
steras
es
Prote
ases
/ Pep
tidas
es / A
mida
ses
Amida
ses
Nitrila
ses
Epox
idase
s
Haloa
lcoho
l deh
aloge
nase
s
D-Hy
danto
inase
s
L-Hyd
antoi
nase
R-HN
L
S-HN
L
Ammo
nia ly
ases
Haloh
ydrin
e deh
aloge
nase
Deca
rboxy
lases
Nitrile
hydra
tases
>700 >500 >1500 >300
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Page 17 Page 17
Homogeneous Catalysis
conversion e.e.
A B C D 1 2 3 4 5 6 7 8
A B C D 1 2 3 4 5 6 7 8
0 10 20 30 40 50 60 70 80 90 100
(%)
• Screening for the right catalyst from our HomCat Platforms using HTS
• Custom made Monophos® catalysts for asymmetric reduction
• Aromatic substitutions: C-C couplings and amine synthesis
• Rapid identification of the best catalytic system using all available
catalysts: proprietary ones AND commercially available ones
• HomCat process development and full scale implementation
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Page 18 Page 18
Flow Chemistry - Micro Reactors Process Intensification
Substrate
• Flow process development from lab to pilot to full-scale plant …in a variety of reactor concepts
• Integration of reaction and work- up • Rapid reaction optimization and numbering up …using various hazardous reagents
…under cGMP conditions
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Page 19 Page 19
Micro process technology
Learning from nature
Evolution
Pulmonary vesicle 400 µm 15000m2/m3
Capillary vains 10 µm
400.000 m2/m3
Enhanced transfer by decreased transfer distance
δ δ
Enhanced transfer by increased transfer area
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Page 20 Page 20
Process typicals • Batch operation at low temperature (2 oC) • Exothermic (nitration, neutralization) • HNO3 (high concentration, excess) • Danger (runaway, explosive) • Low productivity (diluted) • Unstable product
Business needs • Intrinsic safety • High productivity • High selectivity • GMP production
Extraction decomposition
Challenge: Selectively get mono-nitro product
Objective: Manufacture the desired quality and yield at full GMP level.
Example: Micro reactor in production
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Page 21 Page 21
Downstream equipment:
• continuous extraction columns
• centrifugal extractors
• distillation
Good mixing
Micro reactor in production
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Page 22 Page 22
Micro reactor in production
2 towers with 4 lines each 96 micro reactors in total
Scale-up to 800 T/a
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API Synthesis Examples @DSM
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Page 24
3a. Almorexant Catalytic asymmetric hydrogenation
Key step for Almorexant – Actelion Pharmaceuticals
Insomnia treatment
Clinical phase III
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Page 25
3 Options considered:
Asymmetric transfer Hydrogenation (Noyori et al)
Asymmetric Hydrogenation
Dynamic Kinetic Resolution (similar work from Blacker et al)
Synthesis overview
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Page 26
• 8 vessels (5 ml)
• Indep. P ( 90%
Optimization
the best ligand
conv. and e.e. > 95%
Standard autoclave 100mL, optimal stirring
Typical Asymmetric Hydrogenation@DSM
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Page 27
Asymmetric hydrogenation step Condition screen with 4 representative ligands (High Throughput)
O
OP N
Ph
Ph
O
OP N
Fe
(Cy)2P
P(tBu)2
H
PPO
O
O
O
tBu
tButBu
tBu
2
2
1 2 3 4
ee EtOAc MIBK DCM IPA
none Pht. I2 none Pht. I2 none Pht. I2 none Pht. I2
4B 17 16 1 3 3 1 3 4 0 1 43 1
3B 1 1 -25 22 20 -22 31 31 -40 8 8 -26
2B 31 30 28 28 32 29 53 40 -11 20 25 17
1B -44 -41 -44 -28 -24 -27 -33 -27 -24 -26 -12 -47
4A 10 10 0 -2 -1 0 6 1 12 22 15 6
3A 67 66 76 28 34 35 15 18 36 31 41 49
2A 64 62 51 35 34 45 27 17 32 -2 -2 43
1A -56 -66 -49 -54 -55 -31 -57 -57 -26 -27 -27 -51
A: [Ir(COD)Cl]2
B: Ir(COD)2BF4
Pht. = Phthalimide
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Page 28
Chiral phosphoramidite ligands
O
OP N
R1
R2
• Monodentate ligands
• Much easier to prepare than bisphosphines
• Chirality from diol and/or amine
• Modular ligand: diversity from the diol and/or amine part
• Till 2000 not known for asymmetric hydrogenation
DSM and University of Groningen collaboration, see e.g.: Adv. Synth. Cat. 2003, 345, 308;
Org. Proc. Res. Dev, 2007, 11, 585; Org. Proc. Res. Dev, 2010, 14, 568.
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Page 29
Further exploration MonoPhos family
O
OP N
tBu
tBu
O
OP N
O
OP N
O
O
Ph
Ph
Ph
Ph
bn
O
OP N
O
O
Ph
Ph
Ph
Ph
O
OP N
Ph
O
OP N
Ph
Ph
79% ee32% ee10% ee
3% ee 29% ee 17% ee
Zenith of ee
for MonoPhos
library
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Page 30
Further exploration JosiPhos lead
28 Solvias Ligands: Josiphos, Walphos, Mandyphos, and Taniaphos types
[Ir(COD)Cl]2 and I2 in EtOAc
Taniaphos Ligand SL-T002-2 gave the best ee
O
ON CF3 NH
O
OCF3
free base
1. [Ir(COD)Cl2], DCM, I2 S/C = 200 2. EtOAc, 5 bar H2
Process was selected for scale up and piloting (1 m3)
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Page 31
Process optimization
Solvent Acid ee of Salt ee of ML
EtOAc MeSO3H 87% 68%
Toluene MeSO3H 93% 30%
Toluene TsOH - -
Toluene CH3CO2H 99.7% 65%
Toluene / Heptane CH3CO2H 93% 12%
Toluene / Heptane HCO2H 88-93% 72-77%
All solvents gave similar ee’s in IPC (83-90%)
O
ON CF3 NH
O
OCF3
NH
O
OCF3Acid
Salt
Solvent
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Page 32
Full scale protocol
Achievements
• Commercially viable S/C was reached on 6 m3 scale
• Acetate salt
- single crystallization
- non-corrosive
• Robust crystallization procedure
• Isolated yield was increased (compared to Noyori)
O
ON CF3 NH
O
OCF3
NH
O
OCF3
CH3COOH1. aq. NaOH, toluene2. Catalyst preparation3. r.t., 5 bar H2, 3h
Catalyst preparationTaniaphos SL-T002-2, [Ir(COD)Cl2], DCM, I2
1. CH3COOH2. -5 °C
CH3SO3H
98% Conversion90% e.e.
>99% e.e.
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Page 33
0
25
50
75
100
125
150
Reagentskg/kg API
Solventskg/kg API
# Solvents # SolventSwaps
Waste Waterkg/kg API
10
120
8 4
146
4
20
5 1
17
Waste reduction
Phase I
(ATH)
Phase III
(AH)
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Page 34
Almorexant - Summary
Catalytic asymmetric hydrogenation step replaces 5 steps for 1
(3 steps for the chiral resolving agent)
Applied at multi ton scale
Unfortunately the drug development has been abandoned
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Page 35
3b. Statins by Aldolase technology
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Page 36
3b. Statins, e.g. Atorvastatin (Lipitor)
Best selling drug for last 5 years (fighting obesity)
Patent expired in 2012
Several ‘second at market’ analogues
Lowest cost route required
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Page 37
Retrosynthesis
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Page 38
DERA Catalyzed Tandem Aldol Reaction
Published information
• Low product concentration: 1-2 (w)%
• Low enzyme stability
• Work-up not suitable for scale-up
• By-product formation is ‘black-box’
Wong et al. (1995) J.Am.Chem.Soc., 117, 3333
Enzymatic synthesis of a deoxysugar
Cl
O O
+ 2 Cl
OH ODERA DERACl
OH OH O
Cl
O
OH
OH
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Page 39
DERA is Type I Aldolase: Mechanism
172
102
137
201
167
Active site with Lys172, Lys201, Lys167, Lys137, Cys47 and Asp102
Lys167 with carbinolamine intermediate
H
H OPO32-
HNLys167
O
OH
O OH
OPO32-
OH
H
H
HNLys167
O
Cl
O OH
Cl
H
H
HNLys167
O
OH OH
Cl
Cl
OH
O
Heine et al. (2001) Science 294, 269 - thanks to Jan Metske van der Laan
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Page 40
Reaction Intermediates and Products
Cl
OO OH
OH
Cl
Cl
OOH
Cl
OHOH
OH
Cl
OO
OH
Cl
OH
Cl
OO
OH
H2O
H2O
O
OH
OH
O
Cl
OH
OH
+
H2O
O OH
OH
600 MHz 1H-NMR
Reaction is far more complicated than expected
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Page 41
Typical Batch Reaction: 600 MHz 1H-NMR
0
200
400
600
0 100 200 300 time (min)
conc
entr
atio
n (A
U)
mono-aldol intermediate
di-aldol product
acetaldehyde
chloroacetaldehyde
acetaldehyde side product
ClH
O
H
O
+ 2 ClH
OH ODERA DERA
Cl
O
OH
OH
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Page 42
Drawbacks of DERA Reactions
Facts on DERA:
• Low activity for acceptor substrates
• Inactivation by aldehyde substrates
• Therefore: large amounts of enzyme needed
• However, an efficient process is feasible (WO 03006656 to DSM)
Directed evolution to improve DERA
ClH
O
H
O
+ 2DERA
Cl
OH OH
H
O
Cl
O
OH
OH
CAA AA
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Page 43
DERA Limitations: Acceptance of Chloroacetaldehyde and inactivation
Chloroacetaldehyde (mM)
0 100 200 300 400
Initi
al v
eloc
ity (
mAb
s 340
nm/m
in)
0
100
200
300
400
500
600
700
800Chloro-
acetaldehyde
(mM)
Remaining activity of DERA after 5 min exposure to indicated
concentration of
chloroacetaldehyde
0 mM 100%
100 mM 34.7 ± 0.45
200 mM 26.1 ± 0.87
300 mM 13.4 ± 1.07
400 mM 9.9 ± 0.61
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Page 44
Directed Evolution Strategy for DERA
Creation of random genetic diversity using error-prone PCR
Variant DERA expression library
Directed evolution of DERA for increased tolerance
towards chloroacetaldehyde
Screening for more productive DERA variants
Improved DERA variants for the organic synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside
Escherichia coli deoC gene coding for 2-deoxyribose 5-phosphate aldolase (DERA)
More productive DERA variants using chloroacetaldehyde as acceptor
aldehyde substrate
Identification of DERA variants showing improved resistance to
chloroacetaldehyde.
Saturation mutagenesis &
Combination of mutations
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Page 45
Front view
Active site
172
201 167
Side view
Active site
172
5
DERA 3D structure (ribbon presentation)
Lysine residues are shown (18 in total)
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Page 46
Improvement of tolerance to CAA Directed Evolution Strategy
Variant DERA expression library (obtained through error-prone PCR)
Screening of 10,000 randomly chosen DERA variant clones
Identification of 63 stability screening hits
2 rounds of recombination (screening of 3,000 clones per round)
Isolation of 10 stability screening hits
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Page 47
Improvement of tolerance to CAA Application tests
(mm
ol/m
g cf
e)
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
OCl
OH
OH
OClOH
wild
-type
DER
ADE
RAva
r1DE
RAva
r2DE
RAva
r3DE
RAva
r4DE
RAva
r5DE
RAva
r6
Yie
ld (
AU
)
Synthesis conditions:
• 200 mM CAA, 465 mM AA • 0.1 M NaHCO3 (pH 7.2), RT, 16 h. • GC-Analysis for (S)-4-chloro-3-hydroxybutyraldehyde (“monoaldol”)
and (3S,5R)-6-chloro-2,4,6-trideoxy-
hexapyranoside (“acetal”)
• Yields are reported in Arbitrary Units
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Page 48
Screening for variant DERA’ s with Improved Productivity
Variant DERA expression library (obtained trough error-prone PCR)
Screening of 3,000 randomly chosen DERA variant clones using high-throughput
GC/MS
Identification of 9 productivity screening hits
OCl
OH
OH
(3S,5R)-6-Chloro-2,4,6-trideoxyhexapyranoside
OCl
O
O
+Cl
OH OH O
(S) (R)
DERADERACl
OH O
(S)
(S)-4-Chloro-3-hydroxybutyraldehyde
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Page 49
Screening for Improved Productivity
(mm
ol/m
g cf
e)
0.02.55.07.5
10.012.515.017.520.022.525.027.530.032.535.037.540.042.545.047.5
OCl
OH
OH
OClOH
wild
-type
DER
ADE
RAva
r7DE
RAva
r8DE
RAva
r9DE
RAva
r10
DERA
var1
1DE
RAva
r12
DERA
var1
3
Yie
ld (
AU
)
Synthesis conditions:
• 200 mM CAA, 465 mM AA
• 0.1 M NaHCO3 (pH 7.2), RT, 16h
• GC-Analysis for (S)-4-chloro-3-hydroxy-butyraldehyde and (3S,5R)-6-chloro-2,4,6-trideoxy-hexapyranoside
• Yields are reported in Arbitrary Units
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Page 50
• Most productive variants from the two screenings combined
• Test with constant DERA amounts:
Mutations from both screenings are synergistic
Time (h)
0 2 4 6 8 10
mM
( 3
S,5
R
)-6-Chloro-2,4,6-trideoxy-
hexapyranoside 0
50
100
150
200
250
300
350
400
wt DERA
Combination of Beneficial Mutations
F200I
F200I / ext. C-terminus F200I / Y259
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Page 51
Statin intermediate - Summary
DERA enzyme (Aldolase) in our opinion the lowest cost technology
to produce chiral building block for Statins
Subsequent reactions to Statins also performed (and improved)
Better impurity profile than other technologies
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Page 52
DSM’s route scouting and manufacturing services for API synthesis
Desk
Studies
Route
Scouting
Preclinical
&
Early
clinical
Clinical
development/
validation
(phase 2+3)
Registration
&
Launch
InnoSyn®
DSM Pharma Chemicals
ResCom®
DPC Linz + DPC Venlo
InnoSyn® route scouting services
focused & flexible, targeting pre-clinical & phase-I
IP approach: newly generated IP for the customer
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Page 53
4. Acknowledgements
Homogeneous Catalysis
Paul Alsters
Laurent Lefort
Michele Janssen
Lavinia Panella
Ruben van Summeren
Gerard Verzijl
Lizette vd Vondervoort
Hans de Vries
Biocatalysis
Daniel Mink
Martin Schurmann
Michael Wolberg
… …
… …
R&D and production sites in Venlo and Linz