sustainable production of advanced intermediates and api’s ......sustainable production of...

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

[email protected]

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

Company Profile and History

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

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


others

Elastomersothers

Petrochemicals

Engineering Plastic Products


others


others

Annual sales in 2011 was ~9 billion Euro. End 2011, ~22.000 Employees

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

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

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

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

Fermentation &

metabolic engineering

Bioc

atalyt

icco

uplin

g

Chem

istry

& Bi

ocata

lysis

DSM Sinochem Pharmaceuticals Long history in sustainable production of antibiotics

Sustainability Comparison of pure chemical route vs “walking on two legs” route

API synthesis in drug development

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

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

• 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


oxidored

uctases

transfera

ses

hydrolas

eslyas

es


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

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

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

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

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

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

Page 21

Downstream equipment:

• continuous extraction columns

• centrifugal extractors

• distillation

Good mixing

Micro reactor in production

Page 22

Micro reactor in production

2 towers with 4 lines each 96 micro reactors in total

Scale-up to 800 T/a

API Synthesis Examples @DSM

3a. Almorexant Catalytic asymmetric hydrogenation

Key step for Almorexant – Actelion Pharmaceuticals

Insomnia treatment

Clinical phase III

3 Options considered:

Asymmetric transfer Hydrogenation (Noyori et al)

Asymmetric Hydrogenation

Dynamic Kinetic Resolution (similar work from Blacker et al)

Synthesis overview

• 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

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

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.

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

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)

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

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.

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)

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

3b. Statins by Aldolase technology

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

Retrosynthesis

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

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

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

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

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

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

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

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)

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

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

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

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

• 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

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

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

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

sustainable production of advanced intermediates and api’s ......sustainable production of...

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