FINE ENZYMES FOR FINE CHEMICALS

APR. 03RD 2014

PASCAL DUENKELMANN

FROM LABORATORY TO PRODUCTION PLANT; DIFFERENT SCALES – DIFFERENT NEEDS

evocatal facts Introduction

DEVELOPMENT AND PRODUCTION OF INNOVATIVE ENZYMES FOR PHARMA AND FINE CHEMICALS

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

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

• 36 employees

• > 1.000 m2 of labs + pilot plant and offices

• Labs are in S1- and S2-standard

• Sustainably financed: High-Tech Grunderfonds

Sirius Seed Fonds

Business Angels

Series-B financing round closed in May 2013 (Lanxess, NRW.Bank)

Apr. 03rd, 2014 2

evocatal facts Business Model

Customized solutions

Standardized solutions

evocatal has a strong technology background in enzyme production and engineering

evoservices evoproducts

Apr. 03rd, 2014 3

evocatal facts Business Model

evocatal has a strong technology background in enzyme production and engineering

evoservices evoproducts

[…]

Apr. 03rd, 2014 4

evocatal facts Development and core business evocatal GmbH

Core technolgies

Services Exclusive development Research networks

Products Enzymes - Fine chemicals - Screenings

Bio-based chemicals & chiral Pharma-Intermediates

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

Enzyme discovery Enzyme optimisation Catalyst-Production Process development

- Metagenomics

- enzyme engineering

- evocatal Expression-Platform

- Implementation in production

plant

- Development and production

of industrial enzymes

- Chemical and Pharma

- bio-based economy

5 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production Project summary

target reaction Dehydrogenase

NADPNADPH + H+

GlucoseGluconolactone

O OH

API IntermediateSterane with keto-function

3 Enzymes one-pot reaction

- Starting point --> customer brought in two of three enzymes (University Project)

- evocatal designed a one-pot process and provided a third enzyme

- evocatal developed the commercial production for the first two

- evocatal tech-transferred to scale-up to 40 t/a production

Catalysis on two separate positions within the scaffold

6 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production Identification of Enzyme

Customers needs at the beginning of the project

- Time lines: as quickly as possible

- Enzyme: Identification of the most efficient third catalyst

- Analytics: Suitable for high throughput assays

- Raw materials: no special requirements => application of “P.A.

materials”

- Regulatory: no requirements

7 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production evocatal enzyme libraries in numbers

● Libraries and strain collections

- More than 20 Gb cloned eDNA - Enrichment cultures from over 50 extreme habitats - about 5 - 10 Mio gene sequences (pot. enzymes!) ready to screen! ● Proprietary Metagenome sequences for in silico screenings

Apr. 03rd, 2014 8

Case study I – Scale-up of Enzyme Production Tailoring enzymes – customized developments

Gene of interest

screening (activity assay)

collect best variants isolate gene, analyse

create diversity (=library)

1 variant per cell clone into E.coli produce variants

optimal enzyme next cycle

9 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production Process Development

Customers needs at pilot plant scale

- Time lines: as quickly as possible

- Enzyme: Development of a robust production process

- Analytics: reliable and robust

- Raw materials: no special requirements => application of “P.A.

materials”

- Regulatory: low level – e.g. BSE/TSE statement

10 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production evocatal technology platforms

Signal peptide libraries

Chaperone toolbox

Expression hosts

Fermentation parameters

11 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production The evocatal Chaperone Toolbox

Methods of Molecular Biology. Vol.59. Protein Purification Protocols

A) E. coli containing inclusion bodies

B) Isolated

inclusion bodies.

12 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production The evocatal Chaperone Toolbox

GroELS: Xu et al., Nature (1997)

Chaperones aid proteins to get from an

unfolded

or misfolded state to the native conformation

during enzyme expression

Chaperone

13 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production evocatal Signal Peptide Libraries

signal peptide „barcode“

target protein

In Bacillus sp. signal peptides mediate the transport of proteins (enzymes) across the cell membrane to the outer medium. Each signal peptide is highly specific to its corresponding protein and hence a crucial determining factor to the secretion efficiency of an enzyme.

14 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production evocatal Signal Peptide Libraries

5 L - 150 L Fermenters in house

SDS-PAGE (culture supernatant)

enzymatic activity (culture supernatant)

target

0

100

200

300

400

500

600

700

800

rela

tive

act

ivit

y (%

)

--> the best signal peptide/ protein combinations are selected and assayed for performance in fermentations.

15 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production Industrial Production

Customers requirements for large scale production

- Time lines: as quickly as possible

- Enzyme: Reliable and cheap production on cubic meter-scale

- Analytics: reliable, robust and validated

- Raw materials: tap water, technical materials

- Regulatory: - BSE/TSE statement

- Jatropha statement

- HACCP or cGMP statement

- Absence of genetically modified organism

- Absence of genetically modified DNA

- Absence of mutagenic substances

- Absence of endotoxins

- etc. 16 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production Industrial 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

enzyme 1 enzyme 2 enzyme 3

17 Apr. 03rd, 2014

Case study I – Scale-up of Enzyme Production Custom manufacturing of Enzymes

evocatal is currently collaborating with four European CMOs concerning the production of enzymes

Enzyme production in the Far East is not considered

CMOs are selected according to the requirements in the project

The CMOs possess of different quality standards (ISO 9001, GMP, HACCP)

18 Apr. 03rd, 2014

Case study II: Scale-up of Chemical Production Synthesis of an API-building block

Alcohol Dehydrogenase

NADPNADPH + H+

Co-substrateCo-product

N

O

N

OH

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

● Target Drug:

- Solifenacin (Astellas) - becomes generic in 2018 - white spots exist already (e.g. Turkey) - Application: Overactive bladder

● Current synthesis of R-Qol

- Racemic resolution applying chiral auxiliary - Process no longer competitive

19 Apr. 03rd, 2014

N O

O

N

Solifenacin

Case study II: Scale-up of Chemical Production Bench Scale Experiments

Customers requirements for Bench Scale

- Time lines: as quickly as possible

- Chemical: few grams

- Analytics: identification of product sufficient

- Raw materials: all materials usually “P.A.” quality

- Regulatory: none

20 Apr. 03rd, 2014

Case study II: Scale-up of Chemical Production Improvement of enantioselectivity

Customer´s Challenge: Find an enzyme for the synthesis of (R)-3-quinuclidinol (R-Qol)

89 % ee 65 % conv. (24 h) 8 % subst. load

enzyme modeling 6 aa picked for mutagenesis high throughput screening

98 % ee 99% conv. (24 h) 30 % subst. load

99 % ee 99% conv. (24 h) 30 % subst. load

recombination of positive mutations

Alcohol Dehydrogenase

NADPNADPH + H+

Co-substrateCo-product

N

O

N

OH

21 Apr. 03rd, 2014

Case study II – Scale-up of Chemical Production Chemical process development

Customers requirements for Pilot Scale

- Time lines: as quickly as possible

- Chemical: 1 kg of “in Spec” material

- Analytics: identification of product and impurities

- Raw materials: all materials usually “P.A.” quality, starting material

might be from different source

- Regulatory: Specifications must be met

22 Apr. 03rd, 2014

Case study II: Scale-up of Chemical Production Chemical process development

Process development for technical scale

● Optimization of Enzymatic Conversion

- Reactor type: Batch reactor - Enzyme preparation: Liquid crude extract (evo-1.1.200*) - 30 % substrate loading, 30 °C, pH = 7.0

● Goals achieved - Optimized catalyst (enzyme) could be evolved (CP > 99 %, ee > 99 %) - Stable chemical process established

23 Apr. 03rd, 2014

Case study II: Scale-up of Chemical Production Industrial Production

Customers requirements for large scale production

- Time lines: as quickly as possible

- Chemical: Reliable and cheap production on cubic meter-scale

providing “in spec.” material

- Analytics: reliable, robust and validated (suitable for DMF

application)

- Raw materials: technical materials

- Regulatory: - BSE/TSE statement

- Jatropha statement

- HACCP or cGMP statement

- Absence of genes from enzyme production host

- Absence of DNA from enzyme production host

- etc. 24 Apr. 03rd, 2014

Case study II: Scale-up of Chemical Production Custom manufacturing of Chiral Building Blocks

Several Partners for Custom Manufacturing have been established world wide.

Small scale productions are mainly performed in collaboration with European partners

Large scale productions (up to metric ton-scale) are either carried out with European partners or with Indian and Chinese CMOs

All CMOs are ISO 9001- and/or GMP-certified

25 Apr. 03rd, 2014

WE´RE LOOKING FORWARD TO YOUR SUCCESS!

THANK YOU!