University College London

Scale-up Issues for Whole-cell Biocatalytic Oxidation

John M Woodley

Scale-up Issues

Scale

Productivity

Production capacity

Lab equipment Plant equipment

Scale-up and Implementation

Exquisite (chiral) chemistry, under mild conditions but…..

• availability of biocatalysts• integration with chemistry• productivity limitations

Productivity Limitations

• Substrate and product instability

• Substrate and product inhibition / toxicity• Biocatalyst instability• Non-natural substrate access into whole cells

and low rates of reaction

• Aqueous media• Integration with neighbouring operations

Potential Solutions

• Auxiliary phase biocatalysis– two-liquid phase – resin

• Feed and bleed• Catalyst immobilisation• Genetic engineering• Protein engineering

BVMO

Model Baeyer-Villiger Reaction

O

O

H

H

O

H

H

O

+O

O2, NADPH, H+ H2O, NADP+

(-) 1(S), 5(R) 2-oxabicyclo[3.3.0]oct-6-en-3-one bicyclo[3.2.0]hept-2-en-6-one

(-) 1(R), 5(S) 3-oxabicyclo[3.3.0]oct-6-en-2-one

Alphand et al 1989 Tet Lett 30, 3663; Alphand and Furstoss 1992 J Org Chem 57, 1306

BVMO Challenges

• Wild type host is pathogenic, contains contaminating activity and is difficult to grow

• CHMO is susceptible to oxidation and therefore unstable

• Stoichiometric quantities of NADPH are required

• Many reactions suffer from low intrinsic reaction rates and inhibition

• Reactions require molecular oxygen

Recombinant CHMO (pQR239)

Antibiotic – AmpicillinInducer - Arabinose

BVMO Fermentation

0 1 2 3 4 5 6Time (h)

0

20

40

60

80

100

120

140

DO

T (%

), C

ER

, OU

R

0

200

400

600

800

1000

1200

1400

0

4

8

12

16

20

Agi

tatio

n sp

eed

(rpm

)

OD

670n

m

Arabinose induction

1. Choice of Catalyst Form

Does protease inhibition improve

stability

Free enzyme

Does the R-WC suffer reactant or product toxicity

Is the enzyme the cause of the inhibition

R-WC

Does the R-WC have high enough

activity

Low enough contaminating

activity

Is the low activity due to the cell

barrier

Does the R-WC have high enough

stability

Is a R-WC available

Permeabilise

Is the enzyme the cause of the low

stability

Does the WT-WC suffer reactant or product toxicity

Is the enzyme the cause of the inhibition

WT-WC

Does the WT-WC have high enough

activity

Low enough contaminating

activity

Is the low activity due to the cell

barrier

Does the WT-WC have high enough

stability

Is the pathogenicityof the WT-WC

acceptable

Permeabilise

Is the enzyme the cause of the low

stability

Database of enzymes with

known reactions and e.e.

Does the homogenate have

high enough activity

Low enough contaminating

activity

Free enzyme

Purification

Does free enzyme suffer reactant or product inhibition

Is homogenate stability high

enough

Immobilisedenzyme

Protein engineering

Reactant or product inhibition

Protein engineering

Protein engineering

Loss of activity on immobilisation

Catalyst selection software

Process options

air Fermentation+

BiocatalysisPurification

Fermentation Biocatalysis Purification

Fermentation Biocatalystpreparation Biocatalysis Purification

air

airsubstrate

substrate

substrate

air

Product

Product

Product

• Isolation procedure and losses

• Cost of immobilisation support• Number of recycles achievable• Diffusional limitations

(Thiele modulus / Damkohler number)

• Cofactor recycle

Isolated enzyme issues

Process Options

air Fermentation+

BiocatalysisPurification

Fermentation Biocatalysis Purificationair

airsubstrate

substrate

Product

Product

• Clean media for improved DSP

• Optimise production and use of catalyst

• Catalyst concentration independent of fermentation

• Effects on downstream process • Side reactions and over metabolism

• Access of substrate to the enzyme• Need for molecular oxygen • Toxic effects on the host cell

Whole cell catalyst issues

Oxidation activities of different ketones

Ketone Enzyme/Cell

Bicyclo[3.2.0]hept-2-en-6-one 1.7 4-methylcyclohexanone 1.42-hexylcyclopentanone 5.0

Enzyme Microb Technol (2003) 32 347

Oxidation of bicyclo[3.2.0]hept-2-en-6-one

Catalyst %X %ee g/l/h g/l g/gdcw

Cell 85 98 0.55 04.5 02.6 Enzyme 100 98 0.47 11.0 0.38

• Trade off between downstream process (g/l) and fermentation (g/gdcw)

• Potential to overcome fermentation limit by recycle of immobilised enzyme

Biotechnol Prog (2002) 18 1039, Biotech Bioeng (2002) 78 489

Scale-up issues

• Process intensity• Feed-rates of reactant / media• Rates of product removal• Robustness to cope with heterogeneity• Oxygen uptake

2. Oxygen Supply

Evaluation of Biocatalyst Kinetics and Stability

0

10

20

30

40

50

60

70

0 2 4 6

Ketone concentration (g.l-1)

Volu

met

ric a

ctiv

ity (U

.l-1)

OH

H

= 250 µL scale

= 1 L scale

BVMO catalysed lactone synthesis

[Doig et al (2002) Biotech. Bioeng., 80, 41]

Product formation during reaction

Time (min)

Pro

duct

–La

cton

e (g

/L)

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 50 100 150 200 250

Oxygen Supply

[Catalyst]

Rat

e Oxygen supply

Reaction

75L scale (10 g/L dcw)

0 1 2 3 4 5 6Time (h)

0

20

40

60

80

100

120

140

DO

T (%

)

0

200

400

600

800

1000

1200

1400

0

4

8

12

16

20

Agi

tatio

n sp

eed

(rpm

)

OD

670n

m

7 8 9 100

1

2

3

4

5

6

Ket

one

/ Lac

tone

(g/L

)

biomass CHMO reaction

75L scale (5 g/L dcw)

0 1 2 3 4 5 6Time (h)

0

20

40

60

80

100

120

140

DO

T (%

)

0

200

400

600

800

1000

1200

1400

0

4

8

12

16

20

Agi

tatio

n sp

eed

(rpm

)

OD

670n

m

0 1 2 30

1

2

3

4

5

6

Ket

one

/ Lac

tone

(g/L

)

biomass CHMO reaction

Biomass (g/L)

Process limitationsIn

itial

Pro

duct

ion

rate

(g/L

.h)

0

1.0

2.0

3.0

6.0

4.0

5.0

max specific activity(0.65g/g.h)

0 2 4 6 8 10 12

SF/1.5L

1.5LSF75L

1.5LSF75L

% Oxygen in air

Enriched air supplyIn

itial

Pro

duct

ion

rate

(g/L

.h)

0

1.0

2.0

3.0

6.0

4.0

5.0

0 20 40 60 80 100

Biomass (g/L)

Process limitationsIn

itial

Pro

duct

ion

rate

(g/L

.h)

0

1.0

2.0

3.0

6.0

4.0

5.0

max specific activity(0.65g/g.h)

0 2 4 6 8 10 12

SF/1.5L

1.5LSF75L

1.5L – 10%O2

1.5L – 21%O2

1.5L – 40%O2

1.5L – 60%O2

SF75L In

crea

se in

Oxy

gen

Whole Cell Reaction Model

Biomass concentration

Oxy

gen

dem

and

Metabolism

Metabolism+ Reaction

supplyO2

O2 limitation

O2 limitation

Time (min)

Productivity LimitationsOxygen limitation

(Rate)Biocatalyst lifespan

(Time)

Product Inhibition(concentration)

Pro

duct

–La

cton

e (g

/L)

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Biocatalyst concentration

Time (min)

Pro

duct

–La

cton

e (g

/L)

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 100 200 300 400 500

0.2 g/L

1.0 g/L

3.0 g/L5.0 g/L

4.5

7.0 g/L

[Cel

l]

3. Product Concentration

O

O

O

Resin Based Reactor Concept

air

VCYCLE

.

fixed bed of adsorbent

glycerol

biocatalyst/cell exchange

pH-sensorO -sensor2

1M H PO , 4M KOH3 4

Recycle Reactor with Fixed Bed

entry fermenter

outlet fermenter

Oxidation of bicyclo[3.2.0]hept-2-en-6-one

Catalyst %X %ee g/l/h g/l g/gdcw

Cell 85 98 0.55 04.5 02.6 Enzyme 100 98 0.47 11.0 0.38

Cell* 100 98 1.02 20.0 3.40

• Integration with ISPR is critical

4. Process Integration

Bioconversion time

O2 Stability

[Product]

Prod

uct c

once

ntra

tion

[Cell]

Effect of cell concentration

Catalyst Concentration

• Determines interaction with fermentation

• Determines what is limiting productivity in the reactor– catalyst preparation – conversion– downstream processing

Catalyst Preparation

Fermentation Dewater Biocatalysis

Fermentation

Fermentation

Dilution Biocatalysis

Biocatalysis

Concentration

Dilution

Direct

Catalyst Concentration Map

AB

[Catalyst] in reaction

[Catalyst]in

fermentation

ineffective

ineffective

ineffective

Expression

Dilution Concentration

Direct

A – Rate limitedB – Product / Catalyst stability limited

Process Drivers

Catalyst Production

Conversion Downstream Processing

Reaction profiles

AB

C

Time

[Product]

Process Metrics

Metric Cost

g/g catalyst productiong/l/h conversiong/l downstream process

Regime analysis

A B

[Catalyst]

Metric

Stability Product Ratelimited limited limited

Cg/g

g/l/hr

g/l

Process Mapping

Trans I Chem E C (2002) 80 51

CHMO Available….

Products available….

Productivity Targets

gproduct/L

g pro

duct/g

biom

ass

Optimise [Cell]and oxygen

100

10

1.0

0.10 5 10 15 20 25 30

ISPR

Optimise [Cell]and oxygen+ ISPR

Conclusions

• Recombinant Escherichia coli containing CHMO – 300 L scale• Conversion using CHMO – 200 L and 50 L scale / 1 Kg• Oxygen supply – modelled and understood limitations• Product inhibition – modelled and implementation of ISPR

• Scalable process using whole cell CHMO

Future

• Rapid methods of removing product

• Adequate means of oxygen supply

• Modelling for process analysis

• Isolation of product

• Improving stability of whole cells

Acknowledgements

• Jenny Littlechild, Exeter, UK• John Ward, UCL, UK• Dick Janssen, Groningen, NL• Marcel Wubbolts, DSM, NL

• Giacomo Carrea, CNR Milan, IT• Roland Wohlgemuth, Sigma-Aldrich Chemie, CH• Roger Cripps, Consultant, UK• Roland Furstoss, CNRS Marseille, FR

• European Commission• BIO4-CT98-00267• QLK3-CT01-00403

EC BVMO Programme