Process Optimization for Process Optimization for RR-PAC -PAC ProductionProduction

N. LeksawasdiN. Leksawasdi11, ,

M. BreuerM. Breuer22, B. Hauer, B. Hauer22, , P.L. RogersP.L. Rogers11, B. Rosche, B. Rosche11

11BABS, UNSW, Sydney, NSW, 2052, AustraliaBABS, UNSW, Sydney, NSW, 2052, Australia

22BASF-AG, 67056 Ludwigshafen, GermanyBASF-AG, 67056 Ludwigshafen, Germany

What is R-PACWhat is R-PAC

R-PAC is for R-Phenyl-Acetyl-Carbinol

Precursor for production of ephedrine & pseudoephedrine; used to treat asthma and flu symptoms

O

O

CH3

H

PDC catalysed reactionsPDC catalysed reactions

PDC

Process of Process of modelmodel development developmentTheoretical for general model structure

Experimental for model structure modification & evaluation of constants

Combined theoretical & experimental

Confirmation of model by independent batch biotranformation profile

Optimization by designing feeding profile for fed batch system

Theoretical model developmentTheoretical model development

Full form

According to King and Altman (1956)

Simplified form

Theoretical modelTheoretical model

ii

iii

i Bkk

Akkkk

k

EBAkkkk

dt

Pd

5

4

32

32

1

32

32

1

ii dt

Pd

dt

Bd

iiii dt

Rd

dt

Qd

dt

Pd

dt

Ad2

iiiri

EAQVdt

Rd iiiriiq

i

EAQVEAVdt

Qd

Product

Reactants

By-products

Experimental model developmentExperimental model development

Enzyme activity

Substrate concentrations

Enzyme deactivation effect

Batch biotransformations for Overall rate of R-PAC formation

Rate constants of by-products formation

Quantification of kinetics

Enzyme activity effectEnzyme activity effect

y = 0.5508x

R2 = 0.9976

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 1 2 3 4 5 6 7 8 9 10

Enzyme activity (U/ml)

Init

ial r

ate

(mM

per

min

)

0

30

60

90

120

150

180

210

240

Init

ial r

ate

(mM

per

hr)

ii

Edt

Pd

[Benzaldehyde] effect[Benzaldehyde] effect

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 20 40 60 80 100 120 140 160

[Benzaldehyde] (mM)

Init

ial r

ate

(mM

per

min

)

0

12

24

36

48

60

72

84

96

108

Init

ial r

ate

(mM

per

hr)

R2 = 0.9963

hib

hib

i BK

BK

dt

Pd

1

Monod-Wyman-Changeux (MWC) Model

Kb = 1 x 10-4 mM-1.34 h = 2.34

[Pyruvate] effect[Pyruvate] effect

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 30 60 90 120 150 180 210 240 270

[Pyruvate] (mM)

Init

ial

rate

(m

M p

er m

in)

0

12

24

36

48

60

72

84

96

108

Init

ial

rate

(m

M p

er h

r)

Michaelis – Menten kinetics Model

R2 = 0.9973

im

i

i AK

A

dt

Pd

Km = 10.6 mM

Enzyme deactivation effectEnzyme deactivation effect

0

20

40

60

80

100

120

0 20 40 60 80 100 120 140

Time (hr)

Rel

ativ

e en

zym

e ac

tivi

ty (%

)

0 mM

60 mM Bz

0

20

40

60

80

100

120

0 20 40 60 80 100 120 140

Time (h)

Rel

ativ

e en

zym

e ac

tivi

ty (

%)

Enzyme deactivation by benzaldehydeEnzyme deactivation by benzaldehyde

R2 = 0.9827

0 mM

200 mM

Enzyme deactivation effectEnzyme deactivation effect

mMBttEBkK

mMBttEK

tt

dt

Ed

ilagiidd

ilagid

lag

i 20010,;.

100,;

;0

21

1

Kd1 = 2.64 x 10-3 h-1

kd2 = 1.98 x 10-4 mM -1 h-1

tlag = 5.23 h

Overall rate constant & Overall rate constant & by-product rate constants determinationby-product rate constants determination

150 mM Bz150 mM Bz50 mM Bz50 mM Bz

Independent

prediction and

confirmation

k2, Vq, Vr

100 mM Bz100 mM Bz

Batch biotransformationBatch biotransformation

R2 = 0.9857

50 mM Bz ; 60 mM Pyr

0

16

32

48

64

80

0 1 2 3 4 5 6 7 8 9 10Time (h)

Co

nce

ntr

atio

n (

mM

)

0.0

0.8

1.6

2.4

3.2

4.0

En

zym

e ac

tivi

ty (

U/m

l)

[Pyruvate] [Benzaldehyde][Acetaldehyde] [Acetoin][R-PAC] Enzyme Activity

Batch biotransformationBatch biotransformation

R2 = 0.9981

150 mM Bz ; 180 mM Pyr

0

40

80

120

160

200

0 1 2 3 4 5 6 7 8 9Time (h)

Co

nc

en

tra

tio

n

(mM

)

0.0

0.8

1.6

2.4

3.2

4.0

En

zym

e

ac

tiv

ity

(U

/ml)

[Pyruvate] [Benzaldehyde]

[Acetaldehyde] [Acetoin][R-PAC] Enzyme Activity

Overall & by-products rate constantsOverall & by-products rate constants

Overall rate constant (k2 ) = 24.8 mol h-1 U-1

Acetaldehyde rate constant (Vq ) = 0.0156 h-1 (U/ml) -1

Acetoin rate constant (Vr ) = 0.00251 h-1 (U/ml) -1 mM-1

Theoretical & experimental modelTheoretical & experimental model

iim

ih

ib

hib

i

EAK

A

BK

BKk

dt

Pd

1

2

mMBttEBkK

mMBttEK

tt

dt

Ed

ilagiidd

ilagid

lag

i 20010,;.

100,;

;0

21

1

ii dt

Pd

dt

Bd

iiii dt

Rd

dt

Qd

dt

Pd

dt

Ad2

iiiriiqi

EAQVEAVdt

Qd

iiiri

EAQVdt

Rd

Simulation of biotransformationSimulation of biotransformation 100 mM Bz ; 120 mM Pyr

0

24

48

72

96

120

0 1 2 3 4 5 6 7 8 9Time (hr)

Co

nce

ntr

atio

n (

mM

)

0.0

0.7

1.4

2.1

2.8

3.5

En

zym

e ac

tivi

ty (

U/m

l)

.

[Pyruvate] [Benzaldehyde][Acetaldehyde] [Acetoin][R-PAC] Enzyme Activity

Confirmation of simulationConfirmation of simulation

R2 = 0.9953

100 mM Bz ; 120 mM Pyr

0

24

48

72

96

120

0 1 2 3 4 5 6 7 8 9Time (hr)

Co

nc

en

tra

tio

n (

mM

)

0.0

0.7

1.4

2.1

2.8

3.5

En

zym

e a

cti

vity

(U

/ml)

.

[Pyruvate] [Benzaldehyde][Acetaldehyde] [Acetoin][R-PAC] Enzyme Activity

Suggestion of substrates level to be maintained for optimum R-PAC production

Pulse feeding can be designed to achieve optimum R-PAC production

Prediction of fed-batch biotransformation profile

Model application in fed-batch systemModel application in fed-batch system

Simulation for prediction of optimum Simulation for prediction of optimum substrate levelsubstrate level

Hourly feed

1.2 Pyr/Bz

4.0 U/ml PDC

Initial Volume 1.00 L

0

50

100

150

200

250

300

350

0 15 30 45 60 75 90 105 120 135 150

Maintained Bz (mM)

R-P

AC

fo

rme

d, g

;

R-P

AC

pro

du

cti

vit

y, g

/da

y

.

0

20

40

60

80

100

120

140

Tim

e w

he

n r

em

na

nt

PD

C

.

is a

t 0

.5 U

/ml,

hr

.

R-PAC formed, gR-PAC productivity, g / dayTime when remnant PDC is at 0.5 U/ml, hr

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0 4 8 12 16 20 24 28 32 36 40 44 48 52

Time (hr)

Vo

lum

e P

yr a

dd

ed (

ml)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Vo

lum

e B

Z a

dd

ed

(m

l)Feeding profile for 90 mM Bz, 108 mM PyrFeeding profile for 90 mM Bz, 108 mM Pyr

Hourly feed

10.3 M Bz

Hourly feed

1.4 M Pyr

Initial Volume 1.00 L

0

100

200

300

400

500

600

700

0 6 12 18 24 30 36 42 48 54

Time (hr)

Co

nc

en

tra

tio

n (

mM

)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

En

zym

e a

cti

vit

y (

U/m

l)

R-PAC PyruvateAcetaldehyde AcetoinBenzaldehyde Enzyme activity

Predictive fed-batch profilePredictive fed-batch profile

Initial Volume 1.00 L

Final Volume 3.39 L

89 mg/U

ConclusionsConclusions Model provides good prediction for batch

biotransformation system

Model suggests substrate levels in the range of 90 mM Bz & 108 mM Pyr to be maintained in fed-batch system

Potential for 8-fold higher R-PAC per U than in batch system but verification by experiment is necessary

Note : Possible additional effects of inhibition (high R-PAC, acetaldehyde conc.) and inactivation (benzaldehyde droplets) may need to be considered

Professor Peter L. Rogers, Dr. Bettina Rosche

Dr. Russell Cail & Malcolm Noble

Wolfgang Nittel, Sue Jackson

Dr. Vanessa Sandford

Martin Zarka & Tony Gellert

Dr. Christopher Marquis

Mallika Boonmee, Alan Rushby

Royal Thai Government, BASF-AG

Lia, Allen, Cindy, Onn, Ronachai, Apple

AcknowledgementsAcknowledgements

QuestionsQuestions