Process Optimization for R -PAC Production
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Transcript of Process Optimization for R -PAC Production
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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
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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
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PDC catalysed reactionsPDC catalysed reactions
PDC
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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
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Theoretical model developmentTheoretical model development
Full form
According to King and Altman (1956)
Simplified form
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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
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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
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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
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[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
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[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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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QuestionsQuestions