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Uncertainty Analysis of Penicillin VProduction Using Monte Carlo Simulation

Arno Biwer,
1
Steve Griffith,
1,2
Charles Cooney
1
1
Department of Chemical Engineering, Massachusetts Institute of Technology,Massachusetts
02139; telephone: 617-253-3108; fax: 617-258-6876;e-mail: ccooney
@
mit.edu
2
Light Pharma, Cambridge, Massachusetts 02139
Recei ved 8 July 2004; accepted 1 October 2004Published online 28 February 2005 in
Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20359
Abstract:
Uncertainty and variability affect economic andenvironmental performance in
the production of biotech-nology and pharmaceutical products. However, commer-cial
process simulation software typically provides analysisthat assumes deterministic rather than
stochastic processparameters and thus is not capable of dealing with
thecomplexities created by variance that arise in the decision-making process. Using the
production of penicillin V as acase study, this article shows how uncertainty can
bequantified and evaluated. The first step is construction ofa process model, as wel l as
analysi s of its cost structureand environmental impact. The second step is identifica-
t i onof uncer t ai nvar i abl es anddet er mi nat i onof t hei r pr oba - bility distributions
based on available process and literaturedata. Finally, Monte Carlo simulations are run to see
howthese uncertainties propagate through the model and af-fect key economic
and environmental outcomes.
Thus,theoverallvariati onoftheseobjectivefunctionsarequantified,the technical, su
pply chain, and market parameters thatcontribute most to the existing variance are
identified andthe differences between economic and ecological eval-uation are
analyzed. In our case study analysis, we showthat final penicillin and biomass concentrations
in the fer-menter have the highest contribution to variance for bothunit production cost and
environmental impact. The pen-
icill in selling pri ce dominates return on investment vari-ance as well as the variance
for other revenue-dependentparameters.
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2004 Wiley Periodicals, Inc.
Keywords:
Mont e Car l o s i mul at i on; uncer t ai nt y; var i -
ability; penicil lin; economic assessment; environmental assessment
INTRODUCTION
Commercial process simulation software usually
providesanal ys i s t hat as s umes det er mi ni s t i c pr oces s par amet er s . The
software does not consider existing variati ons of tech-ni cal , s uppl y chai n,
and mar ket par amet er s t hat can s i g-
ni f i cant l y al t er oper at i ng deci s i ons and bat ch- t o- bat chexpectations.
However, an understanding of these param-
et er s and t hei r uncer t ai nt y i s es s ent i al f or t he economi cs ucces s of a
pr oces s des i gn and t he anal ys i s of exi s t i ngprocesses. Thus, a methodology
is needed that combinesstandard process simulation software with uncertainty anal-ysis.
In this article, we use a well-characterized process, theproduction of penicill in, to
illustrate how this goal mightbe accomplished.Penicillins produced by
Penicillium chrysogenum
arestill among the most important antibiotics. Peni cill ins be-long to a family of
hydrophobic
h
-lactams. Each
containsa d i f f e r e n t a c y l s i d e c h a i n a t t a c h e d b y a n a mi d e l i n k -
a g e t o t h e a mi n o g r o u p o f t h e p e n i c i l l i n n u c l e u s , t h e 6-
ami nopeni ci l l ani c aci d. Peni ci l l i n G and peni ci l l i n Va r e t h e ma i n c o m
me r c i a l p e n i c i l l i n s . Mo s t o f t h e p e n -
i ci l l i n V ( phenoxymet hyl peni ci l l i n) i s conver t ed t o 6-
ami nopeni ci l l ani c aci d ( 6-
APA) , whi ch i n t ur n i s us edt o make amoxi ci l l i n and ampi ci l l i n ( McCoy, 2
000) . I na d d i t i o n , p e n i c i l l i n V i s u s e d d i r e c t l y a s a n a n t i b i o t i c (
f
1 , 6 0 0 t o n s p e r y e a r ) ( V a n Ni s t e l r o o i j e t a l . , 1 9 9 8 ) a n d r a n k s a mo
n g t h e 1 0 0 t o p p r e s c r i b e d d r u g s i n t h e United States (American
Druggist, http://www.rxl ist.com/ top200a.htm, May 2004).
h
-
L a c t a m a n t i b i o t i c s a m o u n t t o a b o u t 6 0 % o f t h e wor l dwi de an
t i bi ot i cs mar ket ; t hi s was appr oxi mat el y $5 billion per year in sales in 1999
(Demain and Elander,1999). The global demand for
h
-lactams grows by around2% annually, mainly because of rising demand
in countriess u c h a s C h i n a a n d I n d i a ( Mi l mo , 2 0 0 3 ) . L o we ( 2 0 0 1 ) e s t
i ma t e s t h a t t h e wo r l d p r o d u c t i o n o f p e n i c i l l i n wa s 6 5 , 0 0 0 t o n s i
n 2 0 0 1 . A s a r e s u l t o f l a r g e o v e r c a p a c i t y in the market, penicillin
prices have been under continu-ous pressure for several years. Prices have
fallen signifi-cantl y during the last several years from $20/billion units( BU)
dur i ng t he mi d- 1990s t o $12/ BU i n 1997 t o $9/ BUi n 2 0 0 0 ( Mc C o y ,
2 0 0 0 ) . A s o f 2 0 0 3 , t h e p r i c e o f p e n i - cillin G was approximatel y $11/BU,
which is $17–18/kg(Milmo, 2003).Improvements in the penicillin production process
resultprimarily from genetic-based strain i mprovements,
whilet h e p r o c e s s f l o w s h e e t h a s c h a n g e d v e r y l i t t l e ( V a
n Nistelrooij et al., 1998). Although some i mprovement
hasbeen r eal i z ed f r om r ef i nement i n oper at i ng condi t i ons ,
B
2004 Wiley Periodicals, Inc.
Correspondence to:
C. CooneyThis article includes supplementary material available via the Internet
athttp://www.interscience.wiley.com/jpages/0006-3592/suppmat.

these changes are often difficult to observe because vari-ance i n the overall
process masks smal l i mprovements inpr oduct i on. As s uch, i t t akes many
mor e exper i ment s or production runs to statistical ly verify the impact of a
par-ticular process change. In the present work, we assess howvariance in strain
and process parameters affects key eco-nomic and environmental impact metrics.
Because environ-mental concerns have become increasingly important,
weinclude an environmental evaluation in our analysi s.Using a process model for
penicillin V, Monte Carlo sim-ulations are performed to investigate the effect of
param-eter uncertainty on overall process performance. Thereby,we us e a new
appr oach t hat pr ovi des a gener al met hod-
o l o g y f o r c o mb i n i n g p r o c e s s s i mu l a t i o n s o f t wa r e a n d spreadsheet
modeling to conduct high-leverage
uncertaintya n a l y s i s . T h i s o f f e r s a f u n d a me n t a l b a s i s f o r d e c i s i o n ma
king in the design and analysis of bioprocesses.
MATERIALS AND METHODS
The pr oces s model f or peni ci l l i n V pr oduct i on was bui l t using the process
simulator SuperPro Designer version 5.1(Intelligen, Scotch Plain, NJ), whi ch
provides the material balance and key economic parameters of the process.
Toper f or m t he Mont e Car l o s i mul at i ons , key par t s of t hemodel were
transferred to Microsoft Excel and
analyzedv i a Mo n t e C a r l o s i mu l a t i o n , u s i n g C r y s t a l B a l l 2 0 0 0 (Decisio
neering, Denver, CO). Crystal Ball is an ‘‘Add-in’’f or MS Excel t hat enabl es t he
def i ni t i on of t he pr obabi l - ity distributions of stochastic variables, generates
randomnumber s bas ed on t hes e di s t r i but i ons , and s t or es t he r e- sults of
MS Excel cal culations for each trial. Monte Carlos i mul at i ons wi t h 100, 000
t r i al s t ake ar ound 20 mi n ( PC: Pentium III processor, 512 MB RAM). Each run
requiresar ound 5 MB di s c s pace. We not e, however , t hat a
goode s t i ma t e o f t h e s a mp l i n g d i s t r i b u t i o n o f t h e me a n f o r primary
forecast vari ables can often be achieved with thedefault Crystal Ball setting of
1,000
trials.A l l S u p e r P r o m o d e l p a r t s t h a t a r e a f f e c t e d b y t h e uncer
tain parameters were transferred to MS Excel. Sincemost computations in
SuperPro can also be done in spread-
s heet cal cul at i ons , t hi s t r ans f er i s pos s i bl e, but i t i s t hemos t t i me
cons umi ng par t and has a cer t ai n r i s k of t r an- scription errors. Therefore,
it is currently necessary to vali-date the constructed base case spreadsheet
model
againstS u p e r P r o r e s u l t s t o e n s u r e t h a t a l l i n p u t s a r e c o r r e c t . Furth
er work is necessary to develop a direct linkage be-tween the si mulation
software and the Monte Carlo simu-lation tool.For the environmental
assessment, a method developedby Bi wer and Hei nz l e ( 2004) i s us ed. I n
t hi s met hod, awei ght i ng f act or i s cal cul at ed f or ever y i nput and
out put component representing the environmental relevance of thecompound.
These environmental factors (EF) are multi-plied by the amount of the
compound in the mass balancet o obt ai n t he envi r onment al i ndex ( EI ) . The
s um of al l input, respectively, output components gives the EIs of theprocess.
These indices, li ke the economic indicators, rep-
r es ent one of s ever al pos s i bl e i ndi cat or s t o des cr i be t heenvironmental p
erformance of a process. For the economi cevaluation, generall y accepted
indicators are used
and theirdef i ni t i ons can be f ound i n appr opr i at e t ext books ( e. g. , Peters
et al., 2003).
MODELING BASE CASEFermentation Model
I n commer ci al pr oces s es , peni ci l l i n V i s pr oduced as afed-batch
fermentation (Ohno et al., 2002). Regardless of whether a penicil lin producer
uses its own unique strain orone acquired from a common club, fermentation
conditionsand downstream steps are establi shed that are optimal fort he
pr oducer ’ s s t r ai n and f i t wi t hi n t he cont ext of a par - ticular facility.
However, most processes follow a similarstructure and variance is introduced
from operating con-di t i ons . A t ypi cal medi um i s compos ed of gl ucos e,
cor ns t eep l i quor , or anot her compl ex s our ce ( f or ot her pos s i - ble
sources, see Lowe, 2001), mineral salts, and phenoxy-acet i c aci d as a
pr ecur s or f or peni ci l l i n V ( Demai n andEl ander , 1999; Van Ni s t el r ooi j et
al . , 1998; Per r y et al . , 1997).
P. chrysogenum
has difficulty synthesi zing the phe-nol i c s i de chai n f or peni ci l l i n and
phenoxyacet i c aci d i s added continuously to the culture
medium.Peni ci l l i ns ar e s econdar y met abol i t es , gener al l y pr o- duced at low
growth rates (Strohl, 1999). Penicillin synthe-sis starts from three activated amino acids,
involves
severale n z y me s a n d i s o p e n i c i l l i n N a s a ma j o r i n t e r me d i a t e (Strohl,
1999). More details about the penicillin synthesiscan be f ound i n Par adkar et
al . ( 1997) and St r ohl ( 1997) . Key operating parameters requiring optimization
are tem-perature, pH, dissolved oxygen, and assimilable
nitrogen,pr ecur s or , r educi ng s ugar s , and bi omas s concent r at i ons (Van
Nistelrooij et al., 1998).I n t he pr es ent s t udy, we us e a s i mpl i f i ed
f er ment at i onmodel t o des cr i be t he dependence of f i nal pr oduct andbio
mass concentrations on the cell yield and maintenancecoefficient and the specific
product formation rate and yieldcoefficient. The values for the model parameters
(Table I)ar e der i ved f r om a combi nat i on of l i t er at ur e and
pr oces s dat a. Two f er ment at i on s t ages ( gr owt h and pr oduct i onphas e)
ar e as s umed, al t hough i n s ome of t oday’ s hi ghl yproductive fermentations,
such a separation no longer exists(Lowe, 2001). The first (primary) phase lasts about
50 h,and during this time mainly biomass i s produced in a batchculture. After the
biomass formation slows down, penicillinV is produced in the secondary phase (106
h). During theproduction phase, glucose i s fed continuously.
Process Model
The production process model for penicilli n V is based ont he avai l abl e
l i t er at ur e ( Ohno et al . , 2002; Per r y et al . ,
1 6 8 B I OT E C HNOL OGY A ND B I OE NGI NE E R I NG, V OL . 9 0 , NO. 2 , A P R
I L 2 0 , 2 0 0 5

1997; Lowe, 2001; Van Ni s t el r ooi j et al . , 1998) . As penBat ch Pl us and I n
t el l i gen’ s Super Pr o Des i gner wer e t hesoftware packages considered for the
implementation of theprocess model . Although both packages are robust simu-
lation tools, SuperPro Designer was chosen based on theintuitive relationship
between its process representationa n d t h e s p r e a d s h e e t m o d e l t h a t
w a s c o n s t r u c t e d f o r Monte Carlo analysi s.Fi gur e 1 s hows t he pr oces s
f l ow di agr am cr eat ed wi t hthe software SuperPro Designer. Fermenters with a
totalcapacity of 40–200 m
3
are used for production (Ohno et al.,2002; Lowe, 2001; Falbe and Regnitz, 1999;
Perry et al.,1997). We chose a facility with 11 fermenters, each with avolume of
100 m
3
, optimizing the usage of the downstreamequipment. Penicillin V sodium salt is
the final
product.The medi a component s ( phar mamedi a, t r ace met al s , phenoxyacet
ate; S-102 to S-104) are blended in tank P-1and sterilized in the continuous
heat sterilizer P-4. The glu-cose solution is prepared in tank P-2. Medium and
glucoses ol ut i on ar e f ed t o t he f er ment er P- 7 ( gl ucos e s ol ut i on i s fed
continuously only during the production phase). The air(S-113) is compressed (P-5) and
filter sterilized (P-6). Theexhaust air, containing mainly carbon dioxide, is
filtered(P-8) to prevent release of by-products to the environment.
Figure 1.
Process flow diagram of the penicillin V production model (SuperPro Designer, version 5.1).
Table I.
Parameter values of the fermentation model of penicillin V
production.P a r a m e t e r V a l u e Y i e l d c o
e f f i c i e n t s V a l u e
t
exp
(time of exponential growth) (h)50
Y
X/pharmamedia
( g / g ) 2 . 1 4
t
prod
( t i m e o f p r o d u c t i o n ) ( h ) 1 0 6
Y
X/gluc.
( g / g ) 0 . 4 5
X
f
(biomass concentration at t
exp
) ( g / L ) 3 0
Y
pen./gluc.
( g / g ) 0 . 8 1
X
nl
( f i n a l b i o m a s s c o n c e n t r a t i o n ) ( g / L ) 4 5
Y
pen./phenoxyacetic acid
( g / g ) 2 . 0 0
V
in
( i n i t i a l v o l u m e ) ( L ) 5 5 , 0 0 0
Y
X/O2
( g / g ) 1 . 5 6
V
final
( f i n a l v o l u m e ) ( L ) 7 5 , 0 0 0
m
gluc.
(maintenance coefficient)(g glu./g dcw h)0.022
P
final
( f i n a l p r o d u c t c o n c e n t r a t i o n ) ( g / L ) 6 3 . 3
m
O2
(maintenance coefficient)(g/g dcw h)0.023
B I WE R E T A L . : UNC E R T A I NT Y A NA L Y S I S P E NI C I L L I N P R OD UC T I ON
1 6 9


In the agitated fermenter, biomass and penicillin V are pro-duced consuming the
carbon sources, the precursor, and themineral salts.After the fermentation, the
fermenter content is fed to aharvest tank (P-
9).A t y p i c a l d o w n s t r e a m p r o c e s s i s d i v i d e d i n t o t h e following
unit processes: biomass removal, extraction, re-extraction, and crystallization,
filtration and crystal washingand dr yi ng ( Van Ni s t el r ooi j et al . , 1998) . The
f er ment a- t i on br ot h f l ows t o t he r ot ar y vacuum f i l t er P- 20, wher ewash
water (S-150) is used to recover product for the re-tained biomass. The retained
fungal biomass is di scharged(S-151).Bef or e ext r act i on, t he cel l -
f r ee br ot h i s aci di f i ed t o apH of
f
3 in P-22, using sulfuric acid (S-154) and
cooledt o mi ni mi z e degr adat i on dur i ng aci d ext r act i on. I n t hecentrifugal
extraction step (P-23), the penicillin is trans-f er r ed i nt o t he or gani c phas e
( but yl acet at e, S- 157) . Theremaining aqueous solution is discharged and neutralized
inP - 2 4 wi t h s o d i u m h y d r o x i d e ( 1 0 % w/ w, S - 1 5 9 ) . T h e penicillin is re-
extracted (P-25) into acetone/water (S-162),wher e s odi um acet at e i s added ( S-
163) . The s odi um s al t of penicillin V then precipitates. The crystals (S-165)
areseparated and washed in the basket centrifugation (P-26)and conveyed to
the fluid bed dryer (P-31). The remainingwashing solution is discharged (S-173).
The solution sep-ar at ed i n t he cent r i f uge ( S- 168) i s l ead t o P-
27, wher emos t of t he but yl acet at e i s s pl i t of f i n a r ecycl i ng
s t ep( n o t s h o wn i n d e t a i l ) . T h e r e s t i s d i s c h a r g e d a n d n e u -
t r a l i z e d i n P - 2 8 ( Na OH, 1 0 % w/ w; S - 1 7 0 ) . T h e b u t y l acet at e
i s r eus ed i n t he ext r act i on. I n P- 29, f r es h but yl acetate is added (S-156). In
the dryer (P-31), the penicillini s dr i ed wi t h ai r ( S- 175) and t he f i nal
pr oduct s t or ed i ntank P-32.
EVALUATION BASE CASE
A pr oces s s i mul at i on was r un as a bas e cas e t o
es t abl i s ha r ef er ence poi nt f or bot h economi c and envi r onment al assessm
ent.
Base Case Analysis
The aver age pr oduct i on r at e f r om t he f aci l i t y i s appr ox- i mat el y
263 kg peni ci l l i n V s odi um s al t per hour . Thi s results in an annual
production of 2,090 tons with the as-
s umpt i on of 330 oper at i ng days . The i ni t i al f er ment er v o l u m e i s 5 5
m
3
a n d 2 0 m
3
ar e added as nut r i ent andprecursor feeds (36%). The volume added in the
model iswithin the range given by Lowe (2001). Annual productioni s 5 4 6
b a t c h e s a n d i t i s a s s u me d t h a t 1 6 f a i l ( 3 %) . T h e over al l
yi el d of t he f er ment at i on i s 0. 21 g peni ci l l i n/ ggl ucos e. The yi el d acr os s
downs t r eam r ecover y i s 90%. The carbon balance shows that around 25% of
the C-atomsar e conver t ed t o peni ci l l i n, 17% t o bi omas s , and 60%
t ocarbon dioxide.Table II presents the summary material bal ance for
thebas e cas e pr oces s . Al t oget her , 7, 880 kg/ h r aw mat er i al s are needed,
which is 30 kg per kg final product (kg/kg P).The input includes a number of
materials that are typical f or f er ment at i on pr oces s es : a hi gh amount of
wat er , gl u- cos e as car bon s our ce, oxygen, medi a, and t r ace
met al s . Speci f i c t o t he peni ci l l i n pr oduct i on i s t he demand f or phenoxyac
etic acid. Furthermore, relevant amounts of thesolvents butyl acetate and
acetone are needed for extrac-t i on, and a s mal l er amount of s odi um
acet at e t hat f or ms the final product with the penicillin is needed in the crys-
tallization step.Besides the product, the fermentation output consists
of l ar ge amount s of car bon di oxi de and bi omas s . Fur t her -
mor e, s i gni f i cant amount s of unus ed r aw mat er i al s andunrecovered product
leave the process. This model assumesan 80% r ecycl i ng of but yl acet at e ( s ee
al s o Chang et al . , 2002). Acetone (S-167, S-173) is also recycled (70%)
(notshown in Fig.
1).T h e p r o c e s s c o n s u m e s 4 1 G W h e l e c t r i c a l p o w e r (20
kWh/kg P); 4,400 tons steam (2.1 kg/kg P); 6.4 millionm
3
chilled water (3.1 m
3
/ kg P) , and 3 mi l l i on m
3
coolingwat er ( 1. 4 m
3
/ kg P) . The compr es s or and t he f er ment er consume 90% of the electrical
energy required. The ster-i l i z at i on pr oces s ( P-
4) i s t he mai n cons umer of s t eam, al t hough s ome s t eam i s al s o r equi r ed
f or dr yi ng. Chi l l edwat er i s us ed mai nl y i n t he f er ment er and
t he s t er i l i z a-
t i o n s t e p ; a d d i t i o n a l c o o l i n g wa t e r i s u s e d i n t h e c o m- pr es s or P-
5. I n t he ext r act i on s t ep, f r eon i s us ed as heat t r ans f er agent . The
ener gy demand f or t he r ecycl i ng of t he f r eon i s added t o t he el ect r i ci t y
demand. The
r es ul t s o f t h e e n e r g y a n a l y s i s a r e c o n s i s t e n t wi t h Oh n o e t a l . (200
2), who state the energy requirement per kg product
Table II.
Material balance of the model of the penicillin V
production.*C o m p o n e n t I n p u t [ k g / k g P ] O u t p u t [
k g / k g P ] A c e t i c a c
i d —
0 . 1 6 A c e t o n
e 0 . 1 2 0 . 1 2 B
i o m a s s ( d c w ) —
0 . 8 6 B u t y l a c e t
a t e 0 . 2 8 0 . 2 8 C a r
b o n d i o x i d e —
5 . 3 1 G l u c o s e
4 . 9 5 0 . 1 0 O
x y g e n 2
. 5 —
P e n i c i l l i n V ( l o s s )

0 . 1 0 P e n i c i l l i n V s o d i u
m s a l t —
1 . 0 0 P h a r m a m e d i a
0 . 4 6 0 . 0 6 P h e n o x y a c
e t i c a c i d 0 . 5 8 0 . 0 1 S o d
i u m a c e t a t e 0 . 2 3
0 . 0 1 S u l f u r i c a c i
d 0 . 0 5 0 . 0 5 T r a c e
m e t a l s 0 . 6 7 0 .
0 9 S o d i u m h y d r o x i d e
0 . 1 2 0 . 1 2 W a t e r
2 0 . 0 2 1 . 8
T o t a l 3 0 .
0 3 0 . 0 *The recycling of butyl acetate and
acetone is already considered. Fromthe amount of air transported through the
fermenter, only the amount of oxygen consumed is compiled in (kg/kg P) = kg
component per kg finalproduct; final product = penicillin V sodium salt; dcw =
dry cell weight.
1 7 0 B I OT E C HNOL OGY A ND B I OE NGI NE E R I NG, V OL . 9 0 , NO. 2 , A P